gfifigj. M A ‘ .. ' "A '1‘ ' \ . ' Wa r.*~‘.<..\.¢. :‘o k 4... 'I . T .' - O ‘» " ACETYL- CoA SYNTHETASE. . A GLYCOPROTEIN Thesis for the Degree 0151.3. MTCHIGAN STATE UNIVERSITY VASSILTKI STAMOUDTS 1973 ‘J LIBRARY MiChig.f £1 State Univcnity I" smmmav "' w HUM; & SflNS' 800K BINDERY INC LIBP ARY BREW—”S TY ABSTRACT ACETYL-COA SYNTHETASE, A GLYCOPROTEIN By Vassiliki Stamoudis The acetate activation reaction catalyzed by acetyl-CoA syn- thetase is considered to be an important rate-limiting step in acetate utilization by ruminant tissues. In order to study this reaction further acetyl-CoA synthetase was purified from cow and goat mammary gland mitochondria. During these studies the apparent aggregation phenomena and difficulties in purification suggested that the enzymes might be glycoproteins. Consequently, the enzymes were tested for the presence of carbohydrates. Polyacrylamide gel electro- phoresis followed by PAS staining was positive. Sulfuric acid hydrolysis or neuraminidase treatment gave a difference in anodic migration, sug- gesting the presence of N-acetyl-neuraminic acid. The thiobarbituric acid test for N-acetyl neuraminic acid was positive for both cow and goat acetyl-CoA synthetase. GLC analysis showed that the cow enzyme contains fucose, glucose and N-acetyl neuraminic acid. However, the goat enzyme contained fucose, galactose, glucose and N-acetylgalactosamine. The presence of N-acetyl neuraminic acid, in the goat enzyme, was not detected using GLC. These observations were confirmed using mass spectrometry. The role carbohydrates play in determining structural and catalytic properties of acetyl-CoA synthetase is not clear at this time. Previous work has shown that acetyl-CoA synthetase is more active on propionate than on acetate in liver and lung, but is equally active Vassiliki Stamoudis on both substrates in heart and kidney. Also, the enzyme is not active in the non-lactating mammary gland but becomes active after par- turition. The activity decreases with advancing lactation. These dif- ferences in substrate specificity and other phenomena may be explained by differences in the carbohydrate composition of the enzymes in different tissues and under different physiological states. ACETYL-COA SYNTHETASE A GLYCOPROTEIN By Vassiliki Stamoudis A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Biochemistry 1973 To Xenia and Vassilis ii ACKNOWLEDGMENTS I wish to express my gratitude to my academic advisors, Professors Robert M. Cook and John C. Speck for their helpful guidance advice and encouragement during these studies. I also wish to thank Dr. H. Wells, Dr. H. Knull, Dr. _ H. Esselman, Dr. D. Marinez and my husband Vassilis for help- ful discussions. The technical assistance provided by Rosemary Parker, Lorene Scholtz and Jack Harten is greatly appreciated. The financial support provided by Dr. R. M. Cook is also gratefully acknowledged. iii TABLE OF CONTENTS Page ACKNOWLEDGMENTS ........................ iii LIST OF TABLES ........................ vi LIST OF FIGURES ........................ vii INTRODUCTION ......................... l EXPERIMENTAL SECTION ..................... ll A. Enzyme Assay ..................... ll 8. Protein determination ................ 12 C. Gel Electrophoresis ................. 12 D. Enzyme isolation procedure .............. 13 E. DE-23 cellulose chromatography ............ 16 F. Chromatography of acetyl-CoA synthetase on calcium phosphate gel .................... l6 G. Glycoprotein staining following electrophoresis on acrylamide gels ................... 22 H. Thiobarbituric acid assay .............. 26 I. Preparation of l N HCl in CH3OH ........... 27 J. Methanolysis procedure and preparation of methyl- glycoside standards ................. 30 K. Gas liquid chromatography .............. 30 L. Sulfuric acid hydrolysis for removal of sialic acid . 3T M. Removal of sialic acid by the use of neuraminidase. . 3l RESULTS AND DISCUSSION .................... 33 A. Experiments using gel electrophoresis ........ 33 iv TABLE OF CONTENTS (Continued) Page B. Gas chromatography .................. 43 C. Mass Spectrometry analysis .............. 52 D. Discussion ...................... 59 BIBLIOGRAPHY ......................... 82 TABLE 01th 01 TO IT 12 13 LIST OF TABLES The carbohydrate components of some glycoproteins. . . Structures of carbohydrate moieties of glycoproteins . Elution of the calcium phosphate column ........ Staining of glycoproteins according to Hotchkiss . . . Staining of glycoproteins according to R. Kaschnitz et al ......................... N-Acetyl neuraminic acid standard curve ........ Thiobarbituric acid assay for sialic acid content of acetyl-CoA synthetase ............... Optical rotation of the carbohydrates used as standards ....................... Relative retention times of the TMSi derivatives of methylglycosides at 160°C ............. Relative-retention times of the TMSi derivatives of methylglycosides at 190°C ............. GLC analysis of acetyl-CoA synthetase. Relative retention times at 160° and 190°C .......... Selected peaks from the mass spectrum of methyl 2, 3,4,6-tetra-0-trimethylsilyl-a-D-glucopyranoside (I) . Structures of some of the fragments of methyl 2,3, 4,6,-tetra-0-trimethylsilyl-a-D-glucopyranoside (I). . vi Page 21 24 25 28 36 44 45 46 47 52 55 LIST OF FIGURES FIGURE Page 1 Fractionation of bovine mammary gland tissue ...... l4 2 N-acetyl neuraminic acid standard curve ........ 29 3 Polyacrylamide gel electrophoresis of acetyl-CoA synthetase. Schematic presentation of the coomassie blue and PAS staining ................. 34 4 Effect of sulfuric acid hydrolysis on the electrophoretic mobility of acetyl-CoA synthetase ........... 38 5 Effect of 0.1 N sulfuric acid and 7 M urea on the electrophoretic mobility of acetyl-CoA synthetase . . . 39 6 Effect of neuraminidase on the electrophoretic mobility of acetyl-CoA synthetase ................ 41 7 Chromatography of acetyl-CoA synthetase on DE-23 cellulose ....................... l7 8 Chromatography of acetyl-CoA synthetase on calcium phosphate gel ..................... 19 9 Gas chromatogram of a control mixture of TMSi deriva- tives of methylglycosides at 160°C, 3% 0V-I column. . . 48 10 Gas chromatogram of the TMSi derivatives of methyl- glycosides prepared from the bovine mammary gland acetyl-CoA synthetase, at 160°C ............ 49 11 Gas chromatogram of the TMSi derivatives of methyl- glycosides prepared from the bovine mammary gland acetyl-CoA synthetase, at 190°C ............ 50 12 Gas chromatogram of the TMSi derivatives of methyl- glycosides prepared from goat mammary gland acetyl- CoA synthetase, at 190°C ................ 51 13 The gas chromatogram of the TMSi derivatives of methylglycosides prepared from the bovine mammary gland acetyl-CoA synthetase that was used for the mass spectrum analysis ................... 62 vii LIST OF FIGURES (Continued) FIGURE Page 14 The gas chromatogram of the TMSi derivatives of methyl- glycosides prepared from the goat mammary gland acetyl- CoA synthetase that was used for the mass spectrum analysis ........................ 63 15 The mass spectrum of the TMSi derivative of mannitol. . 64 16 The mass spectrum of the TMSi derivative of the methyl- glycoside of N-acetyl galactosamine .......... 65 17 The mass spectrum of the TMSi derivative of the methyl- glycoside of N-acetyl neuraminic acid ......... 66 18 The mass spectrum from scan 4 (Figure 13) of the TMSi derivatives of methylglycosides prepared from the bovine mammary gland acetyl-CoA synthetase ........ -. . 67 19 The mass spectrum from scan 28 (Figure 13) of the TMSi derivatives of methylglycosides prepared from the bovine mammary gland acetyl-CoA synthetase .......... 68 20 The mass spectrum from scan 33 (Figure 13) of the TMSi derivatives of methylglycosides prepared from the bovine mammary gland acetyl-CoA synthetase .......... 69 21 The mass spectrum from scan 48 (Figure 13) of the TMSi derivatives of methylglycosides prepared from the bovine mammary gland acetyl-CoA synthetase .......... 70 22 The mass spectrum from scan 79 (Figure 13) of the TMSi derivatives of methylglycosides prepared from the bovine mammary gland acetyl-CoA synthetase .......... 71 23 The mass spectrum from scan 139 (Figure 13) of the TMSi derivatives of methylglycosides prepared from the bovine mammary gland acetyl-CoA synthetase .......... 72 24 The mass spectrum from scan 41 (Figure 13) of the TMSi derivatives of methylglycosides prepared from the bovine mammary gland acetyl-CoA synthetase .......... 73 viii LIST OF FIGURES (Continued) FIGURE Page 25 The mass spectrum from scan 99 (Figure 13) of the TMSi derivatives of methylglycosides prepared from the bovine mammary gland acetyl-CoA synthetase .......... 74 26 The mass spectrum from scan 3 (Figure 14) of the TMSi derivatives of methylglycosides prepared from the goat mammary gland acetyl-CoA synthetase .......... 75 27 The mass spectrum from scan 25 (Figure 14) of the TMSi derivatives of methylglycosides prepared from the goat mammary gland acetyl-CoA synthetase .......... 76 28 The mass spectrum from scan 36 (Figure 14) of the TMSi derivatives of methylglycosides prepared from the goat mammary gland acetyl-CoA synthetase .......... 77 29 The mass spectrum from scan 42 (Figure 14) of the TM51 derivatives of methylglycosides prepared from the goat mammary gland acetyl-CoA synthetase .......... 78 30 The mass spectrum from scan 47 (Figure 14) of the TMSi derivatives of methylglycosides prepared from the goat mammary gland acetyl-CoA synthetase .......... 79 31 The mass spectrum from scan 90 (Figure 14) of the TMSi derivatives of methylglycosides prepared from the goat mammary gland acetyl-CoA synthetase .......... 