ABSTRACT PURIFICATION AND ROLE OF THE INDUCIBLE SOLUBLE PROTEIN COMPONENT OF THE PHOSPHOENOLPYRUVATE:D-FRUCTOSE l-PHOSPHOTRANSFERASE SYSTEM OF AEROBACTER AEROGENES By Richard Webb Walter, Jr. An inducible soluble protein, involved in phos- phoenolpyruvate (PEP)-dependent phosphorylation of D- fructose, was purified to homogeneity by 100,000 x g, centrifugation, differential ammonium sulfate precipi- tation, chromatography by DEAE cellulose, Sephadex G100, hydroxylapatite columns, and polyacrylamide disc gel electrophoresis. This protein was termed a D-fructose phOSphoryl transfer protein (PTPfru). The molecular weight of PTPfru was determined to be 52,000 by Sephadex 6100 chromatography and the protein contains two 26,000 molecular weight subunits as determined by SDS poly- acrylamide disc gel electrophoresis. PTPfru is required for growth of Aerobacter aerogenes PRL-R3 on low con- centrations of D-fructose. PTPfru significantly increased only the activity of the enzymes II (membrane-bound component of the PEP: D-fructose 1-phosphotransferase system) obtained Richard Webb Walter, Jr. from D-fructose-grown cells when it was added to various enzymes 11 obtained from cells grown on a variety of substrates. Only 100,000 x‘g supernatants obtained from D-fructose-grown cells activated enzyme IIfru' Thus, D-fructose specifically induces both an enzyme II and a soluble component, PTPfru’ which function in a PEPzD- fructose l-phosphotransferase system. This system has 5 M) for D-fructose and does not a low Km (1.6 x 10- require HPr for activity. A second system that does require HPr for activity is constitutive and has a high Km (7.1 x 10"3 M) for D-fructose. Assays were developed to determine the individual enzyme II activities in the presence of each other. The amount of the inducible enzyme II is from 5 to 20 percent of the total activity in enzyme II preparations obtained from cells grown on substrates other than D-fructose, whereas it comprises from 50 to 90 percent of the total activity in enzyme II preparations obtained from cells grown on D-fructose. The inducible enzyme IIfr is U activated by 2-mercaptoethanol. [32 L-malate and was identified by paper chromatography in P] PEP was formed enzymatically from 32P1 and two solvent systems and by enzymatic reactions involving the formation of [32F] ATP and D-fructose l-[32P] phOSphate. A phOSphoryl transfer from [32F] PEP to PTPfru was catalyzed by enzyme I and did not require HPr. The Richard Webb Walter, Jr. 32P bound per [32F] phOSpho-PTPfru formed had two moles mole of 52,000 molecular weight protein, or one mole bound per monomer of 26,000 molecular weight. Enzyme IIfru catalyzed the phosphoryl transfer from [32F] phospho-PTPfru to D-fructose, forming D-fructose l- [32F] phOSphate. Although HPr is not required either for D- fructose phOSphorylation by the inducible system or for phOSphorylation of PTPfru by enzyme I, it does affect the activity of the inducible system. When PTPfru is limiting, HPr increases the velocity of this system; however, at saturating levels of PTPfru the apparent Vmax is decreased by HPr. The RA for HPr of the indu- cible system is 1/50 the Km of the constitutive system for HPr. Increasing the concentration of PTPfru does not affect the apparent KA’ whereas increasing HPr decreases the apparent Km for PTPfru' PURIFICATION AND ROLE OF THE INDUCIBLE SOLUBLE PROTEIN COMPONENT OF THE PHOSPHOENOLPYRUVATE:D-FRUCTOSE l-PHOSPHOTRANSFERASE SYSTEM OF AEROBACTER AEROGENES By Richard Webb Walter, Jr. A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1972 Q‘- ACKNOWLEDGMENTS I would like to thank Dr. R. L. Anderson for his guidance throughout the period during which I worked on this problem. I also want to thank all those who have provided stimulating and fruitful discussions, in particular, T. Hanson, N. Kelker, and J. Markwell. Special appreciation goes to my wife, Christine, who not only endured my "ups and downs" but also helped tremendously in every way possible during the final months of research and in the preparation of this dissertation. Financial support through an NDEA fellowship and departmental assistantships has been very helpful. ii LIST OF TABLES LIST OF FIGURES. ABBREVIATIONS. INTRODUCTION . LITERATURE REVIEW. TABLE OF CONTENTS EXPERIMENTAL METHODS Bacterial Strains Media . Mineral Medium . . . Nutrient Broth Medium. Nutrient Agar Medium . MacConkey Agar Medium. Growth of Cultures. Preparation of Preparation of Preparation Preparation Preparation Preparation Cell Extracts. Enzymes. of Enzyme II of Enzyme I. of HPr . . of D- Fructose 1- -Phosphate Kinase General Assay Procedures. Substrate Assays. Assay for D- Fructose l-PhOSphate Assay for D-Fructose . Assay for D- Fructose 6- -Ph03phate . Assay for DwGlucose l, 6— —Diphosphate. Assay for PhOSphoenolpyruvate. Enzyme Assays Assay for Aldolase . . . Assay for D-Fructose 1- -Phosphate Kinase. Assay for Malate Dehydrogenase Assay for DbGlucose 6- Phosphate Dehydrogenase. iii Page vii ix xii Page Assay for Hemoglobin . . . . . 24 Assay for Enzyme I (D-mannitol continuous) . . 24 Assay for HPr (D- mannitol continuous). . . . 26 General Assay for PEP- Dependent D- Fructose 1- -Phosphate Formation . . . . . . . . . . . 26 a. Assay for Enzyme I. . . . . . . . . . . 29 b. Assay for HPr . . . . . . . . . . . . 29 c. Assays for Enzymes II . . . . . 30 d. Assays for D- Fructose Phosphoryl Transfer Protein (PTPfr u). . . . . . 30 Preparation of [32F] PhOSphoenolpyruvate. . . . . 34 Isolation of Chicken Liver Mitochondria. . . . 34 Synthesis of [32F] Phosphoenolpyruvate . . . . 35 Characterization of [32F] Phosphoenolpyruvate . . 37 Assay for Formation of [32F] ATP from [32P] Phosphoenolpyruvate . . . . . . . . . . . . 37 Other Analytical Procedures . . . . . . . . . . . 38 Reagents. . . . . . . . . . . . . . . . . . . . . 41 RESULTS. . . . . . . . . . . . . . . . . . . . . . . 43 Purification of D-Fructose Phosphoryl Transfer Protein . . . . . . . . . . . . . . . 43 100 ,000 x‘g Centrifugation . . . . . . . . 47 35 to 70 Percent Ammonium Sulfate Fractionation . . . . . . . . 47 DEAE Cellulose Chromatography (I). . . . . . . 48 45 to 65 Percent Ammonium Sulfate Fractionation . . . . . . . . . 49 Sephadex G100 Chromatography (I) . . . . . . . 52 Hydroxylapatite Chromatography . . . . . . . . 52 DEAE Cellulose Chromatography (II) . . . . . . 55 Sephadex G100 Chromatography (II). . . . . . . 58 Polyacrylamide Disc Gel ElectrOphoresis. . . . 58 Other Attempted Purification Procedures. . . . 63 Characterization of D-Fructose PhOSphoryl Transfer Protein . . . . . . . . . . . . . . . 66 Stability of PTPfru' . . . . . . . . . . . . . 66 Molecular Weight Determination by Gel Filtration on Sephadex G100 . . . . . . . . 71 iv Subunit Molecular Weight Determination by SDS Polyacrylamide Disc Gel Electrophoresis Role of D-Fructose PhOSphoryl Transfer Protein. Requirement of PTPfru for Growth on Low D- Fructose Concentrations Lack of Requirement of PTPfr ru for D-[U-lAC] Fructose Binding. . Inducibility of Enzyme II by D- Fructose. Effect of PTPfru on Sephadex G200 Enzyme II Activities. . . . Induction of Both Enzyme II and PTPfr ru by D- Fructose . . . Additivity of Constitutive and Inducible Systems Function of PTPfr ru as a Substrate for Enzyme IIfru' Inhibition of Constitutive Enzyme II Activity by PTPfru' Requirement for HPr of the Constitutive and Inducible Systems Effect of 2-Mercaptoethanol on Enzyme IIfru Activity Formation of Phosphorylated PTPfru a. Preparation and Identification of [32P] PEP . Formation of [32F] ATP . . Paper Chromatographic Identification of [ 32F] PEP. . Formation of D-Fructose l-[32P] PhOSphate b. Requirements for PhOSphoryl Transfer 32 From [ P] PEP to PTPfru c. Determination of Moles 32F Bound Per Mole PTPfru' . . . d. Formation of D-Fructose l-[32P] Phosphate From [32F] PhOSpho-PTPfru \7 Page 75 75 75 82 84 88 91 95 107 111 114 123 125 125 125 128 132 137 146 155 Page DISCUSSION . . . . . . . . . . . . . . . . . . . . . 165 SUMMARY. . . . . . . . . . . . . . . . . . . . . . . 173 BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . 175 vi Table II. III. IV. VI. VII. VIII. IX. LIST OF TABLES Purification of D-fructose hosphoryl transfer protein (PTPfruy . . . Recovery of PTPf"u activity after 48-hour dialysis against water a d variousOO.02 M buffers and storage at 4 C and -18 C for 1 week . . . . . . . . D-[U-lAC] Fructose bound to crude extracts of PRL-R3 grown on D-fructoge and D- glucose and incubated at 30 C and 00 C. Effect of different PTPfru fractions on specific activities of 100,000 x‘g enzyme II precipitates obtained from cells grown on various substrates. Effect of different PTPfru preparations on D- fructose phOSphorylation catalyzed by enzymes 11 purified by Sephadex G200 column chromatography. . . . Comparison of specific activities of dif— ferent enzyme 11 100 ,000 x'g precipitates in the presence of different 100 ,000 x‘g supernatants . . . . . . . . . . Effect of 48- hour incubation of enzyme Ilfr ru in fresh 2-mercaptoethanol . . . . Radioactivity bound to charcoal dependent on ADP and fractions from Dowex 1-X10 column chromatography containing [32F] PEP. Formation of D-fructose l-[32P] phOSphate from [32F] PEP pool. Formation of [32F] phOSpho-PTPfru from 32 [ P] PEP, enzyme 1, and PTPfru vii Page 44 68 83 85 89 94 124 129 133 138 Table Page XI. Formation of [32szphospho-protein from enzyme I and [ P] PEP . . . . . . . . . . 142 XII. Requirements for formation of [32F] phOSpho-PTPfru . . . . . . . . . . . . . . 149 XIII. Formation of D-fructose 1-[32P] phOSphate from [32P] phospho-PTP 152 fru' viii Figure 10. 11. 12. LIST OF FIGURES Effect of PTPfru on PRL-R3 enzyme 11 fru' DEAE cellulose (I) chromatography of 35 to 70 percent ammonium sulfate fraction . Sephadex G100 (I) chromatography of DEAE cellulose (I) pool . . . Hydroxylapatite chromatography of Sephadex G100 (I) pool. . . . . DEAE cellulose (11) chromatography of hydroxylapatite pool . . . Sephadex G100 (II) chromatography of DEAE cellulose (II) pool. . Polyacrylamide disc gel electrOphoresis of PTPfru . Elution profiles of aldolase, D-glucose 6-ph08phate dehydrogenase, D-fructose l-phOSphate kinase, malate dehydrogenase, PTPfru’ and hemoglobin chromatographed on a Sephadex G100 column . Molecular weight determination of PTPfr ru on standard Sephadex G100 column. Molecular weight determination of subunits of PTPfru by SDS polyacrylamide disc gel electrophoresis. A comparison of the growth rates of QQl7 DD31, and PRL-R3 on seven different concentrations of D-fructose . A comparison of the growth rates of QQ17, DD31, and PRL-R3 on seven different concentrations of D-glucose. . ix Page 46 51 54 57 6O 62 65 73 74 76 78 79 Figure 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. Substrate saturation and Lineweaver-Burk plots for growth of 0017, DD31, and PRL-R3 on D-fructose and D-glucose . Saturation curves of constitutive and inducible phOSphotransferase activities. Saturation curves of constitutive and inducible phOSphotransferase activities at low D-fructose concentrations Lineweaver-Burk plot of constitutive and inducible activities at high D-fructose concentrations Lineweaver-Burk plot of constitutive and inducible activities at low D-fructose concentrations Saturation and Lineweaver-Burk plots of inducible activity-dependence on PTPfru' Saturation and Lineweaver-Burk plots showing dependence on HPr concentration of D-fructose phOSphorylation by the constitutive system. . . . . . Saturation and Lineweaver-Burk plots showing dependence of D—mannitol phos- phorylation on HPr concentration . Saturation and Lineweaver-Burk plots showing effect of HPrfru on constitutive and inducible system activities. Saturation and Lineweaver-Burk plots of dependence of inducible system on HPr. Lineweaver-Burk plot showing effect of HPrfru on the apparent Km for PTPfru . Elution of products of [32F] PEP-formation reaction from Dowex 1-X10 column chromatography . . . . . . . . . Scan of radioactivity on paper chromatogram of fractions 80, 170, and pool of 200 through 221 from Dowex 1-X10 column chromatography . . . . . . . . . Page 81 98 100 102 104 110 113 117 119 121 122 127 131 Figure 26. 27. 28. 29. 30. 31. 32. Radioactivity scan of paper chromatogram showing formation of D-fructose 1-[32P] phOSphate dependent on enzyme 11 and HPr Standard Sephadex G25 (0.75 x 56-cm) separation of bound radioactivity from unbound radioactivity. Elution of [32F] phOSpho-PTPfru pool and activity of another sample of PTPfru chromatographed separately on the same Sephadex G100 column . Radioactivity scans of acid chromatograms of pooled bound and unbound radioactivity. Sephadex G100 chromatography of [3 P] phOSpho- PTPfr ru reaction. . . . Radioactivity scans of alkaline chromato- grams of D-fructose l-[32P] phosphate— forming reaction . . . . . . . . . Sephadex G100 column chromatography of large reaction of enzyme 11 and 32 f P] phOSpho- PTPfr u xi Page 135 141 145 154 157 160 163 II "It“ EHZ EH2 8112 E12 En; ADP ATP Bicine BSA DEAE DTT EDTA E1 E11 enzyme enzyme enzyme enzyme enzyme F-l-P GDP HEPES HPr HPrfru ABBREVIATIONS adenosine 5'-diphosphate adenosine 5'-triphosphate N,N-bis(2-hydroxyethy1)g1ycine bovine serum albumin diethylaminoethyl- dithiothreitol ethylenediaminetetraacetate enzyme I of PEP-dependent phOSphotransferase system enzyme 11 of PEP-dependent phOSphotrans- ferase system enzyme II obtained from cells induced on D-fructose enzyme 11 obtained from cells induced on D-glucose enzyme II obtained from cells induced on glycerol enzyme II obtained from cells induced on D-mannitol enzyme 11 obtained from cells induced on nutrient broth D-fructose 1-ph03phate guanosine 5'-diph05phate N-2-hydroxyethy1piperazine-N'-2-ethanesul- fonic acid histidine-containing protein HPr obtained from cells induced on D-fructose xii HPrmt1 NAD NADH NADP+ NADPH 32 PEP PGA PIPES PTPfru SDS TEMED TES Tricine Tris HPr obtained from cells induced on D-mannitol nicotinamide adenine dinucleotide reduced nicotinamide adenine dinucleotide nicotinamide adenine dinucleotide phOSphate reduced nicotinamide adenine dinucleotide phOSphate labeled phOSphoryl group orthOphosphate phOSphoenolpyruvate phOSphoglyceric acid piperazine-N,N'-bis(2-ethane-su1fonic acid) D-fructose phosphoryl transfer protein sodium dodecyl sulfate N,N,N'N'-tetramethy1enediamine N-tris(hydroxymethy1)methy1-2-aminoethane sulfonic acid N-tris(hydroxymethy1)methy1 glycine tris(hydroxymethy1)aminomethane xiii " f) INTRODUCTION The first step in D-fructose metabolism in Aerobacter aerogenes is phOSphorylation with phospho- enolpyruvate to yield D-fructose l-phOSphate (35, 100). The enzyme system that catalyzes this reaction has been resolved by Hanson and Anderson (35) into four protein components. Three of the components, called enzyme 1, enzyme 11, and HPr according to the terminology of Kundig, Ghosh, and Roseman (61), were believed to be constitutive and to participate in the following reactions: E PEP+HPr-———l—) pyruvate+ph03pho-HPr Phospho-HPHD-fructose —-I—I—9HPr+D—fructose 1-phosphate The fourth component, which was specifically induced by D-fructose, was termed a "D-fructose Specifier protein" or a "Km factor" because it increased the maximal velocity and the affinity of the system for D-fructose. A mutant (QQl7) lacking this ”specifier protein" had impaired growth on D-fructose but not on other sugars (33, 35). The purpose of this thesis investigation was to further elucidate the roles of the four components im TEE t1 I'E 2 involved in this PEP-dependent phosphotransferase reaction, particularly with respect to the ”Specifier protein". The results indicate that there are actually two separate systems that utilize phOSphoenolpyruvate to phosphorylate D-fructose at C-1. One system has a low affinity for D-fructose; it involves enzyme 1, HPr, and a constitutive enzyme II. The other system has a high affinity for D-fructose; it involves enzyme 1, the inducible "Specifier protein", and an enzyme II which is also induced by growth on D-fructose. In this thesis, the ”Specifier protein" has been termed a "phosphoryl transfer protein" (PTPfru) because, as the data will show, this more accurately describes its function. Enzyme I catalyzes a phOSphoryl transfer from PEP to PTPfru; however, HPr is not required for this phosphorylation and, further, seems not to be required for the enzyme IIfru-catalyzed phOSphorylation of D-fructose in the presence of PEP, enzyme 1, and PTPfru' LITERATURE REVIEW The phosphoenolpyruvate:sugar phOSphotransferase system was first discovered in 1964 by Kundig, Ghosh, and Roseman (61). Since then there has been an extensive amount of research done in this field, attempting to further explain this complex system which is involved in both the phOSphorylation of sugars and their trans- port into the cell. Much of this work has recently been reviewed (3, 47-49, 83, 93). Consequently, this literature review will only attempt to summarize the major findings, cite critical similarities and differ- ences, and update the previous reviews. As first described, the system in Escherichia coli was comprised of three protein components: (i) Enzyme I, a cytoplasmic protein that catalyzes a phOSphoryl transfer from PEP to a histidine residue of HPr, (ii) HPr, a heat stable, low molecular weight (9,500 daltons) (2) protein that is found in the periplasmic Space (62), and (iii) enzyme II, a membrane—bound protein that catalyzes the phOSphoryl transfer from phOSpho-HPr to the sugar (61, 62). In most cases the products formed are 6-ph03phate esters (62); however, both D-fructose iv the in“ 3D,. II 4 (25, 35) and L-sorbose (54) are converted to 1-phosphate esters. Mutants missing either enzyme I or HPr are pleiotropic in that they do not grow on a wide variety of