John A. Black

Glucose uptake into plasma membrane vesicles from the maternal surface of human placenta

SummaryGlucose uptake into plasma membrane vesicles from the maternal surface of the human placenta was measured with the Millipore filtration technique. Uptake ofd-glucose was dependent on the osmolarity of the incubation medium surrounding the vesicles. Uptake ofd-glucose exceeded that ofl-glucose. The uptake ofd-glucose was not enhanced by placing 100mm NaCl or NaSCN in the medium outside the vesicles (none inside) at the onset of uptake determinations.d-glucose transport was inhibited by cytochalasin B; phloretin, phlorizin, and 1-fluoro-2,4-dinitrobenzene.d-glucose uptake was inhibited by 2-deoxy-d-glucose, 3-O-methyl-d-glucose and to a lesser extent byd-galactose. It was not inhibited by α-methyl-d-glucoside. Cytochalasin B binding to the vesicles was 30% inhibited in the presence of 80mm d-glucose. The results indicate that the system for facilitated transport ofd-glucose at the maternal face of the placenta is distinctly different from that on the brush-border membrane of intestine or renal tubule and more closely resembles that of human erythrocyte.

Activation and inhibition of human erythrocyte pyruvate kinase by organic phosphates, amino acids, dipeptides and anions

Abstract 1. 1.|Human erythrocyte pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.40) has been proposed to conform to the two state conformational model for allosteric enzymes of Monod, Wyman and Changeux. We have examined the kinetic properties of the enzyme in this light and where appropriate made comparative studies on the non-allosteric isozyme from rabbit muscle. 2. 2.|In the pH range from 7 to 8 at low phosphoenolpyruvate concentration the erythrocyte enzyme activity has a strong pH dependence. This behaviour is of practical concern in the conditions used for blood banking. 3. 3.|A number of intracellular organic phosphates were tested. The most likely physiological activators of the erythrocyte enzyme are glucose 6-phosphate and fructose 1,6-diphosphate. Conflicting reports on the effect of 2,3-diphosphoglycerate on the enzyme appear due to differences in the magnesium concentration used in the assays. 4. 4.| l -Amino acids inhibit human erythrocyte pyruvic kinase in a pH and substrate-concentration dependent manner. Various alanine depeptides activate the erythrocyte enzyme, others inhibit. Dipeptides give only inhibition with the muscle enzyme. 5. 5.|Anions inhibit both enzymes. Inhibition of the erythrocyte enzyme is pH and substrate-concentration dependent. 6. 6.|All of these observations are consistent with alterations in the R ⇌ T equilibrium proposed for human erythrocyte pyruvate kinase.

Amino acid compositions and evolutionary relationships with protein families.

Abstract We have examined the merits of the three functions based on amino acid compositions which have been proposed to indicate the similarity in amino acid sequences of two proteins; the difference index, the composition divergence and the composition coefficient. We have taken the amino acid compositions and sequences of 41 cytochrome c s and used the 820 values from all possible comparisons in the evaluation. We conclude that the functions do have a limited value in predicting proteins which are closely related in sequence and that the three functions are equivalent in this predictive ability. We have used the composition divergence values obtained from available pyruvate kinase amino acid compositions to generate a phylogenetic tree for this glycolytic enzyme.

The immunological properties of pyruvate kinase: II. The relationship of the human erythrocyte isozyme to the human liver isozymes

We have examined the hypothesis that the human erythrocyte isozyme of pyruvate kinase (EC 2.7.1.40) is a hybrid of the two isozymes present in liver. Rabbit antiserum against purified human erythrocyte pyruvate kinase inactivates the erythrocyte isozyme and the major liver isozyme from human tissue but does not inactivate the minor liver isozyme. The electrophoretic mobilities of the erythrocyte and major liver isozymes are altered by anti-erythrocyte enzyme antibody while the mobility of the minor liver isozyme is unaffected. Gel diffusion analysis indicates cross-reactivity between the erythrocyte and major liver isozyme but no cross-reactivity with the minor liver isozyme. The hybrid hypothesis would predict cross-reactivity including changes in activity and mobility of all isozymes and we conclude, therefore that the hypothesis is incorrect.

The subunit structure of human muscle and human erythrocyte pyruvate kinase isozymes

In human tissues the pyruvate kinase reaction (ATP: pyruvate phosphotransferase; E C 2.7.1.40) is catalysed by at least three isozymes [l-3] which can be distinguised by their kinetic, electrophoretic and immunological properties: M1 present in muscle, L the major form in liver and Mz found in liver, kidney and adipose tissue. The isozyme present in red cells (R) is kinetically similar to the L isozyme but can be distinguished from it by electrophoresis [2-41. The R isozyme can be converted into a form which is electrophoretically identical to the L isozyme either by treating with a liver homogenate or repeated freezing and thawing [4 ] . The molecular structures and interrelationships of the isozymes are unknown. We have examined the human M1 and R isozymes by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and two dimensional fingerprinting of their cyanogen bromide peptides. The evidence is consistent with identical subunits in the M1 tetramer and two pairs of non-identical subunits in the R tetramer. One of the R subunits may be homologous with the M1 subunit but is probably not identical to it.

