Na(+)-Ca2+ exchange in the rat brain during ontogeny.

A rise of Na(+)-Ca2+ exchange during ontogenic development was found in the rat brain which parallels brain maturation. Nerve endings are the main structure which contributes to the rise of the exchange activity.

Na(+)-Ca2+ exchange in locust striated muscles.

High Na+ + Ca2+ exchange rates comparable with those reported for crayfish striated muscle, rat heart and rat brain, were observed in locust striated muscle homogenates and membrane preparations. The Na(+)-Ca2+ exchange followed the 1st order kinetics with a Km value of 18 mumol.l-1 for Ca, the pH optimum was at 8, the temperature optimum at 30 degrees C, and the exchange was inhibited in the presence of sodium in the incubation medium, with a KiNa of approx. 25 mmol.l-1. The present results suggest a high Na(+)-Ca2+ exchange in locust striated muscles which operate on the calcium electrogenesis principle.

[Biochemical characteristics of voltage-dependent sodium channels]. / Biochemická charakteristika napät'ovozávislého sodíkového kanála.

Present knowledge on voltage dependent sodium channel is summarized in review form. Isolation procedures of channel proteins, subunit composition, posttranslational processing, distribution in various tissues, as well as channel subtypes are being dealt with. Further the properties of the native and reconstituted channels are compared and modifications of channel functions with some pharmacological agents are considered.

Na+-Ca2+ exchanger in the soluble fraction of crayfish striated muscle.

Proteins with Na+-Ca2+ exchange activity from the soluble fraction of crayfish striated muscle were inserted into asolectin proteoliposomes. A pH dependent calcium uptake with an optimum at the alkaline side and inhibition in the presence of sodium or strontium ions in the external medium was observed. When expressed per tissue wet weight the capacity for Na+-Ca2+ exchange of proteoliposomes with inserted soluble proteins was by one half higher than that of the membrane fraction and more than twice higher in comparison with the reconstituted membrane bound exchanger. Using polyacrylamide gel electrophoresis two most prominent proteins with Mr over 200 and 43 kDa could be detected in proteoliposomes with the highest Na+-Ca2+ exchange. It is assumed that protein(s) with Mr 43 kDa could represent the soluble Na+-Ca2+ exchanger in crayfish striated muscle soluble fraction.

Na+-Ca2+ exchange in plasma membranes of crayfish striated muscle.

Na+-Ca2+ exchange rates and some physico-chemical properties of the exchanger were studied in crayfish striated muscle membranes enriched in plasma membranes prepared by differential centrifugation of muscle microsomal fraction on discontinuous sucrose density gradient. The lightest subfraction with the highest Na+, K+-ATPase and Mg2+-ATPase activities also showed the highest Na+-Ca2+ exchange rates. A number of physico-chemical characteristics of the Na+-Ca2+ exchanger found in the present experiments were similar to those reported for excitable membranes of mammals, except for the temperature optimum (20 degrees C for the crayfish).

Effects of sulfhydryl reagents on Na+-Ca2+ exchange in rat brain microsomal membranes.

Effects of six thiol reagents with different physico-chemical properties were tested on the Na+-dependent 45Ca2+ transport into the rat brain microsomal membrane vesicles. The mercurials p-chlormercuribenzoate and Mersalyl effectively inhibited 45Ca2+ uptake with IC50 values in the order of 10(-4) mol X l-1 in the medium. N-ethylmaleimide and its more lipophilic analog N-(4-(2-benzoxazolyl)phenyl)maleimide were much less effective at the same concentrations. 2,2'-dithiodipyridine markedly reduced 45Ca2+ uptake already at concentrations below 10(-4) mol X l-1, whereas 5,5'-dithiobis-2-nitrobenzoate in a concentration range 10(-6)-10(-3) mol X l-1 was a weak inhibitor. Inhibitory effects of the most potent inhibitors p-chlormercuribenzoate and 2,2'-dithiodipyridine were readily reversed by 1 mmol X l-1 dithiothreitol. The results suggest that free SH groups of membrane polypeptides are involved in the functioning of the Na+-Ca2+ exchanger in the nerve tissue cell membranes.

Inhibition of Na+-Ca2+ exchange by calcium antagonists in rat brain microsomal membranes.

