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From page 66...
... this was demonstrated by incubating duck erythrocytes with labeled substrates and then degrading the porphyrin molecule ill a manner by which each carbon atom from a specific position could be isolated. It was found that the carbon atoms of the substrates occupy particular positions in the porphyrin molecule.9~~0 Heme synthesized from glycine-2-C24 was shown to contain eight radioactive 4~~~ carbon atoms in specific positions.9 i~ Samples of heme, synthesized from Calf methyl and C7^J' carboxyl-labeled acetate, which were degraded, revealed a labeling pattern from which it was concluded that the acetate was converted to a four-carbon atom unsymmetrical compound via the citric acid cycled Further, it was concluded that this "active" succinate condensed with glycine, in some unknown manner, to form a precursor pyrrole.
From page 67...
... 1. The relationship of the tricarboxylic acid cycle and porphyrin formation.
From page 68...
... 3~. In order to test this postulate, hemolyzates of duck erythrocytes were inc~bated with b-aminolevulinic acid-5-C 5 and with b-aminolevulinic acid1,4-C~.
From page 69...
... We have found recently that not only can pyridoxal phosphate increase the synthesis of porphyrins as demonstrated by Schulman and Richert,-` but that the cysteine inhibition can be overcome by this coenzyme.'S These experiments point to a necessary activation of the glycine. We have more recently found that formation of b-aminolevulinic acid can be markedly inhibited by aza-L-serine.'0 This latter inhibition was not overcome by the addition of glutamine.~° It may be worth noting that azaserine has no inhibitory effect on the conversion of b-aminolevulinic acid to heme.
From page 70...
... -2~H2 CH2—NH2 Ac P P Ac FIG. 4.—A mechanism of porphyrin formation from the monopyrrole.
From page 71...
... Consistent with this hypothesis is our finding that on the conversion of porphobilinogen to porphyrins, either by heating under acid conditions or by enzymatic conversion in cell-free extracts, formaldehyde i, formed.~5 The formation of protoporphyrin and heme can occur in a cell-free extract of duck erythrocytes. After incubation of cell-free extracts, obtained by centrifugation at 100,000 g, with 6-aminolevulinic acid-5-C ;, the isolated bemire was radioactive.
From page 72...
... C., Lack, L., and Shemin, D.: The mechanism of porphyrin formation. Further evidence on the relationship of the citric acid cycle and porphyrin formation, J
From page 73...
... P., and Rickert, D A.: Heme synthesis in vitamin B.`; and pantothenic acid deficiencies, J
From page 74...
... Shemin and his colleagues relating b-aminolevulinic acid to the Krebs cycle on the one hand and to porphyrin biosynthesis on the other. Also, several laboratories have reportedi~'~3 the isolation of an enzyme which catalyzes the condensation of two molecules of b-aminolevulinic acid to form one of porphobilinogen COOH COOH ,.
From page 75...
... In the initial studies of individual enzymes in this phase of porphyrin biosynthesis,S~9 aqueous extracts of acetone powders of spinach leaf tissue were subjected to ammonium sulfate fractionation. The course of PBG consumption and the appearance of uroporphyrin in the presence of one fraction are shown in figure 3.
From page 76...
... The course of the consumption of PBG and the appearance of uroporphyrin I The reaction shown here was catalyzed by PBG deaminase in a 40-50C/c ammonium sulfate fraction of an aqueous extract of spinach leaf tissue.
From page 77...
... Another enzyme, uroporphyrinogen isomerase, which participates in the synthesis of uroporphyrinogen III from PBG, has been partially purified from aqueous extracts of wheat germ. Crude aqueous extracts catalyze the consumption of PBG and the appearance of uroporphyrin III, sometimes mixed with uroporphyrin I, but two ammonium sulfate fractions are of particular interest.
From page 78...
... Preparations of this fraction vary in their capacity to catalyze the consumption of PBG when incubated alone w-ith this pyrrole; some preparations are slightly active, while others possess no measurable activity. If, however, a preparation which is inactive by these standards is incubated with PBG deaminase prepared from spinach leaf tissue, as well as w-ith PBG, the substrate is consumed at a rate commensurate with the concentration of the deaminase but the III, rather than the I isomer of uroporphyrin, is produced.
From page 79...
... So the possibility of the shifting of acetic and propionic acid side chains as a mechanism of isomerization appears to be as unlikely as would have been predicted. In another series of e~cperimer~ts it was found that the exposure of PBG to the isomerase first and then to PBG deaminase is an equally ineffective means of producing uroporphyrin III.
From page 80...
