scholarly journals Partial purification and characterization of a novel neutral proteinase from human uterine cervix

1980 ◽  
Vol 185 (2) ◽  
pp. 443-450 ◽  
Author(s):  
Akira Ito ◽  
Hiroko Ihara ◽  
Yo Mori
Keyword(s):  
Uterine Cervix ◽  
Gel Filtration ◽  
Reaction System ◽  
Partial Purification ◽  
High Concentration ◽  
Neutral Proteinase ◽  
Bean Trypsin Inhibitor ◽  
Cervical Stroma ◽  
High Concentrations ◽  
Hydrolysed Casein

1. Human uterine cervical stroma was found to contain a Ca2+-independent neutral proteinase against casein and N-benzoyl-dl-arginine p-nitroanilide (Bz-dl-Arg-Nan). This enzyme was tightly bound to an insoluble material (20000g pellet) and was solubilized by high concentrations of NaCl or KCl. High concentrations of them in the reaction system, however, inhibited reversibly the activity of this enzyme. 2. The neutral proteinase was partially purified by extraction with NaCl, gel filtration on Sephadex G-200 and affinity chromatography on casein–Sepharose. 3. The optimal pH of this partially purified enzyme was 7.4–8.0 against casein and Bz-dl-Arg-Nan. The molecular weight of the enzyme was found to be about 1.4×105 by gel filtration on Sephadex G-200. 4. The enzyme was significantly inhibited by di-isopropyl phosphorofluoridate (0.1mm). High concentration of phenylmethanesulphonyl fluoride (5mm), 7-amino-1-chloro-3-l-tosylamidoheptan-2-one (0.5mm), antipain (10μm) or leupeptin (10μm) was also found to be inhibitory, but chymostatin (40μg/ml), soya-bean trypsin inhibitor (2.5mg/ml), human plasma (10%, v/v), p-chloromercuribenzoate (1mm), EDTA (10mm) and 1-chloro-4-phenyl-3-l-tosylamidobutan-2-one (1mm) had no effect on the enzyme. 5. The neutral proteinase hydrolysed casein, Bz-dl-Arg-Nan and heat-denatured collagen, but was inactive towards native collagen and several synthetic substrates, such as 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg, 3-carboxypropionyl-Ala-Ala-Ala p-nitroanilide and 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-d-Arg, and also proteoglycan. The enzyme did not act as a plasminogen activator. 6. These properties suggested that a neutral proteinase in the human uterine cervix was different from enzymes previously reported.

scholarly journals Purification of a neutral proteinase, associated with the actomyosin complex, from uterine myometrium

1984 ◽  
Vol 222 (3) ◽  
pp. 613-620 ◽  
Author(s):  
R Barth ◽  
M Hoechst ◽  
E G Afting
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Thiol Group ◽  
Hydrolytic Activity ◽  
Proteinase Activity ◽  
Molar Ratio ◽  
High Concentration ◽  
Neutral Proteinase ◽  
Actomyosin Complex

We have purified and characterized a neutral proteinase activity from pig uterine myometrium. The proteinase co-purified with the actomyosin complex and could only be separated from it by a high concentration of a chaotropic ion, 3M-NaBr. The proteinase was further purified by gel filtration and affinity chromatography. The purified protein showed a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis corresponding to an Mr of 28 000. Gel filtration on Sephadex G-100 in a buffer containing 3M-NaBr gave an Mr of 27 500. Without the addition of the chaotropic Br- ion, the proteinase aggregates to high-Mr forms of more than 10(6)Da. The proteinase has optimum hydrolytic activity with casein as substrate at pH 7.5-8.0. The thiol-group-blocking reagents p-chloromercuribenzoate, p-chloromercuribenzenesulphonate and Hg2+, as well as soya-bean trypsin inhibitor and 4-aminobenzamidine, inhibited the proteinase. Other bivalent cations, chelating agents and the serine-specific reagents 7-amino-1-chloro-3-tosylamido-L-heptan-2-one and phenylmethanesulphonyl fluoride were without any effect on proteinase activity. The proteinase degraded myosin very rapidly at a molar ratio of proteinase to myosin of 1:50, concomitant with the rate of loss of the ATPase activity. Compared with myosin, actin was only a poor substrate and was degraded at a much lower rate, even at a high molar ratio of the proteinase to actin.