sugars. Pleiotropic mutants isolated from Aerobacter aerogenes and Escherichia coli by Tanaka and co—workers (107, 109) lack the ability to utilize D—glucose, D-fructose, D-mannose, D-glucitol, and D-mannitol; however they do grow on D-galactose. Fox and Wilson (24) reported that an Escherichia coli enzyme 1 negative mutant lacks the ability to utilize twelve sugars and related compounds, including lactose, succinate, and D-galactose. Pleiotropic mutants of Staphylococcus aureus isolated by Egan and Morse (19) do not grow on eight different sugars including D-galactose and lactose, whereas Salmonella typhimurium mutants isolated by Simoni and co-workers (104) have been shown to lack the ability to grow on nine different sugars. Some pleiotropic mutants do not grow on glycerol or succinate even though these compounds are not phOSphorylated by the PEP-dependent phOSphotransferase system. This could implicate a control mechanism involving the phOSphotransferase system (9). Other studies have linked enzyme 1 with both transient repression (116) and inducer exclusion (94), and enzyme 11 with catabolite repression (17, 84). Addition of cyclic AMP relieves both types of repression. This 5 permits enzyme I negative Escherichia coli mutants to grow on lactose (84) and on glycerol (10). Whereas enzyme I and HPr are constitutive and are utilized in the phosphorylation of many sugars, there is a family of enzymes 11, some of which are constitutive (62), while a majority are inducible and sugar-Specific (41, 62, 105). Mutants lacking functional enzymes 11 only lose the ability to grow on a single sugar or closely related sugars, such as lactose (41), D-fructose (21), B-gluco- sides (24), or D-mannitol (8, 108), and therefore are missing components of sugar-Specific enzymes II, rather than constitutive enzymes 11. In 1968 Hanson and Anderson (35) described an inducible fourth component ("Km factor", "D-fructose Specifier protein") which increased the Vmax and decreased the Km of a D-fructose phosphotransferase system in Aerobacter aerogenes. A mutant, QQl7, missing this component exhibited defective growth on D-fructose but grew normally on other substrates. Crude extracts of this mutant induced on D-fructose retained their high Km constitutive-type enzyme 11 activity for D- fructose. This activity was converted to a normal wild- type activity with low Km for D-fructose by addition of partially purified "Km factor". Enzyme 11 isolated from D-mannitol-grown cells contained a constitutive high Km activity for D-fructose phosphorylation which was 6 inhibited by addition of D-mannitol; however, addition of "Km factor” to this enzyme 11 had no effect on the Km or Vmax' Further, D-mannitol did not inhibit D-fructose phosphorylation by enzyme II which had been isolated from D-fructose-grown cells. Thus, enzymes II which were isolated from D-mannitol- and D-fructose- grown cells had different properties. It was proposed ' was constitutive that the enzyme 11 "heavy protein' and that various "factors" induced on different sub- strates ascribed sugar—Specificity to the system. If the D-mannitol-induced factor was tightly bound to the heavy protein, it would not easily be displaced by the D-fructose specifier protein and thus no change in Km would be seen (33, 35). Two four-component systems have been isolated from Staphylococcus aureus. They involve Specifically induced enzymes II for lactose and D-mannitol as well as Specifically induced fourth components (Factors 111) for these two sugars (40, 41, 105). There is a nearly absolute requirement for both components for phosphory- lation of the sugar for which they are Specific. Further investigations of the lactose-Specific Factor 111 have shown that it is a phospho-protein (78) with a molecular weight of 36,000 daltons with three or four subunits of 9,000 to 12,000 daltons (101). It contains two phos- phates per protein molecule and functions as an inter- mediate between phOSpho-HPr and enzyme IIlac (78). 7 Hengstenberg (37) has solubilized the lactose-Specific enzyme II and found that it retains activity after most of the lipid appears to be removed. In investigations of a constitutive phOSpho- transferase system in Escherichia coli, Kundig and Roseman (64-66, 93) have fractionated a solubilized enzyme 11 isolated from D-glucose-grown cells into two protein components (II-A and II-B) and phOSphatidyl- glycerol. II-A has been further separated into three proteins which Specify phosphorylation of D-glucose, D-fructose, and D-mannose, reSpectively. II-B is a protein with a molecular weight of 36,000 daltons and forms an active complex with any of the II-A proteins in the presence of phosphatidylglycerol. This is another four—component system that has sugar-specificity, although the individual II-A proteins are not Specif- ically induced. Also, both protein components are originally membrane-bound, whereas Factors III for lactose and D-mannitol (105) and the "Km factor" (35) for D-fructose are soluble components. Rose and Fox (24, 91, 92) have solubilized a B-glucoside enzyme 11 from Escherichia coli; however, they have not detected any correSponding fourth components. This solubilized enzyme 11 apparently does not require lipid for activity (91), whereas phOSpha- tidylglycerol is required for formation of an active enzyme 11 complex in Roseman's studies (64-66). Palmer K30; SL13- Veg 8 (81) has shown that Aerobacter aerogenes does not contain a PEP-dependent phOSphorylation system for the B-glucoside cellobiose; however, it does have an ATP-dependent B- glucoside kinase (81, 82). Aside from phosphorylation as the initial step in the metabolism of sugars that utilize this system, the phOSphotranSferase system also functions in the uni- directional transport of the sugars it phOSphorylates across the bacterial membrane. This dual function has been termed both group translocation (93) and vectorial phosphorylation (46, 48). Many studies done with whole cells have related uptake of substrates or accumulation of phOSphate esters to the phosphotransferase system (19, 28, 29, 31, 38-41, 53, 55, 102, 107, 111, 120, 129). Kaback (45-47) has shown PEP-dependence of uptake of sugars as their phOSphorylated derivatives into membrane vesicles. Treatment of vesicles with phospholipase D, which specifically hydrolyzes phosphatidylglycerol, inhibits vectorial phOSphorylation of a-methylglucoside. Membrane vesicles isolated from mutants missing enzyme I or HPr were also shown to lack the ability to transport sugars. Osmotically shocked (42, 79, 80) cells have decreased levels of both uptake and phosphorylation activities (43). Addition of HPr to these shocked cells restores both of these activities (62). This phOSphotransferase system that functions both in the phosphorylation and translocation of sugars has mail 9 mainly been studied in Aerobacter aerogenes (33, 35, 52, 53, 109), Escherichia 6611 (1, 2, 20-22, 24-26, 28, 29, 45-48, 60-66, 91, 92, 102, 108, 119-122, 129), Salmonella typhimurium (8, 70, 95, 96, 104, 111), and Staphylococcus aureus (19, 37-41, 76-78, 101, 105). It has also been detected in many other anaerobes or facultative anaerobes including Achromobacter parvulus (90), Bacillus cereus (90), Bacillus megaterium (90), Bacillus subtilis (27, 45, 62, 64, 90), Clostridium perfringens (31), Clostridium thermocellum (85, 86), Corynebacterium ulcerans (90), Lactobacillus arabinosus (61), and Streptococcus lactis (73). The strictly aerobic bacteria investigated by Romano and co-workers (90) showed either very low or no PEP-dependent phOSphorylation of 2-deoxyglucose. Pseudomonas aeruginosa (89) and the fungus Aspergillus nidulans (12) have also been shown to lack the phospho- transferase system. Leighton (68) has presented some qualitative data which imply that Microsporum gypseum contains a PEP-dependent inducible system that phosphory- lates D-fructose and D-mannitol; however, the method by which these data were acquired, and thus their validity, are not clear. Van Steveninck (117) has shown evidence for a maltose-induced transport-associated phOSphorylation of a-methylglucoside in Saccharomyces cerevisiae, but the phOSphoryl donor has not been characterized. Recently, Saier and co-workers (96) reported on a PEP-dependent 10 phOSphorylation of D-fructose in photosynthetic bacteria (RhodOSpirillum rubrum and Rhodopseudomonas Spheroides) which utilized two membrane-bound components, one of which is solubilized by low ionic strength buffer. Neither of these proteins complemented Salmonella typhimurium enzyme 1 or HPr mutants for a-glucoside phOSphorylation. Also, enzyme I and HPr isolated from Salmonella did not substitute for either of the Rhodo- spirillum rubrum components in D-fructose phosphorylation. It has not been demonstrated that this system functions in vectorial phOSphorylation. Weiser (125, 126) has described a PEP-dependent phOSphorylation of D-fructose to D-fructose 1-phOSphate in rat intestine. No evidence has been obtained to indicate that this PEP-activated system is involved in intestinal sugar transport. Ferenci and Kornberg (21, 22) have reported that extracts of Escherichia coli grown on D-fructose contain a PEP-dependent D-fructose phOSphotransferase system that forms D-fructose 1-phosphate at a low concentration of D-fructose (0.04 mM); however at a high concentration of D-fructose (50 mM), D-fructose 6-phosphate is also formed. Neither D—fructose 1-phosphate nor D-fructose 6-phosphate are formed by a mutant apparently missing an enzyme 11 specific for D-fructose. Another system that takes up phOSphorylated hexoses has been studied by Winkler (130, 121) and Ferenci (23). 11 This hexose 6-phosphate transport system is induced by growth on D-glucose 6-phOSphate, D-mannose 6-phos- phate, or D-fructose 6-phosphate, but not by D-fructose l-phosphate. Induced cells, however, were found to transport D-fructose 1-phOSphate for one to one-and- one-half generations. Mutants lacking phOSphoglucose isomerase are not induced by D-fructose 6-phosphate (130) and double mutants lacking phOSphoglucose isomer- ase and D-glucose 6-phOSphate dehydrogenase are not induced by growth on D-glucose (130). Thus, the true inducer is exogenous D-glucose 6-phOSphate (18). There has been no evidence presented to relate this hexose phosphate tranSport system to the PEP-dependent phos- photransferase system. Other transport systems have been elucidated and are discussed in the review articles cited (3, 47-49, 83, 93). The most pertinent of these transport systems is the "D-galactose permease" system originally described by Horecker (44). It has more recently been shown to be an active transport system coupled primarily to a membrane-bound D-lactate dehydrogenase (6, 7, 50, 56, 127, 128). Such coupled transport systems have been characterized in vesicles isolated from a wide variety of aerobic and facultatively anaerobic bacteria (59). This system could be the initial step in the metabolism of sugars in aerobic bacteria replacing the PEP-dependent phOSphotransferase system which serves this function 12 in anaerobic and facultative anaerobic bacteria. Von Meyenburg (118) has isolated a mutant that has trans- port-limited growth rates for sugars, amino acids, and the anions P043- and 8042-. The defect in this mutant has not been characterized; however, it could be related to the D-lactate dehydrogenase-coupled system. As indicated by this literature review, the PEP- dependent phOSphotranSferase system is a complex system which varies widely in its specificity in different species. The research reported in this thesis further elucidates the roles of the four components involved in the PEPzD-fructose l-phosphotransferase system in Aerobacter aerogenes. EXPERIMENTAL METHODS Bacterial Strains Aerobacter aerogenes PRL-R3 (wild type) and a uracil auxotroph, PRL-R3(U-), derived from it were used as the parental organisms. A mutant (strain DD31) missing D-fruetose 1-phOSphate kinase (53) and a mutant (Strain QQl7) missing the D-fruetose "Specifier protein" (35), more correctly termed D-fructose "phOSphoryl transfer protein" (PTPfru)’ were both isolated by mutagenesis of PRL-R3 with ethyl methanesulfonate, treat- ment with penicillin D in D-fructose mineral media, and selection of small colonies on mineral-agar plates containing 0.5 percent D-fructose and 0.005 percent D-glucose. A mutant lacking D-fructose 6-phosphate kinase (strain A9-1) (100) was isolated from PRL-R3(U-) by mutagenesis with ethyl methanesulfonateate and selection for positive growth on D-fructose and Slow growth on D-glucose and D-mannose by replica plating techniques. Pleiotropic mutants were isolated from PRL-R3(U-). Cells from an overnight 7.0 ml nutrient broth culture were harvested by centrifugation, washed twice with 14 m1 of mineral medium, and resuspended in 7.0 ml of mineral medium. Ethyl methanesulfonate (0.03 ml) was 13 14 added to 2.0 ml of the resuspended cells which were then incubated at 32°C on a reciprocal shaker. After 2 hours the cells were harvested, washed three times with 7.0-m1 volumes of mineral medium, resuspended in 7.0 ml of mineral medium containing 0.5 percent D-galactose, and grown for 10 generations. The eXpressed cells were diluted serially to 500 cells per 0.1 ml, assuming 109 cells per ml in a maximally grown culture, and 0.1-ml amounts were plated on MacConkey agar containing 0.5 percent D-mannitol. Nonfermenting pale colonies were then streaked on five individual MacConkey agar plates containing 0.5 percent D-fructose, D-galactose, D-mannose, D-mannitol, and D-glucitol, respectively. Several Strains failed to ferment all the tested sugars except D—galactose and were considered pleiotropic mutants. Crude extracts of each Strain, induced on D-fructose mineral medium for 4 hours after growth on nutrient broth overnight, were assayed for their various phOSphotransferase components by assaying for PEP-dependent D-fructose l-phosphate formation in the presence of added enzyme I, enzyme II, HPr, PTPfru’ or no addition. Only the addition of enzyme I stimulated D-fructose phOSphorylation, and thus it was concluded that all the pleiotropic strains were missing enzyme 1. Strain PL-122 (52) had the highest level of activation and was the strain utilized in this study. 15 Spontaneous revertants were obtained by plating an overnight broth culture of PL-122 on D-mannitol MacConkey agar and selecting red colonies that developed. These revertants were then tested for their uracil requirement for growth to eliminate the possibility of contamination. The revertant (PL-122R) selected for further study phosphorylated D-fructose at wild-type rates without added enzyme I and required uracil for growth. Media For growth of uracil auxotrOphS, 0.005 percent uracil (Sigma) was added to all media described. The pH of all media was adjusted to 7.0 before autoclaving. Mineral Medium This medium consisted of the following components: 0.71 percent of NaZHPOA, 0.15 percent of KH2P04’ 0.3 percent of (NH4)2804, 0.01 percent of MgSOA, 0.0005 percent of FeSO4.7 H20, and 0.5 percent of a Specified sugar (autoclaved separately). Nutrient Broth Medium This medium consisted of 5.0 g of Bactopeptone (Difco) and 3.0 g of Bacto beef extract (Difco) or 8.0 g of Bacto nutrient broth (Difco) in 1.0 l of water. l6 Nutrient Agar Medium This medium consisted of 23 g of Bacto nutrient agar (Difco) in 1.0 1 of water. MacConkey Agar Medium This medium consisted of 40 g of MacConkey agar base (Difco) in 1.0 l of water mixed with an equal volume of 1 percent of a Specified sugar after being autoclaved separately. Growth of Cultures Growth curves were done in 18 x 150-mm culture tubes containing 7.0 m1 of mineral medium which contained 0.3 percent sugar except where Specified otherwise. Inocula were 0.1-m1 amounts of overnight nutrient broth cultures. The tubes were incubated at 32°C on a recipro- cal shaker and Optical density readings were made at 520 nm with a Coleman Jr. Spectrophotometer. Growth of cells for enzyme purification was done either in 1.0 1 of mineral medium in Fernbach flasks on a rotatory shaker at 320C, in 40 l of mineral medium in carboys at 32°C with cotton-filtered air bubbling through at 8 lb pressure, or in 80 l of mineral medium in a New Brunswick Model 130 Fermacell fermenter. For induction studies, cells from overnight nutrient broth cultures were collected by centrifugation and resuspended in mineral medium plus 0.5 percent inducer. 