Multiple forms of calpastatin in pig brain

Pig brain was found to contain two calpain-specific, heat-stable inhibitory fractions which could be separated by DEAE-cellulose chromatography. CS-0.1, which was eluted from the column at 0.1 M NaCl, was identified as an ordinary, well-known calpastatin. CS-0.2, eluted at 0.2 M NaCl, was different from CS-0.1 in that it inhibited calpain 1 more strongly than calpain II and that it did not cross-react with anti-calpastatin antibodies. Partial purification indicated that CS-0.2 contained inhibitor proteins smaller than ordinary calpastatin, but whether they are the products derived from CS-0.1 or entirely different genetic products has not yet been determined.     

Multiple forms of acetylcholinesterase from pig brain

1. A number of methods of solubilization of pig brain acetylcholinesterase (EC 3.1.1.7) were studied. The multiple enzymic forms of the resultant preparations were examined by polyacrylamide-gel electrophoresis. 2. Butanol extraction, Nagarase treatment and ultrasonication proved unsuitable as preparatory methods, but detergent treatment (Triton X-100, Triton X-100-KCl and lysolecithin) gave good yields. 3. Separation of soluble enzyme in three systems of polyacrylamide-gel electrophoresis were compared and the relative advantages are discussed. 4. By using a 6% (w/v) gel and continuous buffer system two forms of acetylcholinesterase were detected in Triton X-100-solubilized enzyme, but the incorporation of a sample and spacer gel and a discontinuous buffer system resolved this into four components. The forms of the soluble enzyme extracted by different methods differed in mobility. 5. With gradient polyacrylamide-gel electrophoresis between two and six forms were detected, depending on the method used for extraction. The average molecular weights of the five forms most frequently found were 60000, 130000, 198000, 266000 and 350000. 6. Treatment of the Triton X-100-extracted enzyme with 2.5m-urea altered the pattern and evidence of dissociation was observed. 7. The results are discussed in the light of present theories on the molecular structure of acetylcholinesterase.     

Multiple forms of pig somatotropin]

The multiple forms of pig somatotropin were investigated. Four monomer hormone variants were isolated and some of their physico-chemical characteristics were determined. It is shown that they differ in their molecular mass, N-terminal amino acid residues and amino acid composition. The results of work permit supposing that the identified somatotropin forms are controlled by two different genes. The age dependence of spectrum of somatotropin monomer forms is demonstrated.     

Two forms of GABA transaminase in pig brain

Two forms of GABA transaminase which could be distinguished by ion-exchange chromatography have been separated and purified in pig brain. The two forms have differentK _m values for -ketoglutarate and show different degrees of inhibition by various salts. Although the two forms are separable, they have identical antigenic properties, pH optima, and NH₂ terminal amino acid composition, and they appear to be of the same molecular size. The biological significance or the relationship between multiple forms of GABA transaminase is not yet understood.     

Multiple forms of human phosphoglycerate kinase.

Phosphoglycerate kinase was isolated by affinity chromatography from human skeletal muscle and erythrocytes. As in the tissue extracts, the purified enzyme showed in Cellogel electrophoresis one major and two minor bands with phosphoglycerate kinase activity. The multiple forms were separated by chromatography on CM-Sepharose. From the three separated forms, A, B, and C, the latter was not detectable in electrophoresis of tissue extracts or in the purified unresolved phosphoglycerate kinase. The faintest, most anodically migrating form observed in the tissue extracts could not be isolated in pure form by chromatography on CM-Sepharose. The electrophoretic mobility of the phosphoglycerate kinase forms depended strongly on the buffer systems used. The different forms had identical molecular weight, substrate affinity, and heat stability and were inhibited to the same extent by antibody. They could also not be separated by column affinity chromatography. Small differences were found in thiol group content and in the specific activity, the latter being a consequence of diminished free sulfhydryl residues. Exposure to either reductive or oxidative conditions changed the specific activity, but did not result in interconversion among the pure forms. The multiple forms probably arise as a result of epigenetic factors occurring after the primary polypeptide chain has been synthesized.     

Multiple forms of cyclic nucleotide phosphodiesterase in pig epidermis.

