Unsaturated fatty acid-activated protein kinase (PKx) from goat testis cytosol.

The cytosolic fraction of goat cauda epididymis possesses a protein kinase (PKx) activity which is stimulated by a number of unsaturated fatty acids of which arachidonic acid is the best activator in absence of cAMP or Ca(2+). Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and diacylglycerol have no effect either alone or in combination. The membrane fraction does not show any appreciable kinase activity even after detergent treatment. PKx migrates as a single band of apparent molecular mass of 116 kDa on 10% SDS-PAGE after sequential chromatographic separation on DEAE-cellulose, phenyl-Sepharose, high-Q anion exchange and protamine-agarose affinity column. PKx phosphorylates histone H1, histone IIIs and protamine sulfate, but not casein. However, the best phosphorylation was obtained with a substrate based on PKC pseudosubstrate sequence (RFARKGSLRQKNV). The kinase phosphorylates two endogenous cytosolic proteins of 60 and 68 kDa. Ser residues are primarily phosphorylated although a low level of phosphorylation is observed on Thr residues also. Ca(2+) and Mn(2+) inhibit PKx activity in the micromolar range. Staurosporine is found to inhibit the PKx activity to a significant level at sub-nanomolar concentration. Lyso-phosphatidylcholine and certain detergents at very low concentrations (<0.05%) stimulate enzyme activity to some extent. The immuno-crossreactivity study with antibody against different PKC isotypes suggests that the protein kinase under study is not related to any known PKC family. Even the antibody against PKN (a related protein kinase reported in rat testis found to be activated by arachidonic acid) does not cross-react with this protein kinase. Hence we believe that the protein kinase (PKx) reported here is different even from the PKN of rat testis. The phosphorylation of endogenous proteins by the protein kinase may be involved in cell regulation including fertility regulation and signal transduction.

An Na+/K(+)-ATPase inhibitor protein from rat brain cytosol.

A protein isolated from rat brain cytosol is found to inhibit Na+/K(+)-ATPase in rat brain and kidney and H+/K(+)-ATPase from toad gastric mucosa, but has no effect on Ca2+,Mg(2+)-ATPase and Ca(2+)-ATPase isolated either from rat testis or goat spermatozoa. The inhibitor has been partially purified by ammonium sulphate precipitation followed by gel-filtration through Sephadex G-100. The inhibitor seems to bind at or close to the ATP binding site of Na+/K(+)-ATPase, such that the binding of the inhibitor to ATPase is reversible and competitive in nature with respect to the substrate. Optimum inhibition is observed at around the phase transition temperature of brain Na+/K(+)-ATPase and the inhibitory activity is only partially dependent on -SH or -NH2 group(s) of the inhibitor protein.

The in vivo inhibition of transport enzyme activities by chloroquine in different organs of rat is reversible.

The antimalarial drug chloroquine is found to inhibit Na+, K(+)-ATPase, Ca2+, Mg(2+)-ATPase, Ca(2+)-ATPase, pNPPase and acetylcholinesterase activities in different organs of rat in vivo when injected for a certain periods of time. The inhibition seems to be due to the changes in the level of phospholipid, cholesterol and the fatty acid of the lipid and the alteration of the fluidity of the microsomal membranes. However, the enzyme activities return to the normal level in about 2-3 weeks after the discontinuation of the drug suggesting that the drug effect is reversible.

The in vivo inhibition of transport enzyme activities in different organs of rat by chlorpromazine is reversible.

Chlorpromazine, an antipsychotic drug is found to inhibit Na+K(+)-ATPase, Ca(2+)-ATPase and acetylcholinesterase activities in the microsomal membranes of rat in vivo, when the drug is injected for certain periods of time. The inhibition seems to be due to the changes in fatty acid composition of lipid and microviscosity of the membranes. However, once the drug has been withdrawn, the enzyme activities are found to return to the normal level in three to five weeks, suggesting that the drug effect is reversible.

Biochemical characterization of a calcium ion stimulated-ATPase from goat spermatozoa.

The goat spermatozoa membranes isolated after treatment with octa (ethylene glycol) mono n-dodecyl ether (C12E8) followed by discontinuous sucrose density gradient centrifugation have been found to contain an ATPase that is stimulated by externally added Ca2+ only. The membrane fraction has also found to contain Mg(2+)-dependent Ca(2+)-ATPase activity, however the former activity is about 2 fold higher than the latter. The molecular weight of the enzyme is found to be about 97,000 on SDS-polyacrylamide gel. The optimum concentration of Ca2+ required for maximum activity is 3 mM for both Mg(2+)-dependent and Mg(2+)-independent Ca(2+)-ATPase. Histidine and imidazole buffers are found to be the most suitable for dependent and independent enzyme activities respectively. ATP with an optimum concentration of 4 mM is observed to be the best substrate than any other nucleotides. The inhibitors like trifluoperazine and vanadate and group specific probes e.g. DTNB and TNBS inhibit these two enzymes but at different rates. Ca(2+)-uptake study shows that the uptake in the presence of Ca2+ and ATP is higher than in the presence of Mg2+, Ca2+ and ATP. The findings lead us to believe that the Mg(2+)-independent Ca(2+)-ATPase has some role in Ca2+ transport like Mg(2+)-dependent enzyme.

Effect of chlorpromazine and gossypol on Ca(2+)-ATPase activity in the microsomal membranes of rat testes.

The microsomal membranes isolated from rat testes have been found to contain a Mg(2+)-dependent and a Mg(2+)-independent Ca(2+)-ATPase. The enzyme activities were inhibited by two contraceptive drugs--gossypol and chlorpromazine. The inhibition by the former was affected by the presence of ligand(s) and not the substrate in the incubation medium, whereas ligand(s)/substrate did not affect the inhibition by chlorpromazine. This may be explained from the fact that the binding of chlorpromazine and ligand(s)/substrate to the enzyme are independent of each other whereas in case of gossypol the ligand(s) compete with the drug at the binding site of the enzyme.