Calmodulin N-methyltransferase. Partial purification and characterization

The distribution, properties, and substrate specificity of S-adenosylmethionine:calmodulin (lysine) N-methyltransferse (EC 2.1.1.60, calmodulin N-methyltransferase) of the rat have been studied. This enzyme is cytosolic and is found at high levels in tissues with high levels of calmodulin and at low levels in tissues with little calmodulin. In liver, heart, and skeletal muscle, which have low levels of calmodulin and very low calmodulin N-methyltransferase activity (a low ratio of calmodulin N-methyltransferase to calmodulin), calmodulin was found to be incompletely methylated, as judged by its ability to act as a substrate for purified calmodulin N-methyltransferase. Calmodulin N-methyltransferase was purified 470-fold with a 33% yield from rat testis cytosol, using ammonium sulfate precipitation and chromatography on DEAE-cellulose, CM-Sepharose, and Sephadex G-100. At pH 7.4, calmodulin N-methyltransferase did not bind to DEAE-cellulose, but bound strongly to CM-Sepharose. The enzyme eluted from Sephadex G-100 with an apparent molecular weight of 55,000. Purified calmodulin N-methyltransferase was incubated with extracts of rat tissues, and [methyl-3H]AdoMet and methylated proteins were resolved by electrophoresis in an attempt to discover substances other than calmodulin, but this enzyme only catalyzed the methylation of calmodulin, indicating a high degree of substrate specificity. Conditions were established for the in vitro preparative methylation of des(methyl)-calmodulin from Dictyostelium discoideum. Three moles of methyl/mol of calmodulin were incorporated into lysine 115 of des(methyl)calmodulin, resulting in the formation of 1 mol of trimethyllysine at the site normally methylated in calmodulins from most species. Activation of cyclic nucleotide phosphodiesterase by des(methyl)calmodulin was indistinguishable from activation by in vitro methylated or sham methylated Dictyostelium calmodulin, indicating that methylation does not affect the ability of calmodulin to activate this enzyme.     

Purification and immunological characterisation of two calcium-activated neutral proteinases from rabbit skeletal muscle

Two Ca2+-activated neutral proteinases have been prepared to a high degree of purity from rabbit skeletal muscle. One, calpain I, is optimally activated by 100 microM Ca2+ and the other, calpain II, by 1 to 2 mM Ca2+. Both enzymes have two subunits of molecular weight 80 000 and 28 000. Antibodies have been raised against the native forms of both enzyme. It was found that the antibody to native calpain I reacted only with calpain I and not with calpain II, and similarly the antibody to native calpain II reacted only to calpain II. This suggested that the epitopes in the two enzymes are located in regions that are structurally different. However, immunoblotting of the denatured calpains after SDS-polyacrylamide-gel electrophoresis revealed cross-reaction between the two subunits for both enzymes. Therefore, although the denatured enzymes have common antigenic sites it would appear that these are not exposed equally in the native proteins.     

Partial purification and characterization of an aldohexose 1-P phosphatase from pig skeletal muscle

An enzyme with a molecular weight of 54,000 which possesses phosphatase activity acting on glucose 1-P, galactose 1-P and mannose 1-P has been partially purified and characterized from pig skeletal muscle. The enzyme is free of phosphoglucomutase and galactokinase activities, and it possesses a neutral optimum pH. Pi acts as an inhibitor; glucose, galactose and mannose do not produce any effect. Divalent cations are required for activity, Mg2+ being the most effective activator. Micromolar levels of fluoride and millimolar levels of chloride act as inhibitors; however, vanadate does not produce any effect. The enzyme may have an important role when galactose accumulates in tissues; for example, in galactosemic patients and in young animals ingesting high-galactose diets.     

Complete monitoring of the purification of the plasma membrane from rabbit skeletal muscle

A fast and reproducible purification procedure for rabbit skeletal muscle plasma membrane is described. Each step was monitored by determination of tetrodotoxin, ouabain and insulin receptors. A ouabain-sensitive K+-stimulated and a Ca2+-dependent phosphatases, probably identical to, respectively the (Na+-K+) and Ca2+-ATPases, were also evaluated. All plasma membrane receptors and the ouabain-sensitive activity accumulated in the lightest fraction separated by sucrose gradient centrifugation (peak at 18% sucrose; purification from crude homogenate, 30-fold).     