... The requirement for the presence, simultaneously, of PBG deaminase, uroporphyrinogen isomerase, and PBG for the enzymatic synthesis of uroporphyrinogen III is apparent. The next question which arises is: Is there any direct interaction between PBG and uroporphyrinogen isomerase ?
From page 81...
... The second, uroporphyrinogen isomerase, appears to be unable to catalyze any modification of PBG when incubated with it alone, but, when this enzyme is incubated with PBG and PBG deaminase, uroporphyrinogen III is produced. These enzymatic products can serve as substrates for enzymes present in frozen and thawed preparations of Chlorella which catalyze the synthesis of porphyrins with fewer than eight carboxyl groups per molecule.
From page 82...
... One of the condensations required is of the sort which PBG deaminase appears to catalyze; the other condensation might possibly also be catalyzable by PBG deaminase, but another enzyme might be required. If this is the mechanism of uroporphyrinogen III biosynthesis, uroporphyrinogen isomerase acts as a transpyrrylase.
From page 83...
... 11. Bogorad, L.: The enzymatic synthesis of uroporphyrin III, Pi.
From page 84...
... To study this first group of enzymes we have followed protoporphyrin synthesis in chicken red cells with inhibitors in order to map out what possible enzyme systems might be involved. We were able to assign the action of these inhibitors to the enzymes that acted between glycine and SAL, or to the enzymes that acted between bAL and PROTO, by making tests with glycine or with bAL as substrates.
From page 85...
... This observation confirms the findings of Schulman and Richer that pyridoxal phosphate is necessary for bAL synthesis. We have also found that pyridoxal phosphate is required for SAL synthesis in homogenates of red cells.
From page 86...
... When chicken red cells are incubated with glycine and arty one of the following amino acids in equimolar concentration: serine, alanine, proline or arginine, the PROTO synthesis is decreased by 30 - 50 per cent; still greater inhibition is found in the presence of cysteine. It is probable that the inhibition is not by way of competition for pyridoxal phosphate since aspartate and glutamate, among others, did not inhibit.
From page 87...
... long. A supernatant solution of a chicken red cell hemolysate was applied as a streak across the width of the block at "Origin." The activities of eluates from 2 cm.
From page 88...
... The relative rates of migration toward the anode of the rabbit enzymes was bAL-ase > UD-ase > PBG-ase; of the chicken enzymes bAL-ase ~ PBGase > UD-ase; and of the human, UD-ase > PBG-ase > DAL-ase.
From page 89...
... . When a specific fraction prepared by zone electrophoresis acts on URO-gen then COPRO-gen is formed.~3 The enzyme fraction is incubated anaerobically in the presence of 0.005M GSH and URO-gen III (prepared from URO by reduction with sodium amalgam)
From page 90...
... The fact that bAL synthesis from glycine requires mitochondrial enzymes and that cell Articulates are involved in the conversion of COPRO-gen to PROTO suggests that the synthesis of PROTO might be a function of such organelles. In this regard it would be interesting to know whether the soluble enzymes, which convert bAL to COPRO-gen, are also concentrated in these bodies.
From page 91...
... 1~. The conversion of bAL to COPRO-gen has been shown to take place via three soluble enzyme fractions which have been separated from red cells by zone electrophoresis (fig.
From page 92...
... was used as a measure of heme synthesis. Our studies have suggested that this reaction is enzyme dependent.
From page 93...
... The rate of heme synthesis at 30 minutes was proportional to enzyme concentration. The preparation was not storable at 5° C., - 3(~° C., or lyophilized and was found to be inactivated after heating at 56° C
From page 94...
... C., Melnick, I., and Klein, J R.: Formation of heme by broken-cell preparations of duck erythrocytes, Arch.
From page 95...
... Such cells have been referred to as sideroblasts when nucleated, and siderocytes when non-nucleated.S 9 These iron stores may be useful to insure adequate available iron during the phase of maximal heme synthesis within the cell. There is evidence that their amount may be influenced by the level of plasma iron, and that they may in turn influence the amount of residual non-heme iron found in the adult circulating erythrocyte.6 In avian and mammalian red cells this fraction may be as much asSor10~.6 it The red cell nucleus would seem to be intimately concerned with hemoglobin synthesis.
From page 96...
... This raises the possibility that iron may be of importance in multiplication of erythroid cells as well as in heme synthesis. In the intact animal a consideration of the role of iron includes a consideration of iron supply to the marrow.
From page 97...
