Purification and some characteristics of a Ca2+-activated proteolytic enzyme from beef cardiac muscle

1981 ◽  
Vol 59 (4) ◽  
pp. 242-249 ◽  
Author(s):  
Susan Tolnai
Keyword(s):  
Enzyme Activity ◽  
Cardiac Muscle ◽  
Specific Activity ◽  
Fold Increase ◽  
Fresh Tissue ◽  
Total Enzyme Activity ◽  
Calcium Dependent ◽  
And Storage ◽  
Assay Conditions ◽  
Salt Fractionation

A calcium-dependent neutral proteinase was purified from beef cardiac muscle. The crude extract prepared from cardiac muscle was subjected to acid precipitation and salt fractionation and then further purified by column chromatography on Sepharose 6B, DE-52, and Sephadex G-200 columns in succession. The final preparation showed an 11 300 fold increase in specific activity of the Ca2+-activated enzyme. Average enzyme protein yield was 2.4 μg/g fresh tissue. The enzyme was maximally active at pH 7.6 in the presence of 4 mM calcium. Proportionality of enzyme activity in partially purified preparations was retained when activity was measured at 25 °C using casein as the substrate. The rate of proteolysis by the purified enzyme was linear for 60 min under similar assay conditions. Fractionation of muscle homogenates showed that 70 to 73% of the total enzyme activity was present in the 24 000 × g and 30 000 × g supernatants. The enzyme was labile in aqueous solutions and storage at 4 °C and −20 °C resulted in considerable loss of activity, unless glycerol (50% v/v) was added to the solution.

5α-Reductase activity in stroma and epithelium of rat prostate and epididymis. A contribution to elucidation of the mechanism for development of hyperplastic growth of prostatic tissue

1983 ◽  
Vol 103 (2) ◽  
pp. 273-281 ◽  
Author(s):  
Ole Djøseland ◽  
Nicholas Bruchovsky ◽  
Paul S. Rennie ◽  
Navdeep Otal ◽  
Sian Høglo
Keyword(s):  
Enzyme Activity ◽  
Specific Activity ◽  
Reductase Activity ◽  
Major Change ◽  
Growth Stimulation ◽  
Total Activity ◽  
Prostatic Tissue ◽  
Ventral Prostate ◽  
Total Enzyme Activity ◽  
Abnormal Growth

Abstract. The 5α-reductase activity was assayed in homogenates of stroma and epithelium in the rat ventral prostate and epididymis. Samples consisting of 0.3 mg/ml tissue protein in TES buffer, pH 7.0 were incubated at 37°C for 30 min in the presence of 50 nm [1,2-3H]testosterone and a NADPH-generating system started with 5 × 10−4 m NADP. The yield of 5α-reduced metabolites, as established by using thin-layer chromatography, gave an estimate of enzyme activity. Whereas the specific activity of 5α-reductase was highest in prostatic stroma and epididymal epithelium, most of the total enzyme activity was associated with the epithelium in both the prostate and epididymis. The effect of dihydrotestosterone on specific activity of 5α-reductase was studied by administering the hormone to 7-day castrated rats. In prostate, the specific activity of both stromal and epithelium forms of the enzyme reached a maximum after 4 days of treatment. In epididymis only the epithelial form of 5α-reductase underwent a major change in specific activity, the latter peaking after 8–12 days of treatment. Furthermore, while the total activity of 5α-reductase in the prostatic tissue fractions could be induced by as much as 4-fold the normal control values, the epididymal enzyme could not be induced above the normal level either in the stroma or the epithelium. This may explain the relative resistance of epididymis to abnormal growth stimulation under the influence of hormones.

scholarly journals Poly(adenosine diphosphate ribose) polymerase activity and nicotinamide adenine dinucleotide in differentiating cardiac muscle

1976 ◽  
Vol 154 (2) ◽  
pp. 387-393 ◽  
Author(s):  
W C. Claycomb
Keyword(s):  
Cyclic Amp ◽  
Dna Synthesis ◽  
Cardiac Muscle ◽  
Dna Polymerase ◽  
Specific Activity ◽  
Polymerase Activity ◽  
Neonatal Rat ◽  
Dibutyryl Cyclic Amp ◽  
Fresh Tissue ◽  
Dna Polymerase Α