Partial purification and properties of the isozymes of S-adenosylmethionine synthetase from sheep liver

Three forms of AdoMet synthetase were separated from sheep liver. The apparent molecular weights of the native isozymes were 122,000, 62,400 and 70,800 for the alpha-, beta 1- and beta 2-form, respectively and beta 1 was the predominant form. The alpha-form exhibited negative cooperativity with [S] 0.5 values of 31 microM for methionine and 62 microM for ATP; while the two beta-forms exhibited positive cooperativity with [S]0.5 values for methionine of 82 microM and 70 microM and those for ATP of 572 microM and 505 microM for the beta 1- and beta 2-form, respectively. Dimethylsulfoxide markedly stimulated the activities of the two beta-forms at low methionine concentrations. However, at high methionine levels, it inhibited the activity of the beta 2-form but not that of the beta 1-form. The effect of dimethylsulfoxide on the alpha-form was not significant. AdoMet was inhibitory at high concentrations. However, it had a slight stimulatory effect on the two beta-forms at low concentrations when methionine level was also low. These results suggest that AdoMet synthetase is a regulatory enzyme and the reaction rate in vivo can be directly influenced by substrate and product concentrations.     

Isolation, purification and structure of molecules of a vasoactive component from rabbit skeletal muscle

A low molecular weight factor with pronounced vasoactive properties has been isolated from rabbit muscle. The procedure for lyophilized tissue extract purification included fractional methanol extraction with subsequent column chromatography on TSK-GEL Toyopearl, DEAE-Toyopearl 650 ion-exchanger and Sephadex G-10. The resulting preparation was homogeneous as evidenced from reversed phase HPLC. The structure of the factor was examined by using UV- and IR-spectrophotometry as well as by PMR and 13C-NMR. It was found that the vasoactive factor includes an inosine nucleotide structure covalently bound to a di- or tri-alanine peptide while the phosphate group is free. It is suggested that the binding of the peptide to the inosine moiety occurs via a C-terminal carboxyl of the peptide and pentose hydroxyls. It seems probable that the vasoactive effect is a result of esterification of the purine nucleotides with the peptide.     

Partial purification of L-asparaginyl-tRNA synthetase from rabbit liver

Asparaginyl-tRNA synthetase activity of both a crude extract of an acetone powder of rabbit liver and also of material fractionated with ammonium sulphate eluted at the void volume when chromatographed on Sephadex G-100. Chromatography on DEAE-cellulose in presence of glycerol and mercaptoethanol led to considerable losses in activity, the loss being related to the precise conditions employed. These losses of activity were due to partial separation of two components, each with a molecular weight in the region of 35,000, required for full enzymatic activity leading to the production of asparaginyl-tRNA.     

Identification,partial purification,and localization of a neutral sphingomyelinase in rabbit skeletal muscle: Neutral sphingomyelinase in skeletal muscle

Transgenic and gene targeting approaches have now been applied to a number of genes in order to investigate the metabolic disorders that would result by manipulating insulin action or pancreatic -cell function in the mouse. The availability of such mutant mice will allow in the future to develop animal models in which the pathophysiologies resulting from polygenic defects might be reconstituted and studied in detail. Such animal models hopefully will lead to better understanding of complex polygenic diseases such as non-insulin-dependent diabetes mellitus (NIDDM).     

Histidine decarboxylase from rat and rabbit brain: Partial purification and characterization

Histidine decarboxylase, the synthetic enzyme for histamine, was partially purified from regions of rat or rabbit brain rich in the enzyme. The enzyme was purified using ion exchange and hydrophobic column chromatography and chromatofocusing. Approximately 70-fold and 110-fold enrichments were attained from rat and rabbit brain, respectively. Rat and rabbit brain histidine decarboxylase had isoelectric points of pH 5.4 and 5.6, Km values of 80 M and 120 M histidine and Vmax values of 210 and 625 pmol histamine formed/hr-mg protein, respectively. The partially purified histidine decarboxylase from both sources was dependent on pyridoxal phosphate for maximal activity and was inhibited by -fluoromethylhistidine, nickel chloride and cobaltous chloride but was not inhibited by impromidine, -methyldopa, DTNB, zinc chloride or mercuric chloride. The enzyme had a broad pH optimum between pH 7.2 and 8.0. These studies provide further information on the characteristics of mammalian histidine decarboxylase from brain.     