17 These suSpensionS were then incubated with agitation at 320C for 4 hours. Preparation of Cell Extracts Cells were harvested by centrifugation at 16,000 x'g in a Sorvall refrigerated centrifuge when grown in tubes or Fernbach flasks. Larger quantities of cells were harvested in a Sharples centrifuge. The cells were washed twice with 0.85 percent NaCl and resuspended in 0.02 M Tris-HCl buffer (pH 7.5) containing 0.028 M 2-mereaptoethanol. For purification of enzymes other than those involved in the phosphotransferase system the cells were resuspended in 0.02 M potassium phosphate buffer (pH 7.5). Small volumes of cells were broken by ultrasonic vibration for 10 minutes in a Raytheon 250-watt, lO-kHz sonic oscillator equipped with an ice-water cooling jacket. Larger cell preparations were broken in a Manton-Gaulin Laboratory homogenizer (Model 15M-8TA) at 6,000 lb pressure. The broken cell suspensions were centrifuged at 45,000 x'g for 15 minutes and the resulting supernatant was the crude extract. All purifications were carried out at 0 to 4°C. l’fi "Q If." 18 Preparation of Enzymes Preparation of Enzyme II The crude extract was centrifuged in a Spinco L2 ultracentrifuge either at 38,000 rpm (100,000 x'g) for 2 hours in a Beckman 40 rotor, or at 28,000 rpm (68,000 x g) for 3 hours in a Beckman 30 rotor. The resulting pellet was resuspended in 0.02 M Tris-HCl (pH 7.5) containing 0.028 M 2-mercaptoethanol and recentrifuged at 100,000 x g for 2 hours. The resulting pellet was resuspended in the same buffer and was the enzyme 11 preparation used in this study. Where noted, this preparation was resonicated for 10 minutes and chro- matographed on Sephadex G200. The enzyme II activity was eluted in the void volume with 0.02 M Tris-H01 (pH 7.5) containing 0.028 M 2-mercaptoethanol and recentrifuged at 100,000 x g for 90 minutes. The re- sulting pellet was resuspended in the same buffer and used as purified enzyme 11. These preparations were stored at 0°C and used for a period of 1 month. Preparation of Enzyme I Enzyme I was prepared from both D-fructose- and D-mannitol-grown cells. The first 100,000 x g_super- natant from the above enzyme 11 preparation was recen- trifuged at 100,000 x g, The resulting supernatant was fractionated by ammonium sulfate precipitation. The 40 to 70 percent saturation precipitate was dissolved am PM fn the Suj 19 in 0.02 M potassium phOSphate buffer (pH 8.0) containing 0.001 M DTT and 10 percent glycerol, dialyzed overnight against the same buffer, and layered on a 4.8 x 12.5-cm DEAE cellulose column. The column was eluted with a 0 to 0.4 M KCl linear gradient in a 0.02 M potassium phOSphate buffer (pH 8.0) containing 0.001 M DTT and 10 percent glycerol. The active fractions (0.3 M KCl) were concentrated by ultrafiltration, layered on a 4.8 x 35-cm Sephadex G200 column, and eluted with the same buffer. The pooled activity was concentrated by ultra- filtration, dialyzed against 0.02 M potassium phosphate buffer (pH 7.5) containing 0.001 M DTT and 0.001 M EDTA, and stored at ~100C. This 60-fold purified enzyme I preparation was devoid of enzyme 11, HPr, PTP D- fru’ fructose 1-phOSphate kinase, and D-fructose l-phOSphate phOSphatase. Enzyme I was stored at -100C and was used for periods of up to 6 months with a loss in activity of 50 percent. Preparation of HPr The 40 to 70 percent saturation supernatant from the enzyme I preparation was saturated wfl:h ammonium sulfate (30) and after equilibrating for 1 hour was centrifuged at 40,000 x g, The resulting precipitate was dissolved in 0.02 M potassium phoSphate buffer (pH 7.5) and layered on a 4.8 x 35-cm Sephadex G75 column. The activity was eluted with the same buffer 20 and layered directly on a 4.8 x 12.5-cm DEAE cellulose column. The activity eluted at 0.1 M KCl with a 0 to 0.2 M KCl linear gradient in a 0.02 M potassium phos- phate buffer (pH 7.5) and was concentrated by 1yophili- zation. The dried protein was dissolved in distilled water and dialyzed against 0.02 M potassium phOSphate buffer (pH 7.5). This l30-fold purified HPr was devoid of enzyme 1, enzyme II, PTPf D-fructose l-phOSphate ru’ kinase, and D-fructose l-phosphate phosphatase. HPr was stored at -100C and used over a period of 2 months. Preparation of D-Fructose 1-PhOSphate Kinase D-fructose l-phOSphate kinase was purified by the method of Sapico (97, 98) through the calcium phos- phate step from crude extracts of strain A9-1 grown on D-fructose. Some D-fructose 1-phOSphate kinase prep- arations were obtained as by-products of the PTPfru preparation. D-fructose l-phOSphate kinase nearly cochromato- graphs with PTPf on both DEAE cellulose and Sephadex ru G200 columns as well as precipitating in the same ammonium sulfate fractions. However, D-fructose 1- phsophate kinase separates from PTPfru on the hydroxyl- apatite column. D-fructose l-phosphate kinase activity was monitored during the purification of PTPfru both as a marker for the location of PTPfru and as a measure of its contamination in the PTPfru pools. If any ATP 21 was present in the assay for PTPfru’ the presence of D-fructose 1-phosphate kinase could convert some of the D-fructose l-phOSphate formed to D-fructose 1,6- diphosphate, and thus affect the measured PTPfru-depen- dent activity. General Assay Procedures All enzyme and end-point assays involved the use of a Gilford Model 2400 absorbance recording Spectro- photometer thermostatically regulated at 300C. Micro- cuvettes with a l-cm light path were used. The oxi- dation and reduction of pyridine nucleotide was measured by monitoring the Optical density changes at 340 nm. In all cases the enzymes were assayed at levels that gave rates proportional to the enzyme concentration. Substrate Assays Assay for D-Fructose l-Phosphate A quantitative end-point assay for D-fructose 1- phOSphate consisted of the following components in a total volume of 0.15 ml: 0.05 umole of NADH, 10 umoles of glycylglycine buffer (pH 7.5), 1.0 umole of MgClz, 0.5 umole of ATP, 5.0 umoles of KCl, and excesses of D-fructose 1,6-diphOSphate aldolase, triose phOSphate isomerase, a-glycerophosphate dehydrogenase, and D- fructose l-phOSphate kinase purified through the calcium phosphate gel step, or an hydroxylapatite column. 22 An aliquot of a solution containing D-fructose 1- phOSphate was added and the reaction started by the addition of D—fruetose l-phosphate kinase or ATP. An optical density change of 0.82 is equivalent to 0.01 umole of D-fructose l-phOSphate (35). Assay for D-Fructose This end-point assay contained the following components in a total volume of 0.15 ml: 10 umoles of glycylglycine buffer (pH 7.5), 1.0 umole of MgClz, 0,5 umole of ATP, 0.2 umole of NADP+, and excesses of hexokinase, phosphoglucose isomerase, and D-glucose 6-phOSphate dehydrogenase. The reaction was started by the addition of ATP. An Optical density change of 0.41 is equivalent to 0.01 umole of D-fructose. Assay for D-Fructose 6—Phosphate This end-point assay was similar to the D-fruc- tose end-point assay except that hexokinase and ATP were omitted. The reaction was started by the addition of phOSphoglucose isomerase. Assay for D-Glucose 6-PhOSphate This end-point assay was similar to the D-fructose assay with ATP, hexokinase, and phosphoglucose isomerase being omitted. The reaction was started by the addition of D-glucose 6-phosphate dehydrogenase. .-.—.. _‘ -— I32- 23 Assay for D-Fructose 1,6-DiphOSphate This end-point assay was similar to the D-fructose l-phOSphate assay except that D-fructose l-phOSphate kinase, ATP, and MgClZ were omitted. The reaction was started by the addition of aldolase. Assay for PhOSphoenolpyruvate This end-point assay contained the following components in a total volume of 0.15 ml: 10 umoles of glycylglycine buffer (pH 7.5), 0.05 umole of NADH, 1.0 umole of MgClz, 0.5 umole of ADP, and excesses of pyruvate kinase and D-lactate dehydrogenase. The reaction was started by the addition of ADP. An Optical density change of 0.41 is equivalent to 0.01 umole of phOSphoenolpyruvate. Enzyme Assays Assay for Aldolase This enzyme assay contained the same components as the D-fructose 1,6-diphOSphate assay with the excep- tion of aldolase which was limiting. The reaction was started by the addition of 1.0 umole of D-fructose 1,6- diphOSphate. Assay for D-Fructose 1-Phosphate Kinase This assay contained the same components as the D-fructose l-phOSphate end-point assay except that 1.0 umole of D-fructose l-phosphate was added and the rate 24 of NADH oxidation was proportional to the amount of D- fructose l-phosphate kinase added (34, 99). Activity was defined as the umoles Of D-fructose 1,6-diphOSphate formed per minute per ml. Assay for Malate Dehydrogenase This assay contained the following components in a total volume of 0.15 ml: 2.5 umoles of potassium phOSphate buffer (pH 7.4), 0.05 umole of NADH, and 0.15 umole of oxaloacetic acid which was used to start the reaction. An Optical density change of 0.41 is equiva- lent to reduction of 0.01 umole of oxaloacetic acid. Assay for D-Glucose 6-Phosphate Dehydrogenase This assay was similar to the end-point assay for D-glucose 6-phosphate except that 0.5 umole of D- glucose 6-phOSphate was added to start the reaction and the rate of reduction of NADP+ was prOportional to the amount of D-glucose 6-phosphate dehydrogenase added. Assay for Hemoglobin The elution of hemoglobin from a Sephadex G100 column, used for molecular weight determination, was monitored by measuring the absorbance at 415 nm (4). Assay for Enzyme I_(D-mannitol continuous) This assay consisted of the following components in a total volume of 0.15 ml: 8.0 pmoles of Tris-HCl buffer (pH 9.0), 0.2 umole of NADP+, 0.2 umole of NAD+, CEY eq; f0] OX' at PT: “l 1’6 Ctj 25 1.0 umole of MgClZ, 1.0 umole of PEP, 0.5 umole of D- mannitol, 0.57 umole of 2-mercaptoethanol, 5.0 ul of enzyme IImtl and excesses of HPr, D-mannitol l-phOSphate dehydrogenase (either purified by the method of L133 (71) or 1.0 ul of a 100,000 X.& supernatant from crude extracts induced on D-mannitol), phOSphoglucose isom- erase, and D-glucose 6-phOSphate dehydrogenase. The reaction was routinely started by the addition of PEP and after a lag of from 1 to 10 minutes (depending on the enzyme 11 preparation) the observed rate of increase in absorbance at 340 nm was dependent on enzyme I con- centration. An optical density change of 0.41 was equivalent to 0.01 umole of D-mannitol l-phosphate formed since the enzyme II preparation contained NADH oxidase. For a more accurate measure of enzyme I activity, sealed cuvettes were evacuated and oxygen present in the reaction mixture was displaced with nitrogen. This process was repeated three times, the reaction was equilibrated at 300C in a thermostated cuvette positioner, and PEP was added to start the reaction. With this anaerobic system there was less of a lag period and 0.01 umole of D-mannitol l-phosphate gave a change in optical density of 0.82. A unit of activity is defined as umoles D-mannitol 1-phOSphate formed per minute per m1. 26 Assay for HPr (D-mannitol continuous) This assay was the same as the enzyme I (D- mannitol continuous) assay except that saturating levels of enzyme I and limiting levels of HPr were used. General Assay for PEP-Dependent D-Fructose erhosphate’Formation This assay consisted of the following components in a total volume of 0.2 ml in a 6 x 50-mm test tube: 8.0 umoles of Tris-HCl (pH 7.5), 0.57 umole of 2-mer- captoethanol, 1.0 umole of PEP, and amounts of MgClz, D-fructose, enzyme 1, HPr, enzyme 11, and PTPfru as noted in the individual assays for the separate com- ponents. The assay was started by the addition of PEP or crude extract (when included in the assay), incubated at 30°C for 10 minutes, and stopped by placing the tubes in a 950C bath for 7 minutes. The denatured protein was removed by centrifugation at 6,000 x.g for 10 minutes and a 50 ul aliquot was assayed for D- fructose l-phOSphate. The amount of D-fructose 1-phOSphate formed in the total reaction was calculated and related to the component being assayed in the form of nmoles of D- fructose l-phOSphate formed per minute per ml or mg of that component. When crude extracts were assayed, low levels of MgC12 (0.01 umole per 0.2 ml assay) were added to decrease the activity of D-fructose l-phOSphate kinase, and NaF 27 (3.0 nmoles per 0.2 ml assay) was added to inhibit the activities of enolase and D-fructose l-phOSphatase. Endogenous contamination by these enzymes would decrease the measured rate of D-fructose l-phOSphate formation dependent on PEP. In reconstituted assays with purified components, which were devoid of D-fructose 1-phOSphatase, ATP, and 2-phosphoglycerate, 1.0 umole of MgCl2 was added and NaF was omitted, because low concentrations of MgCl2 and the presence of NaF in these reconstituted assays were found to inhibit D-fructose 1-phosphate formation. Various D-fructose concentrations were used throughout this thesis research and are noted with each individual experiment. Initially, assays for PTPfru were run at 2.0, 20, and 200 mM D-fructose to Obtain a relative measure of its effect on the Km of the system, and assays for enzyme 1, HPr, and enzyme 11 were run at saturating (200 mM) D-fructose concentrations. Later in the study, when it became apparent that there were two enzyme 11 activities with high (7 mM) and low (0.02 mM) Km's for D-fructose, 100 mM D-fruetose was used in assaying for levels of enzyme 1, HPr, and the consti- tutive high Km enzyme 11, whereas 1.0 mM and 0.5 mM D-fructose were used in assaying for the inducible low Km enzyme IIfru and PTPfru in the presence of enzyme IIfru’ The inducible system is nearly saturated at 0.5 28 mM D-fructose and so, by altering the concentration from 2.0 to 1.0 to 0.5 mM would not affect the actual activity of the inducible system; however, the apparent inducible activity at 2.0 mM would increase because of the presence of the constitutive system whose activity would neces- sarily increase. A problem inherent in assaying for PTPfru and in determining the Km of the inducible system for D-fructose was the low level of substrate. In the normal D-fructose l-phOSphate end-point assay utilizing a 50 pl aliquot of the D-fructose l-phOSphate-forming assay solution, utilization of 25 percent of 0.5 mM D-fructose would give an Optical density change of 0.51. Thus, all assays had to be performed at low levels of enzyme 11 activity to prevent depletion of D-fruetose and con- sequent nonlinear formation of D-fructose l-phOSphate. In the experiment described by Figure 17, where the Km for D-fructose of the inducible system was determined, the effect of depletion of D-fructose on the rate of the reaction was corrected for by measuring the initial and final D-fructose concentrations of each assay, taking their averages, and using these average concen- trations in plotting the rates of D-fructose l—phos- phate formation (67). For more accurate determination of D-fructose l- phOSphate formed at low D-fructose concentrations, a radioactive assay is being develOped in this laboratory. 29 . . . . . . l4 Employ1ng h1gh Spec1f1c act1V1ty-D-[U- C] fructose as the substrate, low levels of enzyme 11 can be used to measure accurate rates of D-fructose l-phosphate forma- tion without utilizing a large percentage of the available substrate. a. Assay for Enzyme I When purified components were not available, a Specific assay for enzyme I included the components of the crude extract assay outlined in the general assay above, 50 ul of a fresh crude extract of PL-122 induced on D-fructose, and the sample containing enzyme 1. When purified components were available, satur- ating amounts of HPr and enzyme IIfru were included with the components of the reconstituted assay described above, and D-fruetose l-phOSphate formation was measured at 100 mM D—fructose. A unit of activity is defined aS the amount of enzyme I that catalyzes the formation of 1.0 nmole D-fructose 1-phOSphate per minute. b. Assay for HPr This assay was the same as the reconstituted enzyme I assay; however, enzyme I was saturating, and limiting levels of HPr were used. A unit of activity is defined as the amount of HPr that catalyzes the formation of 1.0 nmole D-fructose l-phOSphate per minute. A more accurate assay for HPr would be Similar to the "half-maximal saturation" assay developed for PTPfru’ si th C— 85 CE 85 ar. We th ac 118 in 30 since both HPr and PTPfru function as substrates for their reSpective enzymes 11 (inducible and constitutive). c. Assays for Enzymes II There are two enzyme II activities (which can be assayed independently) in extracts of D-fructose—grown cells. The constitutive activity was found in extracts of cells grown on a wide variety of substrates and was assayed at 100 mM D-fructose (20 umoles in a 0.2 ml assay) using the components of the general reconstituted assay described above, saturating enzyme I and HPr, and no added PTPfru' The controls for this activity were assays minus PEP at 100 mM D-fructose and minus HPr at 0.5 mM D-fructose (which would correct for activity of the inducible system). The enzyme IIfru (inducible) activity was assayed at 0.5 mM D-fructose (0.1 umole in a 0.2 ml assay) in the presence of saturating PTPfru and enzyme 1. The activity in the absence of PTPfru at 0.5 mM D-fructose was used as the control and was equivalent to the activity in the absence of PEP. A unit of activity is defined as the amount of enzyme IIfru that catalyzes the for- mation of 1.0 nmole D-fructose 1-phosphate per minute. d. Assays for D-Fructose PhOSphoryl Transfer Protein (PTPfru) In the course of this thesis investigation, three methods of quantifying the amount of PTPfru were 31 developed. The presence of PTPfru in fractions from columns was measured by the increase in D-fructose l- phOSphate formed per minute per ml in an assay that contained the general components of the reconstituted assay, saturating levels of enzyme I and HPr, and limiting levels of PTPfru and PRL-R3 enzyme IIfru' These assays were initially run at 2.0 mM D-fructose; however, in later experiments, 1.0 mM D-fructose was used. By lowering the D-fructose concentration, the activity in the absence of PTPfru was decreased; how- ever, the D-fructose l—phOSphate formed that was dependent on PTPfru remained the same, Since the Km of the inducible enzyme IIfru which requires PTP for fru activity is 0.02 mM D-fructose. The background activity of the PRL-R3 enzyme IIfru is a result of both the constitutive activity (which has a Km of 7 mM) and residual levels of PTPfru in the enzyme IIfru 100,000 x g_precipitate preparation which allows the enzyme IIfru (inducible) to function at a low level. Changing the amount of inducible enzyme 11 alters the amount of D-fructose l-phOSphate formed when a given amount of PTPfru is added. Thus, this assay is only relatively quantitative and units cannot be correlated from one enzyme IIfru to another; however, it is a quick and simple method for detecting the presence of PTPfru in various fractions from columns or other Steps of purification. 