Interactions of acyl-coenzyme A with phosphatidylcholine bilayers and serum albumin.

Interactions of oleoyl- and octanoyl-coenzyme A (CoA) with phosphatidylcholine (PC) vesicles and bovine serum albumin (BSA) were investigated by NMR spectroscopy. Binding of acyl-CoA to small unilamellar PC vesicles and to BSA was detected by changes in 13C and 31P chemical shifts relative to the chemical shifts for aqueous acyl-CoA. When oleoyl-CoA (less than or equal to 15 mol %) was added to preformed vesicles, the 13C thioester signal (200.1 ppm) was upfield from the signal for micellar oleoyl-CoA (201.7 ppm), suggesting decreased H-bonding (partial dehydration) at the carbonyl group upon binding to the bilayer. When vesicles were prepared by cosonication of oleoyl-CoA and PC, a second peak (199.8 ppm) was seen. The major peak at 200.1 ppm broadened and shifted after addition of Dy(NO3)3 and was not seen after addition of BSA, while the peak at 199.8 ppm was unaffected by either perturbation. Thus, oleoyl-CoA in each bilayer leaflet was distinguished, and transbilayer movement was shown to be slow (t 1/2 greater than or equal to hours). PC vesicles remained intact with less than or equal to 15 mol % oleoyl-CoA, while higher oleoyl-CoA proportions produced mixed micelles. In contrast, 13C spectra revealed rapid exchange (ms) of octanoyl-CoA between the aqueous phase and PC vesicles and a low affinity for the bilayer. Thus, the binding affinity of acyl-CoA for PC bilayers is dependent on the acyl chain length. Oleoyl-CoA in the presence of BSA (1 mol/mol) gave rise to three carbonyl signals at 197.2-203.6 ppm. With 2-5 mol of oleoyl-CoA/BSA, 1-2 additional signals were observed. None of the signals corresponded to unbound oleoyl-CoA. Addition of [13C]carboxyl-enriched oleic acid to oleoyl-CoA/BSA mixtures revealed simultaneous binding of oleic acid and oleoyl-CoA to BSA, with some perturbation of binding interactions. Thus, BSA contains multiple binding sites for oleoyl-CoA and can bind fatty acid and acyl-CoA simultaneously.

Progressive enzyme changes within anatomically defined segments of rabbit nephron: demonstration with a new technique.

The kidney is an extremely heterogeneous organ, with morphological, physiological, and metabolic changes occurring from segment to segment along each nephron. To determine the heterogeneity that might exist within discrete anatomical segments of rabbit nephron, we developed a technique for making quantitative enzyme assays in serial samples, about 100 micron long, along identified segments of the nephron. Results for three enzymes in proximal convoluted and straight tubules show that adenylate kinase, an enzyme of high-energy phosphate metabolism, gradually decreases along the S1 and S2 segments of the proximal tubule, with no abrupt changes. Fructose bisphosphatase, a gluconeogenic enzyme, is high along the major portion of the proximal tubule but plummets along the final millimeter of S3. Conversely, phosphofructokinase, a glycolytic enzyme, is very low along the proximal tubule but increases sharply within the final millimeter. These data underscore the biochemical heterogeneity of the nephron, illustrating the enzyme levels may change markedly even within anatomically defined regions. They also suggest the importance of further studies of this type and demonstrate a practical means for such studies.

Bioactive cardiac substances: potent vasorelaxant activity in mammalian atria.

Mammalian atrial extracts possess natriuretic and diuretic activity. In experiments reported here it was found that atrial, but not ventricular, extract also causes relaxation of isolated vascular and nonvascular smooth muscle preparations. The smooth muscle relaxant activity of atrial extract was heat-stable and concentration-dependent and could be destroyed with protease. Rabbit aortic and chick rectum strips were used for the detection of atrial biological activity. The atrial activity was separated by column chromatography into two peaks having apparent molecular weights of 20,000 to 30,000 and less than 10,000. The atrial substance that copurified with the smooth muscle relaxant activity in both peaks caused natriuresis when injected into conscious rats. It appears that atria possess at least two peptides that elicit smooth muscle relaxation and natriuresis, suggesting an endogenous system of fluid volume regulation.

Distribution of enzymes of adenylate and guanylate nucleotide metabolism in rat nephron.

In a previous study of discrete segments of rat nephron, we reported the levels of high-energy adenylate and guanylate phosphates to be highest in the distal straight and convoluted tubules. Those findings stimulated the study of the distribution of seven enzymes involved in the following metabolic pathways of these nucleotides [Formula: see text]. The patterns of distribution of enzymes in each pathway differed greatly. The phosphodiesterases, 1 and 2, were high in glomeruli and distal tubular segments and low in proximal segments. Adenylate kinase, 3, in contrast, was high in glomeruli, proximal segments, thick ascending limb of Henle, and distal convoluted tubules. Guanylate kinase levels, 4, however, were similar in all segments. The pattern of nucleosidediphosphate kinase, 5, was high in proximal convoluted, thick ascending limb, and distal convoluted tubules. The pattern of the degradative enzyme, 5'-nucleotidase, 6, whose levels were highest in proximal segments, was opposite from that of AMP deaminase, 7, highest in the distal nephrons. These dissimilar patterns underscore the extent of nephron heterogeneity.