Partial purification and characterisation of a 2,4,5-trichlorophenol detoxifying O-glucosyltransferase from wheat

An enzyme preparation with UDP-glucose-dependent O-glucosyltransferase (OGT; EC 2.4.1.-) activity toward 2,4,5-trichlorophenol has been purified 215-fold from wheat shoots. The OGT co-purified with the major extractable glucosylating activity toward the flavonol quercetin and was characterised as a monomeric 53 kDa protein. Among the xenobiotic phenols tested, the purified enzyme preparation showed at least a 10-fold preference for 2,4,5-trichlorophenol. When assayed with flavonoids, the OGT was active toward flavonols and coumestrol, showing a clear preference for 3-hydroxy flavone when incubated with a range of monohydroxylated flavonoids. It was concluded that the major 2,4,5-trichlorophenol-detoxifying OGT in wheat shoots is most probably a flavonol-3-O-glucosytransferase.     

Cysteine proteinase inhibitors from rabbit skeletal muscle

1. Two cysteine proteinase inhibitors, I-T (Mr = 29,000) and I-S (Mr = 10,700), were isolated from rabbit skeletal muscle by means of succesive extraction with a neutral buffer solution, precipitation at pH 3.7, acetone fractionation and gel permeation on Sephadex G-75. 2. I-T is a formed trimer of a monomeric inhibitor, I-M (Mr = 10,500), through disulfide bonds. 3. I-S is almost completely stable between pH 3 and 8, while I-M is unstable in the same pH range. 4. I-M acts most effectively towards cathepsins H and L, showing moderate activity towards cathepsin B and only weak activity towards papain. I-S acts most effectively towards cathepsin L, followed by, in decreasing order, cathepsin H, cathepsin B and papain.     

The calcium channel antagonists receptor from rabbit skeletal muscle. Reconstitution after purification and subunit characterization

The Ca2+ channel antagonists receptor from rabbit skeletal muscle was purified to homogeneity. Following reconstitution into phosphatidylcholine vesicles, binding experiments with (+)[3H]PN 200-110, (-)[3H]D888 and d-cis-[3H]diltiazem demonstrated that receptor sites for the three most common Ca2+ channel markers copurified with binding stoichiometries close to 1:1:1. Sodium dodecyl sulfate gel analysis of the purified receptor showed that it is composed of only one protein of Mr 170,000 under non-reducing conditions and of two polypeptides of Mr 140,000 and 32,000 under disulfide-reducing conditions. Iodination of the protein of Mr 170,000 and immunoblots experiments with antisera directed against the different components demonstrated that the Ca2+ channel antagonists receptor is a complex of Mr 170,000 composed of a polypeptide chain of Mr 140,000 associated to one polypeptide chain of Mr 32,000 by disulfide bridges. One of the problems concerning this subunit structure of the putative Ca2+ channel was the presence of smaller polypeptide chains of Mr 29,000 and 25,000. Peptide mapping of these polypeptide chains and analysis of their cross-reactivity with sera directed against the proteins of Mr 170,000 and 32,000 demonstrated that they were degradative products of the Mr 32,000 component. Both the large (140 kDa) and the small (32 kDa) component of the putative Ca2+ channel are heavily glycosylated. At least 20-22% of their mass were removed by enzymatic deglycosylation. Finally the possibility that both the 140-kDa and 32-kDa components originate from a single polypeptide chain of Mr 170,000 which is cleaved by proteolysis upon purification is discussed.     

Characterization of sarcolemma from rabbit skeletal muscle: developmental studies

Sarcolemmal membranes were obtained from skeletal muscle of rabbits at different developmental stages. Lipid and protein composition, as well as enzymatic activities, were compared in sarcolemma prepared in vesicular form. During the developmental period no distinct changes in lipid content and composition were found. Some changes in protein pattern and activities of marker enzymes (5′-nucleotidase and ouabain-sensitive Na⁺,K⁺-ATPase) were detected. Also, changes in Mg²⁺-ATPase and Mg²⁺, Ca²⁺-ATPase activities during development were found.