William S. Beck

LEUKOCYTE GLYCOLYSIS: AN INVESTIGATION OF THE FACTORS CONTROLLING THE RATE BEHAVIOR IN MULTIENZYME SYSTEMS*

The fact that the investigation of the metabolism and enzymology of human leukocytes has flourished in recent years is reflected in the several conferences and seminar series that have been held on this subject since 1952. The published proceedings of some of these meeting~l-~ give ample evidence of the increasing vigor and promise of this field of inquiry. It appears that interest in this field is increasing and that a scattering of pioneering investigators has begun to study the biochemistry of those cells that occasionally contaminate our leukocyte preparations: the erythrocytes and the platelets. Despite the recent surge of interest in the leukocyte, the fact is that leukocyte metabolism has been under investigation for well over fifty years. The early classic demonstration that lactic acid was derived from glucose and not from protein metabolism was made on leukocyte-rich exudate materiaL43 In the intervening years a great many observations were made on the glycolysis, proteolytic activity, histochemistry, chemical composition, and miscellaneous enzymology of leukocyte preparations of diverse origin and, although many of these investigations suffered from inadequacies of the isolation methods and other techniques, it became increasingly clear that the blood leukocyte is an exceedingly interesting cell whose remarkable chemistry, biology, and pathology make it a tantalizing object for investigation. With the possible exception of mouse ascites tumor cells, leukocytes are probably the only free-floating nucleated cells that can be isolated easily from mammalian sources. They can be accurately enumerated and are perhaps ideally suited for study under simulated iiz vivo conditions. Unlike ascites tumor cells, malignant leukocytes have clearly defined normal analogues that may serve as useful controls for metabolic investigation. Also ascites tumor cell suspensions are often contaminated with leukocytes, but suspensions of blood leukocytes are almost never contaminated with mouse ascites tumor cells. Finally, leukocytes have a number of fundamental biological properties, such as phagocytosis and motility, with which one may seek to correlate observed biochemical properties, a fact that greatly increases their usefulness and interest. * The work reported in this paper was performed under Contract No. AT(04-1)-GEN-12 between the Atomic Energy Commission, Washington, D. C., and the University of California a t Los Angeles, Lus Angeles, Calif., and was supported in part by grants Iron) the National Cancer Institute, Public Health Service, Bethesda, hld., the American Cancer Society, Inc., and theLeukemia Society, Inc., New York, N. Y. Established Investigator of the American Heart Association.

The metabolic functions of vitamin B12: I. Distinctive modes of unbalanced growth behavior in Lactobacillus leichmannii

Abstract The rod-shaped organism Lactobacillus leichmannii has growth requirements for vitamin B12 or a deoxyriboside, free purines (xanthine and guanine) and a pyrimidine. Varying the concentrations of these nutrients in the medium produced the following distinctive growth patterns which investigation revealed to be different forms of unbalanced growth behavior: (1) vitamin B12 or deoxyriboside starvation produced long non-viable filaments with decreased DNA/RNA and DNA/protein ration; (2) gross excesses of vitamin B12 or deoxyriboside apparently accelerated DNA synthesis producing unusually small viable forms with increased DNA/RNA and DNA/protein ratios; (3) purine starvation in cultures containing adequate concentrations of vitamin B12 or deoxyriboside increased DNA/RNA ratios and in the case of thymidine-grown organisms produced filamentous forms; and (4) replacement of free purines and pyrimidines with the corresponding ribosides in vitamin B12 cultures produced unusually small forms with normal DNA/RNA ratios and elevated DNA/protein and RNA/protein ratios, whereas riboside substitution had no such effect in deoxyriboside cultures. The results support the view that vitamin B12 participates in the conversion of ribosyl to deoxyribosyl groups. The data also provides evidence on the physiological role of trans-N-deoxyribosidase in L. leichmannii.

Peroxidase activity of hemoglobin and its subunits: Effects thereupon of haptoglobin

Abstract Studies of the peroxidase activity of human hemoglobin A and its separated α and β subunits have shown that native hemoglobin possesses greater peroxidase activity than isolated subunits containing an equivalent amount of heme. Upon recombination of unlike subunits, peroxidase activity approaches that of an equivalent quantity of native hemoglobin. Similar results were obtained when hemoglobin H ( β 4 A ) replaced β subunits prepared from hemoglobin A. It is concluded that the peroxidase activity of the heme moiety, like its binding behavior with gaseous ligands, is affected by interactions between unlike polypeptide chains. Experiments are presented which suggest that haptoglobin enhances the peroxidase activity of the heme moiety through conformational effects on the globin moiety.

