Abstract
Abstract Low concentrations of palmitoyl coenzyme A inhibit yeast glucose-6-P dehydrogenase (Taketa, K., and Pogell, B. M. (1966) J. Biol. Chem. 241, 720). This inhibition is prevented or reversed by bovine serum albumin, mycobacterial polysaccharides, or alkylated cyclodextrins. Sephadex chromatography and sucrose density gradient centrifugation show that palmitoyl-CoA inhibits by dissociating the tetrameric dehydrogenase to dimeric, enzymatically inactive subunits. These structural changes are accompanied by firm binding of palmitoyl-CoA to the dimeric subunit and by release of NADP. After removal of the (nondialyzable) inhibitor from the palmitoyl-CoA-dimer complex by alkylated cyclodextrin the subunits reaggregate to enzymatically active tetramer. Reaggregation does not require NADP. In contrast to palmitoyl-CoA, sodium dodecyl sulfate dissociates yeast dehydrogenase to monomers. It is concluded that palmitoyl-CoA, unlike the synthetic anionic detergent, perturbs the dehydrogenase subunit structure in a controlled manner which may be important for regulating the activity of lipogenic enzymes. The dimeric glucose-6-P dehydrogenase from Leuconostoc mesenteroides is inhibited only by high concentrations of palmitoyl-CoA. In this instance palmitoyl-CoA neither binds to the enzyme nor dissociates it. Torulopsis utilis glucose-6-P dehydrogenase, also dimeric, is irreversibly converted to inactive monomers by low concentrations of palmitoyl-CoA.
Highlights
Thereafter, the extent of inhibition became nearly the same as in the experiments in which the preincubation mixture contained NADP. These results show that NADP but not glucose-6-P protects the enzyme against palmitoyl-CoA
When a mixture of SDS (130 PM) and CHI-cyclodextrin (1.3 mM) was added to the assay mixture at the start, no inhibition was observed. These results indicate that SDS inactivates the enzyme irreversibly and that CHa-cyclodextrin protects against this inactivation by complexing the detergent
Yeast glucose-6-P dehydrogenase was chosen for this purpose because it is readily available, well characterized [17,21], and highly sensitive to palmitoyl-CoA [1, 9]
Summary
IMaterials-Materials were obtained from the following sources: glucose-6-P (bakers’ yeast, 270 units per mg of protein), lactic dehydrogenase (rabbit muscle, 750 units per mg of protein), hemoglobin (bovine), crystalline bovine serum albumin, and sodium glucose-6-P from Sigma; T. utilis glucose-6-P (220 units per mg of protein) and palmitoyl-Cob from P-L Biochemicals;L. mesenteroides glucose-6-P dehydrogenase (207 units per mg of protein) from Worthington BiochemicalCorp.; [1-YI?]palmitoyl-CoA (50 mCi per mmole) from NewEngland Nuclear; NADP from Calbiochem.Heptakis(2,6-di-O-methyl&-cyclodextrin (ll), hereafter referred to as CHr-cyclodextrin, and MMP, the 3-0-methylmannose containing polysaccharide from Mycobacterium phlei [12], were prepared as CH 3 - cyclodextrin, heptakis(2,6-di-O-methyl)&cyclodextrin; MMP, 3-0-methylmannose containing polysaccharide from Mycobacterium phlei.2 P. IMaterials-Materials were obtained from the following sources: glucose-6-P (bakers’ yeast, 270 units per mg of protein), lactic dehydrogenase (rabbit muscle, 750 units per mg of protein), hemoglobin (bovine), crystalline bovine serum albumin, and sodium glucose-6-P from Sigma; T. utilis glucose-6-P (220 units per mg of protein) and palmitoyl-Cob from P-L Biochemicals;. L. mesenteroides glucose-6-P dehydrogenase (207 units per mg of protein) from Worthington Biochemical. Corp.; [1-YI?]palmitoyl-CoA (50 mCi per mmole) from New. England Nuclear; NADP from Calbiochem. Methyl&-cyclodextrin (ll), hereafter referred to as CHr-cyclodextrin, and MMP, the 3-0-methylmannose containing polysaccharide from Mycobacterium phlei [12], were prepared as CH 3 - cyclodextrin, heptakis(2,6-di-O-methyl)&cyclodextrin; MMP, 3-0-methylmannose containing polysaccharide from Mycobacterium phlei.
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