Endogenous biotinylated proteins in Dictyostelium discoideum.

Biotin-dependent enzymes are involved in carboxylation, decarboxylation and transcarboxylation reactions. Here, we have used sodium dodecyl sulfate polyacrylamide gel electrophoresis and electroblotting followed by probing with avidin to identify biotin-containing polypeptides in Dictyostelium discoideum. Twenty biotinyl polypeptides were visualized, with a 23 kDa protein appearing transiently. Based upon the molecular mobility of the biotinyl polypeptides, D. discoideum may contain the biotin-dependent enzymes acetyl CoA carboxylase, proprionyl CoA carboxylase, pyruvate carboxylase, and 3-methylcrotonyl CoA carboxylase.     

Acetyl CoA carboxylase in cultured fibroblasts: differential biotin dependence in the two types of biotin-responsive multiple carboxylase deficiency

In biotin-responsive multiple carboxylase deficiency, a characteristic organic aciduria reflects in vivo deficiency of mitochondrial propionyl CoA carboxylase, 3-methylcrotonyl CoA carboxylase, and pyruvate carboxylase. A possible primary or secondary defect in biotin absorption leads to an infantile-onset syndrome, while abnormal holocarboxylase synthetase activity has been identified in the neonatal-onset form. While distinct mitochondrial and cytosolic holocarboxylase synthetase biotinylation systems may exist in avian tissues, the system has not been characterized in humans. Toward this objective, we studied the biotin dependence of a cytosolic carboxylase, acetyl CoA carboxylase (ACC), in cultured skin fibroblasts of both types of multiple carboxylase deficiency. ACC specific activities in control and infantile-onset cells were not distinguishable at all biotin concentrations: with decreasing biotin availability (+ avidin), there were only modest decrements in ACC activity in both these cell types. In contrast, there were pronounced declines of ACC activity in neonatal-onset (holocarboxylase synthetase-deficient) cells after growth in low biotin concentrations, and activity was undetectable in + avidin. ACC activity was rapidly restored with biotin repletion to biotin-starved holocarboxylase synthetase-deficient cells, and this restoration was largely independent of protein synthesis. The behavior of the cytosolic carboxylase, ACC, is in all these respects identical to that of the mitochondrial carboxylases, an observation consistent with the existence of similar biotinylation mechanisms in the two cell compartments. Further, the data support the notion that at least some components of the holocarboxylase synthetase system are shared by mitochondria and cytosol in humans, and are consistent with the suggestion that restoration of activity in biotin-depleted cells represents biotinylation of preexisting enzyme protein. The modest decrements in ACC activity in normal and infantile-onset cells may be related to the compromised epidermal integrity observed in that form of multiple carboxylase deficiency. Finally, ACC and mitochondrial carboxylase activities were compared in cells from mutants representing a spectrum of clinical severity. Cells from later-onset patients of intermediate clinical severity were ultimately classifiable as putative holocarboxylase synthetase-deficient cells on chemical criteria. Accurate etiologic classification cannot be based on clinical presentation alone, and biochemical studies should be performed on all patients. Accordingly, we propose a classification of multiple carboxylase deficiency based on biochemical criteria.     

Distribution and degradation of biotin-containing carboxylases in human cell lines.

Incubation of cultured cells with [3H]biotin leads to the labelling of acetyl-CoA carboxylase, pyruvate carboxylase, propionyl-CoA carboxylase and methylcrotonyl-CoA carboxylase. The biotin-containing subunits of the last two enzymes from rat cell lines are not separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, but adequate separation is achieved with the enzymes from human cells. Since incorporated biotin is only released upon complete protein breakdown, the loss of radioactivity from gel slices coinciding with fluorograph bands was used to quantify degradation rates for each protein. In HE(39)L diploid human fibroblasts, the degradation rate constants are 0.55, 0.40, 0.31 and 0.19 day-1 for acetyl-CoA carboxylase, pyruvate carboxylase, methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase respectively. A similar series of rate constants is found for AG2804 transformed fibroblasts. The degradation rate constants are decreased by 31-67% in the presence of 50 micrograms of leupeptin/ml plus 5 mM-NH4Cl. Although the largest percentage effect was noted with the most stable enzyme, propionyl-CoA carboxylase, the absolute change in rate constant produced by the lysosomotropic inhibitors was similar for the three mitochondrial carboxylases, but greater for the cytosolic enzyme acetyl-CoA carboxylase. The heterogeneity in degradation rate constants for the mitochondrial carboxylases indicates that only part of their catabolism can occur via the autophagy-mediated unit destruction of mitochondria. Calculations showed that the autophagy-linked process had degradation rate constants of 0.084 and 0.102 day-1 respectively in HE(39)L and AG2804 cells. It accounted for two-thirds of the catabolic rate of propionyl-CoA carboxylase and a lesser proportion for the other enzymes.     