scholarly journals Partial purification and properties of l-asparagine synthetase from mouse pancreas

1979 ◽  
Vol 181 (1) ◽  
pp. 51-59 ◽  
Author(s):  
Harry A. Milman ◽  
David A. Cooney
Keyword(s):  
Specific Activity ◽  
Asparagine Synthetase ◽  
Partial Purification ◽  
Synthetase Activity ◽  
Mouse Pancreas ◽  
Purification And Properties ◽  
Bean Trypsin Inhibitor ◽  
Thiol Reagent ◽  
High Concentrations ◽  
Glutaminase Activity

l-Asparagine synthetase was partially purified from mouse pancreas to a final mean specific activity of 0.10 unit/mg of protein. The enzyme exhibited an l-glutaminase activity which was not affected by l-asparate, NH4Cl, ATP–MgCl2, l-glutamate, AMP (sodium salt) or sodium pyrophosphate. The l-glutamine-dependent l-asparagine synthetase activity of the partially purified enzyme from mouse pancreas was markedly decreased by freezing for 7 days at −87°C in the presence of 1mm-dithiothreitol, but effectively protected from inactivation by high concentrations (10mm) of the thiol reagent. The l-glutaminase activity of the enzyme was inhibited by antagonists of l-glutamine (e.g. 6-diazo-5-oxo-l-norleucine, 5-chloro-4-oxo-l-norvaline, 5-diazo-4-oxo-l-norvaline and NSC-163501) and thiol-reactive compounds (e.g. 2-amino-4-arsenophenol hydrochloride, maleimide, mucochloric acid and ZnCl2), but not by aminomalonic acid, the next lower homologue of l-aspartate, nor by l-homoserine β-adenylate, an analogue of the presumed transitory covalent intermediate. The complete forward reaction catalysed by l-asparagine synthetase from mouse pancreas appears to be irreversible and essentially stoicheiometric under the conditions examined. Mouse pancreas contains a proteolytic inhibitor of l-asparagine synthetase separable from the enzyme by ion-exchange column chromatography. The inhibitor is activated by incubation at 4°C for 110h and inactivated by soya-bean trypsin inhibitor, di-isopropyl phosphorofluoridate and boiling.

Partial Purification and Characterization of an ADP Phosphohydrolase from Human Plasma

1971 ◽  
Vol 26 (01) ◽  
pp. 177-191 ◽  
Author(s):  
I Holmsen ◽  
H Holmsen
Keyword(s):  
Substrate Concentration ◽  
Platelet Rich Plasma ◽  
Gel Filtration ◽  
Partial Purification ◽  
Low Concentrations ◽  
Isotope Technique ◽  
High Concentrations ◽  
Hydrolysis Of ◽  
Plasma Adenosine

SummaryPlasma contains enzymes capable of dephosphorylating ADP, ATP and AMP (adenosine di-, tri- and monophosphate). In platelet-rich plasma these enzymes are important for the regulation of the levels of (platelet-aggregating) ADP and (aggregation-inhibitory) adenosine.Plasma ADPase and ATPase were studied at 1 (µM substrate concentration using an isotope technique. Both enzymes were precipitated from plasma at 45-65% saturation with (NH4)2S04 and emerged together by gel filtration on Sephadex G-200 and from DEAE-Sephadex (0.12-0.20 M Cl-, pH 8.2). In combination these procedures gave 1,500-1,800 times purification of ADPase relative to plasma. The purest fraction contained ATPase, ADPase and AMPase in a 0.17:1.00:2.92 proportion, quite different from their 5.34:1.00:5.34 proportion in plasma. Adenosine deaminase and adenylate kinase were not present in the purest fraction, whereas nucleoside diphosphokinase appeared to be present.The purified ADPase was stimulated by low concentrations of Mg2+ and Mn2+, whereas high concentrations were inhibitory. This inhibition could not be explained by an increase in the ionic strength. Ca2+ and Zn2+ were inhibitory at all concentrations used (0-3 mM). Lineweaver-Burke plots were linear for both ADPase and ATPase in the 0−4 x 10-5 M substrate range, and both enzymes had Km = 1.1 x 10−5 M. Increase of the substrate concentration above 4 x 10−5M gave deviation from MichaelisMenten kinetics, and Eadie-Hofstee plots indicated the presence of “high-Km” ADPase and ATPase. The latter enzymes were not studied.Déphosphorylation of 3H-ADP by purified “low-Km” ADPase was reduced by nonradioactive diphosphates of guanosine, inosine, cytidine and uridine in the same way as when nonradioactive ADP was used. Nonradioactive AMP also reduced dephosphorylation of 3H-ADP, whereas nonradioactive ATP did not.Cyanide, cysteine and tartrate inhibited “low-Km” ADPase whereas p-chloromercuribenzoate, p-chloromercuribenzoesulphonate and N-ethylmaleimide had no effect. EDTA inhibited the enzyme activity in a way that could not be abolished by excess Mg2+. Purified plasma “low-Km” ADPase thus appears to be an unspecific enzyme, as one and the same active site does not seem to distinguish between the base moiety of nucleoside diphosphates, and catalyzes hydrolysis of phosphate esters as well as pyrophosphate bonds. The relation between plasma ADPase and ATPase remains unclear.