32 To correct for the variability in the above assay, activity was calculated as the "fold increase" over the background enzyme Ilfru-dependent D-fructose phOSphory- lation. The net increase in units divided by the back- ground units represents the ”fold increase" in activity. The amount of PTPfru that increases a background of 0.5 units of enzyme IIfru activity per reaction to 3.0 units, yields a net increase of 2.5 units. This is a 5-fold increase and thus, the amount of PTPfru that effects this net increase contains 5 "fold increase" units. These "fold increase” units were utilized throughout a majority of this thesis study; however, when it became apparent that there were two separate systems that utilize PEP to phosphorylate D-fructose in Aerobacter aerogenes, a new method of calculating units of PTPfru was devised. The inducible enzyme IIfru has an absolute require— ment for PTPfru which acts as a substrate for enzyme IIfru and diSplays saturation-type kinetics. QQl7 enzyme IIfru is used in assays at 0.5 mM D-fructose and the amount of PTPfru that gives one-half the maximal activity of a Single concentration of enzyme IIfru is defined as a "half-maximal saturation" unit. This value was determined by assaying four or five different concentrations of PTPfru, plotting the reciprocals of the velocities obtained versus the reciprocals of PTPfru can the def thi the hm Thi use pre Can the Uni II inc Dac Pre it 902 am Wit 33 concentrations, and extrapolating the curves to Obtain 12l1£3 apparent Km. This derived amount of PTPfru is (fleazfined as one unit of PTPfru' To Obtain units per m1, t:11;is amount (in ul) is divided into one ml. Doubling t211ools of activity when PRL-R3 enzyme IIfru was used. A chird, more accurate and reproducible, method can be 11sed to calculate the PTPfru activity when 0017 enzyme 11 fru is used in a purified assay system. the amount of PTP In this case, fru that gives one-half the maximal enzyme IIfru activity is defined as a "half-maximal saturation" unit of activity. Preparation of [32P] PhOSphoenolpyruvate The method for the preparation of [32P] PEP was a modification of the procedure described by Mendicino and Utter (74). Isolation of Chicken Liver Mitochondria One and one-half week-old Leghorn Cockerels, donated by Dr. W. W. wells, were starved for 15 hours. Their livers were excized, minced, and homogenized with a Potter-Elvehjem homogenizer in four volumes of a cold 35 sscrlution (pH 7.5) consisting of 0.25 M sucrose and 22 )< 10.4 M EDTA. The suspension was centrifuged at ‘75515 x g_for 20 minutes. The resulting supernatant was (2(3t1trifuged at 10,800 x g for 15 minutes. The pellet vvéizs resuSpended in the above solution, centrifuged at 75 5 x g for 15 minutes and the supernatant was recen- tirrifuged at 10,800 x g for 15 minutes. The resulting I>€211et was resuspended in two volumes of 0.002 M HEPES IDuffer (pH 7.5) containing 0.07 M sucrose, 0.001 M iEIDTA, and 0.22 M D-mannitol. This suspension was cen- tlrifuged at 6,780 x g for 15 minutes and the pellet was lfesuspended in 2.0 m1 of the same buffer. This suSpen- sion of mitochondria was estimated to contain 75 mg protein per ml. Synthesis of [32P] Phosphoenolpyruvate Each of two [32F] PEP-forming reactions included the following components in a total volume of 1.51 ml: 100 umoles of Tris-HCl (pH 7.5), 100 umoles of sucrose, 8.0 umoles of MgClZ, approximately 0.5 mCi of 32Pi (carrier free), and 30 mg of coupled mitochondria in the main section of a Warburg flask; 0.1 m1 of 10 percent KOH plus pleated filter paper were in the center well to absorb C02. The side arm contained 10 umoles of L-malate, 0.1 umole of ADP, and 0.1 umole of GDP. After equilibration at 30°C the reaction was initiated by adding the contents of the Side arm. The reaction 36 VVEIS terminated after 45 minutes by adding 3.0 m1 of 0.6 Pi trichloroacetic acid. The reaction flasks were Viéashed twice with buffer and twice with trichloroacetic eaczid to get total transfer of the radioactive compounds. 1Tt1is reaction could be run directly in a centrifuge tube, tZEIuS avoiding transfer of the product, since without aacided carrier Pi the amount of 02 uptake was found to 13€2 too low to be measured by the Warburg apparatus. 'iThe protein was removed by centrifugation in an Inter- tlational desk top clinical centrifuge and trichloroacetic eacid was removed from the supernatant by four extractions Vvith anhydrous ether. The ether was removed by bubbling nitrogen through the solution which was then titrated to neutrality with dilute ammonium hydroxide. The solution was applied to a 1.6 x 12-cm column of Dowex l-XlO (200-400 mesh) in the bicarbonate form. The column was washed with 50 ml of water prior to eluting with a step-wise gradient consisting of 300 ml of 0.15, 0.30, 0.40, and 0.45 M (65) triethylammonium bicarbonate buffers (pH 7.5) which were prepared using the procedure of Smith and Khorana (106). The radioactivity in the fractions was measured by using a Geiger-Muller tube and three major peaks were detected as Shown in Figure 24. In future preparations of [32P] PEP it would be best to monitor the eluted radioactivity and ensure that the 0.40 M peak is completely eluted from the column prior to 37 adding the 0.45 M buffer. The third major peak that was eluted with 0.45 M tZITiethylammonium bicarbonate was identified as [32F] PEP by its ability to make [32P] ATP (Table VIII), by F>éiper chromatography (Figure 25), and ability to make [)-fructose 1-[32P] phOSphate using the constitutive Eirlzyme II assay (Figure 26). Fractions 200 through 221 chere pooled, concentrated at 300C in a BOChi rotary ervaporator, and excess triethylammonium bicarbonate Vvas removed by repeated addition and removal of water. ijis labeled product, carrier free [32P] PEP, was dis- Esolved in 1.0 ml of water and stored at -100C; it was (diluted with unlabeled PEP immediately prior to the individual experiments in which it was utilized and the cpm's per nmole were measured using Cerenkov radiation in a liquid scintillation counter (36). These experi- ments extended over a 6-week period, thus only 12 per- 2 cent of the original [3 P] PEP formed was present when the last experiment was performed. Characterization of [32P] Phosphoenolpyruvate Assay for Formation of [32P] ATP from [32P] Phosphoenolpyruvate Aliquots of fractions from the Dowex column of 32 products from [ P] PEP-forming reactions were assayed for their ability to form [32P] ATP using pyruvate kinase. The complete assay contained 10 umoles of 38 Tfirfiis-HCl (pH 7.5), 0.1 umole of PEP, 4.0 umoles of MgC12, 2 - O umoles of ADP, 40 umoles of KCl, 10 ul of a 1/10 (iZLLIution of pyruvate kinase, and a 50 ul aliquot of the iflfeaction being assayed in a total volume of 0.5 ml. After 20 minutes at 30°C the reaction was Stopped by aa110tometrically with the aid of a nomograph (courtesy c>jE Calbiochem) based on the data of Warburg and Christian (T]_23) and by the absorption at 210 nm (114) using bovine Siearum albumin as the Standard. The method of Lowry (72) was also used to check the protein concentration 1111 crude extracts. Proteins were concentrated by ultrafiltration tihrough a UM—10 membrane in an Amicon Model 50 ultra- ifiltration cell. Reagents PEP, D-glucose 6-phOSphate dehydrogenase, pyruvate ‘kinase, lactic dehydrogenase, HEPES, aldolase, B- lactoglobulin, and DTT were purchaSed from Calbiochem, Los Angeles, California; phOSphoglucose isomerase, L- malate, uracil, D-fructose, protamine sulfate, yeast hexokinase, 2-phOSphoglyceric acid, 3-phOSphoglyceric acid, D-fructose 1-phOSphate, D-glucose 6-phosphate, GDP, D-fructose 1,6-diphosphate, ATP, D-galactose, D-glucitol (sorbitol), D-mannitol, L-sorbose, a-glycerophOSphate, Trizma-Base, a-glycerOphOSphate dehydrogenase-triose- phosphate isomerase, lysozyme, trypsin, carbonic anhydrase, and 2-mercaptoethanol from Sigma Chemical Company, St. Louis, Missouri; ethyl methanesulfonate from Eastman Organic Chemicals, Rochester, New York; enzyme- grade ammonium sulfate from Schwarz/Mann, Orangeburg, 9.: 42 New York; NAD+, NADH, NADP+, NADPH, and ADP from P-L Biochemicals, Milwaukee, Wisconsin; Dowex 1-X10 (Cl-) and Dowex 50W-X8 from J. T. Baker Chemical Company, Phillipsburg, New Jersey; D-mannose and D-ribose from General Biochemicals, Chagrin Falls, Ohio; L-arabinose from Pfanstiehl Laboratories, Waukegan, Illinois; hydroxylapatite was prepared by R. L. Anderson by the method Of Levin (69, 113); calcium phOSphate gel was Prepared by the method Of Hartree (l4); carrier free 32 P- (9.1 MCi per mole) in the form of H332P04 in 0.02 1 N HCl from New England Nuclear Corporation, Boston, Massachusetts; malate dehydrogenase and lactate de- hydrogenase from Worthington Biochemical Corporation, Freehold, New Jersey; acrylamide, N,N'-methy1enebis- acrYlamide, N,N,N' ,N'-tetramethylethylenediamine from Conalco, Bethesda, Maryland; D-fructose 6-phosphate from Boehringer Mannheim Corporation, New York, New York; D-mannitol 6-phOSphate was prepared by D. Allison (51) ; SDS from Mallinckrodt Chemical Works, St. Louis, Missouri; triply distilled water was used for all buffers and 0 1: her solutions . RESULTS Purification of D-Fructose Phosphoryl Transfer Protein Throughout this study many different methods of Purification were investigated and utilized in various Sequences. The method reported here was used several times yielding equivalent results. Table I summarizes a tYpical purification. The assays in this purification SChEHne utilized PRL-R3 enzyme 11, and thus PTPfru actiJIities are in terms of "fold increase" units as described in Methods. Figure 1 Shows a validation of these units using different levels of enzyme IIfru and PTPfru' Figure 1A shows the actual data with PTPfru caUsing increases in the level of D-fructose l-phOSphate fOITHed per assay. Figure 1B Shows this data plotted as "fR31d increase” units versus the PTPfru concentration. In tile: linear portion of the saturation curve, these "fOICi increase" units are proportional to the amount Of PTPfru ls re ached, they no longer are proportional. In all However, as saturation of the enzyme IIfru Ca 0 O I Ses where these un1ts were used, the act1V1ty was efigured 1n the l1near port1on of the saturation of the lndiVidual enzyme IIfru being used. 43 -~ 4 Plv . ~l AHV ”VINI 44 CH >uw>wuom .cflmuoua wE\muHcDo .xmmmm muscHE 0H pumpCmum one pcsouwxomn m:o¢mwopcm ecu um>o meMom mumcdmonana mmOuosumuQ CH Ommmuocw paomna m mm>ww umsu summHm mo uG:oEm one mm pmcflmmp Own: :Ommmuocw paom:n .OOLOOE %u3oq kn pmumEHummm 00m 00a.~ 0.4a 00N.0H mm 0.0 xHHv 0000 xmnmramm 00H 000.H 0.HH 000.0H 0.0 0.0 xHHv whoasaamo 0000 0.04 000 0.0a 000.00 00 m.mN H600 muwumamaxxoupmm ma.0 0.H0 5.00 005.00 00 000 AHV 00H0 x60mr060 N.NH 000 0.04 000.00 0H 004 mumwasm E:adoEEm xmonmq ka.m p.00 0.40 000.00 000 00k.m AHV 6604:4066 m<00 00.H 0.0a n.00 000.000 aka 000.0 mummasm 80060656 N0a-mm 0.H H0.0 00H 000.0HH 00N.H 000.NH 00606006000 m x 000.000 0600H00 06000000 sua>auo< x Amuacsv Aaav mAwev paom owmwomam xuo>oomm muw>wuo< mesao> Camuoum cowuomum H6009 H0009 iii sum A memv samuona HOMmcmuu quocmmocm mmOuozum-Q MO cowemOfiMwusm .H magma .ACESHOO ooNU xmpmnamm m EOHMV 300090 HE\wE o©.N paw oumHH mexmcm mmnqmm HE\wE w.mm wo mucsoem pmuoc ecu paw .H mechm mo ma mm .um: mo we ma .mmouooumum mo mmaoej «.0 wGHUOHOc0 .mpocumz CH OmbHHOmmp ACO0umEHOm mumnamonaua mmouosumum pampcmmmpummm pom xmmmm kumcowv xmmmm meOu0uchomu mnu mo mucmcanOO OmC0mucOo mmmmm< .4 .900 mam 00 0006: :mmmmuocw paow: pmumasoamo MO muwnmmC0A .m .500000 00 20000 080660 mmuqmm mo ch0um00cmocoo mso0um> mo Co0um050mm .< .00000 090N00 00-000 00 0000.00 00 060000 .0 005000 46 Figure l :0 0010 5000.0.0 0010 0000 ONH ow 00 o ONH ow Cd 0 _ _ 0‘ p \\) xlw. b HH mahwcm H1 0 H1 OH moo \ wmoo HH mfixuam HH memucm H1 m.~ / HH mEkwsm H1 m I tfi 03 I <3 N nIJdld 10 SLINfl “HSVHHONI 010.1H H1 m.~ HH 05%Ncm E S .< LP. 0‘. r-i mm.~ l O 0‘ m (I-:[UIZ° [_u1m salomu) ELVHJSOHd-I HSOIOHHJ-O mm.q 47 1004 000 x g Centrifugation Crude extracts of PRL-R3 cells grown on D-fructose were centrifuged at 38,000 rpm (100,000 x g) in a Beck- man 40 rotor in a Spinco L2 ultracentrifuge for 2 hours. The resulting supernatant was recentrifuged for 2 hours and this second 100,000 x g supernatant was diluted with .37! 0.02 M Tris-HCl buffer (pH 7.5) containing 0.028 M 2- mercaptoethanol to a protein concentration of 10 mg 1?. per ml . This solution, termed the diluted 100,000 x g supernatant, was generally devoid of enzyme 11. In some cases, where very heavy crude extracts were pre- Pared in order to have a Smaller volume, all of the enzyme II Was not removed by this ultracentrifugation. The PreSence of this contaminating enzyme 11, which makes the PTPfru saturation curves nonlinear, was corrected for in calculating the "fold increase" units of PTPfru' % 70 Percent Ammonium Sulfate Fractionation The ammonium sulfate concentration was brought to 35 Percent saturation (30) by adding 18-8 g 0f powdered amnion ium sulfate for each 100 m1 of diluted supernatant Over a period of 30 minutes. After stirring for another hour, the solution was centrifuged at 16,000 x g for 20 minutes. The resulting precipitate was discarded and the Supernatant was brought to 70 percent saturation by Slowly adding 23.7 g of ammonium sulfate per 100 m1 of 48 original solution. The solution was equilibrated by stirring for 1 hour prior to centrifugation at 16,000 x g for 20 minutes. The resulting precipitate was dis- solved in 0.02 M potassium phosphate buffer (8.0) containing 0.001 M DTT and 10 percent glycerol and was dialyzed overnight against this same buffer to remove ammonium sulfate which inhibits both the phosphotrans- ferase activity and the binding of PTPfru to the DEAE cellulose column. The PTPfru in this solution, termed the 35 to 70 Percent ammonium sulfate fraction, was 1.85-fold puri- fied over the 100,000 x g supernatant. This fraction COrltained enzyme 1 and D-fructose l-phOSphate kinase; however, a majority of contaminating HPr (which precipi- taties out mainly in the 70 to 100 percent ammonium sul fate fraction) was removed. Furthermore, any enzyme 11 Which was not removed by ultracentrifugation was removed in the 0 to 35 percent ammonium sulfate fraction. WCellulose Chromatograjfliy (I) The 35 to 70 percent ammonium sulfate fraction was layered onto a 4.8 x 12.5-cm DEAE cellulose column which Was then washed with 0.02 M potassium phosphate buffer (pH 8 .0) containing 0.001 M DTT and 10 percent glycerol before starting a 2.0 1 linear gradient of 0 to 0.4 M potaSsium chloride in the same buffer. Fractions of 16.5 ml Were collected and PTPfru was eluted under a broad In 49 protein peak at 0.16 M potassium chloride, thus yielding very little purification. However, it was separated from contaminating HPr and enzyme 1 which elute at 0.1 M and 0.3 M potassium chloride, respectively. D-Fructose l-phosphatase activity is also removed by this step; however, D-fructose l-phOSphate kinase is still a con- taminant. Fractions 67 through 89 were pooled (Figure 2). Q to 65 Percent Ammonium Sulfate Fractionation A second ammonium sulfate step was performed at this point not only to purify the PTPfru’ but also to Concentrate it prior to Sephadex G100 chromatography. To each 100 m1 of pooled activity, 21.9 g Of ammonium sulfate were slowly added to attain 40 percent saturation. After 1 hour equilibration, the resulting precipitate was removed by centrifugation at 48,200 x g for 10 mirlutes. The resulting supernatant was brought to 65 perCent saturation by slowly adding 16.8 g of ammonium Sulfate and equilibrated for 1 hour. The solution was centrifuged at 48,200 x g for 10 minutes and the resulting prec ipitate was dissolved in 0.01 M potassium phOSphate buffer (pH 8.0) containing 0.001 M DTT and 10 percent glycerol. A sample of this fraction was dialyzed over- night against this same buffer and assayed for PTPfru actiV’ity. This step yielded a four-fold purification of PTPfru with very little loss in activity. I ..-‘.n 50 .qummua 00 madam @0303 00m 000:3 mmumc uw0mmp A.....v mCHH pmuuop mLH .omuo: m00>0uom 0:0 000 zmmmm one CH pmppm 50000600 06 00a 005:05 poo pmeuom uospoua mo moaoec 00 mmHoE1 mo mEpmu CH mum m0000>000< .cOHUOmum OumeSm E:0CoEEm oomoumd cm 00 mm mo zcamuwoumEO0so AHV mmOHoHHmo m c> co c> c> c: .5 a; 0: .4 (1-Im .[_u:tm 8910111“) HSVNIX HIVHJSOHd-I msommu-a FRACTION NUMBER 3'. '2’.“- . 52 Sephadex 0100 Chromatography (I) The 45 to 65 percent ammonium sulfate fraction was layered onto a 4.8 x 35-cm Sephadex G100 column, chromatographed with 0.01 M potassium phOSphate buffer (pH 8.0) containing 0.001 M DTT and 10 percent glycerol, and 5.0-ml fractions were collected. This lower con- centration of potassium phOSphate was used so that the eluted Sephadex G100 pool of activity would have a lower phosphate concentration. This procedure would allow a Shorter dialysis time for diluting the phosphate concentration to below 0.005 M so that the PTPfru could bind to hydroxylapatite. However, at this lower concen- tration there was a great loss in PTPfru activity resulting in a net decrease in the specific activity of this Sephadex G100 pool (fractions 77 through 95, Figure 3). Other purifications utilizing a Sephadex G200 column at 0.02 M potassium phosphate concentration had little loss in total units with a greater degree of PUrification. mxylyatite Chromatography The Sephadex 6100 (1) pool was concentrated and dialyzed against 0.002 M potassium phosphate buffer (pH 8.0) containing 0.001 M DTT and 10 percent glycerol using an Amicon UM-lO ultrafilter. The resulting protein solution was applied to a 2.5 x 9.5-cm hydroxylapatite Column and 5.0-ml fractions were eluted with a 400 ml 53 .pmuoc >00>0uom 0:0 000 ammmm 050 CH pmppm cowuowuw HE 00a muda0E Hod omepom uospoud mo mmHoEc no mmaoE: mo mEumu £0 mum mm0u0>wuo< .HOOQ aHv mmOHOHHmO m00>0uom 050 now xmmmm 050 C0 pmoom COHuomuw HE 00d 003506 00m meHom uooooua mo mOHOec no 000083 mo 06000 O0 mum mm0u0>0uo< .HOOQ AHV ooao xmomcowm mo mnemuwoumaoaso mu0umamaxxouomm .q ousw0m 57 Figure 4 PROTEIN (mg/ml) no I\ \D In \T m N H c: c: c3 c: c: c: <3 <3 T I 1 I T' 1 r l O 4 " 4:?" o 00% ‘ .