scholarly journals Purification and characterization of a multicatalytic high-molecular-mass proteinase from rat skeletal muscle

1985 ◽  
Vol 228 (1) ◽  
pp. 161-170 ◽  
Author(s):  
B Dahlmann ◽  
L Kuehn ◽  
M Rutschmann ◽  
H Reinauer
Keyword(s):  
Skeletal Muscle ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Hydrolytic Activity ◽  
Maximum Activity ◽  
Disc Electrophoresis ◽  
Polyacrylamide Gels ◽  
Rat Skeletal Muscle ◽  
Muscle Extract

A proteolytic enzyme was purified from the post-myofibrillar fraction of rat skeletal muscle. The purification procedure consisted of fractionation of the muscle extract by (NH4)2SO4, chromatography on DEAE-Sephacel, fast protein liquid chromatography on Mono Q and gel filtration on Sepharose 6B. The enzyme preparation appeared to be homogeneous as judged by disc electrophoresis in polyacrylamide gels and by immunoelectrophoresis. The isoelectric point of the proteinase is at 5.1-5.2. The enzyme has an Mr of about 650 000 and dissociates into eight subunits of Mr 25 000-32 000 when subjected to electrophoresis in sodium dodecyl sulphate/polyacrylamide gels. The proteinase contains hydrolytic activity against N-blocked tripeptide 4-methyl-7-coumarylamide substrates with an arginine or phenylalanine residue adjacent to the leaving group. Maximum activity with the first group of substrates was at pH 10.5, and this activity was inhibited by leupeptin, chymostatin and Ca2+. Maximum activity with the latter group of substrates was at pH 7.5, and was also inhibited by the two microbial inhibitors, but was activated by Ca2+ ions. By using [14C]methylcasein as a substrate, maximum activity was observed at pH9.0, and this proteolytic activity was not affected by leupeptin, was enhanced by chymostatin and inhibited by Ca2+. Similar effects were observed when benzyloxycarbonyl-Leu-Leu-Glu 2-naphthylamide was used as a substrate. These enzymic activities were abolished by p-hydroxymercuribenzenesulphonic acid or mersalyl acid, whereas a small activation was observed with cysteine or dithiothreitol.

Purification and characterization of an endo-β-1,3-glucanase from Trichoderma harzianum

1996 ◽  
Vol 42 (10) ◽  
pp. 1039-1044 ◽  
Author(s):  
Eliane Ferreira Noronha ◽  
Cirano José Ulhoa
Keyword(s):  
Trichoderma Harzianum ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Hydrophobic Interaction Chromatography ◽  
Maximum Activity ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Isolated Cell

β-1,3-Glucanases are produced by Trichoderma harzianum when it is grown in the presence of chitin or isolated cell wall from fungi. An endo-β-1,3-glucanase from the culture filtrate of T. harzianum was purified by gel filtration on Sephacryl S-200, followed by hydrophobic interaction chromatography on phenyl-Sepharose. A typical procedure provided 134-fold purification with a 3.6% yield. The molecular mass of the purified endo-β-1,3-glucanase was found to be approximately 36 kDa, as estimated by sodium dodecyl sulfate – polyacrylamide gel electrophoresis on a 10% w/v slab gel. The enzyme was active toward glucans containing β-1,3-linkages and hydrolysed laminarin to form oligosaccharides. The Km and Vmax values for β-1,3-ghicanases, using laminarin as substrate, was 1.18 mg∙mL−1 and 1.26 U∙mL−1, respectively. The pH optimum for the enzyme was pH 4.4 and maximum activity was obtained at 45–50 °C. Enzyme activity was strongly inhibited in the presence of HgCl2 and stimulated by cations such as Zn2+ and Ca2+.Key words: endo-β-1,3-glucanase, Trichoderma harzianum, purification, characterization.

scholarly journals Purification and some properties of an alkaline proteinase from rat skeletal muscle

1978 ◽  
Vol 171 (3) ◽  
pp. 803-810 ◽  
Author(s):  
B Dahlmann ◽  
H Reinauer
Keyword(s):  
Skeletal Muscle ◽  
Enzyme Activity ◽  
Proteolytic Activity ◽  
Metal Ion ◽  
Gel Filtration ◽  
Disc Electrophoresis ◽  
Exchange Chromatography ◽  
Myofibrillar Proteins ◽  
Rat Skeletal Muscle ◽  
Alkaline Proteinase