80 32 The mass spectrum from scan 144 (Figure 14) of the TMSi derivatives of methylglycosides prepared from the goat mammary gland acetyl-CoA synthetase .......... 81 ix INTRODUCTION Acetate is an important source of energy in ruminants and plays a major role in lipogenesis comparable to the one played by glucose in non-ruminants. This is because the activity of ATP- citrate lyase is negligible in ruminants. Therefore, glucose can only supply limited amounts of acetyl-CoA for fatty acid synthesis. It is important to understand major factors that control the utili- zation of acetate by ruminants tissues. Before being utilized by the cell acetate must be activated (covalently linked to the thiol group of coenzyme A). The acetate activation reaction catalyzed by acetyl-CoA synthetase [acetate: CoA ligase (AMP) (6.2.l.l)], according to the following reaction is considered to be an important rate limiting step in acetate utilization by ruminant tissues(2,3). Mg++ CH3C00H + ATP + CoA-SH=CH3COSCoA + AMP + P-Pi The distribution of acetyl-CoA synthetase in several ruminant tissues has been established (2,3). The enzyme is localized pre- dominantly in the cytoplasm in heart and mammary gland and is almost equally divided between mitochondria and cytoplasm in kidney. However, the enzyme is localized predominantly in the mitochondria in lung and in liver (1). Acetyl-CoA synthetase is not very active in the liver cytoplasm but it is known that ruminant liver uses mostly propionate and butyrate as major precursor for lipid synthesis. The enzyme activity is low also in rumen epithelium and in lung. Acetyl-CoA synthetase has been generally shown to be active on acetate and propionate. In bovine fetal tissue the enzyme prefer- entially activates propionate rather than acetate, a situation in contrast to the adult enzyme (4). Another interesting observation is that acetyl-CoA synthetase is not active in non-lactating mammary gland. The enzyme becomes active at parturition in goats and cows and the activity decreases as the lactation progresses (4). The enzyme from bovine mammary gland has been extensively purified (4). The data indicates that acetyl-CoA synthetase exists in multiple molecular forms. The nature of these forms is not clearly understood. Iso- zymes might exist or there may be aggregated forms of the enzyme. Huang and Stumph (17), report 5 isozymes of acetyl-CoA synthetase in potato tuber, all with similar kinetic properties. Multiple forms of the enzyme were also reported in yeast by DeVincenzi (18). Activity that emerges as a single peak from a DE-52 cellulose column separates into four protein peaks using a calcium phosphate column and different ionic strengths of phosphate buffer. Two of the four protein peaks had enzyme activity. Each of the two protein peaks upon gel electro- phoresis gives 4 bands, 2 of which are found to be associated with enzymatic activity. This data suggests the existence of isozymes. Also the fact that the fetal heart enzyme activates propionate but not acetate in contrast to the adult enzyme, can be explained on the basis of isozymes. That is isozymes exist in fetal tissues that preferentially activate propionate whereas an isozyme in adult tissue predominates that activates acetate. 0n the other hand, much of the data suggests that the enzyme aggregates to yield multiple forms. Acetyl-CoA synthetase activity decreases in concentrated solutions perhaps as the extent of aggregation increases. The presence of ammonium sulfate known to prevent aggregation has a beneficiary effect on the stability of the enzyme. Sedimentation equilibrium studies indicate the presence of at least 2 molecular species one with a molecular weight of 84,500 and one with a molecular weight of 49,000. This higher value could represent a dimer form. However, the molecular weight value of the enzyme from sucrose density gradient centrifugation is esti- mated to be 62,000 (4). The minimum molecular weight reported for acetyl-CoA synthetase from bovine heart mitochondria is 31,000 to 34,000 (47). If it is assumed that the enzymes from both tissues have similar molecular weight, then the mammary gland enzyme could possibly have been aggregated to a dimer form under the conditions of the experiment to give a value to 62,000. It is known today that some glycoproteins exist as isoglyco- enzymes. These isozymes differ only in carbohydrate content. Also it is known that the presence of a small molecule such as sugar, especially when it is charged, can contribute to interactions between subunits. The thought that the enzyme might be a glycoprotein and that this could explain much of the data already known, led to these studies. The function at the molecular level of the carbohydrate resi- dues in glycoproteins fall