.23 BIOSYNTHESIS OF HEMOGLO-BIN: ATTACHMENT 0F HEME TO GLOBIN Thesis for the Degree of M. S. MICHEGAN STATE UNIVERSITY Katherine Liang 19.66 THESIS h“. rpm“ § , “k 1 .‘,.' H"?- . ,t‘ 7'. 3V1~1t§<£r$ :3 - :12 h) V. '- A '1‘”: in L: . J» tilt” a LIBRARY Michigan Sta. U . . t5. .rnLSIs OF 33; GLOBIyz AETAcnxaLT CF HEnE TO 4“ .2] I Katherine Liang A TfSS 5 Submitted to Iichigan State University in partial fulfillment of the requirements for the degree of vn~mw2 c? QCIV‘C” .'410 4. -1. g n) 4;: J.) Department of iiochemistry '1966 J "LC Y’ M “L: ABSTRACT BIOSYNTHESIS OF HEMDGLOBIN: ATTACHMENT OF HENE T0 GLOBIN by Katherine Liang The biosynthetic pathways of heme and globin are known. The details of the regulation and coordination of heme and globin synthesis remain to be investigated. The purpose of this study was to investigate the stage of heme- globin biosynthesis at which the attachment of heme to globin chains occurs in rabbit reticulocytes. Cluc/Zaminolevulinic acid (o/-ALA), a known precursor of heme, was used to introduce a radioactive label into heme in lysates of rabbit reticulocytes. The C1” label associated with the ribosome frac- tion was analyzed on sucrose density gradients. The release of C14 labelled material from ribosomes in the presence of protein synthesis was studied. The effect of puromycin on the release of C14 labelled material was also investigated. Finally Cluc/LALA.1abelled ribosomes were dissociated and analyzed on ECTEOLA cellulose columns for the presence of C14 heme-peptidyl sRNA. Results from sucrose density gradient analyses showed a small amount of radioactivity from.Clqo/-ALA attached to the polysomal frac- tions. Studies on the release of C1]+ labelled material on ribosomes and the effect of puromycin indicate that this 014 labelled material (heme or heme intermediates) is not attached to the globin chains on polyb $017163 0 Katherine Liang Finally, the absence of heme-peptidyl-SREA from dissociated 014 or-ALA ribosomes indicate that heme is attached to globin chains after the latter are released from polysomes. To Peter ACKNOWLEDGMENTS The author wishes to express her sincere appreciation to Dr. Allan J. Morris for his valuable counsel, continual interest, and timely encour- agement, particularly with regard to these investigations. She would also like to thank Miss Ann Stevens, Mr. Bruce MacDonald, Mrs. Linda Seifert, Dr. Fritz M. Rottman and other friends who have gener- ously given advice, suggestions, or assistance. She also gratefully acknowledge the financial assistance from Michigan State University and from the National Institutes of Health. ii TABLE OF COHTEE‘I‘S ACKI‘IOVHJSDEPEEITS o o o o o o o o o o o o o o o e o LIS rI. OF TAELES O O O O O O O O O O O O O O O O 0 LIST OF b‘IGURC-ZS O O O O O O O O O O O O O O O O O IN‘I‘PuODUCI‘ICI‘:oooeoeoooeoooooooo ESTORICMJ O O O O O O O O O O O 0 O O O O O O O EPERII’IEE‘JTALeoooooeoooeoeoeooo I. II. III. IV. Analytical Procedures . . . . . . . . . A. Preparation of Protoporphyrin . . . . . B. Extraction of Hemin and Porphyrins from Incubation Mixtures . . . . . . . . C. Analysis of Extractable Products by Paper Chromatography . . . . . . BiOlogj—cal I‘laterifls O O C O O O O O O A. Preparation of Rabbit Reticulocytes B. Prelabelling of nibosomes . . . . . Sucrose Density Gradient Analyses . . . A. Analyses of Polysomes . . . . . . . Bo AnalySeSOfSRi‘i‘A oooeooooo Polyacrylamide Gel Electrophoresis of supernatant HemoglObin o o e o o o A. Preparation of Methemoglobin from Supernatant . . . . . . . . . . B. Polyacrylamide Gel Electrophoresis of l‘:ethem0g10bin o e o o o o o o 0 iii 0000 CD O\U'\ Kn U\ P F’ {1’ 41‘ 10 TABLE OF CONTEI‘CTS (CONTINUED) V. Releaifi of Protein Bound Radioactivity from C or A. B. C. -aminolevulinic Acid Ribosomes . . . . . . In a Complete Cell-Free System of Protein SyntheSiS................. Effect of Deoxycholate Treatment of Ribosomes . Effect of Incubation With Puromycin . . . . . . VI. Column Chromatography of C1l‘oV-aminolevulfmic Acid RESULTS . . DISCUSSION BIBLIOGRAPHY Labelled Ribosomes (cf-ALA Ribosomes) . . . . . iv 12 12 12 13 13 11+ ’40 Table I. II . III . VII. LIST OF TABLES Incorporation of‘of-aminolevulinic Acid (cf-ALA) Into EXtraCtable PmdUCts o e o o o o o o o 0 Paper Chromatographic Analysis of Hemin Extracts Polyacrylamide Gel Electrophoresis Analysis of Supernatants From Rabbit Reticulocytes . . . Releaze of Protein Bound Radioactivity From C J-ALA Ribosomes . Release of Protein Bound Deoxycholate-Treated C dioactivity From (Jr-ALA Ribosomes . . Effect of Puromycin on the Relgase of Protein Bound Radioactivity From C or-ALA Ribosomes Release of Protein Bound Radioactivity From C14 of -ALA Ribosomes Preincubated in the Presence of Protein Synthesis 15 17 24 27.. 29 30 32 LIST OF FIGURES Incorporation of Ciuor-aminolevulinic Acid.(dr-ALA) Into EXtraCtable PrOduCtS VS. Time 0 o e o o e o e o Sucrose Density Gradient Analysis of Ribosomes Fran a Rabbit Reticulocyte Lysate Incubated With C1 J-ALAforSFfimteSoooeoooooooooeoo Sucrose Density Gradient Analysis of Ribosomes Fffim a Rabbit Reticulocyte Lysate Incubated With C J-ALAforLl'omnutes00000000000000. Radioactivity Associated'With Ribosomes After Succes- sive'Washings by ResuSpension and Resedimentation . SucroEe Density Gradient Analyses of Soluble RNA From C1 J-ALARibosomeSoeoooooooooeoooo ECTEOLA - Cellulose Column Chromatography of ewe/-ALA Ribosomes 16 20 22 25 33 36 II‘GTRODUC TIOI“? Studies on the physical and chemical structures of the vertebrate hemoglobins, their mode of synthesis and functioning, and on their genetic control have occupied many years of research. A fUll understanding of the mechanism and regulation of their biosynthesis has been the goal of these studies. The biosynthetic pathway of heme, the prosthetic group of hemoglobin, is well defined; the complete amino acid sequence of the four polypeptide globin chains and their tertiary and quarternary structures are also known. However, the details of the regulation and coordination of heme synthesis and globin synthesis remain to be investigated. This study was to investigate the stage of hemoglobin biosynthesis at which the attachment of heme to globin chains occurs in rabbit reticulo- cytes. The ultimate goal of this investigation and other investigations of a similar nature in relating the syntheses of heme and globin are two fold: (a) To determine, in general, how prosthetic groups of proteins are com- bined with their apOproteins during synthesis and (b) to obtain an insight into the control of protein synthesis at the molecular level. HISTORICAL Borsook and Kruh (1) first described the close coordination of heme and globin syntheses in the intact rabbit reticulocytes. They also demonstrated (2) that although added iron might be essential for optimal synthesis of hemoglobin, it did not stimulate synthesis in the absence of certain essential amino acids. Similar parallelism in the rates of heme and globin syntheses was also reported by Morell and his coworkers (3) in rabbit bone marrow and by hizet (4) in dog reticulocytes. Hammel and Bessman (5) noted an increase in protein synthesis when hemin and other porphyrins were added to avian erythrocyte nuclei. The degree of stimulation was dependent on the concentration of hemin. They postulated that porphyrins stimulated amino acid incorporation into globin. Gribble and Schwartz (6) have reported that protoporphyrin enhanced the release from ribosomes of newly formed globin chains in their cell- free system. They suggested that such a release might involve the coiling of the globin chains around the protoporphyrin and/or heme groups. Bruns and London (7) showed that hemin, at a concentration of 10'53, inhibited the utilization of glycine into hemin by rabbit reticulocytes, 14 and also increased the incorporation of C valine into newly formed hemo- globin. Rabinovitz and Naxman (8) studied the influence of iron on the state of aggregation and activity of the ribosomes in the intact reticulocytes. Their data indicate that iron or hemin could bring about reaggregation of polysomes which have become disaggregated