... The plasma iron turnover, i.e., the amount of plasma iron being fed to tissues, has been shown largely to reflect marrow uptake.~7 The amount of iron appearing in circulating erythrocytes indicates how much of this iron was synthesized into hemoglobin within viable erythrocytes. The marrow transit time of radioiron indicates the time required for the process of iron incorporation and red cell maturation.iS Studies of this type indicate that in certain anemias—such as thalassemia, pernicious anemia and some "refractory" anemias with cellular marrow— the iron uptake by the marrow far exceeds the production of viable red cells.~'3 Further study is required to determine to what extent this "ineffective" erythropoiesis reflects a defect in fabrication of the cell as compared to abnormality in heme synthesis.
From page 98...
... Ire recent studies carried out in collaboration with Dr. Phillip Sturgeon, it has been found that iron turnover, presumably reflecting for the most part heme synthesis, reached maximal levels in thalassemia despite the marked hypochromia of this diseased The relation of microcytosis without hypochromia to heme synthesis is even less clear.
From page 99...
... A.: Erythrokinetics IV. Plasma iron turnover as a measure of erythropoiesis.
From page 100...
... The type of anemia which develops is microc~rtic and hypochromic, as indicated by a substantial decrease in the mean corpuscular volume (M.C.V.) and in the mean corpuscular hemoglobin concentration (M.C.fI.C.)
From page 101...
... M.C.~., mean corpuscular volume; M.C.H.C., mean corpuscular hemoglobin concentration; V.P.R.C., volume of packed red cells; R.B.C., red blood cells; Hb, hemoglobin; W.B.C., total leukocytes; P.M.~., polymorphonuclear leukocytes. (Lahey et al., Blood 7: 1053, 1952.
From page 102...
... PFe, plasma iron; PCu, plasma copper; M.C.V., mean corpuscular volume; V.P.R.C., volume of packed red cells; W.B.C., total leukocyte count; P.M.N., polymorphonuclear leukocytes. (Lahey et al., Blood 7: 1053, 1952.
From page 103...
... refers to volume of packed red cells M.C.~. refers to mean corpuscular volume M.C.X.C.
From page 104...
... In general, the hemoglobin level increases more slowly than does the V.P.R.C., with the result that the microcytosis disappears before the mean corpuscular hemoglobin concentration returns to normal. The plasma copper level increases significantly within 24 hours and reaches the normal level in about five days.
From page 105...
... ~g/lOO ml iron in the form of colloidal saccharated oxide of iron were administered intra1 efers to volume of slacked red cells. refers to mean corpuscular volume.
From page 106...
... ~ Time required for one-half of the isotope to disappear from the plasma. The plasma iron turnover rate in copper-deficient pigs is even greater than in normal pigs and the amount of iron turned over through red cells per day was about twice as great in the deficient pigs as in the normal control animals.
From page 107...
... Furthermore, both ferrokinetic studies and chromium erythrocyte survival studies indicate that the life-span of the erythrocyte in copper deficiency is shortened. It seems, therefore, that anemia develops in the absence of copper because of a limitation of the capacity of the marrow to produce cells and because of a shortened erythrocyte survival time.
From page 108...
... 3. Showing the development of anemia (V.P.R.C., volume of packed red cells)
From page 109...
... That the activity of all heme chromoproteins is not uniformly depressed suggests that not all of the metabolic pathways of iron in the body are impaired. Furthermore, our recent observation that the daily turnover rate of iron through the plasma and into the red cells is increased rather than decreased in copper-deficient swine suggests that movement of iron within the body is not curtailed.
From page 110...
... XVIII. Skeletal changes associated with copper deficiency in swine, 97: 405, 1955.
From page 111...
... Reticulocytosis was produced in adult rabbits by a modification of the method of London et al.4 One milliliter of a neutralized 2.5 per cent aqueous solution of phenylLydrazine hydrochloride divas injected subcutaneously each day for a week; over 90 per cent of the circulating red cells were then reticulocytes. Labeled amino acids.
From page 112...
... of 35 per cent trichloroacetic acid. If the reaction mixture contained plasma or some other proteins the cells were first separated by centrifugation and washed twice with saline, before the trichloroacetic acid precipitation.
From page 113...
... It is seen that iron and glucose, separately or together, had little accelerating effect. The amino acid mixture alone caused an increase of 70 per cent; amino acids and iron, but not amino acids and glucose, acted synergistically; glucose acted synergistically when added with amino acids and iron.
From page 114...
... Amino acids vary in the degree to which they are limiting; histidine is the most limiting. In a reaction mixture from which one of the limiting amino acids is withheld, the rate is at a characteristic suboptimal level from the beginning and persists so.
From page 115...
... ,, ,, ,, 1 447 94 185 176 215 235 257 262 304 2 3 450 489 87 94 170 181 183 181 227 220 229 237 267 264 280 295 339 353 4 475 96 187 200 228 222 285 297 366 TABLE II. The complete reaction mixture contained the amino acid mixture described in the text, 5 hum.