into two categories, biological and structural. One widely accepted role for carbohydrate residues, is in the transport of glycoproteins through cellular membranes (5). It is believed that sugar is added to many polypeptide chains to facilitate export of the protein from the cell. A large number of extracellular proteins are glycoproteins. Carbohydrates may complex with receptors and other carrier substances in the cellular membrane. This may result in conformational changes allowing the passage of the macromolecule. The catabolism of some of the serum glycoproteins and hormones may be regulated through their carbohydrate moieties. Removal of sialic acid from orosomucoid, fetuin ceruloplasmin, haptoglobin, human chorionic gonadotropin, follicle stimulating hormone, led in each case to the production of materials which after injection into rats, were removed from the circulation much more rapidly than were the original glycoproteins (6). In this process neuraminic acid (7) and D-galactose residues (8) appear to be important. Removal of sialic acid leaves galactose in non-reducing terminal position and it is these galactose residues which are involved in that recognition process, which leads to the removal of the molecules from the circulation. Glycoproteins secreted by the cell often function as antigenic substances (9). But the carbohydrate component appears to play only a minor role in the immunological process (10). The level of plasma protein bound carbohydrate is elevated in a number of pathological states, but the biological significance of this interesting phenomenon is still not clear. Carbohydrates have a significant effect on several of the physical properties of the glycoproteins. The intrinsic viscosity, frictional ratio, diffusion coefficient and solubility are all affected by the presence of carbohydrates. Changes in these properties have been noted, espec- ially for porcine ribonuclease (11). A protective role of the carbohydrate residues is shown by the fact that glycoproteins are resistant to hydrolysis by proteolytic enzymes (12, 13), and they are remarkably stable on storage and at elevated temperatures (14, 15). Carbohydrates function as stabili- zers of the tridimensional structure of proteins (14) especially for enzymes having a molecular architecture similar to that of glycoamylase. Here many carbohydrate side-chains are present on the surface of the molecule, positioned in such a way as to minimize molecular transformations. This is the case for pepsinogen (16). Glycoenzymes often occur in multimolecular forms and evidence is accumulating that such forms are isoglycoenzymes, differing only in the carbohydrate portion of the molecules. Examples are: ribo- nucleases A and B from bovine pancreas (19). Ribonuclease 8 contains an appreciable proportion of carbohydrate whereas the A form does not. The two enzymes possess identical catalytic properties and they have the same amino acid composition. The differences in their electrophoretic and chromatographic properties are apparently due to the presence of carbohydrate in one of them. The monosacharides most often found in glycoproteins are D- mannose and 2-acetamido-2-deoxy-D-glucose. Also D-glucose, D-galac- tose, D-xylose, L-arabinose, L-fucose and sialic acid are often present. Carbohydrates are attached to the protein by two types of linkages, the N—glycolyl and the 0-qucosy1. The N-glycosyl linkage occurs between the reducing end of the carbohydrate chain and an L-asparagine residue of the protein, whereas the O-glycosyl linkage is found between the reducing end of the carbohydrate chain and the hydroxy group of a serine or threonine residue in the protein. There is evidence showing that the attachment of the bridge carbo- hydrate residues to the polypeptide chain, occurs while the chain is still attached to the ribosomes (22, 23, 24). Several of the bridge carbohydrates have been identified and some glycosyl transferases have been purified. For example, 2-acetamido-2-deoxy-D-glucose is one of the bridge carbohydrate residues of glycoproteins. Nucleotidyl transferases are responsible for the activation and transfer of the hexosamine to the polypeptide chain. Other glycosyl transferases are res- ponsible for attachment of carbohydrate moieties to hexosamine (26, 27). For glycoenzymes containing D-mannose and D-xylose as bridge carbohydrates the appropriate enzymes for formation of GDP-mannose and UDP-xylose have been purified (27, 28). Table 1 gives the carbohydrate components of some glycoproteins (29). It can be seen that the carbohydrate content varies widely from one protein to another. Table 2 gives the structures of carbo- hydrate moieties of some glycoproteins (30). The asparagine in- volvement for the N-glycolyl linkage is clearly shown. N-acetyl- glucosanine is very often the first carbohydrate attached to the polypeptide chain and sialic acid usually is at a terminal position. This study was conducted to determine whether or not acetyl-CoA synthetase is a glycoprotein. :85» «cvuuognouapu «saw an «uneconaou ouaeuagoneou ugh muooacougon so .