Calcium transport and Ca2+-ATPase activity in ram spermatozoa plasma membrane vesicles

Plasma membrane vesicles, isolated from ejaculated ram sperm, were found to contain Ca²⁺-activated Mg²⁺-ATPase and Ca²⁺ transport activities. Membrane vesicles that were exposed to oxalate as a Ca²⁺-trapping agent accumulated Ca²⁺ in the presence of Mg²⁺ and ATP. The $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ for Ca²⁺ uptake was 33 nmol/mg protein per h, and the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for Ca²⁺ and ATP were 2.5 μM and 45 μM, respectively. 1 μM of the Ca²⁺ ionophore A23187, added initially, completely inhibited net Ca²⁺ uptake and, if added later, caused the release of Ca²⁺ previously accumulated. A Ca²⁺-activated ATPase was present in the same membrane vesicles which had a $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of 1.5 μmol/mg protein per h at free Ca²⁺ concentration of 10 μM. This Ca²⁺-ATPase had $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values of 4.5 μM and 110 μM for Ca²⁺ and ATP, respectively. This kinetic parameter was similar to that observed for uptake of Ca²⁺ by the vesicles. The Ca²⁺-ATPase activity was insensitive to ouabain. Both Ca²⁺ transport and Ca²⁺-ATPase activity were inhibited by the flavonoid quercetin. Thus, ram spermatozoa plasma membranes have both a Ca²⁺ transport activity and a Ca²⁺-stimulated ATPase activity with similar substrate affinities and specificities and similar sensitivity to quercetin.     

Legumin Synthesis in Developing Cotyledons of Vicia faba L

The synthesis of legumin in developing cotyledons of Vicia faba L. has been examined as a potential system for approaching the problem of differential gene expression. The pattern of legumin synthesis was determined during the growth of the cotyledon by microcomplement fixation which provided a sensitive and specific assay for legumin in the presence of vicilin. Legumin was detected even in young cotyledons. However, when the cotyledons were about 10 millimeters long, and cell division was essentially complete, there was a sharp increase in the rate of legumin accumulation.     

Role of sodium in determining alternate pathways of aerobic citrate catabolism in Aerobacter aerogenes

In contrast to the absolute Na(+) requirement for anaerobic growth of Aerobacter aerogenes on citrate as sole carbon source, aerobic growth of this microorganism did not require the presence of Na(+). However, Na(+) (optimal concentration, 10 mm) did increase the maximal amount of aerobic growth by 60%, even though it did not change the rate of growth. This increase in growth was specifically affected by Na(+), which could not be replaced by K(+), NH(4) (+), Li(+), Rb(+), or Cs(+). Enzyme profiles were determined in A. aerogenes cells grown aerobically on citrate in media of varying cationic composition. Cells grown in Na(+)-free medium possessed all the enzymes of the citric acid cycle including alpha-ketoglutarate dehydrogenase, which is repressed by anaerobic conditions of growth. The enzymes of the anaerobic citrate fermentation pathway, citritase and oxalacetate decarboxylase, were also present in these cells, but this pathway of citrate catabolism was effectively blocked by the absence of Na(+), which is essential for the activation of the oxalacetate decarboxylase step. Thus, in Na(+)-free medium, aerobic citrate catabolism proceeded solely via the citric acid cycle. Addition of 10 mm Na(+) to the aerobic citrate medium resulted in the activation of oxalacetate decarboxylase and the repression of alpha-ketoglutarate dehydrogenase, thereby diverting citrate catabolism from the (aerobic) citric acid cycle mechanism to the fermentation mechanism characteristic of anaerobic growth. The further addition of 2% potassium acetate to the medium caused repression of citritase and derepression of alpha-ketoglutarate dehydrogenase, switching citrate catabolism back into the citric acid cycle.     

Stereospecificity of citrate synthetase in relation to glutamate biosynthesis by extracts of Chloropseudomonas ethylicum

The synthesis of citric and glutamic acids by extracts of Chloropseudomonas ethylicum was studied with labeled precursors. When acetyl-coenzyme A-1-(14)C was used as substrate, only 0.1% of the total radioactivity was found in the C-5 position of citric acid; whereas, with oxalacetate-4-(14)C as substrate, 100% of the total radioactivity was found in C-5. These results demonstrated that the Chloropseudomonas citrate synthetase had an absolute stereospecificity, identical to that of the pig heart synthetase. The distribution of radioactivity in the glutamic acid synthesized from acetyl-coenzyme A-1-(14)C was 0% in C-1 and 94.0% in C-5; whereas the glutamic acid formed from oxalacetate-4-(14)C contained 89.6% in C-1 and 0.5% in C-5. This distribution is entirely consistent with the biosynthesis of glutamic acid from citric acid via aconitase, d(s)-isocitrate, and l-glutamate dehydrogenases. The presence of l-glutamate dehydrogenase in extracts was demonstrated. The stereospecificity of the citrate synthetase and the pattern of glutamate labeling further establish that the aconitase of Chloropseudomonas is completely stereospecific.