1. Rat skeletal muscle was homogenized in 0.05M-Tris/HCl, pH 8.5, containing 1M-KCl. Myofibrillar proteins were precipitated by addition of (NH4)2SO4 (33% saturation). 2. The alkaline proteolytic activity that was precipitated with the myofibrillar proteins was solubilized with trypsin (conjugated to Sepharose) and further purified by affinity chromatography, ion-exchange chromatography and gel filtration. 3. The purified enzyme migrates as a single band in polyacrylamide-disc electrophoresis, and has optimum hydrolytic activity with azocasein and [14C]haemoglobin as substrates at pH 9.4 and 9.6 respectively. Its apparent molecular weight, as determined by gel filtration on Sephadex G-75, is 30800. 4. The purified alkaline proteinase is strongly inhibited by equimolar amounts of soya-bean trypsin inhibitor and ovomucoid, whereas di-isopropyl phosphorofluoidate and alpha-toluenesulphonyl fluoride have no effect. On the other hand N-ethylmaleimide and p-chloromercuribenzoate have inhibitory effects on the enzyme activity. 5. Bivalent metal ions (Fe2+, Co2+, Zn2+, Mg2+, Mn2+) diminish the proteolytic activity, at 1mM concentrations. Ca2+ ions and the metal-ion-chelating agent EDTA are without effect on enzyme activity. 6. The enzyme is part of the alkaline proteolytic activity that appears to be associated with myofibrillar proteins.

Characterization of Different Deoxyribonucleases in Human Lymphocytes

1975 ◽  
Vol 30 (11-12) ◽  
pp. 781-784 ◽  
Author(s):  
E. Jürgen Zöllner ◽  
Hans Störger ◽  
Hans-Joachim Breter ◽  
Rudolf Zahn
Keyword(s):  
Human Lymphocytes ◽  
Disc Electrophoresis ◽  
The Other ◽  
Lower Activity ◽  
Nuclear Fraction ◽  
Polyacrylamide Gels ◽  
Cytoplasmic Fraction ◽  
Acid Deoxyribonuclease ◽  
Deoxyribonuclease Activity

Abstract Deoxyribonucleases, Disc Electrophoresis, Lymphocytes Four groups of deoxyribonuclease activities from human lymphocytes have been characterized by deoxyribonuclease assay in DNA-containing polyacrylamide gels following their separation by disc-electrophoresis. All activities hydrolyse DNA endonucleolytically. One neutral deoxyribo­ nuclease found in the cytoplasmic fraction prefers native or UV-irradiated DNA over denatured DNA as substrate and is a 5′-monoester former. Two groups of acid deoxyribonuclease activities are detectable in the nuclear fraction. Both are 3′-monoester formers. One is as well active with denatured DNA as with native DNA, the other one shows the same activity with native and UV-irradiated DNA but lower activity with denatured DNA. An alkaline deoxyribonuclease activity, also localized in the nucleus, is a 5′ -monoester DNA as substrate.

scholarly journals Characterization of insulin-stimulated seryl/threonyl protein kinases in rat skeletal muscle

1993 ◽  
Vol 268 (18) ◽  
pp. 13203-13213
Author(s):  
Y.J. Hei ◽  
J.H. McNeill ◽  
J.S. Sanghera ◽  
J. Diamond ◽  
M. Bryer-Ash ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Protein Kinases ◽  
Rat Skeletal Muscle

scholarly journals Partial purification and biochemical characterization of a new highly acidic NYSO laccase from Alcaligenes faecalis

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Soad A. Abdelgalil ◽  
Ahmad R. Attia ◽  
Reyed M. Reyed ◽  
Nadia A. Soliman
Keyword(s):  
Enzyme Activity ◽  
Sodium Dodecyl ◽  
Alcaligenes Faecalis ◽  
Maximum Activity ◽  
Sodium Oxalate ◽  
Partial Purification ◽  
Bromophenol Blue ◽  
Bromocresol Purple ◽  
Laccase Enzyme