upon incubation of reticulocytes with an amino acid mixture and glucose. They also reported (9) that cells incubated with iron and transferrin had an increased polysome con- tent. Their results were inconsistent with the tape theory of protein synthesis, which included the concept that terminal events, such as the association oflx and‘g polypeptide chains or the insertion of heme have no role in the peptide forming process. host recently Grayzel and his coworkers (10) reported that in reticulocytes of iron-deficient rabbits there occurred an increase (in the presence of added hemin) in size and proportion of polysomes, in specific activity of the polypeptide chains attached to the polysomes and in the Specific activity of soluble hemoglobin. They postulated a mechanism in which heme might attach to nascent chains of globin on polysomes and thereby promote conformational changes in the polypeptide chains. Towards the end of the present studies, Felicetti and Baglioni in Italy (11) reported their findings that an added F659 label does not seem to be present in heme in a heme-nascent globin form while the glo- bin molecules were being synthesized on the polysome. They also postu- lated thatd and fl globin subunits are probably intermediates in the assembly of hemoglobin. .‘ .. ,_,..___ -LIL'JLI'! T¢AL [‘3 "3', :L-CP I. AnaLgtical Procedures A. Preparation of Protoporphyrin: The Ferrous Sulfate Method A modification of the procedure by Falk (12) was used. One hundred ml. of glacial acetic acid was added to a solution of 10 mg. of crystalline hemin in 1 ml. of pyridine. The reac- tion was performed at room temperature and was carried out in an athSphere of nitrogen. A freshly prepared solution of 0.4 g. of ferrous sulfate in 0.4 ml. of concentrated hydrochloric acid was added to the reaction mixture. The passage of nitrogen over the solution was continued for 5 minutes. The mixture was then transferred to a separatory funnel containing a solution of 70 ml. of 4.55 sodium acetate (haAc) and 500 ml. of fresh ether. The mixture was shaken vigorously. ProtOporphyrin was transferred to the ether phase. The aqueous phase was reextracted with 100 ml. of fresh ether. Thirty ml. of SE hydrochloric acid were added to the combined ether extracts and the mixture shaken vigorously. The aqueous layer was removed and the pH of the solution adjusted to 4. Protoporphyrin was precipitated from the aqueous solution atthisgfih The brown precipitates were collected by centrifuga- tion and dried.ip;ygggg in the absence of light. 3. Extraction of Hemin and Porphyrins fromglggubation Mixtures The incubation mixtures were stirred overnight with 50 nflm of ethyl acetatezacetic acid solution (3:1 v/v) and filtered. l4, The filtrate was washed with water, to remove residual cflamino- levulinic acid GLALA), and transferred to a separatory funnel. The ethyl acetate phase was then concentrated to dryness. The residue was taken up in 10 rd, of formic acid, 0.5 ml. aliquot of which was plated with 5 mg. of carrier hemin onto a tared aluminum plachet, dried ig'vacuo, and radioactivity determined in a low background Iuclear Chicago Automatic Geiger counter. Results have been corrected for self absorption of the A par- ticles by the sample. ‘ C. A alysis of Extractacle Products by Paper Chromatography The residue from the ethyl acetate phase during extraction was taken up in 0.5 ml. of 2,6 lutidine. Two pl of the solution was spotted on a 30 x 40 cm. Jhatman To. 1 paper chromatogram. The solvent System used was a mixture of 2,6 lutidine:H20 (1:1 v/v) (13). The chamber was saturated with 25$ ammonia. The chromatogram was developed descendingly for 18 to 20 hours, dried and cut in 2 inch strips and radioactivity determined in a Packard Fodel 7201 radiochromatogram scanner. II. Biological Eaterials A. Preparation of Rabbit Reticulogytes hale New Zealand white rabbits weighing, six to eight pounds, were made reticulocytic by four daily subcutaneous injec- tions of 0.175 ml./pound body weight of 2.5% neutralized phenyl- hydrazine. On the sixth day after