¢.ouosa we opauopal so: «cauvmos Go .02 n._ u4m oxocm a o»-¢.ouosa n a omen—u u - ~ . ~ n . - - m coo.pn «aucucoa xo -aconvgaxoou ~ a - u u u m - - . m ao~.e— «nosucog no a anon—uacon*5 mosx~cu _ uoao.mo¢ u.u< o=.saa zpam zw mucosa m oak pow co: .9: ._o: mosaom c.o»oga «mapu .oz anam uvpo.m -oxo: —oeu=oz _.uoh pouch page» all mm aq— coo.— um.mo o~p.v com nm.~ Nu u~.p .Aonmpv .mN ._o> .saguowm .Eagu .goneau =3 .>u< .Eamaaazaz .< .Fpmsmeaz .n .m »a cabamuuu~..xul .53“ Queen ou_:n o'eumom cuss: firs—uuumhu co¢5u>o ooo.o—— ooo.ooo.— menu’s xo 5:3...qu2 < ~ c.0uocaooA—o aw Asa—p—xgupzu me uvcunqc oucqunnau-< ocaeaEus acoeonon «ca—a m:.u=x NN —~ ow «— m— NF 2.: .3 .amfluqz—s mum 2282 a: 5: N1 25 f... 3 P a u 8 5JK\ v Lo m m cmmm .c:< .ngmgmz .o .m so: vmuamge TO 53938.53 2.71258 Xi Paw zmumogtucs. .S a m uoa mo :ovuacorpuaem ”H “mama; xgamemopmsoezo Pom mumcamoga save—mo an umzoppoe xgaeemoumsoggu cszpoo mmopappmu Aueuumwvv mNumo m.m In gmmmsa Puzimweh c? a: cmxmh pcmumcewgam mumuvawuwea 15 .AA .cws op Noe mxooo.ON Na mmaeNENcao .N Fe oo_\¢omNA¢=zv Augmumwuv _m MN 53?: New o» apaesbmm .F mumprqwomem ucmumcemaam \/ .CNE op toe mxooo.ON pm aaaevepcau .N e N e Ape ooFNG m.NNv Auemumwuv om A Izv 53?; New 03 apneaumm .. mumpwavomga Auomgpxm opwsv unnumcgmnam 16 20,000 x g for 45 min. The supernatant was adjusted to pH 8 (and to 0.1 M in 2-mercaptoethanol). To the fraction thus obtained, the mitochondrial extract, 21 g of ammonium sulfate per 100 ml were added slowly and with stirring. The solution was adjusted to pH 8 with l N NH40H, stirred for one hour and then centrifuged at 20,000 x g for 10 min. An additional 23.5 g of ammonium sulfate were added to each 100 ml of the super- natant. The precipitate obtained was recentrifuged to remove excess ammonium sulfate, and was then taken up in Tris-buffer pH 8.5 and stored at -60°C. E. DE-23 cellulose chromatography DE-23 cellulose was washed according to Whatman's procedure (31) and then equilibrated in 0.005 M Tris-HCl buffer pH 7.5. The column dimensions were 1.7 cm x 42 cm. The flow rate was 15-20 ml/hour. The ammonium sulfate precipitate was dialyzed for 30 min., diluted to 10 mg of protein/ml with 0.005 M Tris-HCl and added to the column. The column was then washed with 140 m1 of 0.005 M Tris-HCl buffer and then with 160 ml 0.01 M Tris-HCl buffer pH 7.5. The activity was eluted with 600 ml of a linear KCl gradient of 0 to 0.6 M in 0.01 M Tris-HCl buffer pH 7.5. Six ml fractions of eluate were collected. The tubes with the highest specific enzyme activity were pooled and concentrated in a dia—flow cell. The concentrated enzyme protein was stored at ~60°C. F. Chromatography of acetyl-CoA synthetase on calcium phosphateggel The calcium phosphate gel was made by slowly adding equal volumes of 1 M K2 HP04 and 1 M CaCl2 to a large beaker. Mixing was accomplished l7 .m.N In emeeaa _NI weak 2 Po.o c? z m.c op o No “cavemea _8¥ amm=N_ a L0 F5 com 58?; aapapa we; NNN>Npom asp .m.N Ia Laeezn _u: met» 2 Po.o No FE om_ sue; cash .m.N In eaeezn _UI met» 2 moc.o Lo PE 03. sup: “nave eagmmz mm; =E=_ou age .mmo~:P_mo NN-mo co ammumcpcam pmom mo agnmeaoumsoezu “w mmawHu 20 A” «use awAzua <--) 0 fl 1 O Q 1 on: O 0 F l J O O N J Iw/n ecu emu eeW. e2 1 . .: v.1... :1 _._ 02 00.. 0:. ZO__—u<¢u cup 21 TABLE 3: Elution of the calcium phosphate column Molarity of potassium phosphate buffer pH 7.0 Amount used for elution, mls OOOOOOO .001 .010 .030 .050 .065 .070 .080 .100 .200 .500 100 100 100 100 ' 100 100 100 100 150 200 22 using a magnetic stirrer. About 50 mg of the protein, purified from the DE-23 column were diluted to 150 ml with 0.001 M potassium phos- phate buffer. The protein solution was equilibrated for 30 to 60 min. in the cold room and then was layered on the column. The column dimensions were 3 x 12 cm. A buffer schedule of increasing concentra- tion of potassium phosphate buffer pH 7.0 was used to elute the protein. Table 3 gives the molarity and the amount used of each buffer. The eluate was collected in 5 ml fraction. G. Glycoprotein staininggfollowing electrophoresis on acrylamide gels The principle of the periodic acid - Shiff (PAS) technique for detection of glycoproteins following electrophoresis on acrylamide gels is the oxidation by periodic acid of the carbohydrate components of the protein to give polyaldehydes which yield violet-red compounds with Schiff's reagent,fuchsin sulfate, according to the following scheme: [__ [----CH I l-————('3H 0 H- '- OH I 0 0 CHO O F(503H)2 HO-C-'H 0 H104 CHO _:N ._ 1 / i e" ’ l 9” Fuchsin 9” TH Sulfite CHZOH CHZOH polyaldehyde 