Regulation of the structure and activity of pyruvate carboxylase by acetyl CoA

In this review we examine the effects of the allosteric activator, acetyl CoA on both the structure and catalytic activities of pyruvate carboxylase. We describe how the binding of acetyl CoA produces gross changes to the quaternary and tertiary structures of the enzyme that are visible in the electron microscope. These changes serve to stabilize the tetrameric structure of the enzyme. The main locus of activation of the enzyme by acetyl CoA is the biotin carboxylation domain of the enzyme where ATP-cleavage and carboxylation of the biotin prosthetic group occur. As well as enhancing reaction rates, acetyl CoA also enhances the binding of some substrates, especially HCO3-, and there is also a complex interaction with the binding of the cofactor Mg2. The activation of pyruvate carboxylase by acetyl CoA is generally a cooperative processes, although there is a large degree of variability in the degree of cooperativity exhibited by the enzyme from different organisms. The X-ray crystallographic holoenzyme structures of pyruvate carboxylases from Rhizobium etli and Staphylococcus aureus have shown the allosteric acetyl CoA binding domain to be located at the interfaces of the biotin carboxylation and carboxyl transfer and the carboxyl transfer and biotin carboxyl carrier protein domains.     

Different types of mitochondria in parenchymal and non-parenchymal rat-liver cells.

1. Intact and pure parenchymal and non-parenchymal cells were isolated from rat liver. The specific activities of several mitochondrial enzymes were determined in both parenchymal and non-parenchymal cell homogenates to characterize the mitochondria in these liver cell types. 2.In general the activities of mitochondrial enzymes were lower in non-parenchymal liver cells than in parenchymal cells. The specific activity of pyruvate carboxylase in non-parenchymal cells expressed as the percentage of that in parenchymal cells was onlu 2% for glutamate dehydrogenase 4.3% and for cytochrome c oxidase 79.4%. Monoamine oxidase, as an exception, has an equal specific activity in both cell types. 3. The activity ratio of pyruvate carboxylase at 10 mM pyruvate over 0.1 mM pyruvate is 3.35 for parenchymal cells and 1.50 for non-parenchymal cells. This indicates that non-parenchymal liver cells only contain the high affinity form of pyruvate carboxylase in contrast to parenchymal cells. 4. The ratio of glycerol-3-phosphate cytochrome c reductase over succinate cytochrome c reductase activity differs from parenchymal (0.01) and non-parenchymal cells (0.10). This might indicate that the glycerol-3-phosphate shuttle, which is important for the transport of reduction equivalents for cytosol to mitochondria is relatively more active in non-parenchymal cells than in parenchymal cells. 5. The activity pattern of mitochondrial enzymes in parenchymal and non-parenchymal cell homogenates indicates that these cell types contain different types of mitochondria. The presence of these different cell types in liver will therefore contribute to the heterogeneity of isolated rat liver mitochondria in which the mitochondria from non-parenchymal cells might be considered as "non-gluconeogenic".     

Autotrophic CO2 fixation in Acetobacterium woodii

Earlier labeling experiments have shown that autotrophically grown Acetobacterium woodii assimilates cell carbon via direct acetyl CoA formation from 2 CO₂, rather than via the Calvin cycle. Cell extracts contained the enzymes required for biosynthesis starting from acetyl CoA and CO₂. Notably, pyruvate synthase, pyruvate phosphate dikinase, and phosphoenolpyruvate carboxytransphosphorylase were present in sufficiently high activities. Ribulose-1,5-bisphosphate carboxylase activity could not be detected. The observed enzyme pattern was consistent with the postulated biosynthetic pathway as deduced from ¹⁴C-labeling experiments.     

Immunofluorescent localization of acetyl CoA carboxylase, fatty acid synthetase and pyruvate carboxylase during the adipocyte conversion of 3T3 fibroblasts

Three enzymes involved in the conversion of 3T3-L2 fibroblasts into fat cells, acetyl CoA carboxylase (ACC), fatty acid synthetase (FAS) and pyruvate carboxylase (PC) have been localized by immunofluorescence techniques. The method enables the identification of cells undergoing the conversion while they are still fibroblastic in appearance, often before the obvious appearance of fat droplets. Specific fluorescence for each enzyme can be seen in "clones" of cells derived from single cells, which may undergo an event during logarithmic growth, which programs the cells to differentiate subsequent to confluence of and addition of induction medium.     

