scholarly journals Deoxycytidylate deaminase. Properties of the enzyme from cultured kidney cells of baby hamster

1974 ◽  
Vol 141 (1) ◽  
pp. 211-217 ◽  
Author(s):  
Hilary A. Rolton ◽  
Hamish M. Keir
Keyword(s):  
Molecular Weight ◽  
Ethylene Glycol ◽  
Substrate Concentration ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Kidney Cells ◽  
Maximal Velocity ◽  
Ph Optimum ◽  
Hyperbolic Curve ◽  
Km Value

dCMP deaminase was partially purified from BHK-21/C13 cells grown in culture. The molecular weight of the enzyme was estimated by gel filtration and gradient centrifugation to be 130000 and 115000 respectively. The enzyme had a pH optimum of 8.4. Its activity versus substrate concentration curve was sigmoid, the substrate concentration at half-maximal velocity being 4.4mm. dCTP activated the deaminase maximally at 40μm, gave a hyperbolic curve for activity versus dCMP concentration and a Km value for dCMP of 0.91mm. dCTP activation required the presence of Mg2+ or Mn2+ ions. dTTP inhibited the deaminase maximally at 15μm; the inhibition required the presence of Mg2+ or Mn2+ ions. The enzyme was very heat-labile but could be markedly stabilized by dCTP at 0.125mm and ethylene glycol at 20% (v/v).

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

scholarly journals Soluble neutral and acid maltases in the suckling-rat intestine. The effect of cortisol and development

1974 ◽  
Vol 144 (2) ◽  
pp. 281-292 ◽  
Author(s):  
G Galand ◽  
G G Forstner
Keyword(s):  
Molecular Weight ◽  
Gradient Centrifugation ◽  
Heat Sensitivity ◽  
Adult Rats ◽  
Ph Optimum ◽  
Maltase Activity ◽  
Acid Maltase ◽  
Membrane Bound ◽  
Sepharose 4B ◽  
The Relationship

The 100000g supernatants from 13-day-old suckling-rat intestinal homogenates contained 43.5% of the total intestinal maltase activity, compared with 7.1% in weaned adult rats aged 40 days. The soluble maltase activity was separated on Sepharose 4B into two quantitatively equal fractions at pH6.0, one containing a maltase with a neutral pH optimum and the other a maltase with an acid pH optimum. The neutral maltase was shown to be a maltase–glucoamylase identical with membrane-bound maltase–glucoamylase in molecular weight, heat-sensitivity, substrate specificity, Km for maltose and Ki for Tris. The soluble enzyme was induced by cortisol, but the ratio of the soluble to bound enzyme fell during induction. Solubility of the neutral maltase was not accounted for by the action of endogenous proteinases under the preparative conditions used. It is postulated that the soluble neutral maltase is a membrane-dissociated form of the bound enzyme and that the relationship between these two forms is modulated by cortisol. The acid maltase generally resembled acid maltase of liver, muscle and kidney. It was shown to be a maltase–glucoamylase with optimal activity at pH3.0, and molecular weight of 136000 by density-gradient centrifugation. At pH3.0 its Km for maltose was 1.5mm. It was inhibited by turanose (Ki=7.5mm) and Tris (Ki=5.5mm) but not by p-chloromercuribenzoate or EDTA. Some 55% of its activity was destroyed by heating at 50°C for 10min. The acid maltase closely resembled β-glucuronidase and acid β-galactosidase in its distribution in the intestine, response to tissue homogenization in various media, and decrease in activity with cortisol treatment and weaning, indicating that it was a typical lysosomal enzyme concentrated in the ileum.

Partial purification and properties of an L-arabinose dehydrogenase from Azospirillum brasiliense

1983 ◽  
Vol 29 (2) ◽  
pp. 242-246 ◽  
Author(s):  
Norman J. Novick ◽  
Max E. Tyler
Keyword(s):  
Molecular Weight ◽  
Divalent Cation ◽  
Gel Filtration ◽  
Reducing Agent ◽  
Partial Purification ◽  
Ph Optimum ◽  
Glycine Buffer ◽  
Purification And Properties ◽  
Filtration Technique

An L-arabino-aldose dehydrogenase responsible for the oxidation of L-arabinose to L-arabino-γ-lactone has been purified 59-fold from L-arabinose grown cells of Azospirillum brasiliense. The dehydrogenase was found to be specific for substrates with the L-arabino-configuration at carbons 2, 3, and 4. Km values for L-arabinose of 75 and 140 μM were found with NADP and NAD as coenzymes, respectively. The enzyme had a pH optimum of 9.5 in glycine buffer and was stable when heated to 55 °C for 5 min. No enhancement of activity in the presence of any divalent cation or reducing agent tested was found. L-Arabinose dehydrogenase had a molecular weight of 175 000 as measured by the gel filtration technique.

scholarly journals Association of newly synthesized islet prohormones with intracellular membranes.

