Allan Fenselau

Structure-function studies on glyceraldehyde-3-phosphate dehydrogenase. III. Dependency of proteolysis on NAD+ concentration

Abstract Mild treatment of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase with chymotrypsin, trypsin, subtilisin, or Pronase results in a substantial loss in enzymatic activity of the apoenzyme and not the holoenzyme. The products from proteolytic digestion differ in chemical and physical properties, suggesting that peptide bond cleavages have been introduced in quite different regions of the protein Particularly in the cases of chymotrypsin and trypsin binding of NAD + drastically reduces the susceptibility of all these bonds to hydrolysis. The degree of protection from inactivation is greatest in the case of all proteolyses when less than two coenzyme molecules are bound to a molecule of the tetrameric enzyme. These results are considered in terms of understanding the mechanism for the homotropic effects observed in coenzyme binding.

Structure-function studies on glyceraldehyde-3-phosphate dehydrogenase. II. The effects of S-carboxymethylation on enzymatic activity.

Abstract Enzymatic activity of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase genase was correlated with the number of active site cysteine residues alkylated by iodoacetate under various conditions. A one-to-one correspondence between loss of dehydrogenase activity and incorporation of [14C]carboxymethyl groups (up to 3.5 groups per enzyme molecule) was observed at pH 8.0 and 7.0 in the presence of glyceraldehyde 3-phosphate, arsenate or NAD+. Comparable studies using enzyme completely or partially acylated with 3-phosphoglyceryl groups provided identical results. The conclusion based on these observations is that the four alkylatable cysteine residues in the tetrameric protein are intrinsically equivalent in the sense that each can function in the enzymatic reaction.

On the extracellular synthesis of adenosine triphosphate by mammalian cells

Previous reports from another laboratory indicate that two glycolytic enzymes, glyceraldehyde phosphate dehydrogenase (EC 1.2.1.12) and phosphoglycerate kinase (EC 2.7.2.3), are ecto-enzymes, i.e., they are located to some extent on the outer periphery of various mammalian (as well as yeast) cell membranes [l-6] . The evidence that supports this conclusion has been derived from the ability of ‘intact’ cells to synthesize extracellular ATP from the exogenous substrates for the two enzymes (glyceraldehyde-3phosphate, NAD’, orthophosphate, and ADP). The actual cells examined included Ehrlich mouse ascites tumor cells, HeLa cells, and several human cells (embryonic lung and adult skin fibroblasts, sarcoma cells, glia-like cells, glioma cells and erythrocytes). Since no function for this arrangement of the enzymes has been suggested or established, we decided to study the effects of these substrates on various properties of mammalian cell cultures. However, our unsuccessful attempts to reproduce these earlier results using a cell line (BALB/c 3T3 mouse fibroblast) that differs from the cells previously examined prompts us to communicate the following.

Ping-pong chromatography a novel purification of CoA-transferase

Summary Succinyl-CoA:acetoacetate CoA transferase, which forms a covalent intermediate with CoA, can be readily purified from rat kidney, heart, brain, and skeletal muscle using an acetoacetyl-CoA-agarose column. Significant purification (20–180 fold) with low losses in activity was achieved in each case, although minor contaminants were detected by SDS-gel electrophoresis. The isolated material appears to have a mol. wt. of 55–58,000 daltons, suggesting a dimeric structure for the native enzyme. Thus, ping-pong chromatography, based on the formation of a covalent enzyme-substrate intermediate on a solid support, can be used in the selective purification of certain enzymes.

Ketone body oxidation by mouse hepatoma BW7756

Abstract Studies on the mouse hepatoma BW7756 reveal that mitochondria from the neoplastic tissue, unlike those from normal liver tissue, are able to utilize ketone bodies as respiratory substrates and contain succinyl-CoA: acetoacetate CoA transferase. This enzyme is rerequired for ketone body utilization and is virtually absent in normal liver mitochondria. These results suggest that malignant hepatocytes, as a consequence of their production and release of large quantities of cholesterol, derive metabolic advantage over neighboring normal hepatocytes by being able to consume ketone bodies synthesized by normal cells in response to the elevated cholesterol levels.