Abstract Background Due to the multitude industrial applications of ligninolytic enzymes, their demands are increasing. Partial purification and intensive characterization of contemporary highly acidic laccase enzyme produced by an Egyptian local isolate designated Alcaligenes faecalis NYSO were studied in the present investigation. Results Alcaligenes faecalis NYSO laccase has been partially purified and intensively biochemically characterized. It was noticed that 40–60% ammonium sulfate saturation showed maximum activity. A protein band with an apparent molecular mass of ~ 50 kDa related to NYSO laccase was identified through SDS-PAGE and zymography. The partially purified enzyme exhibited maximum activity at 55 °C and pH suboptimal (2.5–5.0). Remarkable activation for enzyme activity was recognized after 10-min exposure to temperatures (T) 50, 60, and 70 °C; time elongation caused inactivation, where ~ 50% of activity was lost after a 7-h exposure to 60 °C. Some metal ions Cu2+, Zn2+, Co2+, Ni2+, Mn2+, Cd2+, Cr2+, and Mg2+ caused strong stimulation for enzyme activity, but Fe2+ and Hg2+ reduced the activity. One millimolar of chelating agents [ethylenediamine tetraacetic acid (EDTA), sodium citrate, and sodium oxalate] caused strong activation for enzyme activity. Sodium dodecyl sulfate (SDS), cysteine-HCl, dithiothreitol (DTT), β-mercaptoethanol, thioglycolic acid, and sodium azide caused strong inhibition for NYSO laccase activity even at low concentration. One millimolar of urea, imidazole, kojic acid, phenylmethylsulfonyl fluoride (PMSF), H2O2, and Triton X-100 caused activation. The partially purified NYSO laccase had decolorization activity towards different dyes such as congo red, crystal violet, methylene blue, fast green, basic fuchsin, bromophenol blue, malachite green, bromocresol purple eriochrome black T, and Coomassie Brilliant Blue R-250 with various degree of degradation. Also, it had a vast range of substrate specificity including lignin, but with high affinity towards p-anisidine. Conclusion The promising properties of the newly studied laccase enzyme from Alcaligenes faecalis NYSO strain would support several industries such as textile, food, and paper and open the possibility for commercial use in water treatment. It will also open the door to new applications due to its ligninolytic properties in the near future.

scholarly journals Purification and subunit structure of argininosuccinate lyase from Chlamydomonas reinhardi

1977 ◽  
Vol 167 (1) ◽  
pp. 71-75 ◽  
Author(s):  
R F Matagne ◽  
J P Schlösser
Keyword(s):  
Enzyme Activity ◽  
Crude Extract ◽  
Enzyme Preparation ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Activity Analysis ◽  
Disc Electrophoresis ◽  
Subunit Structure ◽  
Argininosuccinate Lyase

Argininosuccinate lyase (EC 4.3.2.1) was purified by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and gel filtration on Sephadex G-200. The final enzyme preparation was purified 46-fold compared with the crude extract. Electrophoresis of this preparation revealed three bands, the major one having the enzyme activity. Analysis of the enzyme by gel filtration and by disc electrophoresis (in two different concentrations of acrylamide) gave mol.wts. of 200000 (+/- 15000) and 190000 (+/- 20000) respectively. Treatment with sodium dodecyl sulphate and mercaptoethanol dissociated the enzyme into subunits of mol.wt. 39000 (+/-2000). The results are indicative of the multimeric structure of the enzyme, which is composed of five (perhaps four or six) identical subunits.

Characterization of β-adrenoceptors on rat skeletal muscle cells grown in vitro

1990 ◽  
Vol 40 (5) ◽  
pp. 1043-1048 ◽  
Author(s):  
Marie-Helene Disatnik ◽  
Sanford R. Sampson ◽  
Asher Shainberg
Keyword(s):  
Skeletal Muscle ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Rat Skeletal Muscle

Purification and characterization of tuberculin-active components from BCG

1975 ◽  
Vol 21 (12) ◽  
pp. 2019-2027
Author(s):  
M. Laguerre ◽  
R. Turcotte
Keyword(s):  
Physicochemical Properties ◽  
Guinea Pigs ◽  
Gel Filtration ◽  
Biologically Active ◽  
Polyacrylamide Gels ◽  
Active Components ◽  
Purification And Characterization ◽  
Molecular Weights ◽  
Antigenic Activity

The tuberculin activity of protoplasmic extracts isolated from living BCG was purified successively by gel filtration on Sephadex G-100 and G-75, and by electrophoresis on 7.5% and on gradient (6–18%) polyacrylamide gels. The tuberculin-active fractions, as determined in BCG-sensitized guinea pigs, were used as the starting material for each of the following fractionation steps.The physicochemical properties and the antigenic activity of the biologically active fractions have shown that a single component, or only a few ones with similar properties, possessed high tuberculin activity. These active components were proteins having relatively high molecular weights (about 72 000) and could behave as antigens.

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