Canine erythrocyte pyruvate kinase. II. Properties of the abnormal enzyme associated with hemolytic anemia in the basenji dog

We have compared the solubility, kinetic, immunological, and electrophoretic properties of erythrocyte pyruvate kinase from normal dogs and Basenji dogs with congenital hemolytic anemia due to pyruvate kinase deficiency. Differences can be detected between the two enzymes by all methods. The enzyme from the affected animals has a greater solubility in ammonium sulfate. It has a lower Km for phosphoenolpyruvate, while the Km for ADP is increased. This enzyme is not inhibited by ATP or activated by fructose 1,6-diphosphate. The enzyme from the affected animals has none of the allosteric properties characteristic of the normal canine enzyme. No difference can be detected by enzyme inactivation with rabbit antiserum against the human erythrocyte enzyme, but a slight spur is observed on comparison of the two enzymes by Ouchterlony immunodiffusion. The enzymes also differ in their electrophoretic mobilities on starch gel electrophoresis.

The immunological properties of pyruvate kinase—I: Mammalian erythrocyte enzymes

Abstract Antiserum to purified human erythrocyte pyruvate kinase was used to identify immunologic relationships among erythrocyte pyruvate kinases from several phylogenetically distant mammals: humans, rhesus monkey, beef, rat and two marsupials opposum and phalanger. Each of the enzymes tested shared partial identity with the human enzyme and with the enzyme of every other species by gel diffusion analysis. No two species shared complete identity. Thus each species shares antigenic determinants with the human enzyme which are either missing or unreactive in the other species. All of the enzymes were inactivated by the antibody with the relative efficiency of inactivation being in general agreement with previously established phylogenetic relationships. The one exception was the phalanger which showed a closer relationship to man than expected. We conclude that mammalian pyruvate kinases are related in amino acid sequence and three dimensional structure as predicted by their similar kinetic properties. We further conclude that during evolution from the common ancestor each species has undergone a mutational loss of antigenic determinants so that each bears only a percentage of the ancestral determinants and that these are shared at random with the human enzyme which is presumed to have undergone a similar mutational process. On this basis we suggest that erythrocyte pyruvate kinase has probably evolved at a faster rate than previously thought.

Canine Erythrocyte Pyruvate Kinase. I. Properties of the Normal Enzyme

We have compared the kinetic, immunological, and electrophoretic properties of human and canine erythrocyte pyruvate kinase. Both enzymes are allosteric and subject to positive and negative regulation. The allosteric properties of the canine enzyme are more pronounced than those of the human enzyme; however, the properties of both enzymes are consistent with a regulatory function in the glycolytic pathway of their respective erythrocytes. Antiserum against the human enzyme gives precipitin lines of partial identity between the human and canine enzymes on immunodiffusion. The anti-human serum inactivates the enzymatic activity of both enzymes, although it is more effective against the human enzyme than the canine. The two enzymes have slightly different mobilities on starch gel electrophoresis. While we have demonstrated differences between erythrocyte pyruvate kinase from dogs and that from humans, we conclude that the enzymes are sufficiently similar in properties and function to allow use of the dog as a model for human erythrocyte pyruvate kinase deficiency.

Comparative kinetic and electrophoretic properties of erythrocyte pyruvate kinases

Abstract 1. 1. We have determined the effect of variation of phosphoenolpyruvate ADP and ATP concentrations on the kinetics of erythrocyte pyruvate kinase from man, rhesus monkey, rabbit, dog, phalanger, beef, and oppossum. 2. 2. The enzymes show a wide range of kinetic and electrophoretic properties indicating the incorporation of genetically determined structural alterations in the various molecules during evolution. 3. 3. We propose that the functional consequences of these alterations can be interpreted on the basis of a molecular model for erythrocyte pyruvate kinase similar to that of hemoglobin.

The action of proteolytic enzymes on human erythrocyte pyruvate kinase

Abstract Human erythrocyte pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40) is inactivated at low salt concentration by pronase, chymotrypsin, and trypsin. The kinetics of inactivation are pseudo-first order. We were unable to identify any discrete, large molecular-weight intermediates of the inactivation process by polyacrylamide gel electrophoresis. The evidence suggests random proteolytic cleavage after the initial inactivation step. Nonspecific protection against the inactivation is observed at higher salt concentrations. Specific protection is provided by low concentrations of phosphoenolpyruvate, ADP-Mg, ATP-Mg, and fructose-1,6-diphosphate. Pyruvate kinase exposed to trypsin in the presence of phosphoenolpyruvate is indistinguishable from the native molecule on polyacrylamide gel electrophoresis, but does have altered thermostability We interpret the results in terms of a flexible model of the enzyme which is stabilized in an active configuration by interaction with substrates and other physiological ligands.