the initial injection the animals received a solution containing 2000 i.u. of heparin and 100 mg. of Nembutol by intravenous injection. Blood was col- lected immediately by heart puncture. The red blood cells were separated from the plasma by centrifugation for 20 minutes at 2000 x g. in a Servall centrifuge. The plasma was decanted and its volume recorded. The cells were resuSpended in KKH solution (a solution containing 0.13; L‘JaCl, 0.00 533 KCl and 0.0075; I'fgClZ), using a volume equal to the plasma. The suspension was filtered through glass wool. The filtrate, containing the cells, was centrifuged (20 min. x 2000 g.). The cells, which sedimented, were resuSpended in fiK£.solution and the suSpension was centri- fuged once more. The supernatant was removed. The packed cells were lysed by adding two volumes of a 0.0025fl MgCl2 solution and stirring gently for 10 minutes. After centrifugation at 15,000 g. for 10 minutes, the supernatant was decanted as the lysate. Prelabellingiof Ribosomes Two preincubation procedures were used, one in the presence of active protein synthesis and the other in its absence. 1. The incubation medium contained, per ml. of lysate, 0.1 pC Clucieminolevulinic acid GIALA) (specific activity 24.5 mC/mg) and buffered with 5 pmoles Tris-HCl buffer pH 7.5. Reaction mixtures were incubated for various times at 37°C and the reaction was stopped by the addition of two volumes of a cold solution containing 0.25% sucrose, 0.0175; KHCOB and 0.002g th12 (medium E). Ribosomes were isolated from the reticulo- cytes by centrifugation at 78,000 g. for 90 minutes in a 2. Beckman L-2 ultracentrifuge. The aqueous phase was decanted as "supernatant." Ribosomal pellets thus obtained were designated once sedimented (1):) C1“ aCALA ribosomes. The pellets were resuspended in a small volume of 0.253 sucrose by gently homogenizing with a glass homogenizer and a Teflon pestle. The ribosomal suSpension thus obtained was either directly layered on a sucrose density gradient and analyzed or diluted with medium E (to a concentration of 0.5 mg. ribosomes/ml. medium E) and centrifuged at 78,000 g. for 90 minutes to yield 2X ClucfiALA ribosomes. The ribosomal sus- pension was centrifuged at 15,000 g. for 10 minutes to remove any insoluble material. The ribosome concentration was determined by its absorption at 260 mu. (1 mg. ribosomes/ ml. = 11.3 0. D.) Reticulocyte lysates were incubated in the presence of active protein synthesis by a modification of the method of Lamfrom and Knopf (14). The incubation mixture contained, per ml. of lysate, 0.1 11C. CluoCALA (specific activity 24.5), 4 moles EgClZ, 50‘umoles KCl, 0.05‘umoles each of an equimolar mix- ture of 20 amino acids, 2 pmoles Tris-RC1 buffer pH 7.5, 0.2 pmoles ATP, 0.05 pmoles GTP, 1 pmole phosphoenolpyruvic acid and 10 pg. pyruvate kinase. Other details of the preincuba- tion procedure were identical to those in procedure 1. III. Sucrose Density Gradient Analysis A. B. Analysis of Polysomes Linear sucrose gradients containing 15 to 30% sucrose, 'gfl Tris pH 7.5 were prepared 1.5 x 104g MgClZ, 104.13 KCl, 10 at 4°C. The ribosomal suSpension was gently layered onto the gradient contained in a 30 ml. ultracentrifuge tube. The tubes were then placed in an S.W. 25.1 rotor and centrifuged for 3% hours at 75,500 g. at 4°C. Contents of each.tube were then analyzed for materials absorbing at 260 mu by pumping through a Gilford Spectrophotometer equipped with a flow cell (0.5 cm. path length). Results were plotted automatically by a Sergeant Medal SR recorder. Flow rate through the cell was maintained at 5 ml. per minute using a Buchler polystaltic pump. Effluent from the flow cell was collected in 1.0 ml. portions with.a.Packard model 231 fraction collector. To each fraction was added 1 mg. of bovine serum.albumin and the pre- cipitate which formed in 10% TCA solutions was collected on nitrocellulose filters. The filters were washed twice with 5 ml. of 10% TCA. The dried filters were placed in a counting vial, 15 ml. of toluene, PPO, POPOP counting fluid was added