scholarly journals Characterization of two acid proteinases found in rabbit skin homografts

1978 ◽  
Vol 169 (2) ◽  
pp. 287-295 ◽  
Author(s):  
B Jasani ◽  
M K Jasani ◽  
M D Talbot
Keyword(s):  
Molecular Weight ◽  
Cathepsin D ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Effect Of Temperature ◽  
Heat Inactivation ◽  
Acid Proteinase ◽  
Polymorphonuclear Leucocytes ◽  
Ph Optimum ◽  
Rabbit Skin

Two types of acid proteinase activity found in rabbit skin homografts were characterized by studying the effect of temperature, pH and polyacrylamide-gel electrophoresis. Their chromatographic behaviour was characterized on DEAE-cellulose, Sephadex G-75, G-100 and G-200, and their molecular weights were estimated by gel filtration. One of the acid proteinases in the homograft resembled cathepsin D (EC 3.4.23.5) of normal skin. The other acid proteinase differed from cathepsin D with respect to heat inactivation, pH optimum and molecular weight; it was not inactivated on heating at 60 degrees C for 60 min, its pH optimum was 2.5 and its molecular weight measured by Sephadex G-100 chromatography was 100 000. In all these respects, the heat-stable proteinase resembles cathepsin E (EC 3.4.23.5) of rabbit polymorphonuclear leucocytes.

scholarly journals The partial purification and characterization of cytosol alcohol dehydrogenase from Astasia

1974 ◽  
Vol 141 (2) ◽  
pp. 469-475 ◽  
Author(s):  
Rolf Morosoli ◽  
Nicole Bégin-Heick
Keyword(s):  
Molecular Weight ◽  
Alcohol Dehydrogenase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Total Activity ◽  
Growth Conditions ◽  
Partial Purification ◽  
Heat Inactivation ◽  
Kinetic Properties ◽  
Ph Optimum

1. The cytosol alcohol dehydrogenase (alcohol–NAD oxidoreductase, EC 1.1.1.1) of Astasia longa was partially purified and characterized from cells grown in the presence of air+CO2 (95:5) or of O2+CO2 (95:5). 2. Under both these growth conditions, the cells contained a fraction, ADHII, which was characterized by its electrophoretic properties, by a high degree of resistance to heat inactivation, by a sharp pH optimum at 8.2 and by its kinetic properties. The estimated molecular weight of this fraction was approx. 150000, which is similar to that of yeast alcohol dehydrogenase. 3. Cells grown in air+CO2 (95:5) contain another fraction, ADHI, which can be further separated into two subfractions by polyacrylamide-gel electrophoresis and by DEAE-cellulose chromatography. This was termed fraction ‘ADHI-air’. 4. In addition to fraction ADHII, cells grown in the presence of O2 have a twofold increase in fraction ADHI-air activity as well as two new fractions that could not be demonstrated in air-grown cells. These new fractions which we have called fraction ‘ADHI-O2’, account for about 10% of the total activity. 5. The ADHI fractions (air) and (O2) have similar broad pH–activity curves and similar kinetic properties, both having a lower Km for ethanol and NAD than fraction ADHII. However, they differ from each other with respect to their activity with various substrates. The estimated molecular weight of these two ADHI fractions and their chromatographic behaviour on hydroxyapatite and on DEAE-cellulose also distinguish them.

scholarly journals Two-step affinity-chromatographic purification of cathepsin D from pig myometrium with high yield

1981 ◽  
Vol 197 (2) ◽  
pp. 519-522 ◽  
Author(s):  
E G Afting ◽  
M L Recker
Keyword(s):  
Molecular Weight ◽  
Concanavalin A ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Cathepsin D ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
High Yield ◽  
Acid Proteinase ◽  
Chromatographic Purification

Cathepsin D was purified by two-step affinity chromatography on concanavalin A- and pepstatin-Sepharose. The main purification was achieved by washing the enzyme bound to the pepstatin-Sepharose column with buffered 6 M-urea. This step separated cathepsin D from all low- and high-molecular-weight impurities. Although the 1700-fold purified acid proteinase was homogeneous on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, it still showed microheterogeneity.

scholarly journals Purification and properties of adenylyl sulphate:ammonia adenylyltransferase from Chlorella catalysing the formation of adenosine 5′-phosphoramidate from adenosine 5′-phosphosulphate and ammonia

1981 ◽  
Vol 195 (3) ◽  
pp. 545-560 ◽  
Author(s):  
Heinz Fankhauser ◽  
Jerome A. Schiff ◽  
Leonard J. Garber
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Low Ph ◽  
Polyacrylamide Gel ◽  
Ph Optimum ◽  
Unknown Compound ◽  
Reactive Blue 2