From page 116...
... avian cells, the amino acid mixture required to be added to the reaction mixture may be different from that necessary for rabbit reticulocytes. The literature on the relation between amino acids and blood formation TABLE IV EFFECT OF PARTIAL PHENYLALANINE DEFICIENCY.
From page 117...
... The list agrees surprisingly well with the amino acids accelerating incorporation of labeled amino acids into rabbit reticulocyte proteins. Nizet and Robscheit-Robbins3S found that dog reticulocytes did riot mature so quickly in vitro in the blood of dogs with hemorrhagic anemia and hypoproteinemia as in normal blood, unless a mixture of the ten essential amino acids and glycine was added to the anemic blood.
From page 118...
... Correspondingly a single amino acid, whether injected or fed, will be extensively incorporated because protein synthesis never stops in Volvo. In this sense the results in tables II - ~ and XIII are analogues of experiments in which animals are maintained on suboptimal amounts of indispensable amino acids (or incomplete proteins)
From page 119...
... It is seen that during the first hour the addition of a very small amount of iron, between 0.017 and 0.044 Insoles, had a pearl', maximal effect; whereas over a four-hour period between 0.089 and 0.179 Moles were required for a maximum increase in hemoglobin iron of only 0.022 ~moles. The sensitivity of rabbit reticulocytes in vitro to such low concentrations of iron raises the question whether or not iron is necessary for hemoglobin synthesis in addition to its participation in the structure of heme.
From page 121...
... RESULTS EXPRESSED AS PER CENT OF VALUE IN- OTHERWISE COMPLETE REACTION MIXTURE. Protein added None Plasma ,, i, ,, ,' Transferrin ,' ,' ,, ,, Rabbitt serum albumin Bovine " " Human ~~ i, .
From page 122...
... It is surmised that the accelerating effects of fructoseamino acids and citrate are for the same reason, table XII. The maximum accelerating effect is obtained with about 10-6 M transferrin and 5 x 10-4 NI fructose- or tagatose-amino acids or citrate.
From page 123...
... Table XIII shows the accelerating effect of glucose added to the reaction mixture. It is seen that the effect was little in the first hour of incubation and became progressively greater, presumably as the carbohydrate initially in the reticulocytes was consumed.
From page 124...
... protein 0 0 18.4 2.8 x 10 - 3 0 11.6 0 0.25 x 10 - :, 19.4 0 1.0 " 19.1 2.8 x 10 - 3 0.25 " 17.3 2.8 " 1.0 " 19.7 TABLE XVI. The basic saline solution was as in Table XIII.
From page 125...
... RESULTS EXPRESSED AS PER CENT INHIBITION. Concentration: molal lo - 3 Ammonium Alum Potassium Alum Antimony Potassium Tartrate Cupric Chloride Gold Lead Acetate Mercuric Acetate 17 10 98 16 96 98 100 2 4 93 o 84 96 92 10—~ 1 10—~ 1 10 - '3 1o 4 5 o 2 79 5 o o o o o 18 o TABLE XIX EFFECTS OF SQME AMINO ACID, PURINE AND PYRIMIDINE ANALOGS AND ANTIBrOTICS.
From page 126...
... This was observed with rates of hemoglobin synthesis which were made to vary widely by variatiorls in the iron fructose-amino acids in the reaction mixture. This result would not have occurred if there had been a pool of significant size of unlabeled intermediates of either heme or of globin, or of free heme or globinO The rates of synthesis of the two parts of hemoglobin under our experimental conditions, i.e.
From page 127...
... H.: Incorporation in vitro of labeled amino acids into proteins of rabbit reticulocytes, J
From page 128...
... 19. Matsuoka, A., and Nakao, T.: Uber die Wirkung des Methyltryptophans auf kunstliche Anamie und Ernahrung, Z
From page 129...
... R., and Greenberg, D M.: Effect of amino acid deficiencies on incorporation of radioactive carbon-labeled amino acids into animal tissue proteins, Proc.
From page 130...
... H.: Pructose-amino acids in liver: Stimuli of amino acid incorporation in rvilro, J
From page 131...
