Conditions for reproducible detection of calmodulin and S100 beta in immunoblots

The conditions used in some immunoblot procedures can fail to detect calmodulin, S100 proteins, and other proteins with similar physical properties. We describe here some of the basis of this difficulty, and provide an immunoblot protocol that allows the rapid and reproducible detection of calmodulin and S100 beta in crude biological samples. These proteins are rapidly transferred from sodium dodecyl sulfate-polyacrylamide gels to membrane matrices, and retention on the matrix is enhanced by a glutaraldehyde fixation step. Either nitrocellulose or a positively charged membrane filter (ZetaProbe) can be used as the immobilizing matrix. By combining microslab gel electrophoresis, 30 min electrophoretic transfer, and glutaraldehyde fixation of nitrocellulose paper, an immunoblot analysis can be done in an 8-hr day.     

Three-step preparation and purification of phosphorus-33-labeled creatine phosphate of high specific activity

Rabbit heart mitochondria were used as a source of enzymes for the synthesis of phosphoruslabeled creatine phosphate. This method is based on the coupled reaction between mitochondrial oxidative phosphorylation and mitochondrial-bound creatine kinase. It is possible to convert more than 90% of the inorganic phosphate (P_i) to creatine phosphate. The method used only small amounts of adenine nucleotides which led to a product with only slight nucleotide contamination. This could be removed by activated charcoal extraction. For further purification, a method for the removal of residual P_i is described.     

Changes in key regulatory enzymes of hepatic carbohydrate metabolism after glucose loading of starved rats

When glucose was given to starved rats there was an increase in both 6-phosphofructo 2-kinase and pyruvate kinase activity and a decrease in fructose 2,6-bisphosphatase activity 30 min and 60 min later. These changes were accompanied by an increase in glycogen deposition and by modest, but significant increases in fructose 2,6-bisphosphate levels at the same time. Metabolite measurements indicated that flux through 6-phosphofructo 1-kinase and pyruvate kinase were increased. These results suggest that although glycogen deposition may occur via the gluconeogenic pathway, glycolysis is activated at the same time by changes in the phosphorylation state of key regulatory enzymes as well as by the small rise in fructose 2,6-bisphosphate.     

Carrier mediated GABA translocation into rat brain mitochondria

GABA added to rat brain mitochondria causes oxidation of intramitochondrial NAD(P)H as well as inducing glutamate efflux from the mitochondrial matrix. The rate of NAD(P)H oxidation shows saturation characteristics, depends on GABA transport across the mitochondrial membrane and is inhibited by non-penetrant compounds and by the metal-complexing agent bathophenanthroline. These results show the existence of a specific GABA carrier. Inhibition studies strongly suggest the existence of two separate binding sites, namely the GABA binding site and the dicarboxylates binding site, as well as suggest the presence of a metal ion (ions) at GABA binding site. The occurrence of a GABA/GLUTAMATE antiport is proposed which allows a cyclical route to account for GABA synthesis and degradation in brain.     

Fructose 2,6-bisphosphate, sugar phosphates and adenine nucleotides in the regulation of glucose metabolism in the lactating rat mammary gland

Fructose 2,6-bisphosphate is present in the rat mammary gland, rising from a value of 1.4 nmol/g in pregnancy to 4.3 nmol/g tissue at 14 days lactation; the equivalent values calculated/ml intracellular water are 5.2 and 11.6 nmol, respectively. The tissue content of fructose 6-phosphate, fructose 1,6-bisphosphate, ATP and phosphoenolpyruvate remain relatively constant in the transition from pregnancy to the height of lactation. The changes in AMP, cyclic AMP, and citrate content of the mammary gland during lactation are such as to promote an increase in fructose 2,6-bisphosphate formation and flux through phosphofructokinase.     

Inositol trisphosphate activates pyruvate dehydrogenase in isolated fat cells

Inositol trisphosphate, when added to permeabilized rat fat cells, led to a several-fold increase of pyruvate dehydrogenase activity due to conversion of the inactive phospho form (PDHb) to the active, dephospho form (PDHa). It is suggested that inositol trisphosphate, probably through intracellular Ca2+-mobilisation, acts as a physiological mediator of insulin for activation of the mitochondrial PDH-complex.     

Lectin-like binding of Bacillus thuringiensis var. kurstaki lepidopteran-specific toxin is an initial step in insecticidal action

The two delta-endotoxins comprising the Bacillus thuringiensis var. kurstaki HD1 insecticidal protein crystal were separated. The lepidopteran-specific protoxin was activated in vitro and its mechanism of action investigated. Toxicity towards Choristoneura fumiferana CF1 cells was specifically inhibited by preincubation of the toxin with N-acetylgalactosamine and N-acetylneuraminic acid. The lectins soybean agglutinin and wheat germ agglutinin, which bind N-acetylgalactosamine, also inhibited toxicity. Since N-acetylneuraminic acid is not known to occur in insects, these results suggest that the toxin may recognise a specific plasma membrane glycoconjugate receptor with a terminal N-acetylgalactosamine residue.     

Microbial mineralization of organic phosphate in soil

Summary Phosphate-dissolving microorganisms were isolated from non-rhizosphere and rhizosphere of plants. These isolates included bacteria, fungi and actinomycetes. In broth cultures, Gram-negative short rod,Bacillus andStreptomyces species were found to be more active in solubilizing phosphate thanAspergillus, Penicillium, Proteus, Serratia, Pseudomonas andMicrococcus spp. The sterile soils mixed with isolated pure culture showed slower mineralization of organic phosphate than that of non-sterile soil samples at all incubation periods. Maximum amount of phosphate mineralization by isolated microorganisms were obtained at the 60th and the 75th day of incubation in sterile and non-sterile soils respectively. The mixed cultures were most effective in mineralizing organic phosphate and individuallyBacillus sp. could be ranked next to mixed cultures. Species ofPseudomonas andMicrococcus were almost the same as that of the control under both sterile and non-sterile conditions.     

Localization of pyrodoxal phosphate binding site on the mero-receptor domain of the glucocorticoid receptor

Previous studies have demonstrated that the vitamin pyridoxal phosphate can alter the physicochemical properties of glucocorticoid receptors. We now report the localization of a pyridoxal phosphate binding site within the mero-receptor domain of this glucocorticoid receptor. Mero-glucocorticoid receptors that are generated by trypsin (10 μg/ml) or chymotrypsin (100 μg/ml) digestion of intact receptors sediment as 2.6 S species on 5–20% sucrose gradients in the presence or absence of pyridoxal phosphate. Mero-glucocoritcoid receptors prepared by exogenous proteinases are hydrophobic and show no affinity for DEAE Bio-Gel A. Treating either trypsin-generated or chymotrypsin-generated mero-receptors with pyridoxal phosphate rapidly converts the proteins (60 and 35%, respectively) into forms that bind to DEAE Bio-Gel A. Induction of DEAE binding is specific to pyridoxal phosphate, for treating mero-receptors with pyridoxal, pyridoxamine or pyridoxine phosphate is ineffective. Furthermore, DEAE binding cannot be induced by adding other pyridoxal phosphate-treated cytosols to untreated mero-receptors. High-resolution polyacrylamide gel isoelectric focussing studies indicated that treating mero-receptor generated by either proteinase with pyridoxal phosphate shifted the isoelectric points of lower pH values. The conversion of the mero-receptor to a more acidic form also occurred when the intact glucocorticoid receptor was treated with the vitamin prior to proteolysis. These studies localize at least one pyridoxal phosphate binding site on the mero-receptor domain of the rat thymocyte glucocorticoid receptor.