Extracts of Chlorella pyrenoidosa, Euglena gracilis var. bacillaris, spinach, barley, Dictyostelium discoideum and Escherichia coli form an unknown compound enzymically from adenosine 5′-phosphosulphate in the presence of ammonia. This unknown compound shares the following properties with adenosine 5′-phosphoramidate: molar proportions of constituent parts (1 adenine:1 ribose:1 phosphate:1 ammonia released at low pH), co-electrophoresis in all buffers tested including borate, formation of AMP at low pH through release of ammonia, mass and i.r. spectra and conversion into 5′-AMP by phosphodiesterase. This unknown compound therefore appears to be identical with adenosine 5′-phosphoramidate. The enzyme that catalyses the formation of adenosine 5′-phosphoramidate from ammonia and adenosine 5′-phosphosulphate was purified 1800-fold (to homogeneity) from Chlorella by using (NH4)2SO4 precipitation and DEAE-cellulose, Sephadex and Reactive Blue 2–agarose chromatography. The purified enzyme shows one band of protein, coincident with activity, at a position corresponding to 60000–65000 molecular weight, on polyacrylamide-gel electrophoresis, and yields three subunits on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of 26000, 21000 and 17000 molecular weight, consistent with a molecular weight of 64000 for the native enzyme. Isoelectrofocusing yields one band of pI4.2. The pH optimum of the enzyme-catalysed reaction is 8.8. ATP, ADP or adenosine 3′-phosphate 5′-phosphosulphate will not replace adenosine 5′-phosphosulphate, and the apparent Km for the last-mentioned compound is 0.82mm. The apparent Km for ammonia (assuming NH3 to be the active species) is about 10mm. A large variety of primary, secondary and tertiary amines or amides will not replace ammonia. One mol.prop. of adenosine 5′-phosphosulphate reacts with 1 mol.prop. of ammonia to yield 1 mol.prop. each of adenosine 5′-phosphoramidate and sulphate; no AMP is found. The highly purified enzyme does not catalyse any of the known reactions of adenosine 5′-phosphosulphate, including those catalysed by ATP sulphurylase, adenosine 5′-phosphosulphate kinase, adenosine 5′-phosphosulphate sulphotransferase or ADP sulphurylase. Adenosine 5′-phosphoramidate is found in old samples of the ammonium salt of adenosine 5′-phosphosulphate and can be formed non-enzymically if adenosine 5′-phosphosulphate and ammonia are boiled. In the non-enzymic reaction both adenosine 5′-phosphoramidate and AMP are formed. Thus the enzyme forms adenosine 5′-phosphoramidate by selectively speeding up an already favoured reaction.

Properties and characteristics of partly purified glutamate dehydrogenase from sheep rumen mucosa

1981 ◽  
Vol 46 (11) ◽  
pp. 2766-2773
Author(s):  
Katarína Holovská ◽  
Viera Lenártová ◽  
Ivan Havassy
Keyword(s):  
Molecular Weight ◽  
Glutamate Dehydrogenase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Enzymatic Reaction ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Ph Optimum ◽  
Relative Molecular Weight ◽  
Sheep Rumen ◽  
The Individual

The purification of glutamate dehydrogenase from sheep rumen mucosa on DEAE-cellulose afforded two enzyme fractions with glutamate dehydrogenase activity. The enzyme fraction II (tissue glutamate dehydrogenase) was freed of contaminating proteins in the subsequent purification step on Sephadex G-200. The approximate relative molecular weight (260 000) of tissue glutamate dehydrogenase (fraction II) was determined by gel filtration on Sephadex G-200 and the approximate relative molecular weight of its polypeptide chain (48 000) was established by polyacrylamide gel electrophoresis in SDS. The pH-optimum of fraction II was 7.9. The effect of substrate concentration on the rate of the enzymatic reaction was examined and the following apparent Michaelis' constants were found for the individual substrates: NADH 6.25 . 10-5 mol/l, 2-oxoglutarate 4.5 . 10-3 mol/l, and NH4+ 77 . 10-3 mol/l.

Characterization of an enzyme from Rhizoctonia praticola which polymerizes phenolic compounds

1979 ◽  
Vol 25 (2) ◽  
pp. 229-233 ◽  
Author(s):  
Jean-Marc Bollag ◽  
Roy D. Sjoblad ◽  
Shu-Yen Liu
Keyword(s):  
Molecular Weight ◽  
Phenolic Compounds ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Phenol Oxidase ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Rhizoctonia Praticola

An extracellular phenol oxidase from the fungus Rhizoctonia praticola which polymerizes various xenobiotic phenols was isolated and characterized. The enzyme was purified by DEAE-cellulose and Sephadex G-200 chromatography followed by preparative polyacrylamide gel electrophoresis. Atomic absorption and EPR spectroscopy indicated the presence of copper, and SDS gel electrophoresis revealed a molecular weight of 78 000. With 2,6-dimethoxyphenol as substrate, the enzyme showed a pH optimum of 6.7–6.9, and a temperature optimum of 40 °C. According to these and additional characteristics it appears that the enzyme belongs to the class of laccases.