—4 4 I m < , I "*3 . . A as '- r- OM ° N8 U H 00) O - 5&4 0m 0 I “."S. . . Q0) 0 b o. - —I\_r é: ' . .4 I "‘ I O _ .- —E:: O K' T 0 h- ‘l—l -S s 0 14.4 m \\\ _ 51 ll. 1 l 14. .3 h- an O\ nn .4 a: 0: <4 c: c> <3 c: c> c> 7‘5 <5 53/ (N) 0d)! 0 H a. c> — _¢ C? n. :z c: T‘ “‘0: l l l 00 O \‘T \‘f \T N (I [w I uim sa[omu) “JJJLd l l l l 1 L O In C ‘O 04 c: :2 SR ;3 <3 .4 F4 04 <5 C; <5 ([_1w 1 _u1m SQIOMU) HSVNIX HLVHdSOHd'I HSOIOOHJ-O FRACTION NUMBER 58 0.3 M potassium chloride in a 0.02 M potassium phOSphate buffer (pH 8.0) containing 0.001 M DTT and 10 percent glycerol. Five-m1 fractions were collected and the peak fractions (46 through 54, Figure 5) were pooled and concentrated to 3.5 ml by ultrafiltration. Very little PTPfru activity was lost and a greater than 2- fold purification was attained. Sephadex G100 Chromatography (II) The concentrated protein solution was then care- fully layered on a 2.5 x 42-cm Sephadex G100 column and subsequently eluted with 0.02 M potassium phOSphate buffer (pH 8.0) containing 0.001 M DTT and 10 percent glycerol, 3.2-m1 fractions were collected, and fractions 30 through 38 were pooled (Figure 6). This step also resulted in very little loss in PTPfru activity with another two—fold purification, yielding a total purifi- fication of 309-fold with 11.6 percent recovery of total PTPfru activity. The actual yield and purification may have been greater if 0.02 M potassium phosphate buffer had been used for the first Sephadex G100 Step. Polyacrylamide Disc Gel Electrophoresis Polyacrylamide disc gel electrophoresis at pH 9.0 ‘with 7.5 percent acrylamide gels was performed on samples Of PTP from the final steps of a purification in fru which the second DEAE cellulose column and second Sephadex G100 column were run prior to a second hydroxylapatite 59 ~0 .p0pom CO000000 HE 00a 005:05 00a 008000 000:& umonaua 0000oo0wun m0aoEc mo 08000 C0 00 >00>00om 300090 .HOOQ 0000000000000»: 00 >£Q00wo0meo0no aHHV 0000:0000 m00>0000 00009m .0000 AHHV 0000:0000 m0000w000500£o AHHV OQHQ x0pmsm0m .o 00Dw0m 62 6 F i gun- PROTEIN (mg/m1) no.0 00.0 m0.o ON.O mmmZDz ZOHHommm 00 on ON 00 _ _ . . .ll. . o SHHME (I-Im I_u;nn salomu) HJJJLd 63 column (see hydroxylapatite chromatography step). The gel shown in Figure 7A contained 70 ug of protein from a DEAE cellulose (II) pool, and the gel in Figure 7B contained 18 ug of protein from the hydroxylapatite pool and was similar to a gel of the Sephadex GlOO pool (not shown). Another gel was over- loaded with 200 ug of protein from the hydroxylapatite pool, sliced, and protein was eluted with 0.02 M potas- sium phOSphate buffer (pH 8.0) containing 0.001 M DTT and 10 percent glycerol. The eluted fractions were assayed for PTPfru activity and a sample (containing approximately 25 ug) of the active fraction was concen- trated by 1y0philization and run on a second gel. The specific activity and recovery of this homogeneous PTPfru (Figure 7C) was not calculated due to the low concen- trations of protein. Other Attempted Purification Procedures Other purification steps which were investigated included carboxymethyl cellulose and phOSpho-cellulose columns. A Sephadex GZOO pool of PTPfru was adjusted to pH 6.5 and applied to both types of columns. In both cases all of the protein, including the PTPfru activity, failed to be adsorbed to the column. Several attempts at acid precipitation were also attempted on the Sephadex G200 pool of PTPfru‘ The pH of the protein sample was adjusted by addition of dilute acetic acid. 64 Figure 7. Polyacrylamide disc gel electrophoresis of PTPfru' A. 70 ug of protein from a DEAE cellu- lose pool. B. 18 ug of protein from a second hydroxylapatite pool C. Approximately 25 ug of PTPfru from an overloaded disc gel of the hydroxylapatite pool. Further details are given in the text. 65 66 A 1.8-fold purification of PTP was attained in the fru pH 4.7 precipitate which was resuSpended in 0.02 M potassium phOSphate buffer (pH 8.0) containing 0.001 M DTT and 10 percent glycerol; however, only 50 percent of the activity was recovered. Due to this loss in activity, this step was not utilized. A calcium phosphate gel purification was attempted; however, the activity eluted in several successive step- wise fractions and thus, the more successful hydroxy- lapatite column was used to replace this step. Another sample of the Sephadex G200 pool was heated at 500C for varying periods of time. There was a loss of 50 percent of PTPfru activity during a 10 minute period of time with no concomitant purification. Characterization of D-Fructose Phosphoryl Transfer Protein Stability of PTPfru It became apparent during purification that PTPfru loses activity upon storage at low concentrations of buffer and at low protein concentrations. Experiments to stabilize the activity involved dialysis of aliquots of a Sephadex GZOO pool of PTPfru against various buffers including 0.02 M Tris-HCL, 0.02 M potassium phosphate, 0.02 M glycylglycine, 0.02 M HEPES, 0.02 M Tricine, 0.02 M TES, 0.02 M PIPES, and 0.02 M Bicine at different pH levels (7.0 to 9.0), and in the presence of various 67 combinations of 0.001 M DTT, 0.001 M EDTA, 0.028 M 2- mercaptoethanol, 0.02 M sodium chloride, 0.02 M potassium chloride, and 0.02 M ammonium sulfate. The best recovery of activity after 48-hour dialysis and storage at 40C for one week was achieved with 0.02 M potassium phosphate buffer (pH 8.0) and with this buffer at pH 7.5 in the presence of 0.001 M DTT (Table II). In another experiment, a sample of PTPfru eluted from a calcium phosphate gel with 0.015 M potassium phosphate containing 0.001 M DTT, lost 50 percent of its activity in three days at 0°C, whereas a duplicate sample which was stored at -180C, retained 95 percent of its activity. Three aliquots from another Sephadex 6200 pool were incubated for 2 days with 5.0 mg per ml of bovine serum albumin, 5 percent glycerol, and 1 mM D-fructose in an attempt to stabilize activity at 00C. The sample incubated with BSA lost 80 percent of its activity; the sample stored in D-fructose lost 50 percent of its activity; and the sample stored in 10 percent glycerol did not lose any activity. These data show that 0.02 M potassium phOSphate, 0.001 M DTT, and 10 percent glycerol stabilize PTPfru at 00C. Thus, for all stages of purification following the first step, the protein was stored in 0.02 M potassium phOSphate buffer (pH 8.0) containing 0.001 M $(HIS‘ ULwr»: ICUEquCIv TrCQZ U .0 0...... Cwyu:r£.rw\ (s. kUH>AUU< .x003 H HCH U‘Q Um UWHOUW mQB NHWSIUCO :rvNCMW "v.53 Nanm.:mwmuCMv£U H0 HHS—LIQCAQ mwfimxflfinufiuu u V—aJnJB H -‘N FJMfi 1v m. ~, I hue-Z. ..U Q ‘u nu. mwvx~V14an .ru ~1— Mwfld o A.» laughaai :q FVK/ ud-uuh read u aw} an «sq-N P... vufl..¥ .I. tux/I .- u-\ ~-!- ‘--.~.-! ~-. \I. cuts). i.vV\ 3me L .Jukz.‘ —«-.-§ Iluudlfiiujfi— iL-wmkfnn...-v .q-oauuvloxs...‘ (drduI-\ 68 o.wn «.mm m.m Hm>ooom unmoumm .wE nod wows: :mmmmuocH paom: m.HN mmz mHm%Hme on “Oahu zufi>wuo< .xmmB H now 0 ¢ um pmuoum mw3 mamsuoco paw cmuouw mm3 mHaEmmomsu mo mamnumco mflmzamwn umum< .xmm3 H now 0 man paw o a um owmuoum paw mummmdn 2 No.0 msmwuw> pom mmum3 umamwm mHmHHmHn unonumq umumm >uw>Huom summHm mo >um>oumm .HH memH 69 on- o.mm o.w mcHoHuH 0-- a.mw o.m mamas on: N.Nw o.m mcHowaonwa 0.0m 0.0m m.m Homz 2 No.0 .mumnamosa EsHmmmuom m.mm moH m.n «Ham :5 H .Hem :5 H amumnmmona EnHmmmuom m.~ H.wo m.H «Ham as H .Hocmsum noummoumEnN XE mm .mumcamosa EnHmmmuom o.oo m.mw m.m oumm uamoumm E .umscHucoo--.HH mHnme 70 .pmcHEHmumU uoc mmHchmenu o .summem we won muHc: :mmmmuocH oHom: o.MN.mH uqu>Huom Hmouomn .summem me you muHc: :mmmmuocH pHom: ¢.MH.mH nqu>Huom Hm5u0oomm quonm .wmscHucoo-.HH mHan 71 DTT and 10 percent glycerol. Other PTPfru samples were stored at -180C for 6 months with 50 percent loss of activity. Molecular Weight Determination by Gel Filtratibn onfiSephadex G100 The molecular weight of PTPf was determined by ru Sephadex G100 chromatography as described by Andrews (4). A sample of PTP from a 45 to 65 percent ammonium fru sulfate step was mixed with the following proteins: aldolase (MW 149,000) (110), D-glucose 6-ph05phate dehy- drogenase (MW 128,000) (4), D-fructose l-phosphate kinase (MW 75,000) (98), malate dehydrogenase (MW 70,000) (112), and hemoglobin (MW 32,000) (103). A total volume of 0.7 ml was carefully layered on a 2.5 x 42-cm Sephadex G100 column which was eluted with 0.02 M potassium phOSphate buffer (pH 8.0) containing 0.001 M DTT and 10 percent glycerol. One-ml (BO-drop) fractions were collected and assayed for the individual activities as described in Methods. The elution profiles of relative activities are shown in Figure 8. From a plot of log molecular weight of the standards versus the fraction number of the center of each activity peak (Figure 9), the molecular Weight of PTP was estimated to be 52,000 daltons. fru 72 .CESHoo OOHO xmp umsamm m co nmsamuwoumEouno cHnonoEmn cam .summem .mmmme noucxnmo mumHmE .mmmcHx mumnamonmuH mmouodumuo .mmmcmwopv nmnmp mumnamonano mwoousuo .mmmHopHm mo mmHHmoua COHuDHm .w mHDme 73 Figure 8 mmmZDz ZOHHo El __ ' (18, 00) 1.7—. .H 106— — 1°5__ __ Lysozyme —§U 1,4— (14,400) - J l l l J 0.3 0.4 0.5 0.6 0.7 Rf Figure 10. Molecular weight determination of Subunits of PTPfru by SDS poly- acrylamide disc gel electrophoresis. Details are given in the text. 77 shown to be missing PTPfru and has a slow growth rate only on D-fructose (35). To see if the growth rate of 0017 was dependent on the concentration of D-fructose, QQl7, DD31, and PRL-R3 were grown on seven different concentrations of D-fructose and D-glucose (Figures 11 and 12). Growth of 0017 was dependent on D-fructose con- centration (Figure 13A) and had an apparent Km of 7.4 mM for D-fructose (Figure 13C). Strain DD3l (lacking D-fructose l-phOSphate kinase) had slow growth on D- fructose; however, increasing the D-fructose concentration had no effect on its rate of growth. By comparison, 0017 and DD3l had higher growth rates than PRL-R3 on D-glucose (Figure 13B). Thus, it appears as though growth on D-fructose is dependent on a constitutive PEP-dependent phospho- transferase system that is functional in QQl7 in the absence of PTPfru and has an apparent Km of 7.4 mM for D-fructose. Further evidence that D-fructose is phos- phorylated by this system in 0017 is the fact that growth on D-fructose does not induce high levels of D- fructokinase, whereas DD31, which cannot directly utilize the D-fructose l-phOSphate formed by its PEP system, induces a D-fructokinase to a level six times that of 0017 or PRL-R3 (33, 53). If D-fructose was entering 0017 by facillitated diffusion, it would have induced this enzyme to a higher level. 78 .mwOHUDHMIQ mo mCOHumuucmocoo “Gouwwpr Cm>om :o mmuHmm cam .Hmmm .mHoo mo moumh Luzouw osu mo somHumafiou < HmmDOIV m2 :- .HH oustm 3. «23 3. (”“029 ALISNBG WVOLLdO O Q‘QV: O. O @ ¢ 0 O. 0 w ¢ N O O. 0 o O H . . H H . H H . IHIWIHI" “I" III '0‘ @N.N :51 I a ATflmfimw. ¢n1¢xAM x\ \- an“ I. L y\°\° Q. X\ x on .w x. u 1 mmw . I nfie. C\ xx \ r c n \\ h L «\u u n - at. u on.» no .6 - J “V\ - x. a n H. $0.. a n \D \\Q\ I|.\ .0 nail-I 8.4.. \k V\\0 q 3.. o J 0 Q - . - 01¢ avxx.onv - \\ 2. .0. 9!- .n on.» .\ o nfi¢ - I o w. oo\\ r «.14 ¢ - _wo_ - x a on.“ Illafllv\ . o as 1 “RV l MAN 1 osu AW\ n Qw. U SE 1 m;¢ G - SN @wa - H z... - n._¢ 1 ono.ost.-o I unoHoa..-o n 26 n u ouo.o:.-o mm-Jma Soc 500 H -\L P L- _ IL . _ . _ L‘ .mmoousaQ mo mcoHumHHCmocoo HcoponHp Cm>mm so manmm cam Hmoo .mHoo mo moumu Luzon» 659 HO comHumano < .NH mustm HmmDOIV m2...- 79 O. m o v N .u o. o w Hy N my 0. o w e. N O H H H H H H H H -1- 1H H H H H1 H :2 . w I l . . NO. .. I. I. .l N \\\; l m H x.» J H... H. m u H “mm 0 n . ax .-. u #9. 3 3.. an... N S 8.. sun...- o~.~ out I T @N.N .l.l.l I. 1 JON. IIA. . Hr -4\ m. a. 4nd .. .- - I WW.“ ulflllxlxu I. 00 .n x l.- x Ixinxlx‘ «V- ) .I 1 Cxlo .1 10?. C.— u . 1 ad. a! . s... u l l u we. a $ .- 05 a .HH: .- poo. 2 HI 0NN ¢‘& 1 . o 0 o I. I. O .- 93. 1 n .v . u 1 ca. U n SE n IE 1 uoo..w 3320-0 082.35 I\ m m u Jma _m 00 - P h F p p — n p b 80 .NH cam HH monomHm Eopw mum muma .HOHQ Musmuum>mmBmcHH mmoousaQ .uOHd xusmuum>mm3mcHH mmouosumua .m>udo COHumusumm omoousuQ .m>uso COHumHSuwm mmouosumuo . «mom .mmouus-n cam amouosHH-a so mm-Hmm cam .Hmno .HHoo Ho LuBouw How muOHa xnsmuum>mm3mcHH paw COHumHSuMm muwuumnsm .mH mustm 81 Figure 13 MIN/GENERATION mm om mm OCH OCH oom com -29 mmouaHu-n Axe. mmoomHu-a H 00. me. om. mH. o as on ON oH H H . . . . _ . I J l ..mwmmwmnnlAwnflnAw¥ I. @131. a a. x - .- r. Hum-HE 1 47 J57 4‘1 {Job-o . Hmna .H-ze 9.. nvw- w -nw- Maw» tunnwnmwl %.i? 19:1 H-ze mmoeoamH-o HHoo H259 mmoeosmH-m m cm ma. om. mH. o as om OH OH H H H H -1 H- H H .< OH mH om NIN/SNOILVHHNHS C_0I p1 fx t1 I‘E It) 157* w} 56 82 Thus, QQl7 apparently metabolizes D-fructose by a phosphotransferase system that has a high Km for D- fructose and does not require PTPfru for activity and therefore is a constitutive system. For normal growth rates at low D-fructose concentrations, induction of active PTPf is required and thus, PTPfru functions in I'll an inducible phosphotransferase system. Lack of Requirement of PTPfru for D- [U-IAC] Fructose Binding PTPfru was shown to increase the apparent affinity of the phOSphotransferase system for D-fructose (35). To determine if this effect was due to a PTPfru-dependent change in affinity of enzyme IIfru for D-fructose, the 14 amount of D-[U- C] fructose bound to a component(s) in crude extracts of Aerobacter aerogenes was measured. Crude extracts of PRL-R3 and 0017 induced on D-fructose were incubated in 10'4 MID-[U-IAC] fructose at 300 C for 10 minutes and then chromatographed over Sephadex G25 to separate free D-[U-IAC] fructose from that which was bound to a high-molecular-weight component(s). 0017 5 and PRL—R3 extracts had 1.16 x 10.5 and 1.06 x 10- umoles of D-[U-14 C] fructose bound per mg of protein, respec- tively. Thus, the presence or absence of PTPfru has no effect on the amount of D-fructose that becomes bound. When the bound radioactivity was chromatographed on Sephadex G200, the peaks of radioactivity eluted in the 83 void volume, indicating that it was bound to a high- molecular-weight particle (probably membrane vesicles). Crude extracts isolated from PRL-R3 grown on D- glucose, when incubated at both 00C and 300C with D- [U_l4 C] fructose, had less bound radioactivity than did extracts of cells grown on D-fructose (Table III). Table III. D-[U-laC] Fructose bound to crude extracts of PKL-R3 grown on B-fructoge and D-glucose and incubated at 30 C and 0 C. a . Growth Substrate CPM /mg Crude Extract Protein 30°C * 0°C D-fructose 560 477 D-glucose 167 207 aCounts per minute in the void volume of a Sephadex G25 column. The data indicate that growth on D-fructose in- creases the capacity of extracts to bind D-fructose. The presence of active PTPfru is not required for this binding; however, QQl7 could contain a defective PTPfru that affects the binding of D-fructose but not its phOSphorylation at low D-fructose concentrations. Another explanation is that a Specific enzyme II is induced by growth on D-fructose that has a higher affinity for binding D-fructose. 84 Inducibility of Enzyme II by D-Fructose Enzymes II (100,000 x g_precipitates) obtained from crude extracts of PRL-R3 cells grown on D- fructose, D-galactose, D-glucose, D-mannitol, cello- biose, and sucrose contained various levels of PEP— dependent D-fructose phOSphorylation activity (Table IV). The activities at high (200 mM) D-fructose concentrations, without added PTP ranged from 1.86 nmoles D-fructose fru’ l-phOSphate formed per minute per mg enzyme II to glu 13.4 nmoles per minute per mg enzyme IImtl' The activities at low (2 mM) D-fructose concentrations also varied; however, the D-fructose-induced enzyme II activity was 2.2 times greater than that of the next highest activity. Thus, the levels of D-fructose phos- phorylation vary with the source of enzyme II. At high D-fructose concentrations the enzyme IIfru is in the middle range of activities. However, at lower D— fructose concentrations, it has a higher activity than the other enzymes II, and thus must have a higher affinity for D-fructose. This higher activity at 2 mM D-fructose is partially due to some PTPfr that is u trapped in the enzyme IIfru vesicles. This residual PTP is removed from the enzyme II by Sephadex G200 fru chromatography as shown in the following section. The results of these control experiments at high D-fructose concentrations show that D-fructose phosphor- ylation is catalyzed by a constitutive enzyme II. This '1‘ c ‘\E ‘\ ‘\ F\ "030 H-HU 0:4 Erii" TC”IH‘~IU\'FC‘ C-f1mnn.» Nun-NUC-Uuwb T.P~A~N luthflrA-AIN ”W4..VN-\--Uudn nfl-~> van-aw n y! ‘.v:l\ ‘.J\ rilwiiifii! .Ivl-N-Jz 6‘ i ii! a C WQUOC mCCfiUWHUCUUCOU WWOUUJHHI Q uzu “W U C «UH-vH-HCQ .UHUI DNMNAW P-dIIH p: de.d~Nd-N.F-H ind-.5; HHW -U .- \ni 5|. P-II\I\ \ o-ll HumH-HWWU.CCJ WLOJUUWQZ HL-H ACCHUH-chHC-H annernHTaJ—HQIH. 40030-03 ~l-H- IQ ubcm-Tuc HUMJUHHU .HUuuFHFVUmUH .vHLU h~PJH mu.dn~ht~nv.ru.4~u nu..- ananm-q..~> FHA. F.§nu-I MLH ~:.v-J CHHu-IJH uv.d-.~ PHHNAHAH wild-H Uni-Ii d,v-V ,il I m ‘:Iu~9.~ on... ’un-nuh U-II-i h i -d||‘\.§i-s§ - la-HJHL AVAHAH I «H...- u \.\u,‘ 85 mm.o Hm.N HH.o mo.N mo.H OON ow.o wN.H oa.o Hm.o mq.o N o.mH mmoposm oN.o Nm.m ON.o- Nm.N NH.m ooN om.o Ho.H mq.o 0H.H HN.o N m.HH mmOEOHHmo no.0 ON.N Ho.H- mm.a Nm.© CON NN.H HH.N mN.o om.H NH.H N m.HH mmouomHmm-a mN.© H.0H Nm.m HH.w NH.H ooN HN.