Partial purification and photoaffinity labelling of sunflower acyl-CoA:lysophosphatidyl-choline acyltransferase

2000 ◽  
Vol 28 (6) ◽  
pp. 715-718 ◽  
Author(s):  
T. Fraser ◽  
K. Stobart
Keyword(s):  
Enzyme Activity ◽  
Liquid Chromatography ◽  
Gel Filtration ◽  
Partial Purification ◽  
Trypsin Treatment ◽  
Photoaffinity Labelling ◽  
Sds Page ◽  
Molecular Masses ◽  
High Concentrations ◽  
Choline Acyltransferase

Previous attempts to purify acyl-CoA: 1-acyl-lysophosphatidylcholine acyltransferase (EC 2.3.1.23) have been frustrated by difficulties in solubilizing the enzyme without inactivation. Microsomal preparations, from the developing cotyledons of sunflower, in high concentrations of urea retain activity. Gel-filtration liquid chromatography followed by trypsin treatment (minus urea) resulted in the removal of many contaminating proteins without loss of enzyme activity. SDS/PAGE showed the presence of two major peptides with apparent molecular masses of 52 and 59 kDa. These polypeptides cross-reacted with the radiolabelled photoreactive substrate 1-azido-oleoyl-sn-lysophosphatidyl-[N-methyl-3H]choline.

scholarly journals Proteoglycan-degrading enzymes of rabbit fibroblasts and granulocytes

1978 ◽  
Vol 173 (3) ◽  
pp. 949-958 ◽  
Author(s):  
Z Werb ◽  
J T Dingle ◽  
J J Reynolds ◽  
A J Barrett
Keyword(s):  
Trypsin Inhibitor ◽  
Gel Filtration ◽  
Synovial Fibroblasts ◽  
Enzymic Activity ◽  
Polymorphonuclear Leucocyte ◽  
Exchange Chromatography ◽  
Soya Bean ◽  
Polymorphonuclear Leucocytes ◽  
Neutral Proteinase ◽  
Bean Trypsin Inhibitor

A neutral proteinase secreted by rabbit synovial fibroblasts in parallel with specific collagenase was partially purified by ion-exchange chromatography. At pH 7.6 this proteinase degraded 35S-labelled bovine nasal proteoglycan and azo-casein. The enzymic activity was inhibited by EDTA, 1,10-phenanthroline and serum, whereas di-isopropyl phosphorofluoridate and soya-bean trypsin inhibitor had little effect. By gel filtration the apparent mol.wt. of the enzyme was 25000. The fibroblast neutral proteinase was compared with the proteoglycan-degrading neutral proteinases of rabbit polymorphonuclear-leucocyte granules. Two distinct activities were found in the granules: one was inhibited by soya-bean trypsin inhibitor and the other by EDTA. The proteoglycan-degrading proteinases of rabbit fibroblasts and polymorphonuclear leucocytes at acid pH also were examined. Both cathepsin D and a thiol-dependent proteinase contributed to the degradation of proteoglycan at pH 4.5.