1984 ◽  
Vol 99 (2) ◽  
pp. 418-424 ◽  
Author(s):  
B D Noe ◽  
M N Moran
Keyword(s):  
Secretory Granules ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Proteolytic Processing ◽  
Membrane Association ◽  
Freezing And Thawing ◽  
Ph Optimum ◽  
Intracellular Membranes ◽  
Membrane Lysis ◽  
Repeated Freezing

Results from recent studies have indicated that pancreatic islet prohormone converting enzymes are membrane-associated in islet microsomes and secretory granules. This observation, along with the demonstration that proglucagon is topologically segregated to the periphery within alpha cell secretory granules in several species, led us to investigate the possibility that newly synthesized islet prohormones might be associated with intracellular membranes. Anglerfish islets were incubated with [3H]tryptophan and [14C]isoleucine for 3 h, then fractionated by differential and density gradient centrifugation. Microsome (M) and secretory granule (SG) fractions were halved, sedimented, and resuspended in the presence or absence of dissociative reagents. After membrane lysis by repeated freezing and thawing, the membranous and soluble components were separated by centrifugation. Extracts of supernatants and pellets were chromatographed by gel filtration; fractions were collected and counted. A high proportion (77-79%) of the newly synthesized proinsulin and insulin was associated with both M and SG membranes. Most of the newly synthesized proglucagons and prosomatostatins (12,000-mol-wt precursors) were also membrane-associated (86-88%) in M and SG. In contrast, glucagon- and somatostatin-related peptides exhibited much less membrane-association in SG (24-31%). Bacitracin, bovine serum albumin EDTA, RNAse, alpha-methylmannoside, N-acetylglucosamine, and dithiodipyridine had no effect on prohormone association with membranes. However, high salt (1 M KCl) significantly reduced membrane-association of prohormones. Binding of labeled prohormones to SG membranes from unlabeled tissue increased with incubation time and was inhibited by unlabeled prohormones. The pH optimum for prohormone binding to both M and SG membranes was 5.2. It is suggested that association of newly synthesized prohormones with intracellular membranes could be related to the facilitation of proteolytic processing of prohormones and/or transport from their site of synthesis to the secretory granules.

scholarly journals Purification and characterization of two fructose diphosphate aldolases from Escherichia coli (Crookes' strain)

1973 ◽  
Vol 131 (4) ◽  
pp. 833-841 ◽  
Author(s):  
Donald Stribling ◽  
Richard N. Perham
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Metal Ions ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Class Ii ◽  
Class I ◽  
E Coli ◽  
Km Value ◽  
Bivalent Metal Ions

Two fructose diphosphate aldolases (EC 4.1.2.13) were detected in extracts of Escherichia coli (Crookes' strain) grown on pyruvate or lactate. The two enzymes can be resolved by chromatography on DEAE-cellulose at pH7.5, or by gel filtration on Sephadex G-200, and both have been obtained in a pure state. One is a typical bacterial aldolase (class II) in that it is strongly inhibited by metal-chelating agents and is reactivated by bivalent metal ions, e.g. Ca2+, Zn2+. It is a dimer with a molecular weight of approx. 70000, and the Km value for fructose diphosphate is about 0.85mm. The other aldolase is not dependent on metal ions for its activity, but is inhibited by reduction with NaBH4 in the presence of substrate. The Km value for fructose diphosphate is about 20μm (although the Lineweaver–Burk plot is not linear) and the enzyme is probably a tetramer with molecular weight approx. 140000. It has been crystallized. On the basis of these properties it is tentatively assigned to class I. The appearance of a class I aldolase in bacteria was unexpected, and its synthesis in E. coli is apparently favoured by conditions of gluconeogenesis. Only aldolase of class II was found in E. coli that had been grown on glucose. The significance of these results for the evolution of fructose diphosphate aldolases is briefly discussed.