Poly(ADP-ribose) polymerase activity in nuclei isolated from differentiating cardiac muscle of the rat has been characterized and its activity measured during development. Optimum enzyme activity is observed at pH 8.5. Poly(ADP-ribose) polymerase is inhibited by ATP, thymidine, nicotinamide, theophylline, 3-isobutyl-1-methylxanthine and caffeine and stimulated by actinomycin D. The activity measured under optimal assay conditions increases during differentiation of cardiac muscle and is inversely related to the rate of DNA synthesis and to the activities of DNA polymerase α and thymidine kinase. When DNA synthesis and the activity of DNA polymerase α are inhibited in cardiac muscle of the 1-day-old neonatal rat by dibutyryl cyclic AMP or isoproterenol, the specific activity of poly(ADP-ribose) polymerase measured in isolated nuclei is increased. The concentration of NAD+ in cardiac muscle increases during postnatal development. In the adult compared with the 1-day-old neonatal rat the concentration of NAD+ relative to fresh tissue weight, DNA or protein increased 1.7-fold, 5.2-fold or 1.4-fold respectively. The concentration of NAD+ in cardiac muscle of the 1-day-old neonatal rat can be increased by approx. 20% by dibutyryl cyclic AMP. These data suggest that NAD+ and poly(ADP-ribose) polymerase may be involved with the repression of DNA synthesis and cell proliferation in differentiating cardiac muscle.

scholarly journals Regulation of pyruvate carboxylase in 3T3-L1 cells

1995 ◽  
Vol 306 (1) ◽  
pp. 205-210 ◽  
Author(s):  
J Zhang ◽  
W L Xia ◽  
F Ahmad
Keyword(s):  
Enzyme Activity ◽  
Relative Abundance ◽  
Pyruvate Carboxylase ◽  
Protein Concentration ◽  
Specific Activity ◽  
Cdna Probe ◽  
Fold Increase ◽  
Spatial Arrangement ◽  
Cellular Protein ◽  
Prosthetic Group

When 3T3-L1 fibroblasts differentiate to adipocytes, the specific activity of pyruvate carboxylase (PC) increases about 25-fold in parallel with its intracellular protein concentration. The increase in PC protein concentration is accompanied by a 9-10-fold increase in the relative abundance of 4.2 kb PC mRNA measured by Northern-blot analysis using a cDNA probe encoding a segment of the PC gene of 3T3-L1 adipocytes. The effects of cyclic AMP (cAMP) alone and together with insulin on levels of cellular protein, PC activity, PC protein and on the relative abundance of PC mRNA were examined in mature 3T3-L1 adipocytes. Adipocytes exposed to cAMP for 24 h exhibited a 25% decrease in cellular protein and marked decreases in enzyme activity (88%) and PC mRNA abundance (98%) compared with untreated adipocyte controls. After 48 h of exposure to cAMP, PC activity and PC mRNA diminished to levels approaching their detection limits. When exposed to medium containing cAMP plus insulin, adipocyte enzyme activity and PC mRNA declined more slowly during the first 24 h exposure (about 20% decrease) but after 48 h fell to values comparable with those of adipocytes exposed to cAMP alone. Despite these decreases in enzyme activity, the PC protein content of adipocytes treated with cAMP alone or cAMP plus insulin are nearly identical with that of control adipocytes. The inactivation of PC in cAMP-treated adipocytes does not involve loss of the prosthetic group from the holoenzyme. Cross-linking experiments suggest that the spatial arrangement of protomers in inactive PC may differ from that in the active tetrameric enzyme. Data presented suggest that, in addition to inducing inactivation, cAMP may also regulate adipocyte PC by decreasing transcription of the PC gene and/or enhancing the rate of degradation of PC mRNA.

scholarly journals A Discussion of Enzyme Reference Materials: Applications and Specifications

1973 ◽  
Vol 19 (1) ◽  
pp. 5-9 ◽  
Author(s):  
Charles F Fasce ◽  
Robert Rej ◽  
William H Copeland ◽  
Raymond E Vanderlinde
Keyword(s):  
Enzyme Activity ◽  
Reference Materials ◽  
Aspartate Aminotransferase ◽  
Specific Activity ◽  
High Specific Activity ◽  
Aminotransferase Activity ◽  
Precision Control ◽  
Clinical Laboratories ◽  
Assay Conditions

Abstract Clinical laboratories estimating enzyme activity in serum are using commercial lyophilized sera for four major purposes. These uses—as a standard and for intermethod, intramethod, and precision control—are segregated, and specifications for each deployment are examined in terms of requirements for the enzyme material: freedom from interfering or indicator enzymes and chromogens; high specific activity; inclusion of optimal cofactor concentrations; commutability, human properties and source; the presence of a single isoenzyme; and stability. The effects of serum matrix and variable assay conditions on the utility of enzyme materials are analyzed. Specifications differ for each enzyme material application. The compatibility of commercial lyophilized sera containing aspartate aminotransferase activity with several cited specifications is assessed