Na+-Ca2+ exchange rates were studied in native and/or pronase pretreated rat brain microsomal membranes in the presence of calcium channel antagonists verapamil, nimodipine and nifedipine. In native membranes all the substances used inhibited Na+-Ca2+ exchange. A relatively stronger inhibition was observed in membranes pretreated with pronase. The values of Ki for nimodipine and nifedipine did not change and it fell to about one half for verapamil. Lineweaver-Burk's plots have revealed that the verapamil inhibition in native membranes as well as in pronase pretreated ones was of a non-competitive type; Km for calcium oscillated around 15 mumol.l-1. It is suggested that the inhibition strength depends on the access of inhibitors to the membrane binding sites as well as on the solubility of inhibitors in membrane lipids.

Na+-Ca2+ exchange in rat brain microsomal membranes pretreated with pronase and/or SDS.

The effects of pronase and/or SDS pretreatment on Na+-Ca2+ exchange were studied in rat brain microsomal membranes. Pronase in concentrations that liberated 11% of the membrane proteins stimulated the Na+-Ca2+ exchange. When about 24% of the proteins were split off, the results did not differ from those in control experiments. When 40% or more of the proteins were solubilized, Na+-Ca2+ exchange was abolished. Pronase pretreatment did not change the Km value for Ca2+, it increased Vmax only. The effect of pronase was partially blocked by Trasylol. Neuraminidase had no effect on Na+-Ca2+ exchange. SDS pretreatment of the membranes inhibited Na+-Ca2+ exchange: when 25% of membrane proteins were solubilized with SDS, the Na+-Ca2+ exchange was abolished while the same amount of proteins split off with pronase did not change the rate of Na+-Ca2+ exchange as related to membrane proteins. Ischaemia lasting for 2-4 h or complete hypoxia which should stimulate endogenous proteinases due to the rise of free intracellular calcium did not influence the Na+-Ca2+ exchange. A decrease in Na+-Ca2+ exchange rate was observed when proteins with molecular weight between 45,000 and 20,000 were split off from the membranes. It is assumed that the Na+-Ca2+ antiporter is a polypeptide from the group of proteins within the above molecular weights.

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.

Alanine aminotransferase and some other enzymes in different populations of free brain cortex mitochondria.

The activity of certain key enzymes involved in glutamic acid metabolism was studied in purified brain mitochondria and in mitochondrial subfractions separated in a discontinuous 1.2--1.6 mol/l sucrose gradient. Alanine aminotransferase and glutamate dehydrogenase were found to be matrix enzymes and aspartate aminotransferase to be associated with the inner mitochondrial membranes. After the purified mitochondria had been separated into 5 subfractions, aspartate aminotransferase and NAD+-dependent isocitrate dehydrogenase were found to be bound to the lighter mitochondrial subfractions settling at the 1.4--1.5 mol/l sucrose boundary while alanine aminotransferase, 4-aminobutyrate transaminase and glutamate dehydrogenase were associated with the heavier subfractions settling below 2.4 mol/l sucrose. The highest specific activity of the given enzymes was found in the subfraction settling at the 1.4--1.5 mol/l sucrose boundary, the only exception being alanine aminotransferase activity, whose maximum was found in the subfractions settling in 1.5 and 1.6 mol/l sucrose. It was concluded that alanine aminotransferase, in conjunction with glutamate dehydrogenase, is linked to NH3 binding and to the oxidation of reduced adenine nucleotides; in addition, alanine aminotransferase is presumed to have the function of transporting glutamate from the mitochondria to the extramitochondrial space.

Subcellular distribution and some properties of alanine aminotransferase in striated muscles of the crayfish, trout, carp, frog, pigeon and rabbit.

Subcellular distribution and some physicochemical properties of alanine aminotransferase in striated muscles of the crayfish, trout, carp, frog, pigeon and rabbit were studied. It was established that: (1) Alanine aminotransferase activity in all mentioned animals occurred almost entirely in the cytosolic fraction of the muscles. Total activity and activity per mg protein were highest in crayfish and pigeon muscles and lowest in carp and trout muscles. (2) The pH optimum for the muscles of homoiotherms and poikilotherms ranged from 7.5 to 8, Km values for L-alanine were of the order 10(-3)--10(-2) M and those for alpha-ketoglutarate 10(-4) M. (3) A 10 degree C temperature increase of the incubation medium was accompanied by a 70--90% increase in activity. (4) The higher the alanine aminotransferase activity of the muscles, the relatively higher their alanine production during electrical stimulation. (5) From the above results it is concluded that alanine aminotransferase in striated muscles regulates the rate of glycolysis and energy production under conditions of anaerobiosis through the formation of alanine.