Transfer of hydrogen from cobamide coenzyme to water during enzymatic ribonucleotide reduction

Abstract A purified preparation of combamide-dependent ribonucleotide reductase from Lactobacillus leichmannii catalyzes an exchange of hydrogen between water and cobamide coenzyme in the course of CTP reduction. The exchange is dependent upon the reductase reaction.

Transfer of hydrogen in the cobamide-dependent ribonucleotide reductase reaction

Abstract A purified preparation of cobamide-dependent ribonucleotide reductase from Lactobacillus leichmannii catalyzes a transfer of hydrogen from H2O to dCTP in the course of CTP reduction. Degradation studies indicate that the incorporated hydrogen is associated exclusively with C-2′ of the deoxyribosyl moiety.

The metabolic functions of vitamin B12. III. Vitamin B12 binding in Lactobacillus leichmannii and other lactobacilli.

Abstract Lactobacillus leichamannii rapidly removed [60Co]vitamin B12 from the medium. The amounts taken up exceeded the quantities needed to satisfy nutritional requirements but bound vitamin was metabolically available if organisms were subsequently placed in medium lacking or deficient in vitamin B12. Binding was affected by the concentration of vitamin B12 in the medium and maximal binding occurred at a concentration of approx. 10 mμg/ml (7.4·10−9 M). Under these conditions, the vitamin has been concentrated about 1000 times within the cells. The binding process seemed independent of the growth phase and was little affected by temperature changes between 4 and 40° or by a number of metabolic inhibitors. The amount of [60Co]vitamin B12 bound was decrease by additions of unlabeled vitamin B12 or the ethylamide, methylamide, anilide and monocarboxylic acid derivatives of vitamin B12. Dimethylbenzimidazole was without effect. Cell-free extracts of L. leichmannii effectively bound [60Co]vitamin B12 and preliminary evidence indicates that a major adsorptive site is a ribosomal glycoprotein. L. acidophilus (ATCC 4357), L. casei and L. lactis also bind [60Co]vitamin B12. Two species of lactobacilli that failed to bind the vitamin, L. delbrueckii and L. acidophilus (ATCC 11506), were also incapable of utilizing vitamin B12 as a growth factor. Of these, only L. delbrueckii demonstrated binding in cell-free extracts.

Deoxyribonucleotide Synthesis and the Role of Vitamin B12 in Erythropoiesis

Publisher Summary This chapter reviews the current knowledge of the several pathways of deoxyribonucleotide synthesis in bacteria and animal cells other than bone marrow and discusses the available evidence bearing on the pathway in human bone marrow. Current knowledge of the pathways of deoxyribonucleotide synthesis has been derived mainly from studies in two strains of bacteria— Escherichia coli , species lacking a nutritional requirement for vitamin B 12 and Lactobacillus leichmannii , a nutritional vitamin B 12 auxotroph. The system from E. coli utilizes ribonucleoside diphosphates as substrates; and L. leichmannii reduces ribonucleoside triphosphates. The more striking difference between the two enzyme systems is the fact that the enzyme from L. leichmannii , but not that from E. coli , has an absolute requirement for the vitamin B 12 coenzyme, 5,6-dimethylbenzimidazolyl 5′-deoxyadenosine cobamide (DBCC). The ribonucleotide reductases from Novikoff hepatoma cells and E. coli have some properties in common that distinguish them from the corresponding enzyme of L. leichmannii , including (1) the preferred substrate is a ribonucleoside diphosphate, (2) no requirement for a cobamide coenzyme has been demonstrated, (3) an absolute requirement for Mg ++ , and (4) similar patterns of allosteric effects, among which is an inhibitory effect of deoxyadenosine triphosphate on the reductions of all four ribonucleotides.

The metabolic functions of vitamin B12: II. Participation of vitamin B12 in the biosynthesis of deoxyribonucleic acid and its acid-soluble precursors

Abstract Addition of excess vitamin B12 to previously limited cultures of Lactobacillus leichmannii increased the concentration of DNA, the ratios DNA/RNA and DNA/ protein, and the concentration per milligram of protein of acid-soluble deoxyribosides dexoyribonucleotides and unidentified compounds containing deoxyribosyl moieties in combined form. Time-course studies of the appearances of the acid-soluble deoxyriboysl compounds favor the hypothesis that they are DNA precursors and that vitamin B12 functions primarily in their biosynthesis. The results also provide additional information on the unbalanced growth state resulting from vitamin B12 or deoxyriboside deprivation. If nutritional repletion takes place after more than 2 h of unbalanced growth in deficient media, filamentous growth is not fully reversed despite extensive replacement of DNA.