Biochemical characterization of biotin-responsive multiple carboxylase deficiency: heterogeneity within the bio genetic complementation group

Three biotin-dependent enzymes, pyruvate carboxylase (PC), propionyl CoA carboxylase (PCC), and beta-methylcrotonyl CoA carboxylase (beta MCC), were biochemically characterized in fibroblasts from two patients with neonatal multiple carboxylase deficiency. Genetic complementation analyses indicated that both cell lines, designated lines 1 and 2, were deficient in the various carboxylase activities and belonged to the bio complementation group. The activities of the three carboxylases became normal when line 2 cells were incubated in medium supplemented with biotin (1 mg/l) for 24 hrs, whereas 4-6 days were required to achieve maximum activities of PC, PCC, and beta MCC (57%, 46%, and 29% of mean normal enzyme activity, respectively) in line 1 cells incubated in medium containing up to 10 mg/1 biotin. Furthermore, PC activity in line 2 continued to increase under apparent gluconeogenic conditions in culture, but not in line 1. Thermostability studies suggested that biotin stabilizes PC and beta MCC in both cell lines. PC in line 1 cells incubated with or without biotin was less stable than that in normal or line 2 cells, and the less than normal increase of enzyme activities in line 1, especially that of PC, may represent incomplete biotination. These results indicate that there is biochemical heterogeneity within the bio complementation group. Immunotitration with antibodies prepared against purified pig heart PCC demonstrated normal quantities of cross-reacting material in both lines and no differences in the amount of this material after incubation with supplemental biotin, despite the seven- to 20-fold increase in PCC activity. Thus, the increase in carboxylase activity in both bio lines appears to represent activation of rpe-existing apocarboxylase rather than de novo enzyme synthesis. The primary defect in this form of multiple carboxylase deficiency may be in a common holocarboxylase synthetase or in biotin transport. If the defect is in the synthetase, the differences noted between the two bio lines could be explained by a difference in the enzyme's Km for biotin.     

Evidence for two genetic complementation groups in pyruvate carboxylase-deficient human fibroblast cell lines

We have examined genetic complementation in pyruvate carboxylase deficiency by comparing the enzyme activity in polyethylene glycol-induced heterokaryons with that in unfused mixtures of fibroblasts from three affected children. Complementation, manifested as a three- to sevenfold increase in pyruvate carboxylase activity, was observed in fusions between a biotin-responsive multiple carboxylase (pyruvate carboxylase, propionyl CoA carboxylase, and -methylcrotonyl CoA carboxylase) deficient fibroblast line and two other lines deficient only in pyruvate carboxylase activity. Kinetic analysis of complementing pyruvate carboxylase deficient lines, measured by the rate of restoration of enzyme activity as a function of time, revealed that maximum restoration was achieved within 10–24 hr after fusion. This profile is similar to those observed for fusions between the multiple carboxylase deficient line and two lines deficient in propionyl CoA carboxylase activity that are known to represent different gene mutations. Although the patients with pyruvate carboxylase deficiency had similar clinical findings, our studies indicate that pyruvate carboxylase deficiency is genetically heterogeneous, with at least two distinct, probably intergenic, complementation groups.This work was supported by an NIH research grant (AM 25675) and an A. D. Williams research grant (6-48360). B. Wolf is the recipient of an NIH Research Career Development Award (AM 00677) and is aided by a Basil O'Connor Starter Research Grant from The National Foundation-March of Dimes (5-263). G. Feldman is the recipient of an NIH predoctoral training grant (GM 07492). This article is No. 100 from the Department of Human Genetics at the Medical College of Virginia.     

Kinetic characterization of yeast pyruvate carboxylase isozyme pyc1

Yeast (Saccharomyces cerevisiae) is unusual in being the only organism thus far identified as having two genes for pyruvate carboxylase. The expression of the two isozymes Pyc1 and Pyc2 appears to be differentially regulated, and since both are expressed cytoplasmically, this suggests that they have different properties. To the present, little has been done to characterize these isozymes, and almost all of the published kinetic information on yeast pyruvate carboxylase comes from measurements of enzyme prepared from bakers' yeast which is likely to be a mixture of both isozymes. Here we have measured basic kinetic parameters for Pyc1 and found that the K(a) of this isozyme for acetyl CoA is in the order of 8-10-fold higher than previously recorded, suggesting that Pyc1 and Pyc2 may be differentially regulated by this effector. Pyc1 is highly dependent on the presence of acetyl CoA for activity and in this respect is similar to chicken liver pyruvate carboxylase. However, unlike the chicken liver enzyme, the quaternary structure of the enzyme is quite stable in the absence of acetyl CoA, and the major locus of action of this effector appears to lie outside of the stimulation of the biotin carboxylation reaction.     