23 _____J I 2“~F O O poly-substituted dye-compound CH OH The protein samples were stained according to the procedures of Hotchkiss (34) and Kaschnitz et a1. (32). The details are outlined in Table 4 and 5 respectively. Basically the two procedures differ only as to the agent used for fixation of the protein on the gel. The solutions used for the staining according to Hotchkiss (34) were prepared as follows. Periodic acid solution: 400 mg periodic acid, dissolved in 10 cc distilled water, 5 cc of M/5 sodium acetate (equivalent to 135 mg of the hydrated crystalline salt) and 35 cc ethyl alcohol. This solution may be used for several days if protected from exposure to light. Reducing rinse: One 9 potassium iodide and l g sodium thiosul- fate pentahydrate were dissolved in 20 cc distilled water. 30 cc ethyl alcohol were added and then 0.5 cc 2N HCl. A precipitate of sulfur Slowly forms and is allowed to settle out, the solution may be used immediately. (This is designed to be an iodide thiosulfate 24 TABLE 4: Staining of glycoproteins according to Hotchkiss (34) Step Gel treatment Time interval min. 1 Bring the gel into 70% alcohol 10 2 Leave at room temperature in periodic acid 5 solution 3 Flood with 70% alcohol, transfer to reducing 5 rinse 4 Flood with 70% alcohol, leave in fuchsin- 15-45 sulfite 5 Wash 2-3 times with SOz-water 6 Store in 3-5% acetic acid 25 TABLE 5: Staining of glycoproteins according to R. Kaschnitz, et a1. (32) Step Gel Treatment Time interval min. 1 Immerse in 12.5% TCA (25-50 mg/gel) 30 2 Rinse lightly with distilled water 1 3 Immerse in 1% periodic acid (made in 3% 50 acetic acid) 4 Wash 6 times for 10 min. each in 200 ml 60 or distilled water/gel with stirring or Shaking overni ht or wash overnight with a few changes 9 If 60 min. washing was used check last wash with 0.1 N AgNO3 and when test is negative for 103'; continue washing with 2 more changes 5 Immerse in fuchsin-sulfite stain; store in 50 the dark 6 Wash with freshly prepared 0.5% metabisulfite 3O 3 times for 10 min. each (25-50 ml/gel) 7 Wash in distilled H20 with frequent changes and overnight motion until excess stain is removed 8 Store in 3 to 5% acetic acid 26 solution containing the maximum amount of mineral acid compatible with the thiosulfate; when it ceases to be acidic, it should be re-acidified or replaced. Sulfite wash water was prepared by adding 0.5 m1 of concentrated HCl and 0.2 g of potassium metabi- sulfite to 50 ml of distilled water. The fuchsin-sulfite solution that was used for both procedures was prepared according to McGuckin and McKenzie. To 1 liter of water 8 g potassium metabisulfite and 10.5 ml of concentrated HCl were added. When solution was obtained 4 g of basic fuchSin were added and the mixture was stirred gently with a mechanical stirrer for 2 hours at room temperature. At this time the dye was in solution. After standing for 2 hours a small amount of charcoal (Darco-G-60) was added and the solution was filtered within 15 min. The resultant colorless reagent was stored at 5°C. It was stable for several months. H. Thiobarbituric acid assay (36) This method determines only free sialic acid, consequently when employed for the measurement of this sugar in glycoproteins sialic acid must first be released by weak acid hydrolysis or neuraminidase. Reagents: 1) Sodium metaperiodate, 0.2 M in 9 M phosphoric acid stored in an amber glass bottle. 2) 10% sodium arsenite in a solution of 0.5 M sodium sulfate and 0.1 N H SO . 3) Thiobarbituric 2 4 acid, 0.6% in 0.5 M sodium sulfate. Procedure: Sialic acid must first be released from the protein by hydrolysis in 0.1M H2S04 at 80° for 1 hour. The samples and 27 standards should contain 2 to 18 pg of sialic acid dissolved in 0.2 ml of water. To each tube as well as to 0.2 ml water blank, is added 0.1 m1 of the periodate solution. The tubes are shaken and allowed to stand at room temperature for 20 min. One ml of the arsenite wolution is then added. The tubes are shaken until the yellow-brown color has disappeared. Then 3 ml of the triobarbituric acid solution is added to each tube, the contents are mixed by shaking, the tubes are capped with glass bulbs and heated in a vigor- ously boiling water bath for 15 min. They are cooled in a water bath for 5 min. Then the entire 4.3 ml of aqueous solution is ex- tracted with 4.3 ml of cyclohexanone. The tubes are vigorously Shaken and then are centrifuged. The top cyclohexanone phase is transferred to cuvettes and the optical density is determined at 549 mu. (Table 6). Color production varies linearly with increasing concen- trations of N-acetyl neuraminic acid (NANA) over the range usually used, 0.01 to 0.06 pmole (Figure 2). A strongly acidic environment is required to obtain a maximal molecular extinction coefficient. Strong acid is probably required to remove the