Variations of intracellular deoxyribosyl compounds in deficiencies of vitamin B12, folic acid and thymine

Abstract Intracellular deoxyribosyl compounds were measured collectively and individually in vitamin B12-deficient Lactobacillus leichmannii, wherein DNA synthesis is impaired by defective deoxyribonucleotide synthesis, and in folate-deficient L. leichmannii and L. casei and thymine-deficient Escherichia coli 15 T−, wherein DNA synthesis is impaired by lack of thymidylate. Effects of the two types of deficiencies differed strikingly. Pool sizes were decreased by vitamin B12 deficiency and increased by deficiencies of folic acid or thymine. All major deoxyribosyl compounds were uniformly decreased in vitamin B12 deficiency, levels rising again after repletion. A number of deoxyribosyl compounds increased in folate and thymine deficiency, although responses of individual compounds differed in folate-limited L. leichmannii and thymine-limited E. coli 15 T−. Preliminary attempts to demonstrate accumulations of deoxyribosyl derivatives in folate-deficient humans and chicks were unsuccessful. A dTDP derivative, tentatively identified as a sugar complex, is the major intracellular deoxyribosyl compound in optimally nourished L. leichmannii. In folate deficiency, other intracellular thymine compounds disappear, but the dTDP derivative accumulates. A similar phenomenon was not observed in thymine-limited E. coli 15 T−. The results suggest that (1) vitamin B12 participates in deoxyribosyl synthesis; (2) folate deficiency in the organisms studied is the equivalent of thymine deficiency; (3) a thymine derivative, probably dTTP, governs the repressor system controlling ribonucleotide reductase synthesis; and (4) the level of a major thymine derivative is controlled by factors other than those controlling the levels of the immediate DNA precursors.

Studies of the ribosome-associated vitamin B12s adenosylating enzyme of Lactobacillus leichmannii☆

Abstract The ribosomes of Lactobacillus leichmannii (ATCC 7830) are the loci of an enzyme system that converts vitamin B 12 (cyanocobalamin, CN-Cbl) to adenosylcobalamin (AdoCbl) in two steps, reduction of B 12a (Co III) to B 12s (Co I) by B 12a reductase and the adenosylation of B 12s to AdoCbl by an adenosylating enzyme. The vitamin B 12 reductase, as in other organisms, is unstable. Adenosylating enzyme, however, is readily demonstrable. Reported experiments deal primarily with that enzyme. Evidence of the association between ribosomes and adenosylating enzyme was found in sucrose density gradient analyses. Intact, washed ribosomes yielded an enzyme activity profile that coincided with the ultraviolet maximum of 70S reference ribosomes. When intact ribosomes were exposed to 2.5 m CsCl so that 70% of ribosomal protein was recoverable in the 144,000 g supernatant fraction and >90% of RNA was in the pellet, adenosylating enzyme was found in the supernatant fraction. “Stripped” ribosomes had low levels of enzyme activity and could reassociate with free enzyme protein. Stripped ribosomes remained competent in protein synthesis. Hence, adenosylating enzyme is not an integral ribosome component. Partially purified ribosome-associated B 12s adenosylating enzyme has requirements for vitamin B 12s , ATP, and Mn 2+ , though Mn 2+ could be partially replaced by Mg 2+ . Isotopic studies showed that ATP is the source of the adenosyl moiety of AdoCbl and that inorganic tripolyphosphate is a reaction product. Substantial adenosylating activity is associated with ribosomes only in the vitamin B 12 -requiring lactobacilli, L. delbrueckii (ATCC 9649) and L. leichmannii . Surprisingly, L. casei (ATCC 9595) ribosomes displayed a measurable, if low, level of activity. L. acidophilus (ATCC 11506) ribosomes had no detectable activity. The bulk of the activity in Clostridium tetanomorphum (ATCC 3606) and Propionibacterium shermannii (ATCC 9614) is in the 144,000 g supernatant fraction. Ribosomes from animal cells (liver, reticulocytes, and Ehrlich ascites tumor) were without detectable activity.