Pig epidermal cyclic nucleotide phosphodiesterases (EC 3.1.4.16) have been partially purified by DEAE-cellulose column chromatography. At least three different forms of the epidermal phosphodiesterases were identified. They were cyclic GMP-specific, cyclic GMP- and cyclic AMP-hydrolyzing and apparently a cyclic AMP-specific enzyme: the first two forms were soluble and the last was the particulate enzyme. The cyclic GMP-specific soluble fraction had a relatively low Km, the cyclic GMP- and cyclic AMP-hydrolyzing fraction had a high Km for the respective substrates and the third particulate enzyme had both high and low Km values for cyclic AMP. The cyclic GMP-hydrolyzing enzyme was localized almost entirely in the soluble fraction, whereas cyclic AMP-hydrolyzing enzyme was distributed to both soluble and particulate fractions. Thus, our studies show that the multiple forms of pig epidermal enzyme differ distinctly in their substrate affinity, specificity and subcellular distribution.     

The glycine-rich loop of adenylate kinase forms a giant anion hole

The conformation of the glycine-rich loop of adenylate kinase is described in detail. It forms a giant anion hole for a sulfate ion, which presumably mimicks a nucleotide phosphoryl group. This loop had been called flexible, because at pH values of 6 or below it is displaced in the crystal. In the region of this loop the adenylate kinases are probably homologous to the p21 proteins. Is is known that a mutation in this loop at residue 12 of p21 causes cell transformation and therefore cancer. Other potentially homologous proteins are indicated.     

Multiple molecular forms of rat brain enkephalinase

Rat brain enkephalinase has been partially purified by ion exchange chromatography, chromatofocusing, and affinity chromatography on immobilized lectins. Ion exchange chromatography resolved two principle forms of enkephalinase designated A1 and A2. Both enkephalinase A1 and A2 are bound to immobilized lentil lectin while chromatography on immobilized wheat germ lectin resolved each of the principle forms into two subforms, A1, 1, A1, 2, A2, 1, and A2, 2. All four enkephalinase forms have similar, if not identical kinetic properties. The possible implications of multiple molecular forms of enkephalinases are discussed.     

Phosphorylation of pig brain diacylglycerol kinase by endogenous protein kinase

Pig brain diacylglycerol kinase did not catalyze autophosphorylation. However, the kinase was phosphorylated on serine, when immunoprecipitated from the partially purified enzyme preparation preincubated with Mg2+ and [gamma-32P]ATP. The action of the endogenous protein kinase phosphorylating diacylglycerol kinase was independent of cyclic nucleotides and Ca2+, and became maximum at pH 5.5. Although the extent of enzyme phosphorylation was limited (maximally about 0.25 mol Pi incorporated per mol kinase), the results show that diacylglycerol kinase can be a phosphoprotein.     

Multiple forms and inhibitors of uridine-cytidine kinase in neoplastic cells

1. Two forms (isozymes) of uridine (urd)-cytidine (cyd) kinase are present in the 30-50% ammonium sulfate fraction of the cytosols of L1210 ascites leukemia cells and a human malignant lymphoma. 2. These findings confirm those which described multiple forms of urd-cyd kinase in tumors with rapid growth rate. 3. Studied of inhibitors (nucleoside analogs) of urd-cyd kinase derived from L1210 and 6410 leukemia cells resulted in the finding of four possible inhibitors of this enzyme.     

Creutzfeldt-Jakob infection increases adenylate cyclase activity in specific regions of guinea pig brain

Creutzfeldt-Jakob disease is a slow, infectious, progressive neurological disorder which results in human dementia. Synaptic membranes from various brain regions of guinea pigs infected with Creutzfeldt-Jakob disease show increased guanyl nucleotide- or 5-hydroxytryptamine-mediated activation of adenylate cyclase. This increased enzyme activity appears due, primarily, to facilitated 'coupling' between the GTP-binding protein which stimulates adenylate cyclase (GNs) and the catalytic moiety of that enzyme rather than increased sensitivity to 5-hydroxytryptamine. It is possible that this phenomenon is due to direct effects of the Creutzfeldt-Jakob infectious agent, or a pathological product resulting from that agent, upon synaptic membrane adenylate cyclase.