to each.vial and radioactivity was determined by a Packard Mbdel 3003 liquid scintillation Spectrometer. Analyges of sRNA A modification of the procedure of Traut and Monro (15) was used. The method was identical to the one just described except for the following: Linear sucrose density gradients containing 5 to 20,3 sucrose, 0.11_~1 LiClZ, 0.53 sodium dodecyl sulfate (SDS), and 5 x 10'3M Tris-RC1 buffer pH 7.0 were pre- pared at room temperature. Raterials to be analyzed were adjusted to 0.5} SDS and layered onto a gradient of 56 ml. Centrifugation was carried out for 48 hours at 20°C in a S.W. 25.2 rotor at 75,500 g. IV. Polyacrylamide Gel Electrophoresis of Sgpernatgnt hemoglobin A. Preparation of Methemoglpbin from Supernatant The concentration of hemoglobin in the supernatant was determined by the method of Drabkin and Austin (16). Potas- sium ferricyanide (KBFe(CK)6) was added to a sample contain- ing 1.5-3.2 mg. hemoglobin/ml. to a final concentration of 6 x 10-E§;K3Fe(CL)6. The reaction mixture was stirred and allowed to stand for 2 minutes to permit complete formation of methemoglobin from hemOglobin. Potassium cyanide (KCL) was then added to a final concentration of 8 x 10‘93 and allowed to stand for 2 minutes to convert methemoglobin to the methemoglobin-CH complex. The solution was measured for absorb- ance at 540 mp. (16 mg. hemoglobin/ml. = 11.5 O.D.) To pre- pare methemoglobin from supernatant, the concentration of which was determined as described above, one equivalent of K3Fe(CN)6 was added to the solution dropwise and allowed to stand for 30 minutes at room temperature. The solution was then dialyzed against distilled water overnight. B. 10 Polyacrylamlde Gel Electrophoresis of Rethemgglobin from.Canal Industrial Corp., Bethesda, Rd. and 9 x 1 cm. glass columns were used. Procedures for preparing the sample gels were obtained A 7% gel solution The following stock solutions were necessary to prepare the separating and stack- ing gel solutions: (A) 1£_HC1 Tris Tetramethylethylenediamine H20 to make 1; RC1 Tris Tetramethylethylenediamine H20 to make Acrylamide Rethylene bisacrylamide H?O to make Acrylamide hethylene bisacrylamide H20 to make Riboflavin 320 to make Sucrose H20 to make Ammonium Persulfate H20 to make “8 In]... 36.3 g- O.23 ml. 100 ml. (pH 8.8-9.0) I48 TIL-Lo 5.98 g. 0.46 ml. 100 ml. (pH 6.6-6.8) 28 g. 0.735 g. 100 ”1].. 10 g. 2.5 g. 100 ml. 4.0 mg. 100 ml. 40 g. 100 ml. 0.14 g. 100 ml. The following working solutions were prepared from the stock solutions: (A) Separating gel solution: 1 part A 2 parts C 1 part H20 11 The above was mixed immediately before use with an equal volume of a freshly prepared solution of ammonium.persulfate (G). The mixture was used to fill a glass column (9 x 1 cm.) to about 3 cm. and allowed to stand for 40 minutes for polymerization. (B) Stacking gel solution: 1 part B 2 parts D 1 part E 4 parts F The solution was layered on top of the separating gel solution and exposed to fluorescent light to facilitate polymerication. The methemoglobin sample was then mixed with 1 ml. of the stack- ing gel solution and layered over the stacking gel. After poly- merization, the sample tube was placed between the two chambers of the electrophoresis apparatus. The chambers contained a Tris-glycine buffer (.373 Tris, 1.4% gly). The current was main- tained at 5 milliamperes per tube for the 60 minute running time. The sample tubes were then removed from the apparatus and the gel columns extruded from the glass cylinder. The major methemoglo- bin band was cut from the column and eluted with a small amount of Tris-glycine buffer. The eluent was then filtered through glass wool. The filtrate was read for absorbance at #09 mp. 15 mg. of carrier bovine serum albumin was then added to the fil- trate and the solution precipitated with 5% TCA. The solution was centringed and the pellets dissolved in 0.25 ml. 1§;NaOH and transferred to scintillation counting vials. 