© H®.m mo.q mm.N om.N N o.HH mmouosHH-a u :0 GBOHw mHHmo Eoum wmchuno mmumuHaHomua HH 65%Ncm.w x ooo.ooH mo mmHuH>Huum onHomam so mCOHuowum summem HamHmmme mo Homwwm .>H mHan 86 .suwmem venom mo mocmmmna cH zuH>Huom onHomdm paw HouucOo mo qu>Huom onHommm cmm3um£ mommummep mmHchmeo .HH mezmcm Hume HucHE mumcamonanH mmouosumso mmHoEc mo mEHmu CH zuH>Huom onHomamn .vmnem :HHHHH ozm oo.H «.HH om.ou H.MH q.MH oom mo.o do.H qm.o mo.m Hm.H N m.w HouHCCmEuQ 00.0 oq.m mo.ou mw.H ow.H com NN.o OH.H mq.o wm.o mm.o _ N N.mH mmoous-o oH anmH 87 activity varies over a six-fold range and is present even in extracts obtained from cells grown on substrates not utilized by this phophotransferase system (D- galactose, cellobiose, and sucrose). When PTP was fru added, the enzyme II specific activities at 2 mM and 200 mM D-fructose either increased equally or to a greater extent at low D-fructose than at high D-fructose concentrations. The increases, caused by addition of either PTPfru sample, in the enzyme IIfru activity at both D-fructose concentrations were much greater than any of the other enzymes II. However, it is evident that PTPfru did activate all of the enzymes II tested at 2 mM D-fructose. This was first thought to be an effect of lowering the Km for D-fructose which would increase the activity at 2 mM D-fructose. Any increases in activity at 200 mM D-fructose were explained as a secondary role of PTPfru’ that of an activator (35). Another hypothesis which fits the data is the activation by PTPfru of a separate system which has a low Km for D-fructose, thus effecting equal increases in D-fructose l-phosphate formed at both 2 and 200 mM. Since enzyme IIfru has a much greater increase in activity, D- fructose could be inducing a component of this separate system, namely a D-fructose enzyme II. The lower increases at 200 mM could be due to inhibition of some part of this complex system. The high activity of the enzyme IImtl at 200 mM 88 D-fructose may be due to an induced D-mannitol enzyme II (105) that also has a low affinity for D-fructose. D-Fructose phosphorylation catalyzed by enzyme IImtl is inhibited by D-mannitol, whereas D-fructose phOSphor- ylation by enzyme IIfru 18 not (35). Effect of PTP on Sephadex fru G200 Enzyme II Activities The relationship between PTPfru and enzyme IIfru was further studied with more purified enzyme II samples. Crude extracts were prepared from D-fructose- and D-mannitol-induced PRL-R3. They were chromatographed on Sephadex G200 and fractions containing maximal enzyme II activity were pooled and used for assaying the effect of (i) a Sephadex G200 pool of PTPfru and (ii) the 35 to 55 percent saturated ammonium sulfate fraction of this pool on high (200 mM) and low (2 mM) D-fructose concentrations. Enzyme I and HPr which had been purified by Sephadex G200 and DEAE cellulose column chromatography were present at saturating levels. The specific activities at 200 mM D-fructose of PRL-R3 enzyme IIf and enzyme IImtl were approximately ru equal (Table V). When the Sephadex G200 fraction of PTPfru was added to the PRL-R3 enzyme IIfru the actual increase in activity was almost exactly the same as in Table IV. Thus, with equal additions of the same sample of PTPfru to two different enzymes IIfru the total 89 .mmcHEumumw uoc mmHchme .260 0 Hem 0600a mo monommua CH muH>Huom oHHHommm 0am HouucOo mo NuH>Huom onHomam me3umn mucoummmHu mmHchmeo .HH mechm Hqu anHE wumcdmonauH mmouosumuo mmHoEG mo mEumu CH qu>Huom onHomamn .umnum :HHHHH 02m 00.0 00.m 0:: 00.N 00N No.0 mm.H 0n- Hq.0 N H.NH HouHccmEno q.NN N.mN m0.m 0N.0 mw.N 00N m.qN 0.0N 00.0 mH.m 00.0 N m.HH mmouosumua oH mHan mm 65mm mnu mum chHuommu 65H .xnamuwoumEopno CEdHoo 00N0 xmwmnamm N0 vamHusa HH mmezucm >0 Umukaumo GOHumHmuosdmosm mmouosumum so mCOHumumamua summHm ucmummme mo uommmm .> chmH 90 activity increases to an equal extent. The "fold increase" over the background was much greater, however, with the Sephadex G200 enzyme IIfru' This method of preparing enzyme II removes residual PTPfru’ whereas ultracentrifugation preparations of enzyme II retain varying levels of PTPfru' This residual level of PTPfru increases the activity at 2 mM D-fructose as seen in comparing the data in Tables IV and V. When the ammonium sulfate fraction was added to the enzymes II described in Table V, the increase in activity of PRL-R3 enzyme IIfru was 24 times greater than the increase in activity of enzyme IImtl; however, the increases at 2 and 200 mM D-fructose were equal for both enzymes II. These data, obtained with more puri- fied enzymes II, fit the hypothesis that D-fructose induces to higher levels an enzyme II which is acti- vated by addition of PTPfr and has a low Km for D- u fructose. Thus, the net effect of activation is addition of equal amounts of D-fructose l-phosphate-forming activity at both 2 and 200 mM D-fructose. The fact that the increase in PRL-R3 enzyme IIfru activity was greater at 2 mM than at 200 mM D-fructose could be due to the inhibition of one system, either the "constitutive" phOSphotransferase system (measured in the absence of PTPfru) or the "inducible" phosphotransferase system (activated by addition of PTPfru), by the components required for the other system. 91 These data indicate that the role of PTPfru is that of a Specific requirement for a separate system with a low Km for D-fructose. This system could involve an inducible enzyme II which Specifically inter- acts with the PTPfru in this low Km system. The level of this enzyme II varies with the growth substrate. In these experiments the D-fructose-induced activity (the increase in 2 mM D-fructose phOSphorylation affected by addition of PTPfru) was 89.6 percent of the total activity (induced activity plus 200 mM constitutive activity measured in the absence of PTPfru)’ while in D-mannitol-induced cells the level of this specific enzyme II was only 27 percent of the total enzyme II. Other data (discussed in a later section, Figure 19) have indicated that this inducible enzyme II activity is only 2 to 8 percent of the total enzyme 11 activity in enzymes II obtained from substrates other than D- fructose. Induction of Both Enzyme II and PTPfrubnyAFructose The data in the previous two tables (IV and V) indicated that a D-fructose-Specific enzyme II with a high affinity for D-fructose is induced by growth on D-fructose and that this enzyme II is activated by the addition of a fraction isolated from D-fructose-grown cells. The induction of both PTP and enzyme II was fru examined by assaying combinations of 100,000 ng 92 supernatants and precipitates isolated from cells grown on different substrates. Crude extracts of PRL-R3 cells grown on nutrient broth, D-mannitol, D-mannose, D-glucitol, and D-fructose were centrifuged at 100,000 x g, The resulting pellets [resuSpended in 0.02 M Tris- HCl buffer (pH 7.5) containing 0.028 M 2—mercaptoethanol] and the upper portions of the supernatants were recen- trifuged at 100,000 x‘g for 2 hours. The second centri- fugates were resuspended and adjusted to approximately equal protein concentrations as noted in Table VI. The supernatants were chromatographed over Sephadex G25 columns to remove endogenous low-molecular-weight compounds which might affect the activity and then were adjusted to approximately equal protein concentrations by dilution with 0.02 M Tris-HCl (pH 7.5) containing 0.028 M 2-mercaptoethanol. Assays were then run with saturating levels of HPr and enzyme I at 200 mM D- fructose and with limiting levels of enzyme II. Controls were run without adding the enzyme II fractions. The only change in the activities of the enzymes II which was interpreted to be Significant occurred when the D-fructose supernatant was combined with the enzyme IIfru’ which resulted in a 160 percent increase in the Specific activity of the enzyme II (Table VI). This supernatant had little effect on the activities of the other enzymes II. The only other noticeable effects were minor, namely (i) a 56 percent stimulation of 93 .mnamuwoumEouso CEDHoo mmOHsHHmo mu30H ma woumEHumo mm3 GHmuonm .mNu xmnmsamm Co vocamuwoumaouno 0cm mm x 000.00H um moH3u 0mmSHHHu5mov mucmumcumasm msu mo H: om 0am .Aw.x 000.00H um moH3u wowsm -Huquov HH mmE%NCm Hm50H>H0cH ocu mo m1 0H Humm mo we NH.0 HH mexmcm we we 00.0 .mmz mo mmHoel 0.m . How: mo mHoEJ H.0 .wmou nosumum mo mmHoEJ 00 .HocmsumouamoumEuN mo mH081 00.0 .Am.n mav HomanHH mo mmHoEJ 0.0 vmchucoo AHE N.00 xmmmm 60H .mucmumc nquSm_w x 000.00H ucmummeU mo moCmmmum mnu SH mmumuHaHomua.w x 000.00H HH mENNcm ucmummmHv mo mmHuH>Huom onHoon 00 somHHmanu .H> mHHMH .0m00m unmumcumasm oz 94 a .HH mEzmcm HnwE HucHE mumnamonauH omouosumun mmHoEc mm vochQO NH.H wq.H N0.H 0N.H om.H 0N.H He\we N0.ON Luopm ucmHHusz NH.H 00.0 wo.N ON.0 mN.© wN.N He\we om.oN HouHccmEnQ HN.0 No.0 0N.0 mH.0 m0.0 No.0 He\we oo.NN HOHHosHm-a wm.m No.0 0N.m HH.m 00.0 oN.0 He\we mm.ON mwofifimena Ho.N Hm.N oH.m N0.N HH.m mm.H He\me 0N.mH mmouUS-HMIQ HE\wE HE\wE HE\wE HE\wE HE\wE nHouucou mumaHomum mo.m mN.m 0m.m HN.m NH.H HH masucm Luoum ucmHuusz HouHGCmEuo HouHodeuQ mmoccmEuQ mmouosumua unmumcquSm m x 000.00H mNHH>Huo< UHHHumam .H> mHan 95 enzyme IIfru by the D-glucitol supernatant, (ii) a 49 percent stimulation of enzyme II by the nutrient broth NB supernatant, and (iii) a 19 percent inhibition of enzyme IImtl by the nutrient broth supernatant.~ The data in these experiments Show that only growth on D-fructose induces a component (PTPfru) in the 100,000 X.8 supernatant that significantly increases the PEP:D-fructose l-phosphotransferase activity of enzyme IIf This supernatant had no effect on the ru activity of other enzymes II and the other supernatants had little effect on any of the enzyme II activities. Thus, the cytoplasmic component, PTPfru’ induced by D- fructose only interacts with the D-fructose-induced enzyme 11. The sugar-specificity of these two inducible components has not been rigorously studied; however, pre- liminary data have shown that PTPfru does not increase the rate of PEP-dependent phosphorylation of D-mannose, D-mannitol, or D-glucitol catalyzed by enzymes 11 isolated from extracts of cells induced on D-fructose, D-mannose, D-mannitol, and D-glucitol. Additivity of Constitutive and Inducible Systems If these two systems contain separate enzymes II, their activities should be additive. Data in Tables IV and V showed that addition of PTP effected an fru increase in activity that appears to be additive. It was later discovered that the high affinity system 96 functions in the absence of HPr, and thus can be assayed in the absence of HPr. The constitutive activity can be assayed in the presence of HPr and absence of PTPfru‘ These assays, which are described in Methods, were used to determine the effect of D-fructose con- centration on reaction velocity (Figures 14-17). It was determined, using these assays and QQl7 enzyme IIfru obtained by 100,000 x g centrifugation of a pool of enzyme II activity from a Sephadex 0200 column, that the Vmax and Km of the constitutive system were 5.34 nmoles D-fructose 1-phosphate formed per minute per 3 mg and 7.14 x 10- M D-fructose, reSpectively (Figure 16) - The V and K of the inducible system were max m S M D-fructose in 5 determined to be 3.78 and 1.6 x 10- the presence of HPr and 3.15 and 1.9 x 10- M D-fructose in the absence of HPr (Figure 17). The above six values were obtained by assaying D-fructose l-phosphate for- mation at D-fructose concentrations from 0.01 to 90 mM in the presence and absence of HPr and PTPfru' The activity of the reaction in the absence of both HPr and PTPfru (0.2 to 0.3 nmoles D-fructose l-phOSphate min-1 mg- enzyme II) was subtracted from the activities found when (i) both HPr and PTPfru’ (ii) only HPr, or (iii) only PTP were included. The three resulting fru Values were termed (i) constitutive + inducible (+HP1'): (11) ConStitutive, and (iii) indUCible ('HPI‘), respec- tively (Figures 14 and 15). The final D-fructose 97 .HH mE>Ncm Hume ICHE mum0mm000uH mmOHUSHHnQ meoEG 0.0 on N.0 EOHH wmwcmu :meHm 0cm nmm 0000 Ho mocom0m m0u CH mosHm> HamHm .>uH>Huom Aumm+v mH0HoswcH + m>HusuHumcoo $00 EonH >uH>Huom m>Hu:uHucho m0u wcHuomHu0sm >0 UmumHsono mw3 m>uso Aumm+v mH0HosvaH 00H .uxmu m0u CH mum mHHmumU Hm0uusm .0m05HoaH mmB mGESHoo mmOHsHHmo mHuow m>HunuHumcoo Mom .UmwsHocH mmB mGESHoo 00N0 xmwmzamm 00m mmOHs -HHmo mHuom mH0H650CH mom .>0mmumoumEou0o CESHou 00N0 xmvmnamm 0am meHaHHwo m0 UmHHHusa H wE>Ncm we we mmH.0 00m CESHoo 00N0 memHQmm m no 0m0amuwoumEou0o HHHHHH mE>Ncm 0cm >HOO Ho we mMH.0 umchucoo mCOHuommu wwm0H .mmHuH>Huom mmmumm umcmuuonamo0a mH0HosucH 0cm m>HuDuHumcoo Ho mm>pso COHumusumm .0H mustH 98 J 4 Figure A250 mmOHOOmm-O OOH OO OO H H H H m HOSE H mm+0 H0. O H-It 00 OIJ Jl‘ AHHm-v «HOHosvcH-Jz .H‘ m- \ m>HuDuHumcoo .- \. Humm+0 OHHHosccH + m>HHsHHuchO INK UIm saIOWU) EIVHJSOHd-I asomonaa-a [— 8w I- (11 amxzus 99 .uxmu m0u CH mum mHHmumv um0uudm .0H mustH CH 0m0HHomm0 mm mum mucmcoaaoo .mCOHumHucmocoo mmouosumnm 30H um mmHuH>Huom mmmummmcmuu uo0amo0a mH0HodvcH 0am m>HusuHumcoo Ho mm>Hso SOHumusumm .mH mustH 100 J "A 2 Pi guru HZEV mmOHUHHm-Hum 00.N 0m.H 00.H 0m.o H H H E m>HusuHHmcoo AHAHHHIV m 7H USHEH ( o 3.30 22.. 265 Z Aumm+v mHOHuducH -JLi\\\\\\\)SIJ +.m>Hu:HHumcou J¢ m.H 0.m H.0 ”*1 amfi‘dua [film I UIUI 891mm) HIVHJSOHJ'I 330131300 101 .ume m0u CH 0m0Huomm0 mm Humm+v mH0HoswcH + m>HusuHumcoo pom mmump 0mumH56Hmo msu mucmmmuaou mcHH 0HHom msu H>uH>Huom Humm+v mH0HoswcH + m>H050Humcoo m0u Mom mucHom mumv mum A00 meowHHu ammo $09 .0H mustm EouH mum muma .mGOHumuuchCOo wmouosumum 0wH0 um meuH>Huom mH0HosncH 00m o>HusuHucho me uon Husmuum>mm3mcHH .OH mustH 102 16 J“ 3.5 ure AH 250 mmOHOOmm-O N H H H humm+v 0H0Ho§0aH + o>HusuHumcoo vmumHfionu ./ Aumm+v oHOHoswaH + m>HusuHumcooJ< Humm+v mHHHoswcH\ \- Aumm V mH0HosnaH OHHHOHHHOOOQ lrl'l' 1. h ,H; o , cs awAzua 8m aim) HIVHdSOHd-I asomonua-a 0m. ( [—Saloluu II 103 .uxou 000 CH mum mHHmum0 Hm0uusm .mH mustm Eoum mum mumm .mGOHumHHCmocoo omouodumna 30H um mmHuH>Huom mH0HosncH 0am m>HuduHumcoo Ho uoHa Husmnum>mm3mcHH .HH must-H 104 J 7 Figuro AH-zev mmoaoamm-O 00H 00 00 00 ON 0 ON: 00: 30: cm: . H H . H H \ H Tammi-v 04 4 \ mHnHosocH .1 H. a O c AhmmnTv I1 I] mHHHOsOcH + 0>HusuHumaoo a. p Humm-H «HHHuavcH I. II. o>HuDuHumaoo . . . . . HT . . . x? (”O O O 00.0 N0.H 0m.H 0>.H (I-Satomu II awfizua 3m UIm) ELVHdSOHd—I SSOLOHHJ~O 105 l-phOSphate and D-fructose concentrations in each individual reaction were determined and the sum of these was used as the total initial D-fructose concen- tration. The initial concentration had to be measured because reactions without added D-fructose contained 6.4 nM D-fructose, which was most likely bound to the enzyme II vesicles. The average D-fructose concentra- tion of the reaction was determined and used as the actual concentration in plotting the curves and cal- culating the data for this eXperiment (67). This procedure was necessary because at the lowest D-fructose concentration, 60 percent of the available D-fructose was converted to D-fructose l-phOSphate during the 10 minute reaction period (see Methods). The activity of the inducible enzyme IIfru in the presence of HPr was determined by subtracting the constitutive activity from the constitutive + inducible (+HPr). An apparent inhibition of the calculated inducible (+HPr) activity was obtained at higher D- fructose concentrations (Figure 14); however, there was no inhibition of the inducible activity in the absence of HPr. Therefore, either HPr was inhibiting the inducible activity, or the PTPfru was inhibiting the constitutive activity. Further experiments (to be described later and which are depicted in Figure 19) using enzyme II isolated from QQl7 indicate that, in gly 106 fact, the PTPfru does inhibit the constitutive enzyme II activity. In the assays shown in Figures 14-17, the inhibition of the constitutive activity by PTPf is approximately ru 40 percent. To determine if the separate inducible and constitutive D-fructose 1—phosphotransferase activities were additive when corrected for this 40 percent inhi- bition, a theoretical curve was generated from the following equation: max1 0'6 vmax2 v: K + K “‘1 m2 “Ts? 1+TSTJ— where v is the velocity, [S] is the D—fructose concen- tration, V and K are the kinetic constants for max1 m1 the inducible system in the presence of HPr, and Vmax 2 and Km are the kinetic constants for the constitutive 2 system. V is multiplied by 0.6 to correct for max2 inhibition of the constitutive system by PTPfru The resulting calculated velocities (Figure 16) superimposed over the actual data points. Thus, the separate inducible and constitutive systems are additive in their formation of D-fructose 1-phOSphate when the inhibition of the constitutive system by PTPfru is corrected for. 5 The Km of 1.6 x 10- M D-fructose in the inducible 107 5M, system is similar to the Km (0.66 to 1.8 x 10- depending on Source of enzyme II) for D-mannitol phos- phorylation studied by this author (unpublished results), and lower than Km values for other phOSphotransferase systems reported in the literature (78, 92, 108). The 3 M D—fructose) Km of the constitutive system (7.1 x 10- is nearly equal to the apparent Km for growth of 0017 on D-fructose (7.4 x 10-3, Figure 13C). This is consis- tent with the supposition that 0017 utilizes the consti- tutive phosphotransferase system in its metabolism of D-fructose, that the rate of growth of 0017 on D-fructose is limited by the rate of phosphorylation of D-fructose by enzyme IIfru (constitutive), and that the constitutive system does function when PTPfru is absent. The fact that PRL-R3 has a higher rate of growth than that attained by 0017 could be evidence for the in 2112 additivity of the inducible system (which functions in PRL-R3) and the constitutive system (which functions in both PRL-R3 and 0017). Function of PTPfru as a Substrate for Enzyme IIfru When it was determined that there were two separate systems involved in PEP-dependent D-fructose phOSphory- lation and that PTPfru is an absolute requirement for activity in the inducible system, a new assay had to be developed to replace the "fold increase" units of activity. 