Proteinase activity in the plerocercoid of Proteocephalus ambloplitis (Cestoda)

Parasitology ◽  
1994 ◽  
Vol 109 (2) ◽  
pp. 209-213 ◽  
Author(s):  
M. Polzer ◽  
R. M. Overstreet ◽  
H. Taraschewski
Keyword(s):  
Molecular Weight ◽  
Intestinal Tract ◽  
Chelating Agents ◽  
Gel Filtration ◽  
Proteinase Activity ◽  
Host Liver ◽  
Ph Optimum ◽  
Chromatographic Techniques ◽  
Tissue Migration ◽  
Enzyme Class

SUMMARYHost invasion and tissue migration of several helminths have been linked to expression and release of parasite-derived proteinases. The plerocercoid of the cestode Proteocephalus ambloplitis can migrate into the visceral organs or, in the case of bass, from them into the intestinal tract of the same individual fish. It does this within a few hours, aided by secretion of a substance from its apical gland. Proteinase activity in this plerocercoid, obtained from the host liver, was defined by pH optimum, by substrate and inhibitor specificity, and by electrophoretic and chromatographic techniques. Homogenates of plerocercoid contained a metalloproteinase exhibiting a molecular weight of 30000 determined by gelatin substrate gel electrophoresis. Peak activity of this proteolytic enzyme in gel filtration fractions when azocoll was used as substrate then corresponded to a molecular weight of 31500. The proteinase showed collagenolytic, haemoglobinolytic and slight elastinolytic activity, and it had a pH optimum at 9·0. Enzyme activity could be inhibited by various chelating agents. The metalloproteinase identified in this study constitutes the only enzyme class present in this larval stage of P. ambloplitis. We suggest that the plerocercoid's metalloproteinase is the substance secreted from the apical organ, necessary for the previously recognized tissue migration phase. This enzyme might also have a nutritional function.

scholarly journals Partial purification and characterization of chick-embryo prolyl 3-hydroxylase

1979 ◽  
Vol 183 (2) ◽  
pp. 303-307 ◽  
Author(s):  
K Tryggvason ◽  
K Majamaa ◽  
J Risteli ◽  
K I Kivirikko
Keyword(s):  
Molecular Weight ◽  
Chick Embryo ◽  
Ethylene Glycol ◽  
Affinity Chromatography ◽  
Concanavalin A ◽  
Gel Filtration ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Chick Embryo Extract

Prolyl 3-hydroxylase was purified up to about 5000-fold from an (NH4)2SO4 fraction of chick-embryo extract by a procedure consisting of affinity chromatography on denatured collagen linked to agarose, elution with ethylene glycol and gel filtration. The molecular weight of the purified enzyme is about 160000 by gel filtration The enzyme is probably a glycoprotein, since (a) its activity is inhibited by concanavalin A, and (b) the enzyme is bound to columns of this lectin coupled to agarose and can be eluted with a buffer containing methyl alpha-D-mannoside. The Km values for Fe2+, 2-oxoglutarate, O2 and ascorbate in the prolyl 3-hydroxylase reaction were found to be very similar to those previously reported for these co-substrates in the prolyl 4-hydroxylase and lysyl hydroxylase reactions.

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.

5-ENE-3β-HYDROXYSTEROID DEHYDROGENASE OF HUMAN AND BOVINE ADRENOCORTICAL ENDOPLASMIC RETICULUM: SOLUBILIZATION AND FRACTIONATION

1977 ◽  
Vol 72 (2) ◽  
pp. 225-233 ◽  
Author(s):  
A. R. EASTMAN ◽  
A. M. NEVILLE
Keyword(s):  
Molecular Weight ◽  
Adrenal Glands ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Hydroxysteroid Dehydrogenase ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Microsomal Membranes ◽  
Molecular Sizes ◽  
Triton X

SUMMARY Protein moieties of various molecular sizes and possessing 5-ene-3β-hydroxysteroid dehydrogenase activity have been successfully solubilized from the microsomal membranes of both bovine and human adrenal glands using a combination of Triton X-100 and sonication. These moieties have been studied by gel filtration, sucrose density gradient centrifugation and isoelectric focusing, and were shown to possess a minimum molecular weight of about 118000, with an isoelectric point between 7·2 and 7·4. The molecular weight was dependent upon the concentration of Triton X-100 used during fractionation. No separation of dehydrogenase activities toward the three steroid substrates, pregnenolone, 17α-hydroxypregnenolone and dehydroisoandrosterone, was observed. Changes in the relative activities for the steroid substrates during fractionation were observed, but have been attributed to the formation of allotypes rather than to the existence of separate dehydrogenases with restricted substrate specificity.

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.

Sign in / Sign up

Export Citation Format

Share Document