15 ml. of thixotropic counting fluid was added to each vial and the radioactivity deter- mined in a liquid scintillation Spectrometer. V. A. 5. Release of Protein Bound Radioac 'vi 12 In a Complete Cell-Free System of Protein §ynthesis In the complete cell-free system, the incubation mixtures contained, per ml. of assay, 0.25 pmoles GTP, 1 pople ATP, 5 umoles phOSphoenolpyruvate, 40 ug. pyruvate kinase, 50 umoles Tris-Cl buffer pH 7.5, 20 pmoles glutathione, 0.05 umoles of an equimolar amino acid mixture (10'3g), 50 pmoles K01, 4 pmoles thlZ, 6 mg. of a 40—705 ammonium sulfate precipitated enzyme from supernatant, and 2 mg. of CllfLALA ribosomes. The reaction was carried out at 37°C for the time periods indicated. Each assay was then chilled and transferred to a # ml. Spinco tube, the tube filled with a 0.25; sucrose, 0.015 ligCl2 solution and the mixture was centrifuged at 100,000 g. for 60 minutes. Super- natants were decanted into reSpective tubes and 15 mg. of carrier bovine serum albumin was then added to each assay. The precipi- tate which formed in 5£ TCA was centrifuged and reprecipitated with 55 TCA three times. Each pellet was dissolved in 0.25 ml. of 1§.haOH and transferred to a scintillation counting vial. 15 ml. of thixotropic counting fluid was added to each vial and the radioactivity determined by a liquid scintillation Spectrom- eter. Effect of Deoxycholate Treatment of Ribosomes Six mg. of ClldLALA ribosomes were suSpended in a 0.53 sodium deoxycholate solution at 0°C for 10 minutes then diluted to 30 ml. with medium E. Ribosomes were reisolated by centrifu- gation at 75,000 g. for 90 minutes. The pellet was homogenized 13 in a small volume of 0.255 sucrose and used in a release assay as described above. C. Effect of Incubation with Puromycin One pmole of puromycin, pH 7.0, was added to the release assay as described in A. Column Chromatography of Glut/LALgééabelled Ribosomes A modification of the procedure by Ganoza and hakamoto (24) was used. An ECTEOLA cellulose column (3 x 1 cm.) was packed under a pressure of 0.5 lb. per square inch. The column was equilibrated with a solution of 0.1g jaCl, 0.13 NH4C00H pH 4.7 and 0.5% Brij-35 at room temperature. Before loading, the ClucYLALA ribosomes were diluted to 1 ml. with the buffered detergent, 2 mg. of carrier sdhA added and incubated at 0°C for 10 minutes. The reaction mixture was loaded onto the column and eluted with 80 ml. of a linear gradient containing 0.1;; BlaCl to 2.0;; I‘EaCl and 0°13: E-EEILFCOOH pH ’4 .7'. 0.55 Brij-35. Flow rates were maintained at 1-2 ml. per minute. Eluants were collected in 3.0 ml. portions. Each fraction was determined for absorbence at 260 mp. To each fraction was added 1 mg. bovine serum.albumin and the precipitate which formed in 10$ TCA solution was collected on nitrocellulose filters. Fifteen ml. of toluene, PPO, POPOP counting fluid was added and each vial containing a dried filter, and radioactivity determined by a liquid scintillation Spec- trometer. RESULTS It has been established by Shemin, heuberger and Scott (15, 16) that.01aminolevulinic acid (o/-ALA) originating from glycine and suc- cinyl CoA is a specific precursor of heme and porphyrins. In the present studies, rabbit reticulocytes were incubated with c” J-ALA in m. Incubation mixtures were extracted with ethyl acetate-acetic acid mixtures and the extracts, containing hemin, por- phyrin and possibly other heme intermediates were analyzed for radio- activity. The results, in Table I, show that CluchALA was incorporated better into extractable porphyrins by lysates than by whole cells of rabbit reticulocytes. This result is probably due to poor penetration of the h.) 5010 54¢ 5 3225 I 00 050 OOWO 43 I5 3%: B FRACTION NUMBER 0.5- l5 FRACTION NUMBER 3| T 0.5 - 25.: 00$ >._._mzmo 1.42.7.0 3;: 003 fthuo 442:5 OJ“- 35 chromatographs, A modification of the procedure according to Ganoza and Nakamoto (2h) was used. Soluble RNA and peptidyl-SHEA were eluted from the column at approximately 0.6% NaCl, while soluble methemoglobin was eluted at 0.131 N aCl. Figure 6 shows the analysis of Clue/LALA.ribosomes in an ECTEOLA cellulose column. Clearly. all of the radioactivity from the ribosomes was associated with the fraction corresponding to globin and no radio- activity was associated with the sRNA (peptidyl-SHEA) peak. These results provided more conclusive evidence that heme intermediates are not attached to globin chains at the polysomal stage of globin synthesis. FIGURE 6 ECTEOLA—CJLU 313 com»: CEZROLCATOGLLQPL—TI 05‘ c1 of-ALA RIBCSOLES (Solid lines denote optical density at 260 rm. Dotten lines denote radioactivity in counts/ min.) m cwmzaz 20:041.... ¢N NN ON 0. w. v. N. o. o w v N 'C’C‘IIC'IICII'.