Characterization and distribution of arginine kinase in the tissues of the scorpion, Palamneus phipsoni

1985 ◽  
Vol 63 (10) ◽  
pp. 2262-2266 ◽  
Author(s):  
A. V. Arjunwadkar ◽  
S. Raghupathi Rami Reddy
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Alimentary Canal ◽  
Polyacrylamide Gel Electrophoresis ◽  
Arginine Kinase ◽  
High Specificity ◽  
Ph Optimum ◽  
High Concentrations ◽  
Claw Muscle ◽  
Slight Inhibition

Arginine kinase in claw muscle extracts of the scorpion, Palamneus phipsoni, was characterized. The enzyme, with a pH optimum of 8.5 in the direction of phosphoarginine synthesis, showed activation by Mg2+, high specificity towards L-arginine as the guanidino substrate, slight inhibition by high concentrations of L-arginine and ATP, and a molecular weight of 33 500. On polyacrylamide gel electrophoresis at pH 8.3 the enzyme migrated to the anode as a single molecular species. In addition to the claw muscle, the enzyme activity was also found to be present in the heart, alimentary canal, hepatopancreas, and nervous system. In general, scorpion muscle arginine kinase appears to be similar in its properties to the enzyme from other arthropods.

Guanosine 3’:5’-Cyclic Monophosphate -Dependent Particulate Protein Kinase Activity from Yeast (Saccharomyces cerevisiae)

1999 ◽  
Vol 54 (1-2) ◽  
pp. 84-93 ◽  
Author(s):  
Hans Eckstein ◽  
Birgit Flügge
Keyword(s):  
Protein Kinase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Temperature Tolerance ◽  
Maximum Activity ◽  
Partial Purification ◽  
Protein Kinase Activity ◽  
Ph Optimum ◽  
Yeast Saccharomyces Cerevisiae ◽  
High Concentrations

Continuing our studies on cGMP in growing yeast we detected a particulate cGMPdependent protein kinase (Pk-G), which was solubilized by detergents and NaCl. It achieves maximum activity at 25 °C and pH = 6.8, high concentrations of substrate proteins or cGMP produce saturation. Casein and histones are appropriate substrates, phosphatase-pretreated histone H-2a provokes outstandingly high activity. Pk-G differs from cAMP-dependent protein kinase (Pk-A) with respect to pH optimum, temperature tolerance above 50 °C, and stability. Partial purification is achieved by chromatography with DEAE-cellulose, Sepharose, and cGMP-substituted Sepharose. The latter step also markedly removes Pk-A. At least three proteins with Pk-G-activity and high cGMP-affinity are separated by polyacrylamide-gel-electrophoresis. Their apparent molecular masses, as deduced from comigrating marker proteins, differ considerably from those of other Pk-G’s, but also of Pk-A’s

Effect of somatic cell count and polymorphonuclear leucocyte content of milk on composition and proteolysis during ripening of Swiss-type cheese

2000 ◽  
Vol 67 (2) ◽  
pp. 301-307 ◽  
Author(s):  
SIOBHÁN COONEY ◽  
DENISE TIERNAN ◽  
PATRICK JOYCE ◽  
ALAN L. KELLY
Keyword(s):  
Somatic Cell ◽  
Cell Count ◽  
Cathepsin D ◽  
Somatic Cell Count ◽  
Proteolytic Enzymes ◽  
Raw Milk ◽  
Yield Potential ◽  
Acid Proteinase ◽  
Polymorphonuclear Leucocytes ◽  
Texture Characteristic