Studies on an Inhibitor of Plasminogen Activation in Human Serum

1973 ◽  
Vol 30 (02) ◽  
pp. 414-424 ◽  
Author(s):  
Ulla Hedner
Keyword(s):  
Ion Exchange ◽  
Human Serum ◽  
Antithrombin Iii ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Specific Adsorption ◽  
Partial Purification ◽  
Plasminogen Activation ◽  
Preparative Electrophoresis

SummaryA procedure is described for partial purification of an inhibitor of the activation of plasminogen by urokinase and streptokinase. The method involves specific adsorption of contammants, ion-exchange chromatography on DEAE-Sephadex, gel filtration on Sephadex G-200 and preparative electrophoresis. The inhibitor fraction contained no antiplasmin, no plasminogen, no α1-antitrypsin, no antithrombin-III and was shown not to be α2 M or inter-α-inhibitor. It contained traces of prothrombin and cerulo-plasmin. An antiserum against the inhibitor fraction capable of neutralising the inhibitor in serum was raised in rabbits.

Plasminogen-Activator in Human Early Milk: Its Partial Purification and Characterization

1981 ◽  
Vol 45 (02) ◽  
pp. 121-126 ◽  
Author(s):  
Utako Okamoto ◽  
Noboru Horie ◽  
Yoko Nagamatsu ◽  
Jun-Ichiro Yamamoto
Keyword(s):  
Plasminogen Activator ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Partial Purification ◽  
Order Kinetic ◽  
Diisopropyl Fluorophosphate ◽  
Step Procedure ◽  
Activity Band ◽  
C 50 ◽  
Gel Isoelectric Focusing

SummaryMilk plasminogen-activator was partially purified from human transitional milk collected at about 10 days after delivery, by a five-step procedure involving chloroform treatment, ammonium sulfate precipitation, and column chromatography on Sephadex G-150, CM Sephadex C-50 and DEAE Sephadex A-50. This gave milk-activator with a maximum purification factor of about 2,400-fold with respect to the skimmed milk. The CM Sephadex-step preparation showed, on polyacrylamide gel electrophoresis, a single plasminogen-activator activity band located between the bands of albumin and prealbumin of human serum. This preparation exhibited no kinin forming activity. The activator hydrolyzed acetyl-glycyl-L-lysine methyl ester with similar order kinetic constants to urokinase, and was inhibited strongly by diisopropyl-fluorophosphate. The molecular weight of the activator as estimated by gel filtration was approximately 86,000, the isoelectric points as estimated by gel isoelectric focusing were pH 7.2, 6.9 and 6.6, and the activator activity was not quenched by antiurokinase globulin, indicating that the milk-activator is a different entity from urokinase.

The Toxicity of Castor Beans and its Treatment with Doxycycline in Local Rabbits

2018 ◽  
Vol 9 (2) ◽  
Author(s):  
Nael Mohammed Sarheed ◽  
Osamah Faisal Kokas ◽  
Doaa Abd Alabas Muhammed Ridh
Keyword(s):  
Castor Bean ◽  
The Body ◽  
Control Group ◽  
High Concentration ◽  
Biochemical Tests ◽  
Doxycycline Treatment ◽  
High Concentrations ◽  
Physiological Tests ◽  
Animal House ◽  
Physiological And Biochemical

The plant of castor is widely spread in the Iraqi land, and characterized with containing ricin toxin, which has a very serious effects, and because the seeds of this plant scattered in the agricultural soil and rivers water, which increases the exposure of humans and animals to these beans. Objective: This experiment was designed to study the effect of high concentration of castor bean powder in some physiological and biochemical parameters and changes in some tissues of the body, as well as trying to use doxycycline to reduce the effects of ingestion of these seeds. Materials and Methods: In the experiment, 24 local rabbits were raised and fed in the Animal House of the Faculty of Medicine / Al-Muthanna University, then divided into four groups and treated for three weeks (21 days), Control group: treated with normal saline solution (0.9) orally throughout the experiment, G1: was treated orally with a concentration of 25 mg / kg of castor bean powder daily during the experiment, G2 : orally treated 25 mg / kg of castor bean and 25 mg / kg of doxycycline, G3: orally treated 25 mg / kg of castor powder with 50 mg / kg of doxycycline daily throughout the trial period. Results: The results of the experiment showed significant changes (P less than 0.05) in all physiological and biochemical blood tests when compared with control group. There was a significant decrease in PCV, Hb, RBC, T.protein and body weights, while demonstrated a significant increase in WBC, Urea, Creatinine, ALT, AST and ALP, with distortions in liver and kidney of animals that treated with Castor beans. In contrast, the treatment with doxycycline and caster beans showed significant improvement reflected by a normal proportion in physiological tests and biochemical tests with improvement in the tissues when compared to control group. Conclusions: It can be concluded from this study that castor bean has high toxic and pathogenic effects that may be dangerous to the life of the organism. Therefore, it is advisable to be cautious of these pills and avoid exposure to them, also recommended to take high concentrations of doxycycline treatment when infected with castor bean poisoning.