‘ C'IO-'O‘ ’ ’ ' (NdO) ALIMLOVOIOVU 0 o O . 2 ES I T 1 O O n j oomT 0. o o o In G“ .5 N 0. on 0. p «“3092 IN ALISNBO ‘IVOlldO TVLOL I 0. o 5. a DISCUSSION The present studies have shown that heme or heme intermediates are not combined with globin chains when the latter are being synthesized on polysomes. The attachment, therefore, is believed to occur at a later stage in the synthesis of hemoglobin. The above conclusion is based on the following data: 1) The absence of a release of the CluzflALA labelled material bound to ribosomes when completed globin chains attached to these ribosomes were released from the ribosomes. 2) The absence of a release of the C11+ labelled material when nascent polypeptide chains were released from the ribosomes in the presence of puromycin. 3) The absence of heme-peptidyl-SREA by direct analysis of peptidyl-531A. It is therefore sugrested that heme interacts with globin after the latter has been released from the polysome. Winterhalter and Huehns (21) have reported the presence of free globin in the red cell supernatant. They have also shown (22) that heme can promote the conversion of d'plus figlobin dimers of globin to the stable hemoglobin tetramer. In the light of the present studies, such a role of heme may be postulated for its regulation of globin synthesis. However, it does not seem.likely that heme enhances the release from ribosomes of globin chains by coil- ing around them and causing conformational changes on the polysome as proposed by Gribble and Schwartz (6). 39 Karibian and London (25) reported that hemin inhibits the incor- poration of glycine into heme and postulated a control mechanism which involves a feedback inhibition by heme in the conversion of glycine to dLALA. Granick and Levere (26) have reported in their studies with chicken blastoderm that heme Synthesis is regulated by the first enzyme in its biosynthetic pathway,«Jeaminolevulinic acid synthetase GJLALA synthetase). If indeed heme inhibits JZALA synthetase and free globin is pres- ent in the soluble fraction of the cell, a mechanism of the regulation of heme synthesis by globin can be postulated. Globin which is released from ribosomes combines with heme to form hemoglobin, thus making heme unavailable to inhibitc/LALA synthetase and thereby inhibit heme synthe- sis. However, the reverse mechanism in which heme regulates globin syn- thesis is not known. 11. 12. 13. 11+. 15. 16. 17. 18. BI BLIOG PJXP HY Kruh, J., and Borsook, 31., J. Biol. Chem. 220, 905 (1956). borsook, H., Fischer, E. H... and Keighley, G., J. Biol. Chem. 229, 1059 (1957). Iiorell, EL, Savoie, J. C., and London, I. 16., J. Biol. Chem. 222, 923 (1953). liizet, A., Bull. Soc. Chim. Biol. 32, 265 (1957). Hammel, C., and Bessman, S., Fed. Proc. 22, 317 (196”). Gribble, T. J., and Schwartz, H. C., Biochim. Biophys. Acta 102, 333 (1965). Bruns, G. P., and London, I. 11., Biochem. Biophys. Res. Comm. _1§., 236 (1965). daman, 13.. 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Comm.‘Z, 326 (1962). horris, A. and Schweet, R., Biochim. BiOphys. Acta 4 , 415 (1961). Ninterhalter, K. H., and Huehns, E. 3., J. Clin. Invest. 32, 995 (1963). winterhalter, K. H., and Huehns, E. R., J. Biol. Chem. 222, 3699 (1964). horris, A. J., Biochem. Biophys. Res. Comm. 22, 498 (1966). Ganoza, 1. C. and hakamoto, T., Proc. Nat. Acad. Sci. (U.S.) 55, 162 (1966). Karibian, D. and London, I. h., Biochem. Biophys. Res. Comm. 2%, 243 (1965). Levere, R. D. and Granick, 3., Proc. hat. Acad. Sci. (U.S.) 54, 134 (1965) . ‘