Genetically controlled variation of "acid" beta-galactosidase detected in Rattus norvegicus by isoelectric focusing.

Two genetically variant forms of rat "acid" beta-galactosidase were found to differ in isoelectric point and pH dependence, but not in thermostability or sensitivity to inhibition by p-mercuribenzoate (PMB). The results of two backcrosses and an intercross indicated that the isoelectric focusing phenotypes are controlled by two codominant alleles at a single autosomal locus, for which we propose the name Glb-1. No significant linkage between Glb-1 and albino (LG I), brown (LG II), or hooded (LG VI) was observed. Strain-specific differences in total levels of kidney beta-galactosidase were detected, but it is not yet known whether the variation is controlled by genes linked to Glb-1. Experiments in which organ homogenates were incubated with neuraminidase indicated that the genetically variant forms do not result from differences in sialylation, though sialylation does appear to be largely responsible for the presence of multiple bands within each phenotype and for differences in the banding patterns of beta-galactosidases derived from different organs. The beta-galactosidase present in the bands used for Glb-1 typing resembles human GM1 gangliosidase (GLB1) with respect to pH optimum, substrate specificity, and susceptibility to inhibition by PMB. It also appears that Glb-1 is homologous with the Bgl-e locus of the mouse. In rats as in mice the genetically variant bands of beta-galactosidase are active at acid pH and have relatively high isoelectric points. In both species these bands are readily detectable in kidney homogenates, and can be revealed in homogenates of liver or spleen following treatment with neuraminidase. The presence of the same beta-galactosidase bands in homogenates of rat kidney and small intestine as well as in neuraminidase-treated homogenates of liver and spleen suggests that the Glb-1 variants differ by one or more point mutations in the structural gene for "acid" beta-galactosidase.

Proton flux across artificial vesicle bilayers

The study of proton conductance across artificial membranes has revealed a surprisingly high permeability for H+, (Pnet H+). A high Pnet H+ is difficult to reconcile with the biological requirement for the maintenance of pH gradients across the plasma membranes of cells, organellar study was undertaken to examine the role played by cholesterol and phospholipid fatty acid side chain composition in determining how well a membrane will function as a barrier to acid. The effects of counter-ion movement on acidification rates were examined in order to interpret the data obtained from variations in membrane composition. In phosphate buffered saline solutions, vesicle membranes composed of unsaturated fatty acid phosphatidylcholines proved to be poorer barriers to acid than membranes composed of saturated fatty acids. The barrier properties of these membranes could be ranked in the following order: DPL, (palmitic) $>$ Egg PC, (mixed chains) $>$ DLL, (linoleic), with DPL being the most effective in maintaining a one pH unit gradient near neutrality. Cholesterol decreased acidification rates of membranes made from the unsaturated phosphatidylcholines Egg PC and DLL, but enhanced acidification rates in vesicle membranes composed of the saturated phospholipid DPL. The cholesterol and fatty acid side chain effects were mediated by changes in membrane fluidity, with more rigid bilayers forming better barriers to acid. Experimental evidence was obtained which confirmed the Pnet H+ is very high relative to the permeabilities of other ions. Counter-ion controlled acidification rates depended on the size and charge of the ion which was moving in order to maintain electroneutrality. The biological relevance of a high intrinsic Pnet H+ and the possible role of counter-ion controlled acidification were discussed. ^

Citric acid cycle flux and pressure-volume work in the isolated working rat heart

It has been demonstrated previously that the mammalian heart cannot sustain physiologic levels of pressure-volume work if ketone bodies are the only substrates for respiration. In order to determine the metabolic derangement responsible for contractile failure in hearts utilizing ketone bodies, rat hearts were prefused at a near-physiologic workload in a working heart apparatus with acetoacetate and competing or alternate substrates including glucose, lactate, pyruvate, propionate, leucine, isoleucine, valine and acetate. While the pressure-volume work for hearts utilizing glucose was stable for 60 minutes of perfusion, performance fell by 30 minutes for hearts oxidizing acetoacetate as the sole substrate. The tissue content of 2-oxoglutarate and its transamination product, glutamate, were elevated in hearts utilizing acetoacetate while succinyl-CoA was decreased suggesting impaired flux through the citric acid cycle at the level of 2-oxoglutarate dehydrogenase. Further studies indicated that the inhibition of 2-oxoglutarate dehydrogenase developed prior to the onset of contractile failure and that the inhibition of the enzyme may be related to sequestration of the required cofactor, coenzyme A, as the thioesters acetoacetyl-CoA and acetyl-CoA. The contractile failure was not observed when glucose, lactate, pyruvate, propionate, valine or isoleucine were present together with acetoacetate, but the addition of acetate or leucine to acetoacetate did not improve performance indicating that improved performance is not mediated through the provision of additional acetyl-CoA. Furthermore, addition of competing substrates that improved function did not relieve the inhibition of 2-oxoglutarate dehydrogenase and actually resulted in the further accumulation of citric acid cycle intermediates "upstream" of 2-oxoglutarate dehydrogenase (2-oxoglutarate, glutamate, citrate and malate). Studies with (1-$\sp{14}$C) pyruvate indicate that the utilization of ketone bodies is associated with activation of NADP$\sp+$dependent malic enzyme and enrichment of the C4 pool of the citric acid cycle. The results suggest that contractile failure induced by ketone bodies in rat heart results from inhibition of 2-oxoglutarate dehydrogenase and that reversal of contractile failure is dissociated from relief of the inhibition, but rather is due to the entry of carbon units into the citric acid cycle as compounds other than acetyl-CoA. This mechanism of enrichment (anaplerosis) provides oxaloacetate for condensation with acetyl-CoA derived from ketone bodies allowing continued energy production by sustaining flux through a span of the citric acid cycle up to the point of inhibition at 2-oxoglutarate dehydrogenase for energy production thereby producing the reducing equivalents necessary to sustain oxidative phosphorylation. ^

ISOELECTRIC FOCUSING STUDIES OF STEROID BINDING PROTEINS

Steroid binding proteins are an obvious choice in the search for genetic factors in plasma that might predispose to upper body obesity, a risk factor for non-insulin dependent diabetes and cardiovascular disease. The two steroid binding proteins studied by isoelectric focusing were sex hormone binding globulin (SHBG), the transport protein for sex hormones and corticosteroid binding globulin (CBG), the transport protein for corticosteroids. Auto-radiography and immunoblotting on polyacrylamide gels were used to detect polymorphism in SHBG. Immunoblotting on agarose gels was used to visualize corticosteroid binding globulin. SHBG showed similar structural variation in American Caucasians, American Blacks and Canadian Indians. Two alleles (1, 2) were hypothesized with highly polymorphic frequencies in all three ethnic groups. CBG was not found to be polymorphic, but two variants were found in Caucasian male twins and in a Black individual. The finding of a good assay and a polymorphic system for SHBG are the first steps for additional studies into disease associations. ^