[The role of Na(+) pump inhibition induced by Ca2(+) ions in the development of pathologic changes during calcium overload in myocardial cells]. / Uloha inhibície Na(+)-pumpy indukovanej Ca2(+)-iónmi pri rozvoji patologických zmien pocas pret'azenia myokardiálnych buniek vápnikom.

Ca2+ ions cause marked inhibition of (Na+ + K+)-ATPase. Strontium and barium ions exert a less pronounced inhibition of (Na+ + K+)-ATPase activity. These findings suggest the existence of a regulatory binding site for calcium ions on the (Na+ + K+)-ATPase molecule. Under physiological conditions, this binding site is presumably involved in stopping the activity of (Na+ + K+)-ATPase during depolarization of the sarcolemma. In pathological conditions which are characterized by calcium overload of cardiomyocytes inhibition of (Na+ + K+)-ATPase by calcium ions can significantly contribute to massive damage of myocardial cells. (Fig. 3, Ref. 26.).

The 165-kDa peptide of the purified skeletal muscle dihydropyridine receptor contains the known regulatory sites of the calcium channel.

The dihydropyridine receptor purified from rabbit skeletal muscle yields in the presence of dithiothreitol and sodium dodecyl sulfate on polyacrylamide gels bands of apparent molecular mass 165 +/- 5, 130 +/- 5, 55 +/- 3, 32 +/- 2 and 28 +/- 1 kDa (chi +/- SEM, n = 12). Under nonreducing conditions, the 130 kDa and 28-kDa peptides migrate as a single peptide of 165 kDa. These peptides were separated on a HPLC size-exclusion column. The specific absorption coefficients of the isolated peptides were determined. From these a stoichiometry of 1:1.7 +/- 0.2:1.4 +/- 0.3 (chi +/- SEM of 12 experiments with three different preparations) was calculated for the 165-kDa, 55-kDa and 32-kDa peptides. The relative amount of the 130/28-kDa peptide varied with different preparations. Tryptic, chymotryptic and V-8 protease peptides of the isolated proteins suggested that the 130/28-kDa peptide was not related to the 165-kDa peptide. The dihydropyridine photoaffinity analog (+/-)-azidopine was specifically incorporated only into the 165-kDa peptide with an efficiency of about 2.4%. The azido analog of desmethoxyverapamil, LU 49888, was specifically incorporated into the same peptide with an efficiency of 1.5%. These results suggest that only the 165-kDa peptide contains the regulatory sites detected so far in the voltage-operated L-type calcium channel. They suggest further that the 130/28-kDa peptide, which migrates as a 165-kDa peptide under nonreducing conditions, does not contain high-affinity binding sites for the calcium channel blockers.

Alanine aminotransferase in bovine brain: purification and properties.

Mitochondrial and cytosolic alanine aminotransferases (EC 2.6.1.2) were partially purified (140- and 180-fold), respectively) from bovine brain cortex by means of (NH4)2SO4 precipitation, gel filtration on Sephadex G-150, and in-exchange chromatography on DEAE A-50 and characterized. The enzymes exhibited identical molecular weights (110,000 +/- 10,000) and pH optima (7.8), but were eluted from CM Sephadex C-50 at different ionic strengths. Isoelectric focusing of the enzymes indicated a pI value of 5.2 for the cytosolic enzyme and 7.2 for the mitochondrial enzyme. The Km values of the mitochondrial enzyme were 5.1 mM, 6.6 mM, 0.7 mM, and 0.4 mM and of the cytosolic isozyme were 30.3 mM, 4.3 mM, 0.7 mM, and 0.5 mM for alanine, glutamate, 2-oxoglutarate, and pyruvate, respectively. The results indicated that two forms of alanine aminotransferase exist in nerve tissue, which suggests that they may play different roles in the cellular metabolism of nerve tissue.

Isoelectric focusing of the alanine aminotransferase isoenzymes from the brain, liver and kidney.

1. The isoelectric points of mitochondrial and cytosolic alanine aminotransferases (EC 2.6.1.2) of bovine brain cortex, of rat, guinea-pig and human livers and of rat and pig kidney cortices were estimated. 2. The pI of the cytosolic enzymes were found at about 5.2 and those of mitochondrial ones at 7.2. 3. It is suggested that in the examined tissues except for the human liver there are two different proteins with the alanine aminotransferase activity.