Multiple carboxylase deficiency

1. The multiple carboxylase deficiencies are inborn errors in the metabolism of biotin in which there is defective activity of propionyl CoA carboxylase, 3-methylcrotonyl CoA carboxylase and pyruvate carboxylase. 2. Two distinct disorders have been described. 3. In one the fundamental defect is in the enzyme holocarboxylase synthetase which catalyzes the molecular activation of the apocarboxylase proteins. 4. In the other the fundamental defect is in biotinidase which catalyzes the reutilization of biotin and may be involved in its digestion and intestinal absorption.     

Lipid Synthesis and Abundance of Acetyl CoA Carboxylase in Isochrysis galbana (Prymnesiophyceae) Following Nitrogen Starvation

Fatty acid content and the rate of lipid synthesis were measuredin the marine prymnesiophyte Isochrysis galbana grown undernitrogen starvation and in cultures recovering from nitrogendeprivation. Nitrogen starvation imposed a reduction in cellularsoluble protein content, variation in fatty acid compositionand reduction in the in vitro activity of the enzyme acetylCoA carboxylase. An increase in total fatty acid content isattributed to a differential reduction in cell division andthe rate of lipid synthesis. Recovery from nitrogen deprivationwas characterized by an increase in cellular soluble proteincontent and in the rate of lipid synthesis. Although the invitro activity of acetyl CoA carboxylase increased as the culturesrecovered from nitrogen starvation, the total cellular fattyacid content decreased, evidently due to an acceleration incell division. The relative cellular pool size of acetyl CoAcarboxylase determined by immunoblotting decreased under nitrogenstarvation conditions and increased as cells recovered fromit. Cellular accumulation of acetyl CoA carboxylase during recoveryfrom nitrogen starvation is ascribed to de novo synthesis ofthe enzyme that takes place in the cytoplasm. However, photosyntheticproteins such as ribulose bisphosphate carboxylase are synthesizedearlier than acetyl CoA carboxylase in the recovery process. (Received June 12, 1992; Accepted September 21, 1992)     

The sub-cellular localisation and regulatory properties of pyruvate carboxylase from Rhizopus arrhizus

Cell-free extracts of Rhizopus arrhizus contain exclusively cytosolic pyruvate carboxylase and NAD-glutamate dehydrogenase, a single mitochondrial isoenzyme of NADP-isocitrate dehydrogenase, and both mitochondrial and cytosolic isoenzymes of NADP-malate dehydrogenase (decarboxylating). Other enzymes examined have sub-cellular localisations similar to those characteristic of mammalian liver. Purified preparations of R. arrhizus pyruvate carboxylase are subject to partial regulatory inhibition by L-aspartate and 2-oxoadipate. L-Glutamate acts as a less effective analogue of L-aspartate while 2-oxoglutarate is ineffective. Competition studies indicate the presence of separate inhibitory sites for L-aspartate and 2-oxoadipate. Under routine assay conditions R. arrhizus pyruvate carboxylase shows significant activation by acyl derivatives of coenzyme A with long chain acyl CoA being more effective than acetyl-CoA. This activation is no longer observed in the presence of high concentrations of pyruvate, MgATP2- and HCO-3. The concentrations of L-aspartate and 2-oxoadipate required to give 50% inhibition ([I]0.5), and the maximal extents of inhibition, are increased by addition of acetyl-CoA. Acetyl-CoA increases the sigmoidal character of the relationship: initial rate/[L-aspartate], but decreases this parameter for the relationship: initial rate/[2-oxoadipate]. The studies indicate that R. arrhizus possesses an entirely cytosolic pathway for the conversion of glucose to fumaric acid and that both the organisation of pyruvate metabolism and the regulation of pyruvate carboxylase differ significantly in this organism as compared to that proposed previously for Aspergillus nidulans.     