The Effects of Shock Vancomycin Concentrations on the Formation of Heteroresistance in Staphylococcus aureus

2020 ◽  
Vol 65 (9-10) ◽  
pp. 3-7
Author(s):  
V. V. Gostev ◽  
Yu. V. Sopova ◽  
O. S. Kalinogorskaya ◽  
M. E. Velizhanina ◽  
I. V. Lazareva ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
In Vitro Selection ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
High Concentration ◽  
Short Term ◽  
High Concentrations ◽  
Selection Of ◽  
Selection Of Resistance ◽  
Test Cultures

Glycopeptides are the basis of the treatment of infections caused by MRSA (Methicillin-Resistant Staphylococcus aureus). Previously, it was demonstrated that antibiotic tolerant phenotypes are formed during selection of resistance under the influence of high concentrations of antibiotics. The present study uses a similar in vitro selection model with vancomycin. Clinical isolates of MRSA belonging to genetic lines ST8 and ST239, as well as the MSSA (ATCC29213) strain, were included in the experiment. Test isolates were incubated for five hours in a medium with a high concentration of vancomycin (50 μg/ml). Test cultures were grown on the medium without antibiotic for 18 hours after each exposure. A total of ten exposure cycles were performed. Vancomycin was characterized by bacteriostatic action; the proportion of surviving cells after exposure was 70–100%. After selection, there was a slight increase in the MIC to vancomycin (MIC 2 μg/ml), teicoplanin (MIC 1.5–3 μg/ml) and daptomycin (MIC 0.25–2 μg/ml). According to the results of PAP analysis, all strains showed an increase in the area under curve depending on the concentration of vancomycin after selection, while a heteroresistant phenotype (with PAP/AUC 0.9) was detected in three isolates. All isolates showed walK mutations (T188S, D235N, E261V, V380I, and G223D). Exposure to short-term shock concentrations of vancomycin promotes the formation of heteroresistance in both MRSA and MSSA. Formation of VISA phenotypes is possible during therapy with vancomycin.

Growth responses of periphyton and chironomids exposed to biologically treated bleached kraft pulp mill effluent

1997 ◽  
Vol 35 (2-3) ◽  
pp. 339-345 ◽  
Author(s):  
M. G. Dubé ◽  
J. M. Culp
Keyword(s):  
Kraft Pulp ◽  
Pulp Mill ◽  
Pulp Mill Effluent ◽  
Growth Responses ◽  
High Concentration ◽  
Wood Extractives ◽  
Kraft Pulp Mill ◽  
Bleached Kraft Pulp ◽  
Low Levels ◽  
High Concentrations

Experiments were conducted in artificial streams to determine the effects of increasing concentrations of biologically treated bleached kraft pulp mill effluent (BKPME) on periphyton and chironomid growth in the Thompson River, British Columbia. Periphyton growth, as determined by increases in chlorophyll a, was significantly stimulated at all effluent concentrations tested (0.25%, 0.5%, 1.0%, 5.0% and, 10.0%). Chironomid growth (individual weight) was also significantly stimulated at low effluent concentrations (≤1.0%). At higher concentrations (5.0% and 10.0%), chironomid growth was inhibited relative to the 1.0% treatment streams. Increases in growth were attributed to the effects of nutrient and organic enrichment from BKPME. The effluent contained high concentrations of phosphorus and appears to be an important source of carbon for benthic insects grazing on the biofilm. In high concentration effluent streams, chironomid growth decreased despite low levels of typical pulp mill contaminants. This suggests that other compounds in the effluent, such as wood extractives, may be inhibiting chironomid growth. These results support findings of field monitoring studies conducted in the Thompson River where changes in periphyton and chironomid abundance occurred downstream of the bleached kraft pulp mill.

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