108 This new assay involves calculating the amount of PTPfru that gives one-half the maximal activity of a given amount of enzyme II. The activity is defined by ”half- maximal saturation" units as described in the PTPfru assay in Methods. To demonstrate the validity of this assay, reactions were run at 0.5 mM D-fructose containing saturating enzyme 1, a level of HPr which was half- saturating, 0017 enzyme IIfru’ and varying concentrations of a sample of PTPfru purified through the second DEAE cellulose step. A slight sigmoidal characteristic seen at low PTPfru concentrations (Figure 18A) has not yet been explained. For the following analysis, the sigmoidicity was ignored and the data treated in Line- weaver-Burk fashion. Double reciprocal plots of the data indicate that the apparent Km for PTPfru remained the same for two levels of enzyme IIfru (Figure 18B). Thus, the apparent Km for PTPfru is independent of the amount of enzyme IIfru’ and PTPfru acts as a substrate in the enzyme II-catalyzed phosphorylation of low concentrations of D-fructose. If PTPfru was an integral part of an enzyme IIfru complex, there would be a pr0portional relationship between PTPfru and the enzyme IIf DoUbling the level ru' of enzyme IIfru Should have doubled the amount of PTPfru needed to saturate the system. This effect was not observed. To determine if the data obtained with the "fold 109 .CEDHoo mmOHaHHmo mCamuw noumEou0o >0 anHHusa H mE>NCm we we NH.O .>0@muwoumEou00 mmOHsHHmo muH>Huom mH0HodvcH HH mE>NCm m0u pom mno0umz CH 0m0Hpomwc mm Cap mum3 mCOHuommm .duHmHm Co momeCmamv u>uH>Huom mH0H050CH Ho muOHa Husmuum>mmeCHH 0Cm COHumHDumm .OH mpstC Figure 18 l I ( -9910mu Imz° 111m) mvuasoua-I asomnua-a I - I J— ‘3 \JP_ \0 N (1-1m? 1.31m 881mm) mvnasoua-I ssomonHJ-a 0.075 0.100 0.050 -1 PTPfru (“1 ) 0.025 40 60 80 100 20 PTPfru (“1) 111 increase" units correlates with the ”half-maximal saturation" units, the D-fructose l-phosphate formation catalyzed by 0017 enzyme IIfru in the presence and absence of several purified PTPfru fractions was measured and units of PTPfru were calculated by both methods. The fold purification and percentage recovery obtained with both units were approximately the same. The data in this experiment have Shown that the apparent Km for PTPfru is not altered by changing the enzyme 11 concentration. A method of determining the "half-maximal saturation" units of PTPfru was developed us ing this fact . @hibition of Constitutive Enzyme II Agtivity by PTPfru PTPfru apparently inhibits the constitutive system as mentioned in the section on the additivity of the inducible and constitutive systems. This apparent inhibition of the constitutive activity was further studied using 0017 enzyme IIgly’ which has a relatively lour level of inducible activity (3.5 percent of the total). Assays were run at 100 mM D-fructose with varying levels 0f HPr, obtained from D-fructose-grown PRL'R3’ and PTPfru' A Slight increase (8 percent) in activity was observed When 5.0 ul of PTP were added (Figure 19A). This was fru due to a low level of the inducible enzyme II in enzyme Ilgly Which was activated by levels of PTPfru lower th . . . . . - an those required for inhibition. Higher concentrations 112 OH OH O.m O IJfl-HH OHHCHC C) ‘3 C] CD “nmeoH .HE\wB N.0 UwCHmuCoo mHHmo C3ouwummouosumun EOHH vmumHomH “mm .3on0 wouoC mm 0000m mumB >0amuwoumfiou00 CEsHoo muHumamH>xou0>0 Eoum nmumHomH AHE\wE H.wv Duwmem Ho mucnoE< .>HwHH mE>mCm NHOO we we N>N.0 nCm H mE>NCm me we 00H.0 nmCHmuCoo 0C0 muoHumz CH 060Huomm0 >Mmmm AuCHoaqum mmouosumunv umm 600 wCHmC CDC mum3 m>mmm< .Emum>w m>HusuHum uCoo mLH >0 COHumH>uozmm000 mmouodumna Ho COHumuuCooCoo umm Co moCmvaamc wCH300m muOHa Husmuum>mm3mCHH nCm COHumHSumm .OH mHOmHC 113 Figure 19 AH-He .HH: :3 Ex .OH.O ONOO Ono-O ON0.0 O OO OO O0 ON a H H H , H. _ . H O .HH \ m m \ I NH.H o S 3 "O... .M T. ) r 0 [£0 ..a m I. H I. _. nu DU 4 3 my HN.0 m . H 1. 1 OOH O 30 m ul 20 \1 m3 I 1:6 m 8 0 H1 H1 0ON.O s m 40H; .1 nu n m .A S \H m. 0% .._- o T Lfinow an ...\ m. NNN.O s.”- I I.\ a LO.OH an.“ 2 m 8 m OOHO H. an H H P _ P _ H hm 114 of PTPfru decreased both the maximal velocity by 23 percent and the apparent Km for HPrfru from 0.113 mg to 0.095 mg (Figure 19B). The observed inhibition was most likely due to interaction of PTPfru with the enzyme I; however, this cannot be determined without using separate uncoupled assays for enzyme I and enzyme II. The data in this experiment Show that PTPfru inhibits the constitutive activity by 23 percent and decreases the apparent Km for HPrfru from 0.113 to 0.095 mg. The data also establish the definite require- ment of HPr for activity of the constitutive system. Requirement foerPr of the Constitutive and Inducible Systems HPr was required for activity of the 0017 enzyme II constitutive system (Figure 19) and in the 0017 sly enzyme IIfru activity termed "constitutive" in Figures 14-17. The inducible system functions in the absence of added HPr; however, its activity is increased 20 percent by addition of HPr (Figure 16). To further determine the actual requirement for HPr of the constitutive and inducible activities, two enzymes IIfru were used to measure the apparent Km's for HPrfru of these activities, and enzyme IImtl was used to measure the apparent Km for HPr (obtained from both D-mannitol- and D-fructose-grown cells) of D- mannitol phOSphorylation using the HPr (D-mannitol continuous) assay. (Both HPr's were purified 115 approximately lOO-fold by the procedure described in Methods.) Enzyme 11m 1 had an apparent Km for HPrmtl of t 26.3 pl (27 ug) and an apparent Km for HPrfru of 17 pl (80 ug) at two enzyme II concentrations (Figure 20). 0017 enzyme IIfru assayed at 100 mM D-fructose had an apparent Km for HPrfru of 25 pl (13 ug); when assayed in the presence of PTPfru at 0.5 mM D-fructose (Figure 21), addition of HPr had no affect. Using another 0017 enzyme II preparation, there fru was an increase in activity at 0.5 mM D-fructose with the addition of HPrfru (Figure 22A); however, the appar- ent KA for HPrfru of 0.4 ul (1.9 ug) (determined by subtracting activity in the absence of HPr prior to plotting the data, Figure 22B), is 1/50 of the Km's for HPrfru of the other enzymes II. Also, the level of activation is dependent on the level of PTPfru present. HPr causes a 100 percent activation with limiting (0.25 "half-maximal saturation" units) PTPf and only I'll a 20 percent activation at a four-fold higher level of PTPfru' HPr actually decreases the apparent Vmax at saturating PTPfru (Figure 23). The different levels of PTPfru had no effect on the RA for HPr (Figure 22B); however, increasing HPr decreased the apparent Km for PT (Figure 23). Pfru These data Show that a constitutive enzyme IIgly (Figure 19B), a constitutive enzyme IIfru (Figure 21B), 116 .A>Hm>Huomammu .CHmuoua HE\wE 00.H UCw N.0 wCHCHmuCoo mHHmo CzouwuHouHCCmEuo Ucw ummouosumuo Eoum nmumHOmHv umm 0Cm HuEHH oE>NCm Ho mquoEm vmumoHnCH mnu 0am >0amuwoumEou00 CEDHoo 00N0 xmvmnamm 0am mmOHsHHmo m0 0mHHHu5a HH.HHH mE>NCm we we mm0.0 mCHCHmu uCoo >mmmm AmsosCHuCou HouHCCmEuQV Hmm mCH wCHm: Can mums w>mmm< .COHumuquoCoo um: Co COHumH>uonmmo0m HouHCCmEuQ Ho moCmchamw wCH3o0m muOHm Husmnum>mm3mCHH UCm COHumusumm .ON OHOOHH 117 Figure 20 AH-Hav “Om V HHav Hmm OH.O Om.O ON.O OH.O O O0 O0 ON O H e H H xx. H H H HHHH NHu VHO . . \ Hue HH mE>NCm o u. .0 we 0N.0 ) H W sum . u . . F OCH. IHH 0 m NN H a m HuEHH mE>NCm - , _.l we 2.01/1» H M T- DHMHnHHH 4 1w m m .00 N W n H r. 1H.NH m. O0.0 - r C.. 4T:///// m Hue. s w .L E NC 5 o T HH m m HuwH O [0.OHw 3.0 . AH: m.ONquv “mm m. m (m. .m H H- 0- .ON OO.O (1-1111 91' [311111 satomU) ELI-VHJSOHcI-I 'IOJ-INNVN-CI 118 .mmouosumuo SE 0.0 nCm Dummem we we 000.0 0mCHmuCoo m>mmmm Emum>m mH0HonnCH 609 .nmm: mw3 HH.HHHH mE>NCm HHOO we we N0N.0 umCu unmoxm 0H mustm mm mquCanoo mEmm o0u 0mCHmuCoo m>mmm< .mmHUH>Huom Emum>m oH0HodwCH nCm m>HusuHumCoo Co sumum: Ho uomHHm wCH300m muOHa xusmuum>mo3wCHH 0cm COHumudumm .HN mustH 119 >00 1.00 . m 100.0 HH-HOH Hmm AHJV Hmm OOHO s- OH.O O0.0 O OO O0 O0 ON ._ 0\ m H H H H H . mmouosumun D ‘ a n1 . II T 25 m 0 m H0 HN - H... u a H I mg I a m u 3 .mmw S I lNNmO a a I IT. I. n. m n 0 S r- 1306 0% I wil mmouoH-HHHIQ 0nd :2 OOH 1UI\.$I JOII (1 -- .10NO.O r. m“ n. e r u .wO .m .HH F H H _ _ H P H n1 ( JII amfizua P8111 1511111 831011111) H-I-VIHSOHd-I asomnm-a 120 .0muomuu05m >uH>Huom nanouwxom0 000 m>u50 HmoouaHowu How mmHuHoon> .Umm: mumB H06 0>0.0 0cm we 0H0.0v COHuomHH AHHV mmoHsHHmo mmH 030 UCm meum> mums AHE\0E H.0v Duwumm Ho mucsoEm m0u unmoxm 0H musmHm mm mquCoa quo mEmm m0» cwCHmuCoo m>mmm¢ .umm Co Emum>m mH0H650CH Ho moCmvaamv Ho muOHa Husmuum>mm3mCHH 0Cm COHumusumm .NN mHOmHm 121 Figure 22 In J S. lt‘ — H—1, [\ C) L— "Wi tn I'— —-I. 04 . m 1 l \o o: ‘3 <0 \o H 0 (I_sa{omu “JJII amfizua 3m uim) HIVH&SOHd-I BSOIDHHJ-G l I fl H. _.”2 H o — _H o O C 8.06- 4.03 12.09|,_ n41 . ( ‘II amfizua [_3m I_U!w satowu) aivndsond-I asomonuJ-a HPr (“1’1) HPr (ul) 122 0.372 I I I HPrfru A (Lil H o I m g; 0.298- 53 HS 0 . 0.] a {30.223— O-a '44 IH H+4 5g . 0.5 8 a 0.149— 1.0 g 8 ///////””’- 2 0 Dd .. 22° Q ‘2 . .5 0.074- E 7 0 . I 1 - 0.025 0 0.025 0.050 -] PTPfru(u1 ) Figure 23. Lineweaver—Burk plot showing effect of HPrfru on the apparent Km for PTPfru' 123 and an enzyme 11m 1 used to catalyze D-mannitol phos- t phorylation (Figure 20B), all had similar affinities for HPr obtained from D-fructose-grown cells. Although the inducible system does not require HPr for activity, an enzyme IIfru assayed at 0.5 mM D-fructose had an affinity for this HPr 50 times greater than the other enzymes II. Because of this high affinity it is possible that very low levels of HPr which may be trapped in the enzyme IIf vesicles could be enough ru to support activity of the inducible system. Effect of 2-Mercaptoethanol on Enzyme II‘ru Activity The inducible and constitutive system as described thus far have different affinities for D-fructose and different requirements for HPr and PTPfru for activity. Further evidence for two separate enzymes 11 for D- fructose phOSphorylation was observed when a sample of QQ17 enzyme IIfru (100,000 x g_precipitate of a Sephadex GZOO pool), which had lost appreciable indu- cible activity, was incubated for 48 hours in fresh 28 mM 2—mercaptoethanol at 4°C. After this incubation, the inducible activity was four times greater than the activity of a control that was stored at 4°C without added 2-mercaptoethanol (Table VII). The constitutive activity was not affected by the 2-mercaptoethanol. 124 Table VII. Effect of 48-hour incubation of enzyme IIfru in fresh 2-mercaptoethanol. Assays are as described in Methods for the enzyme 11 recon- stituted system and contained 20 umoles of D-fructose, 0.26 mg of enzyme I, 0.11 mg of QQl7 enzyme IIfru’ and the noted amounts of HPr and PTP fru' Specific Activitya Activity Enzyme IIfru HPr PTPfru b ExperiE Increase Assayed (ug) (ug) Control mental (Z) Constitutive + Inducible 110 32 16.9 28.2 67 Constitutive 110 0 14.5 15.9 10 Inducible 0 32 2.1 10.3 400 _ 8In terms of nmoles D-fructose 1-ph05phate min".1 mg enzyme IIfru at 100 mM D—fructose. bIndicates no 48-hour incubation with 2-mercap- toethanol. CIndicates enzyme IIf was incubated for 48 hours . ru With 2-mercaptoethanol. 125 Thus, the inducible enzyme IIfru is selectively reacti- vated by the addition of 2-mercaptoethanol. Formation of Phosphorylated PTPfru To further determine the requirement for HPr of the inducible system and the actual role of PTPfru as a J substrate for the inducible enzyme IIfru’ a series of experiments utilizing [32F] PEP was designed. 9. m a. Preparation and Identification of [32P] PEP The products of a reaction containing 32P L- i’ malate, and mitochondria isolated from chicken livers, were separated by Dowex 1 chromatography (Figure 24) as described in Methods. The radioactivity that eluted with 0.45 M triethylammonium bicarbonate buffer was identified as [32F] PEP by paper chromatography and its [32 ability to form P] ATP and D-fructose 1-[32P] phosphate. Formation of [32F] ATP—-—Samples of radioactive fractions (170, 190, 206, and 215) from the Dowex 1 column were tested for their ability to form [32F] ATP in the presence of pyruvate kinase and ADP in reactions as described in Methods. Acid-washed charcoal (Darco G-60) was added to the samples, filtered from the solution, and radioactivity bound to the charcoal was then measured by Cerenkov radiation. Only the charcoal from reactions containing samples of fractions 206 and 215 had bound Figure 24. 126 Elution of products of [32F] PEP-formation reaction from Dowex 1-X10 column chroma- tography. Different concentrations of triethylammonium bicarbonate buffer were added as indicated. Further details are given in Methods. 102 COUNTS PER MINUTE 127 Figure 24 ‘1 l T l WOOL 0.15M 0.30 M 0.40 M 0.45 M + P1 1 l 1000- W J a" 300- r PGA PEP 100- 1 Jr 1. 30- . x, 1 ld— J ‘1 J, l ' ’ .‘L 3L > J“ All 14% 1 . 1 l 0 5 100 150 200 FRACTION NUMBER 25 128 radioactivity that was dependent on ADP (Table VIII). These data show that fraction 206 contained a compound that is utilized by pyruvate kinase to make [32P] ATP. Paper Chromatographic Identification of [32P] PEP-—-Samples of fractions 80, 170, and a pool of fractions 200 through 221 from the Dowex 1 column were spotted on Whatman No. 1 filter paper and chromatographed with the alkaline solvent system. The chromatogram was dried and cut into strips which were then scanned for radioactivity (Figure 25). The sample from the pool of fractions 200 through 221 contained a radioactive compound that migrated with standard PEP; fraction 170 contained a major peak of radioactivity that corresponded to phOSphoglyceric acid, and a minor peak that migrated with standard Pi’ The radioactivity scan of fraction 80 had a single peak which migrated with Pi“ The sections of the chromatogram of the pool (fractions 200 through 221) that corresponded to standard Pi and PEP were cut out and counted in glass scintillation vials using Cerenkov radiation. The section correSponding to PEP contained 95 percent of the total radioactivity in the two sections. These data show that 95 percent of the material in the pool of fractions 200 through 221 from a Dowex 1 column is similar in its migration in the alkaline solvent to standard PEP. M ‘- T'7—.‘— _ 3‘ 3 ~ . L 129 Table VIII. Radioactivity bound to charcoal dependent PA- on ADP and fractions from Dowex 1-X10 column chromatography containing [32P] PEP. Procedure is described in Methods. ”—— ran—“rum“ - .I' r T L t r :— Radioactivity Bound to Charcoala Fraction Number +ADP - ADP 170 4,252 5,606 190 3,401 3,619 206 10,784 4,506 215 5,748 3,871 aCounts per minute in total sample of charcoal. Figure 25. 130 Scan of radioactivity on paper chromatogram of fractions 80, 170, and pool of 200 through 221 from Dowex l-XlO column chroma- tography. Standards were detected with acid molybdate Spray. [Note: Scale for fraction 170 is different from scales for pooled fractions (200 through 221) and fraction 80. Extension of each peak remains on its own scale.] 131 Figure 25 ------’.§O‘--~-.u JV n O 0 ' .I' v“ '9’ Fraction 120 . 0 —----"O--" Pool 200 - 221 Fraction 80 1.2— 5sz «ma 92:8 m2 origin 0 ’r 132 Formation of D-Fructose l-[32P] PhOSphate———To further determine that the pool of fractions 200 through 221 contained [32P] PEP, samples of it were added to D- fructose l-phosphate-forming reactions. These reactions Were run at 100 mM D-fructose and are described in Table IX. After the heat-denatured protein was removed by centrifugation at 6,000 x g, 50 pl amounts from each supernatant were assayed for D-fructose 1-phosphate using the D-fructose 1-phosphate end-point assay. The relative level of radioactivity in each reaction was determined by counting 10 ul amounts of this supernatant in a liquid scintillation counter. Another 10 ul amount was chromatographed on paper using the acid solvent. AKLJthentic D-fructose l-phosphate and phOSphoenolpyruvate Were detected with acid molybdate Spray and the radio- aCtive Spots were detected with a strip scanner (Figure 26) . These spots were then cut out and Cerenkov radiation was measured in a liquid scintillation counter. Reactions that contained enzyme 1, enzyme II, and HP]: formed D-fructose l-phOSphate as well as a radio- acltive material that co-chromatographed with standard D“ fructose l-phOSphate on the paper chromatogram (Figure 26) , whereas reactions lacking HPr or enzyme II did not form D-fructose l-phOSphate and only had a radioactive peélk that corresponded to PEP. Addition of PTPfru doubled the level of D-fructose l-phosphate formed; hoWever, the level of radioactivity in the spot 133 000.0 000 000.0 0000.0 00 - 00 000000 manaz : 000.0 000 000.0 0000.0 00 - 000 00002 0 000.0 000.0 000.0 0000.0 00 + : 000.0 000.0 000.0 0000.0 00 - 0 000.0 000.0 000.0 0000.0 00 - 00000000 mmm 0u0£a005muH uC000Cp0asm C000000m ACHZV Du 000aodphua H1 OH 0EHH mmHm 5000000m 0umnamosmuH 0uSCHZ 00m mucsoo 0000050hum 00H0€1 .00050005 003 co000000u >oxC0n0o 0:0 uso 050 0003 mmm 0:0 000LamonauH 0000090Mun ou wcHoCOmm0uuoo 00000 059 .mmm UC0 000samosmnH 0mouosuwum 00000900 00 p0na00woumeouco 0:0 muw>0uo0oww0u H0000 Mom 0000000 0003 0005U0H0 H1 OH .00000 unwoau0C0 000LamonauH 0mouosumnm 0&0 wchs 00050006 003 0umsamosauH 0mouosuwua .0009CHE u now 5003 00mm 0 CH wcflo0am 09 0009508 om no ma u0um0 U0aaou0 0003 0C000000m .00000 003 dummHm mo w: N00 .0000: 000:3 .CEUHoo H x03oo 050 Eoum HNN nwsoncu oom meowuomum mo 0000 0:0 m0 0: 00 000 000 000000000 00 0000: N.o .0mm M0 ma mam 00000000 0000 00 000m00 00 000000000 00000 00% 00 050000 0>0000000000 000 000000000 000000000 .0000 000 00000 0000 000000000 00000-0 00000000.0 00 000005000 .mefiams an. 1.0., uni ri.