... The biosynthesis of heme has been extensively studied ire intact human, rabbit and avian erythrocytes,~ '' and in non-intact preparations of these cells.3~5 Although evidence for the formation of peptide bonds in vitro in the hemoglobin of duck erythrocytes was obtained several years ago with the use of N75-labeled histidine,0 relatively little attention has been given to the usefulness of this system for the study of the formation of protein. Chicken erythrocytes and reticulocytes have been employed for the study of the rates of formation of heme and of hemoglobin.0 The incorporation in vitro or various isotopically-labeled amino acids into tile total protein of reticulocytes obtained from phenylLydrazine-treated rabbits leas been studied; and more recently these observations have been extended to the incorporation of glycine into globin and its utilization for the synthesis of heme in these cells.S As a system for the study of protein synthesis, or for the incorporation of an amino acid into a protein, the immature mammalian or avian erythrocyte affords the advantages of simplicity and ready availability.
From page 132...
... The precipitated material was dissolved in distilled water, precipitated with 14 To trichloroacetic acid, washed twice with 7 ~ trichloroacetic acid and once with distilled water. The globin was then dissolved in 2 ml of 1N NaOH and the precipitation and washing procedures were repeated.
From page 133...
... In preliminary experiments incubation in plasm resulted in a higher rate of incorporation of glycine into globin than incubat~on in 0.9C/o sodium chloride solution, isotonic sodium phosphate buffer pH 7.4 or Krebs-Ringer phosphate buffer plI 7.4. However, in order to eliminate unknown variable factors which might be present in plasma, isotonic phosphate buffer was chosen as the medium.
From page 134...
... . The differential effect of hypotonicity is consistent with the findings that heme synthesis proceeds in non-intact avian erythrocytes3-, and that the incorporation of amino acids into the protein of rabbit reticulocytes ceases if the cells are lysed.7 Influence of Amino Acids and Glucose.
From page 135...
... Since the addition of iron did not increase the incorporation of glycine into globin of duck erythrocytes, whereas Kruh and Borsook had reported that iron increased the formation of globin in rabbit reticulocytes,S experiments TABLE IV THE EFFECTS OF LEAD, COBALT AND IRON ONT SYNTHESIS OF HEME AND INC~RPCRATION OF GLYCINE INTTO GLOBIN Specific activity of gIycine (c.p.m./mM) Metal Added in Robin per cent of control in heme per cent of control "Heme/Globin Ratio" Control Pb+~FeCI2 CoCl 15,200 10,800 15,000 1 5,700 71 100 103 50,500 11,600 96,000 2,030 190 4 3.3 1.3 6.4 0.1 Ferrous chloride, lead acetate and cobaltous chloride were added in concentrations of 5 :; 10~ M
From page 136...
... The Endings in rabbit reticulocvres differed from those in normal duck erythrocytes not only in terms of the effects of iron on glycine incorporation into globin, but also in the "heme-globin ratios." "Heme-globin ratios" of 1.1 and 1.2 in rabbit reticulocytes are similar to those previously described.S In the normal duck erythrocytes, however, the "heme-globin ratios" are generally much higher and more variable. To determine whether the "hemeglobin ratio" might be closer to unity and whether iron might enhance glycine incorporation into globin in more immature duck erythrocytes, the cells of acetylphenylhydrazine-treated ducks were employed.
From page 137...
... TABLE VII EFFECTS OF PURINE RIBOSIDES AND RELATED COMPOUNDS ON HEME SYNTHESIS AND ON GLYCINE INCORPORATION INTO GLOBIN IN DUCK ERYTHROCYTES AND IN IMMATURE ERYTHRCCYTES OF RABBITS Expt.
From page 138...
... The first process has been studied in duck erythrocytes by Christensen, Riggs and Ray who showed that normal duel: erythrocytes take up amino acids from a plasma or saline medium against a concentration gradient.") This concentrative activity is considerably less than that which has been observed for guinea pig brain,~'' rat diaphragm'':; or rabbit reticulocytes.~4 Coupled with the lesser concentrative activity of duck erythrocytes for amino acids is a relative insensitivity of the concentrative process to anoxia and to agents such as cyanide, 2,4 dinitrophenol and other metabolic inhilaitors in high concentrations.
From page 139...
... H.: Incorporation in Vitro of labeled amino acids into proteins of rabbit reticulocytes, J
From page 140...
... N., and Streicher, J A.: Concentration of amino acids by the excised diaphragm suspended in artificial media.
From page 141...
... In the demonstration that the two processes are separable, one may overlook what to me, at any rate, is a remarkable fact that, at least in rabbit reticulocytes, the two go so closely together. There is evidently some kind of interaction.
From page 142...
... Table I presents the results of an experiment which divas performed on a ing periods of time. The data indicate that after one half hour of incubation, the specific activity in the hemin derived from the stroma is much higher than that derived from the hemoglobin solution, the ratio of the specific aciarge sample of duck erythrocytes which were pooled and incubated for varytivities in these two fractions being close to 6.


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