EXTRACTION OF ENZYMES AND ASSESSMENT OF METABOLISM IN BOVINE RUMEN EPITHELIUM

1982 ◽  
Vol 62 (2) ◽  
pp. 429-438 ◽  
Author(s):  
ROY S. BUSH
Keyword(s):  
Enzyme Activity ◽  
Specific Activity ◽  
Total Activity ◽  
Small Contribution ◽  
Bacterial Protein ◽  
Total Enzyme Activity ◽  
Rumen Epithelium ◽  
Bacterial Enzyme ◽  
Protein Contamination ◽  
Total Enzyme

Papillae collected from the rumens of freshly killed cows were used to estimate the most appropriate methods for enzyme extraction from rumen epithelium and the amount of enzymes in extracts which might be of bacterial origin. Extractions of enzymes from fresh and frozen papillae were compared for the Polytron homogenizer (PT), the Potter-Elvehjem homogenizer (PE), the Waring blender, sonication and acetone powdering plus PE. PE extraction yielded solutions with the highest specific activity for each enzyme. PT extraction released the most protein and total enzyme activity into solution. PT extraction was chosen for the remaining tests because of the high total activity released. Mixed rumen bacteria were homogenized by sonication. Electrophoretic examination of epithelial and bacterial extracts showed differential migration for malate dehydrogenase. Lactate dehydrogenase from the epithelium showed four distinct isozymes whereas the bacterial enzyme showed little distinct band development. Contamination of epithelial extracts by bacterial protein was estimated to be less than 5%. The specific activities of 10 enzymes were found to be similar in epithelial and bacterial extracts so that a small amount of protein contamination would result in only a small contribution to total enzyme activity. The presence of the enzymes assayed in this study plus a number reported in the literature showed that rumen epithelial metabolism is more diverse than previously recognized. Key words: Rumen epithelium, enzymes, extraction

scholarly journals Modification of β-galactosidase for use in organic mono-phase hexanol system

2009 ◽  
Vol 28 (1) ◽  
pp. 91 ◽  
Author(s):  
Daniela Nikolovska-Nedelkoska ◽  
Irina Mladenoska ◽  
Filimena Poposka ◽  
Eleonora Winkelhausen ◽  
Slobodanka Kuzmanova
Keyword(s):  
Enzyme Activity ◽  
Water Activity ◽  
Sodium Dodecyl ◽  
Reaction Medium ◽  
Fold Increase ◽  
Total Activity ◽  
Phase System ◽  
Eupergit C ◽  
Total Enzyme Activity ◽  
Micro Emulsion

Different methods for preparation of the Aspergillus oryzae β-galactosidase were evaluated and the total enzyme activity was determined in hexanol mono-phase system using p-nitrophenyl-β-D-galactoside as a substrate. Several supports such as Acurell EP-100, Amberlite IRC (50), Celite, Dowex, Eupergit C and silica gel were tested in order to select the most suitable matrix for immobilization of the β-galactosidase for performing transgalactosylation reactions in hexanol. Celite and Amberlite IRC (50) were selected as the most appropriate carriers. Albumin, PEG 6000, starch and glycine, added prior to the immobilization procedure, acted as stabilizers of the galactosidase. By adding albumin on Celite, a 3.3-fold increase of enzyme activity was achieved. Sodium dodecyl sulphate (SDS), dioctyl sulfosuccinate (AOT), Tween 65 and crown ether were used directly in the reaction medium in order to increase its homogeneity. Addition of SDS to the medium resulted in a 3.65-fold increased activity of the β- galactosidase deposited on Celite. A micro-emulsion system created by addition of AOT resulted in an increased catalytic activity. The β-galactosidase showed enhanced total activity with increased water activity in the system having the highest value for the water activity close to the saturation level (0.92).

scholarly journals Partial purification of (Ca2+ + Mg2+)-dependent ATPase from pig smooth muscle and reconstitution of an ATP-dependent Ca2+-transport system

1981 ◽  
Vol 198 (2) ◽  
pp. 265-271 ◽  
Author(s):  
F Wuytack ◽  
G De Schutter ◽  
R Casteels
Keyword(s):  
Enzyme Activity ◽  
Smooth Muscle ◽  
Transport System ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Partial Purification ◽  
Total Enzyme Activity ◽  
Dependent Atpase ◽  
Specific Activities ◽  
Total Enzyme