Inhibition of pyruvate carboxylase by sequestration of coenzyme A with sodium benzoate

Pyruvate-dependent CO2 fixation by isolated mitochondria was strongly inhibited by sodium benzoate. Pyruvate carboxylase was identified as a site of inhibition by limiting flux measurements to assays of pyruvate carboxylase coupled with malate dehydrogenase. Benzoate reduced pyruvate-dependent incorporation of [14C]KHCO3 into malate and pyruvate-dependent malate accumulation by 74 and 72%, respectively. Aspartate-dependent malate accumulation was insensitive to benzoate, ruling out malate dehydrogenase as a site of action. Inhibition by benzoate was antagonized by glycine, which sharply accelerated conversion of benzoate to hippurate. Assays of coenzyme A and its acyl derivatives revealed inhibition to correlate with depletion of acetyl CoA and accumulation of benzoyl CoA. Depletion of acetyl CoA was sufficient to account for greater than 50% reduction in pyruvate carboxylase activity. Competition between acetyl CoA and benzoyl CoA for the activator site on pyruvate carboxylase was insignificant. Results support the interpretation that the observed inhibition of pyruvate carboxylase occurred primarily by depletion of the activator, acetyl CoA, through sequestration of coenzyme A during benzoate metabolism.     

The activities of pyruvate carboxylase, phosphoenolpyruvate carboxylase and fructose diphosphatase in muscles from vertebrates and invertebrates

1. The activities of pyruvate carboxylase, phosphoenolpyruvate carboxylase and fructose diphosphatase in crude homogenates of vertebrate and invertebrate muscles are reported. 2. Pyruvate carboxylase activity was present in all insect flight muscles that were investigated: in homogenates of bumble-bee flight muscle the activity was inhibited by ADP and activated by acetyl-CoA, and it was distributed mainly in the mitochondrial fraction. This is the first demonstration of pyruvate carboxylase activity in muscle. However, the activity appears to be restricted to insect flight muscle, since it was not found in other invertebrate or vertebrate muscles. 3. Since the three enzymes were never found together in the same muscle, it is concluded that these enzymes cannot provide a pathway for the synthesis of glycogen from lactate or pyruvate in muscle. Other roles for these enzymes in muscle are suggested. In particular, pyruvate carboxylase may be present in insect flight muscle for the provision of oxaloacetate to support the large increase in activity of the tricarboxylic acid cycle which occurs when an insect takes flight.     

Carbon Dioxide Fixation by Extracts of Streptococcus faecalis var. liquefaciens

Extracts of cells of Streptococcus faecalis var. liquefaciens strain 31 incorporated (14)CO(2) into aspartate. Dialyzed extracts produced radioactive oxalacetate in the absence of exogenously added glutamate and pyridoxal-5'-phosphate and produced radioactive aspartate in the presence of these components. Reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate could not be substituted for adenosine triphosphate (ATP); phosphoenolpyruvate even in the presence of nucleoside diphosphates could not replace pyruvate plus ATP; propionate plus coenzyme A (CoA) could not replace pyruvate in supporting CO(2) fixation by cell extracts. Fixation by dialyzed cell extracts required pyruvate, ATP, MgSO(4), and was stimulated by biotin, KCl, 2-mercaptoethanol, CoA, and acetyl CoA. Inhibition of fixation occurred when avidin, NaCl, oxalacetate, or aspartate was added to dialyzed extracts. On the basis of the products formed and the effects of substrates and cofactors on the fixation reaction, it was concluded that pyruvate carboxylase is responsible for CO(2) fixation in this microorganism.     

Activation of yeast pyruvate carboxylase: interactions between acyl coenzyme A compounds, aspartate, and substrates of the reaction

Chicken liver pyruvate carboxylase has an absolute requirement for short-chain acyl coenzyme A (CoA), whereas the same enzyme from yeast has less stringent requirements. The yeast enzyme has now been studied in an effort to elucidate the mechanism by which acyl-CoA stimulates pyruvate carboxylase activity. Yeast pyruvate carboxylase has an apparent basal level of activity above which CoA and acyl-CoAs of 2-20 carbons activate; the concentration of acyl-CoA required for half-maximum activation (K0.5) decreases as the chain length of the acyl moiety increases to 16 carbons. Activation of yeast pyruvate carboxylase by acyl-CoA is brought about in part by increasing the affinity of pyruvate carboxylase for two substrates, bicarbonate and pyruvate. The affinity of pyruvate carboxylase for bicarbonate is also increased by potassium ions. The observation of only low levels of activity in the absence of acyl-CoA or potassium ion leads to the conclusion that the basal activity so frequently referred to is probably due to the presence of activating monovalent cations. Pyruvate carboxylase from yeast probably has an absolute requirement for monovalent cations or acyl-CoA with a combination of the two being required for optimum conditions for maximal activity. Stimulation by acyl-CoA and inhibition by aspartate are mutually antagonistic with each affecting the activation or inhibition constant and the degree of cooperativity brought about by the other. The enzyme from liver is unaffected by aspartate.     