During the manufacture of Swiss-type cheese varieties, such as Emmental, Gruyère and Comté, a high scald temperature is used, which inactivates the added coagulant, decreasing the primary proteolysis of αs1-casein during ripening and resulting in the development of the rubbery texture characteristic of this type of cheese (Kosikowski & Mistry, 1997). Swiss-type cheese may be made from raw, thermized or pasteurized milk, depending on the type of manufacture and the country (Beuvier et al. 1997).The suitability of milk for cheesemaking is often assessed from the somatic cell count (SCC) of the raw milk, with increased milk SCC being linked to impaired cheesemaking properties and cheese yield potential (Barbano et al. 1991; Auldist et al. 1996). Late lactation milk, which often has elevated SCC, also yields poor quality cheese (Lucey, 1996). High SCC milk has elevated levels of the milk alkaline proteinase plasmin (EC 3.4.21.7), but its activity is not generally considered to be detrimental during cheese ripening (Farkye & Fox, 1992; Bastian & Brown, 1996). Milk somatic cells are themselves also associated with a number of proteolytic enzymes of differing properties, the activities of which are ill-defined in dairy products (Verdi & Barbano, 1991), with the exception of the acid proteinase cathepsin D, which has a chymosin-like activity towards αs1-casein (McSweeney et al. 1995). Preliminary evidence of cathepsin D activity in Swiss cheese during ripening has been reported (Igoshi & Arima, 1993; Beuvier et al. 1997).In the present study the effect of milk SCC on the ripening of Swiss-type cheese was examined. Of particular interest was the role of polymorphonuclear leucocytes (PMN), the predominant cell type in high SCC mastitic milk (Heegaard et al. 1994) and late lactation milk, when the frequency of milking varies (Kelly et al. 1998). PMN possess a range of proteolytic enzymes, including cathepsin D (Verdi & Barbano, 1991) and plasminogen activators (PA; Heegaard et al. 1994). The experimental strategy chosen was to examine the effects on the quality and proteolysis during ripening of Swiss-type cheeses of adding high SCC milk, whose cell population is mostly PMN, to creamery milk.

Purification and characterization of the extracellular α-amylase activity of the yeast Schwanniomyces alluvius

1987 ◽  
Vol 65 (10) ◽  
pp. 899-908 ◽  
Author(s):  
F. Moranelli ◽  
M. Yaguchi ◽  
G. B. Calleja ◽  
A. Nasim
Keyword(s):  
Amino Acid ◽  
Amylase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Proteinase K ◽  
Ph Optimum ◽  
Sds Page

The extracellular α-amylase activity of the yeast Schwanniomyces alluvius has been purified by anion-exchange chromatography on DEAE-cellulose and gel-filtration chromatography on Sephadex G-100. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) and N-terminal amino acid analysis of the purified sample indicated that the enzyme preparation was homogeneous. The enzyme is a glycoprotein having a molecular mass of 52 kilodaltons (kDa) estimated by SDS–PAGE and 39 kDa by gel filtration on Sephadex G-100. Chromatofocusing shows that it is an acidic protein. It is resistant to trypsin but sensitive to proteinase K. Its activity is inhibited by the divalent cation chelators EDTA and EGTA and it is insensitive to sulfhydryl-blocking agents. Exogenous divalent cations are inhibitory as are high concentrations of monovalent salts. The enzyme has a pH optimum between 3.75 and 5.5 and displays maximum stability in the pH range of 4.0–7.0. Under the conditions tested, the activity is maximal between 45 and 50 °C and is very thermolabile. Analysis of its amino acid composition supports its acidic nature.

scholarly journals Cathepsin D from pig myometrium. Characterization of the proteinase

1984 ◽  
Vol 219 (3) ◽  
pp. 899-904 ◽  
Author(s):  
R Barth ◽  
E G Afting
Keyword(s):  
Cathepsin D ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Ph Optimum ◽  
Equilibrium Studies ◽  
Main Band ◽  
Km Value ◽  
Ph Range

The purification of cathepsin D from pig uterus by two-step affinity chromatography on concanavalin A- and pepstatin-Sepharose was described previously [Afting & Becker (1981) Biochem. J. 197, 519-522]. In this paper, chemical and physical properties of the proteinase are presented. The purified enzyme showed three bands on SDS (sodium dodecyl sulphate)/polyacrylamide-gel electrophoresis, one main band corresponding to an Mr of 31 000 and two minor bands with Mr values of 43 000 and 15 000 respectively. Gel filtration on Bio-gel P-150 and sedimentation-diffusion equilibrium studies give an Mr for the main band of about 35 000. The pI of the enzyme was determined to be 7.2. Haemoglobin was the best substrate, with a Km value of 6.4 X 10(-6)M. It was hydrolysed with a pH optimum between 3.0 and 3.3 for a substrate concentration of 100 microM. The proteinase was stable over the pH range of 3.5-6.5. At pH 6 the enzyme showed stability up to a temperature of 50 degrees C; at pH 3 the activity was already decreased below 40 degrees C. Carbohydrate studies resulted in the staining of all three bands on an SDS/polyacrylamide gel by thymol/H2SO4. After treatment with endo-beta-N-acetylglucosaminidase H, all three bands were shifted to a region of lower Mr. Of various inhibitors tested, only pepstatin was strongly inhibiting, with a Ki of 2.1 X 10(-9)M.

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