acetyl group before oxidation. The error is considerable if the sample to be assayed is in a volume greater than 0.2 m1. This is because the acidity of the reaction mixture drops. I. Preparation of 1 N HCl in CH OH 3__ Methanol was dried by heating 500 ml with magnesium turnings (3.0 g) and iodine (0.1 9) under reflux for 2 hours. The dry methanol was then distilled using a system to exclude moisture and collected in a clean dry flask. Dry HCl gas (36.46 g per liter) 28 TABLE 6: N-acetyl neuraminic acid standard curve N-acetyl neuraminic acid Optical density ug at 549 mu 2.5 .080 5.0 .145 7.5 .226 10.0 .258 12.5 .340 15.0 .400 17.5 .495 20.0 .530 22.5 .630 25.0 .725 30.0 .820 35.0 .900 29 “>13 33324:» wummmc o>mm . e .m .ocpcweum mwpwmoa m>em T v .o .n .m :N_=no~mouuep-m ”e cmmocwmaxgposzcu "m cmsan~m>o ”u pmue ccmpo NLeEEeE umow ”a wmeumnuczm umum tempo NeeEEme mcw>om "a .mcwcwepm mwueeemme mcee; _— new .m .m ..cwe on Low .Ie m.m use ONm em emeewewEeezec sew: eepeezecw mmeuecucxm NpmNem mcw>ee Eeew eegeemee meewmeoxpm Panama we me>wue>weme wmzw use we mwmx—ece gee .emeuegpexm o Nm .eoeep em ”Newmou»_m Pagpas we me>wue>weme wmzw we weepst Neepceo e we seemepeseese new no mmauNN us: 20:23: 3:32. .p . . a.“ . o p 111 - 0520332.; D 133-2 05203920 C 1.31-2 (2(2 C 828“ rllllw .e. .53... 33:3 00"; 49 .uooop “a .mmmpmzucxm on mga sog$ nmgmamga mmurmooxpm Fxspms we mm>Pum>rLou wmz» as» $o Emgmopmsoggu mmo ”op mmawum Qd at: 20:55.. 2:23: .3 (2(2 3:53: Pl. ououam 50 fucose 4 Mannitol- glucose . N—acetyl. galactosamine ? fl J I I l ‘L0 21) an n eunve RE‘I’ ENU‘ON \ TIM§ FIGURE 11: Gas chromatogram of the TM51 derivatives of methy1 egcosides prepared from the bovine mammary 91and acetyl-CoA synthetase, at 190°C. 51 1 fucose f'__i galactose F""_"l giucose N-acety] galactosamine silicon ? 5 15 1'5 ’ 2'0 2‘5, RETENTION. TIME min. FIGURE 12: Gas chromatogram of the TMSi derivatives of methyl giycosides prepared from goat mammary gland acetyl-CoA synthetase at 190°C. 52 Glucose is present as the minor peak of the glucose standard and N-acetyl galactosamine as the major peak. The last peak of the chromatogram has a relative Rt of 2.9, corresponds to none of the standards and mass spectrum shows a fragmentation pattern charac- teristic of silicon. Silicon bleeding is very often observed from 0V and SE columns. C. Mass spectrometry analysis The data from the GLC analysis was confirmed using mass spectrometry. The studies were conducted with an LKB-9000 gas chromatograph-mass spectrometer using an ionizing energy of 70 EV. The GLC column, 4 feet x 1/8 inch glass was packed with chromosorb G containing 3% SE-30. The temperature was programmed from 160-240° with a 5°/min. increase. The protein samples were prepared exactly the same way as for GLC analysis. At this point, a discussion of the mass spectra of the TMSi derivatives of methyl-glycosides is necessary. Selected peaks from the mass spectrum of methyl 2,3,4,6-tetra-0-trimethy1silyl-a-D-gluco- pyranoside (I) are listed in table 12. TABLE 12: Selected peaks from the mass spectrum of methyl 2,3,4,6- tetra-O-trimethylsilyl-a-D-glucopyranoside(I) m/e m/e m/e m/e m/e 482 393 332 271 133 467 377 319 217 131 451 361 305 204 117 435 345 303 191 89 407 335 287 147 73 53 Molecule I fragments as follows: r T. 6 + CHZOTMST m/e 392 + TMSiOH V/ % m/e 467 + CH5 -————. L m/e 482 “l . m/e 422 + 3 CH3 m/e 377 + TMSiOH m/e 333 m/e 287 + TMSiOH m/e 243 + TMSiOH V ¢ m/e 435 + CH30H m/e 451 + CH30' m/e 451 has the structure: CHZOTM51 54 m/e 451 further fragments to give m/e 361 + TMSiOH, and 361 gives m/e 271 by elimination of TMSiOH. The structure of some of the smaller fragments is listed in Table 13. The fragmentation of the trimethylsilyl ethers of methyl- 2-acetamido-2-deoxy-a-D-glucopyranoside (II) and N-acetyl-neuraminic acid (111) will be discussed shortly. The structures of II and III are given below: TMSiOCH (II) m/e 451 H NHCOCH 9 CHZOTMSi 8 CHOTMSi l 7 CHOTMSi (III) m/e 625 TABLE 13: Structures of some of the fragments of methyl 2,3,4,6- tetra-O-trimethylsilyl-a-D-glucopyranoside (I). m/e 73 103 117 129 133 147 191 Molecular Structure + . 