New experiments of biotin enzymes.

The objects of structural studies on biotin-enzymes were acetyl CoA-carboxylase and pyruvate carboxylase of Saccharomyces cerevisiae and beta-methylcrotonyl CoA-carboxylase and acetyl CoA-carboxylase of Achromobacter IV S. It was found that these enzymes can be arranged in three groups. In the first group, as represented by acetyl CoA-carboxylase of Achromobacter, the active enzyme could be resolved in three types of functional components: (1) the biotin-carboxyl carrier protein, (2) the biotin carboxylase, and (3) the carboxyl transferase. In the second group, as represented by beta-methylcrotonyl CoA-carboxylase from Achromobacter only two types of polypeptides are present. The one carries the biotin carboxylase activity together with the biotin-carboxyl-carrier protein, the other one carries the carboxyl transferase activity. In this third group, as represented by the two enzymes of yeast, all three catalytic functions are incorporated in one multifunctional polypeptide chain. The evolution of the different enzymes is discussed. The animal tissues acetyl CoA-carboxylase is under metabolic control, as known from previous studies. It thus has to be expected that the levels of malonyl CoA in livers of rats in all states of depressed fatty acid synthesis are much lower than under normal conditions because the carboxylation of acetyl CoA is strongly reduced and cannot keep pace with the consumption of malonyl CoA by fatty acid synthetase. A new highly sensitive assay method for malonyl CoA was developed which uses tritiated NADPH and measures the incorporation of radioactivity into the fatty acids formed from malonyl CoA in the presence of purified fatty acid synthetase. The application of this method to liver extracts showed that the level of malonyl CoA which amounts to about 7 nmoles per gram of wet liver drops to less than 10% within a starvation period of 24 hr and even further if the starvation period is extended to 48 hr. A low malonyl CoA concentration is also found in the alloxan diabetic animals and in animals being fed a fatty diet after starvation. On the other hand, feeding a carbohydrate rich diet leads to malonyl CoA levels surpassing the levels found after feeding a balanced diet. These observations reconfirm the concept that fatty acid synthesis is principally regulated by the carboxylation of acetyl CoA.     

Differential Accumulation of Biotin Enzymes during Carrot Somatic Embryogenesis

The activities of four biotin enzymes, acetyl-coenzyme A (CoA) carboxylase, 3-methylcrotonyl-CoA carboxylase, pyruvate carboxylase, and propionyl-CoA carboxylase, and the accumulation of six biotin-containing polypeptides were determined during development of somatic embryos of carrot (Daucus carota). Acetyl-CoA carboxylase activity increased more than sevenfold, whereas the activities of 3-methylcrotonyl-CoA carboxylase, pyruvate carboxylase, and propionyl-CoA carboxylase were relatively unaltered. An increase also occurred in the accumulation of three of the biotin-containing polypeptides (molecular masses of 220, 62, and 34 kilodaltons). Of these, the most dramatic change was in the accumulation of the 62-kilodalton biotin-containing polypeptide, which increased by at least 50-fold as embryogenic cell clusters developed into torpedo embryos.     

Heterogeneity of the glucose transporter in malignant and suppressed hybrid cells

Previous work has demonstrated unequivocally that the kinetics of glucose transport of tumorigenic and suppressed hybrid cells show a consistent difference. This lies in an increased affinity for glucose in the tumorigenic cell lines (i.e., a reduced Km). Evidence has also been presented that the degree of glycosylation of the transporter may affect the Km. When a suitable antiserum to the transporter present in these cells became available, it was of interest to examine the patterns of binding to immunoblots of extracts of the hybrid cell pairs. It became apparent that there was a clear difference between the two parental cell lines and that this difference was reflected in the hybrid cells. Both the tumorigenic parent and the tumorigenic hybrid cell presented a much more heterogeneous distribution of apparent molecular weights than the nontumorigenic parent or suppressed cell line. That this was probably due to differences in glycosylation was indicated by the effect of tunicamycin on the cells; this gave rise to a more homogeneous band of lower molecular weight. The significance of these differences is discussed in relation to results obtained with other tumorigenic cell lines and to the published structure of the human erythrocyte glucose transporter.