|‘l..ll|l' 134 Figure 26. Radioactivity scan of paper chromatogram showing formation of D-fructose 1- [32F] phosphate dependent on enzyme II and HPr. Standards were detected with acid molybdate Spray. 135 Figure 26 Complete Minus HPr Minus Enzyme IIfru fiv— origin 23()00F' 2K300" 11300"- 11300" 3:sz mum 3.20.50 1000“ 136 corresponding to D-fructose l-phOSphate did not increase. This could be due to the fact that the radioactive [32P] PEP was added to the reactions before they were started by the addition of 0.2 umole of unlabeled PEP. This time lag could activity to be beled PEP. In of the 32F was phate, whereas have allowed a majority of the radio- utilized prior to addition of the unla- the complete reaction, over 60 percent transferred to D-fructose 1- [32F] phos- the total amount of D-fructose l-phos- phate formed was only 20 to 35 percent of the amount of unlabeled PEP added. The data in the previous three eXperiments show that a compound that eluted with 0.45 M triethylammonium bicarbonate buffer (pH 7.5) (i) formed a radioactive compound that binds to charcoal [charcoal binds ATP (15)] when incubated with ADP and pyruvate kinase (Table VIII), (ii) migrated with standard PEP on both an alkaline chromatogram (Figure 25) and an acid chromatogram (Figure 26), and (iii) was utilized as a phOSphoryl donor in enzyme I- and HPr-dependent phosphorylation of D-fructose (Figure 26, Table IX). This evidence confirms that 95 percent of the radioactive compounds in the pool of fractions 200 through 221 was [32P] PEP. This [32F] PEP pool was concentrated and was stored at -100C in the carrier free form. Dilutions with unlabeled PEP were made immediately prior to the individual experiments in which the [3 2P] PEP was used and specific activities 137 were noted as cpm (measured by eerenkov radiation) per nmole PEP. The amount of PEP in the diluted sample was measured using the PEP end-point assay and was determined to be equal to the amount of unlabeled PEP added. It was concluded that if the [32F] PEP contained any carrier PEP (due to contamination of the mitochondria by Pi) it was below the level of detection (4 percent of the amount of carrier PEP added) and considered insignificant. b. Requirements for Phosphoryl Transfer From [32F] PEP to PTPfru A sample of PTPfru eluted from a native polyacryl- amide disc gel similar to that shown in Figure 7C was used to prepare [32F] phOSpho-PTPfru in the reactions described in Table X. This homogeneous PTPfru preparation was estimated by 210 nm absorbance to contain approxi- mately 0.075 mg protein per ml. This value was obtained by the difference in absorbance of the sample containing the protein and a solution which it had been dialyzed and concentrated against. The background level absorbance was equivalent to a protein concentration of 0.29 mg per m1, and thus the 0.075 mg per ml could be in error. The reactions described in Table X were incubated for 30 minutes at 300C, cooled on ice, and chromatographed over a 0.75 x 56-cm Sephadex G25 (course) column to separate the [32F] phospho-protein from the unbound [32F] 138 32 fru from [ P] The reactions were Table X. Formation of [32P] phOSpho-PTP PEP, enzyme I, and PTPfru' run for 30 minutes at 30°C and consisted of 20 umoles of Tris-HCl (3% 7.5), 0.1 umole of MgClZ, 0.003 umole of [ P] PEP (119,883 cpm), 86 pg of enzyme I, 1.03 pg of HPr, and 3.75 ug of PTPfru in a total volume of 76 ul. Bound Radioactivity b moles 32P Reaction Totala PTPfru 32P components (cpm) (cpm) (nmoles) moles PTPfru Complete 5,710 2,420 0.061 0.85 " Minus HPr 5,580 2,290 0.057 0.79 " Minus PTPfru 3,290 0 0 0 " Minus c c enzyme I 450 --C —— -_ " 1/10 PEP 2,970 --C --C --C aCounts per minute in fractions 11 through 15. b . . . Total counts per minute of reaction minus total counts per minute of "Complete minus PTPfru" reaction. C-— Signifies not determined. 139 PEP. The elution profile of the complete reaction (Figure 27) was similar to others, all of which had some radioactivity in the void volume. The pool of fractions 11 through 15 was defined as the total radioactivity bound to protein (Table X). In the assay without PTPfru’ radioactivity was bound to enzyme I and was subtracted from the values of the complete reaction and the reaction in the absence of HPr to determine the amount of bound radioactivity which is dependent on PTPf Omitting ru HPr from the reaction had little effect on the radio- activity bound to PTPfru‘ Thus, HPr is apparently not required for phOSphorylation of PTPfru’ whereas enzyme I is definitely required. The amount of radioactivity bound to enzyme I was determined by incubating two concentrations of enzyme I with the [32P] PEP pool in the presence and absence of HPr (Table XI). Doubling of enzyme I doubled the level of [32P] phOSpho-protein that eluted in the void volume from the Sephadex G25 column. Addition of HPr had little effect on the level bound to protein, while increasing the incubation time slightly augmented the amount of bound radioactivity. This series of experiments shows that both the enzyme I and homogeneous PTPfru preparations are phosphory- lated by [32P] PEP in the absence of HPr. Enzyme I is required for the phosphoryl transfer from PEP to PTPfru forming a phOSpho-PTPfru with approximately 0.82 moles 140 .x mHQmH cw pmnwuomop Gowuowmu :mumHmEoo: mnu mo ma COHuSHm umHsoHuuma mHsH .Umuomaaoo mama mcowuomum acupuom .xuw>fluomownmu banana: Eoum muw>wuom0flpmu boson mo sowumuwamm AEonom x mm.ov mmu xmpmsamw pumpcmum .NN madman 141 Figure 27 mumZDz ZOHHUwuomowpmm pcsom O xuw>wuomowpmm masons: II C C _ _ 0H 1 q- HLDNIN.HHJ SlNflOD r01 5 Table XI. Formation of [3 I and [32p] PEP. omitted. 142 P] phOSpho-protein from enzyme Reactions contained the same components as Table X except that PTPfru was Reaction Component Enzyme I HPr Time Bound (pg) (pg) (Min) Radioactivitya 43 0 17 1,450 86 0 17 3,360 86 2.06 17 3,420 86 2.06 35 3,810 aCounts per minute in fractions 11 through 15 from Sephadex 625 column. 143 32F bound per mole of protein of 52,000 molecular weight. This value could be in error due to estimation of protein concentration. The protein-bound radioactivity from the complete reaction and the reaction minus HPr was pooled (Table X), concentrated by ultrafiltration, and chromatographed on Sephadex 6100. The radioactivity in the fractions was measured and two peaks of radioactivity were detected (Figure 28). One peak superimposed with the PTPfru activity assayed on a previous elution of this column. (The amount of PTP in the radioactive fractions was fru too low to detect activity by kinetic assays). The second peak corresponded to the low-molecular-weight region, and is presumed to be 32Pi which hydrolyzed from the PTPfru' Thus, in the 24-hour interim between ultrafiltration of the radioactive sample and elution of the column, approximately 50 percent of the phOSpho-PTPfru was hydro- lyzed. The phospho-histidine isolated by Anderson and co-workers (2) from HPr was found to hydrolyze to P1 in one-week, which would mean 50 percent hydrolysis in a 24-hour period (assuming logarithmic decay). This Sephadex G100 column further documents that 32 P bound to protein was actually bound to PTP and fru not to a protein in the enzyme I preparation which would have eluted close to fraction 40. It appears as though 32 the P bound to the enzyme I protein preparation is 144 .pmppm cowuomum HE you manage pom pmeuom mumnmmozaua mmouosumua mmHoEc mo meumu ca ma zuw>wuom5uwm9m .uxmu mnu CH pmnwuommp mum mawmumo .GE:Hoo ooao xmpmcamm mem mnu co kHMumnmamm pmnamuwoumEouso summHm mo wHaEmm umsuocm mo >uw>wuom pom Hooa dummemuosamosm flmmmu mo coausHm .wN magmam HLHNIN.83d SLNHOO Figure 28 - I T I l :3. H .. _.q- c: H _. __ -H .U o :3 cu p o ‘H -H a. 'o i: L9 r- _g 1.0 __ _‘ c> c: c> c) In 0 Ln 0 tn N N H r-l FRACTION NUMBER 146 more unstable than the [32F] phOSpho—PTPfru since enzyme I originally had approximately the same amount of 32P bound as did PTP and there is no peak where the fru’ enzyme I would have eluted. The fact that the hydrol- ysis rate is approximately the same as the phOSpho- histidine reported by Anderson (2) implies that this D-fructose phosphoryl transfer protein could involve a histidine residue in its active site. 32 c. Determination of Moles P Bound Per Mole PTPfru A second set of experiments was run to form a larger quantity of phOSpho-protein by using [32F] PEP with a higher Specific activity and a sample of protein eluted from a hydroxylapatite column. This PTPfru fraction was judged to be 24 percent pure by running a native polyacrylamide gel, staining with Coomassie blue, and scanning the destained gel at 550 nm. The area under the peak of PTPfru was 24 percent of the total area representing the sum of the total protein peak. An SDS gel was run on the same sample of protein and the area under a peak that corresponded to a molecular weight of 26,000 was 23 percent of the area representing the total protein. The relative staining of proteins by Coomassie blue is accurate at the low concentrations (5 to 10 pg total protein) of protein used in both of these experiments (29a). The protein concentration of 147 this PTPfru sample was estimated to be 0.93 mg per ml by both 210 nm absorption and the Lowry method. Each assay contained 18.6 pg of protein from the hydroxylapatite pool, which was equivalent to 4.46 pg or 0.085 nmole of PTPfru’ assuming a molecular weight of 52,000 daltons. The other components were as described in Table XII except for [32P] PEP as noted. The assays were run for the times listed, then imme- diately chromatographed on a standard Sephadex G25 column (same column as Figure 27), and 20-drop fractions were eluted with 0.02 M potassium phosphate buffer (pH 8.0) containing 0.001 M DTT and 10 percent glycerol. The pool of fractions 11 through 15 was defined as the bound radioactivity fraction. Fractions 16 through 37 were pooled and contained the remainder of the radio- activity that was not bound to protein. The data again indicate that formation of a phos- phorylated PTP is dependent on the presence of enzyme fru 1. Adding HPr actually decreased the amount of radio- activity bound by 5 percent, and thus was not required for PTPfru phOSphorylation. Radioactivity was again bound to enzyme I in the absence of PTPfru' The complete reaction run for 24 minutes shows that the formation of [32F] phOSpho-PTPfru was complete after 15 minutes. The control with 1/6 the level of [32F] PEP shows that PTPfru phOSphorylation has a high affinity for [32F] PEP and actually utilized 50 percent of the available [32F] 148 .Hn co mo mEDHo> kuou m aw .zcamuwoumEouco CEDHoo muwumamaxxoupms Eoum Hooa mam mo w: o.wH new .Aeao cam.omav mum flmNmH mo mmaoe: mm.H .umm mo w: mo.H .H 66%Nc6 mo my mq .Naowz mo mace: H.0 .Am.~ mdv Humumwuh mo mmHoEJ O.N pwckucOo muowuommu mLH .summamuozamosa mmmmw mo CowumEhow pom mucmEmuHsvmm 5pm .HHx mHLmH .pmcfiEumump uoc mmwmwcwwmuup .cowuommu :aummHm mange mumHano: mnu CH mucsoo onu mDCHE cowuommu comm Ca pcsow mausoo pcsomo .AmH zwsousu Ha mCOfiuomumV mEdHo> pwo> mnu CH pmusam umcu monsoon .cEDHoo mmu xmpmsamm Eoum muowuomum mcu Ham CH pmHSmmmE mucdoo Hmuon 149 na.a omH.o oom.m ooo.ma ooo.qaa am mumHQEOU .0 wo.H Nmo.o omo.m omm.a oom.wa ma mum .mmmu mmHOEC mN©.H mDCHE : .m -- -- -- 0mm ooo.naa OH H waswcm a a a wasp: : .q mm.H soa.o ooH.0H oom.mH ooo.wNH NH “mm 94sz : .m o o o 00m.m ooo.mma ma summem mSSHZ : .N NR.H smH.o 000.0 oom.ma ooo.m~a ma momagEoo .H summam mmHoE AmmHoEcv Aanv Aedov AEQQV ACHZV cowuomwm mmm ooamwomam npcnom mamuOH oEfiH mmm mmHoE usummam sua>auomoanmm .HHx mHQmH 150 PEP, although the amount of 32F bound did not attain the same level as with more saturating [32P] PEP. The nmoles of radioactivity bound, dependent on 32 PTP were calculated and the ratio of moles of P fru’ to moles of PTPfru was determined. This ratio ranged from 1.77 for the complete system to 1.93 for the complete system minus HPr. The reaction with 1/6 the level of [32F] PEP had a ratio of 1.08. Thus, it becomes apparent that under completely saturating conditions and with no hydrolysis of the phospho-protein, the PTPfru binds 2 32 moles of P for each mole of protein with a molecular weight of 52,000 daltons. This is equivalent to 1 mole 32P bound per subunit of molecular weight 26,000. Reactions 1, 3, 5, and 6 (Table XII) were pooled, concentrated by ultrafiltration, applied to the standard Sephadex GlOO column (same column as Figure 28), and 60-dr0p (2.0-ml) fractions were eluted with 0.02 M potassium phOSphate buffer (pH 8.0) containing 0.001 M DTT and 10 percent glycerol. Fractions 46 through 60 were pooled, concentrated, and dialyzed against 0.01 M potassium phosphate (pH 8.0) containing 0.001 M DTT. Radioactivity was again found in the dialysates, ultra— filtrates, and the low-molecular-weight fractions eluted from the Sephadex G100 column, thus showing 32P from the [32F] phOSpho-protein. hydrolysis of The concentrated [32F] phOSpho-PTPfru fraction was then incubated with enzyme IIfru and D-fructose, and 151 in some cases HPr, as described in Table XIII. The reactions were stopped by layering them on a 0.75 x 56- cm Sephadex 025 column (same column as Figure 27) and 20-drop fractions were eluted with water. Fractions 11 through 15 were pooled and contained radioactivity that remained bound to protein. The pool of fractions 16 through 26 would contain any D—fructose l-phOSphate formed as well as hydrolyzed 32F. Omitting enzyme 11, adding HPr, or increasing the D-fructose concentration 10-fold, as well as altering the reaction times, had little effect on the ratio of unbound radioactivity (fractions 16 through 26) with respect to radioactivity bound (fractions 11 through 15) (Table XIII). The variability in the total radio- activity (sum of bound and unbound, Table XIII) in reactions 1 through 7 was due to addition of non-uniform amounts of [32P] phOSpho-PTP to the reactions. fru The unbound radioactivity from reactions 1, 4, 5, 7, and 8 in Table XIII was pooled, treated with Dowex 50, lyophilized, dissolved in water, and chromatographed with standard D-fructose l-phosphate and P1 in the acid solvent system. A scan of the radioactivity indicated that greater than 95 percent of the radioactivity in these pools was in the form of 32 P1 (Figure 29). The bound radioactivity from the same fractions was pooled and boiled to denature the protein which was then removed by centrifugation at 40,000 x g, This sample was then Table XIII. 152 Formation of D-fructose 1-[32P] phosphate from [32F] phOSpho-PTPfru. Reactions con- tained 8. O pmoles of Tris- HCl (pH 7. 5), 0. 2 pmole of2 D- fructose, 1. 0 pmole of MgClz, 0. 2 m1 of [3 2P] phOSpho- PTPfru , 20 p1 of enzyme 11, and 1.0 p1 of HPr (where noted). Af er 15 minutes (with exceptions noted) at 30 C, reactions were cooled on ice and chro- matographed on a Sephadex G25 column equili- brated with water. Radioactivity Time Bounda Unboundb Unbound Reaction (Min) (cpm) (cpm) Bound 1 Complete 15 434 487 1.12 2 " Plus HPr 15 344 399 1.16 3 " Plus HPr 15 579 749 1.29 4. Complete 2 348 425 1.22 5 " 30 327 434 1.33 6 " Minus Enzyme II 15 313 370 1.18 7. " 2.0 pmoles D-fructose 15 377 391 1.03 8. N 1/2 [321?] phOSpho-PTP 2 216 163 .75 fru aCounts per minute in pool of fractions 11 through 15. bCounts per minute in pool of fractions 16 through 26. Figure 29. 153 Radioactivity scans of acid chromatograms of pooled bound and unbound radioactivity. The pool of unbound radioactivity from reactions 1, 4, 5, 7, and 8 in Table XIII was treated with Dowex 50, concentrated by ly0philization, and chromatographed ( ). The pool of bound radioactivity from the same reactions in Table XIII was boiled, centrifuged to re- move protein, treated with Dowex 50, concen- trated by ly0philization, and chromatographed ( --------- ). Standards were chromatographed with the readioactive samples and detected with Hanes-Isherwood Spray reagent. 154 Figure 29 150, origin 125L mHDzHZ mum mHZDOU 155 treated in a similar fashion as the unbound sample dis- cussed above. The radioactivity scan had small peaks that co-chromatographed with P1 and D—fructose l-phos- phate, and a larger peak that migrated closer to the origin. Identification of this radioactive compound was not determined. The D-fructose l-phOSphate present in this pool of high-molecular-weight components was possibly trapped inside the membrane vesicles, and thus did not separate on the Sephadex G25 column. d. Formation of D-Fructose l-[32 From [32F] PhOSpho-PTPfru P] Phosphate In the previous experiment the low levels of 32P that were transferred to D-fructose l-[32P] phOSphate may have been due to inactivation of the PTPfru as a result of manipulation. A majority (75 percent) of the 32P had hydrolyzed from the original [32P] phOSpho-PTPfru formed during these manipulations. In this experiment the Sephadex G25 column and subsequent dialysis were omitted, and an enzyme 1, [BZP] PEP, PTPfru reaction was layered directly on the Sephadex GlOO column (Figure 30). Fractions 44 through 66 were pooled and concentrated to 2.0 m1. This [32P] phospho- PTPfru preparation containing 68,000 Cpm was obtained in one day rather than the two days required to prepare the sample in the previous experiment. This preparation was used in four small reactions 156 .Houmoxaw ucmopma 0H paw BBQ 2 Hoo.o wchHmucoo Ao.w mav mumndmosa Ebwmmmuoa 2 No.0 suw3 pmusam mHmB AHEuo.NV muowuomum an .cOHuommu mmamuonamosa fimmmu mo msamuwoumEouco ooau xmpmnamm .om madman 157 Figure 30 mmmzaz onHo