(CaMg)ATPase [(Ca2+ + Mg2+)-dependent ATPase] was partially purified from a microsomal fraction of the smooth muscle of the pig stomach (antrum). Membranes were solubilized with deoxycholate, followed by removal of the detergent by dialysis. The purified (CaMg)ATPase has a specific activity (at 37 degrees C) of 157 +/- 12.1 (7)nmol.min-1.mg-1 of protein, and it is stimulated by calmodulin to 255 +/- 20.9 (7)nmol.min.mg-1. This purification of the (CaMg)ATPase resulted in an increase of the specific activity by approx. 18-fold and in a recovery of the total enzyme activity of 55% compared with the microsomal fraction. The partially purified (CaMg)ATPase still contains some Mg2+-and (Na+ + K+)-dependent ATPase activities, but their specific activities are increased relatively less than that of the (CaMg)ATPase. The ratios of the (CaMg)ATPase to Mg2+- and (Na+ + K+)-dependent ATPase activities increase from respectively 0.14 and 0.81 in the crude microsomal fraction to 1.39 and 9.07 in the purified preparation. During removal of the deoxycholate by dialysis, vesicles were reconstituted which were capable of ATP-dependent Ca2+ transport.

scholarly journals Partial Purification, Characterization, and Application of Extracellular Aspartic Protease from Lactobacillus casei WSP in Producing the Bioactive Peptides with Antibacterial and Antioxidant Activity

2018 ◽  
Vol 22 (2) ◽  
pp. 47
Author(s):  
Akhmad Solikhin ◽  
Apon Zaenal Mustopa ◽  
Suharsono Suharsono ◽  
Wendry Setiyadi Putranto
Keyword(s):  
Enzyme Activity ◽  
Metal Ion ◽  
Lactobacillus Casei ◽  
Specific Activity ◽  
Gel Filtration ◽  
Fold Increase ◽  
Iodoacetic Acid ◽  
Aspartic Protease ◽  
Partial Purification ◽  
Antioxidant Agents

   Lactobacillus casei WSP-derived an aspartic protease was sequentially purified by using chromatography gel filtration sephadex G-50. It resulted in a 22.81-fold increase of specific activity (51.5 U/mg) with a final yield of 1.9%. The estimated molecular weight of the purified enzyme was 37 kDa and showed gelatinolytic activity in zymogram assay. The enzyme exhibited optimum activity at 40ºC and pH 6 with casein as the substrate. Enzyme activity was significantly inhibited by pepstatin A (0.5 mM and 1 mM), confirming that this enzyme is a group of aspartic proteases, while other inhibitors such as EDTA, PMSF and iodoacetic acid showed no inhibition effect on the activity of enzyme. The addition of metal ion to the enzyme decreased enzyme activity, indicating the proteolytic enzyme was metal ion- dependent. Denaturant such as DDT tended to increase caseinolytic activity. Furthermore, this enzyme was capable of generating the new peptides from skimmed milk with the size 8 kDa, 10 kDa and 15 kDa. These peptides have potential as antibacterial and antioxidant agents.

scholarly journals Decolourisation of Reactive Black 5 by Azoreductase Produced by Brevibacillus panacihumi ZBI

2012 ◽  
Vol 59 (1) ◽  
Author(s):  
Masyithah Aalia Mohd Ramlan ◽  
Nadhirah Aminah Azizan ◽  
Bay Hui Han ◽  
Lim Chi Kim ◽  
Shaza Eva Mohamad ◽  
...  
Keyword(s):  
Ionic Liquid ◽  
Enzyme Activity ◽  
Phosphate Buffer ◽  
Culture Supernatant ◽  
Specific Activity ◽  
Enzyme Reaction ◽  
Reactive Black 5 ◽  
Degrading Bacterium ◽  
The Stability ◽  
Assay Conditions

Azoreductases are often associated with decolourisation of non–degradable azo dyes via cleavage of azo bonds. In this study,Brevibacillus panacihumi ZBI, an azo dye–degrading bacterium which has not been reported before, was used for the decolourisation of Reactive Black 5 (RB5) dye. The highest activity of azoreductase was obtained during the end of log phase. Azoreductase produced intracellularly had the highest specific activity of 0.334 U/mg compared to the culture supernatant (extracellular), resting cell and cell debris with low enzyme activity of 0.034 U/mg, 0.010 U/mg and 0.200 U/mg respectively. The optimum assay conditions for the maximum azoreductase activity were at 37°C, pH 7, RB5 dye concentration of 100 mg/L and NADH concentration of 0.2 mM by using phosphate buffer as a medium for the enzyme reaction. Alternatively, the azoreductase assay was also carried out using ionic liquid, [emim][EtSO4] that may function to enhance the activity and stability of azoreductase. Results using phosphate buffer (pH 7) showed higher enzyme activity twice that of the ionic liquid besides enhancing the stability of enzyme. Under the optimum assay conditions up to 93 % of decolourisation was achieved after 8 h of incubation. In addition, growth of bacteria was also concurrently observed during the decolourisation of RB5.

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