51(CH3)3 6 _j . CH2-—081(CH3)3 6 +. (IZH2051(CH3)2 5CH=0 CH=§CH ' 2 5 4 . +CHOTMSi + CH3OCHOTMSi 4. (CH3)3SiOSi(CH3)2 l + HI': =OSi(CH3)3 OSi(CH3)3 TABLE 13: continued 56 204 217 305 319 Molecular Structure +cH ——-?H TMSiO OTMSi 4CH 2: 3CHZCH+ TMSi0 0TMSi OTMSi 1 CH :: C-C+H l 1 TMSiO OTMSi 6 . CH20TM51 51 CH TMSiO __9c 31 + CHOTMSi 57 Molecule II fragments as follows: "' ‘1 II TMSiOCHZ _____3> m/e 435 + CH5 TMSi m/e 404 + CH30H TMSiO \1 L H NHCOCH3 _ m/e 314 + TMSiOH m/e 451 m/e 392 + CH3 c0NH2 m/3 420 + CH30 Further fragmentation of m/e 420 which is a characteristic fragment gives: HZOTMSi OTMS‘i ‘_—9 m/e 330 4' TMSIOH TMSi0 NHCOCH3 m/e 420 The fragment at m/e 173 is an intense ion and has the structure: - + CH-——CH | I m/e 173 TMSiO NHCOCH3 58 The same two carbon unit occurs in N-acetyl-neuraminic acid and, hence, m/e 173 can arrise either from sialic acid residues of the N-acetyl type or from the N-acetyl hexosamine residues. The related ion at m/e 186 can also be formed from either N-acetyl-neuraminic acid or N-acetyl-hexosamine. CH —-C = CH I l 2 NH OTMSi l m/e 186 C = 0 1 CH3 Figures 13 and 14 give the GLC traces of the TMSi derivatives of methyl glycosides prepared from the cow and goat acetyl-CoA synthetase respectively. Each peak of these traces was analyzed by the mass spectrometer. Figures 15, 16 and 17 give the mass spectra of the TMSi derivatives of mannitol and the TMSi derivatives of the methyl glycosides of N-acetyl galactosamine and N-acetyl neuraminic acid respectively. FrOm Figure 13 (cow enzyme) scans 4, 28, 33, 48, 79, 139 (Figures 18, 19, 20, 21, 22, 23 respectively) give fragmentation patterns characteristic of the pyranoside ring. Scan 41 (Figure 24) is mannitol (Figure 15). Scan 99 (Figure 25) gives good evidence that the component is either a N-acetyl sugar or N-acetyl neuraminic acid. This peak eluted at about 220°C where N-acetyl neuraminic acid elutes under the same conditions (38). From Figure 14 (goat enzyme) scans 3, 36, 42, 144 (Figures 26, 28, 59 29, 32 respectively) can be identified as sugars having the pyrano- side ring. Scan 25 (Figure 27) has several of the fragments of the pyranoside ring but only the ones with lower molecular weight. Scan 90 (Figure 31) has fragments characteristic of silicon (m/e 29, 42, 56, 207, 221, 281, 355, 429, 503). Scan 47 (Figure 30) does not show fragments characteristic of sugars. The mass spectrum of the goat enzyme was obtained 8 days after the GLC analysis. Partial hydrolysis of the TMSi derivatives can occur when the samples are stored for several days. N-acetyl neuraminic acid was not found in the goat enzyme using GLC. It is known that N-acetyl neuraminic acid 1‘? is unstable in storage and probably this is the reason of its absence from the gas chromatogram. However, the neuraminidase and thiobarbi- turic acid assay were both positive and we can conclude that N-acetyl neuraminic acid is also present in the goat acetyl-00A synthetase. It is concluded from the above data that fucose, glucose and N-acetyl neuraminic acid are present in the cow enzyme. The goat enzyme con- tains fucose, galactose, glucose, N-acetyl galactosamine and N-acetyl neuraminic acid. D. Discussion The discovery that acetyl-00A synthetase is a glycoprotein may explain many of the questions related to the physical and catalytic properties of the enzyme. However, the exact role that carbohydrates play and the magni- tude of the significance of this role is not clear at this time. Much of the purification data indicates that the enzyme has the ten- dency to aggregate. A charge is very often needed for interaction 60 between subunits. N—acetyl neuraminic acid present in acetyl-CoA synthetase could very well give this charge and direct this apparent aggregation and deaggregation phenomena. Acetyl-CoA synthetase elutes in more than one protein peaks from both DE-23 cellulose column and calcium phosphate gel. All these protein peaks possessed enzymatic activity. This data suggests the existence of isozymes. Several proteins are known to exist as isoglycoenzymes or isozymes that differ only in their carbohydrate content. It is reasonable to think that acetyl-CoA synthetase is a isoglycoenzyme. Acetyl-CoA synthetase from bovine fetal heart tissue preferen- tially activates propionate rather than acetate, in contrast to the adult enzyme. It would be of interest to study the carbohydrate content and molecular weight of the fetal and adult enzymes. It could be determined whether or not isozymic or aggregated forms of acetyl-CoA synthetase are responsible for this difference in sub- strate specificity. There are more points of interest that should be investigated. Acetyl-CoA synthetase is not active in non-lactating mammary gland. The enzyme becomes active after parturition and its activity decreases with advancing lactation. These phenomena described above may be explained by differences in the carbohydrate composition of the enzymes in different tissues and under different physiological states. In summary this work has proven that acetyl-00A synthetase is a glycoprotein and this appears to be a significant discovery that 61 will probably explain many of the physical and catalytic proper- ties of the enzyme. .. 79‘ “ j ’1‘! \‘r-.~t.~'q v. '~ L M 1:“:- g. 62 i a! 3 . .. fl! :11“... 41:15 in» mans-haunt" 51E: .mwmxwmcm szguomgm mmms mg» cow tom: mm: peg“ mmmpmcucxm on mgu sogw vmgmnmgn mmmeooxpm waspms wo mm>wum>wgmu wmzw us» we segmopmsogsu mam mzw umw mmswwd OOfi. . . . . 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