scholarly journals rac-Glycerol 1:2-cyclic phosphate 2-phosphodiesterase, a new soluble phosphodiesterase of mammalian tissues

1978 ◽  
Vol 173 (2) ◽  
pp. 579-589 ◽  
Author(s):  
N Clarke ◽  
R M C Dawson
Keyword(s):  
Molecular Weight ◽  
Cyclic Amp ◽  
Gel Filtration ◽  
Reducing Agents ◽  
Ph Optimum ◽  
Acetone Precipitation ◽  
Nitrophenyl Phosphate ◽  
Mammalian Tissues ◽  
Cyclic Phosphate

1. A soluble phosphodiesterase is present in mammalian tissues which rapidly hydrolyses enantiomorphs of rac-glycerol 1:2-cyclic phosphate, producing rac-glycerol 1-phosphate. 2. The enzyme has been purified up to 1700-fold by a combination of acetone precipitation and chromatography on DEAE-Sephadex A-50, Sephadex G-150 and hydroxyapatite. 3. The Km with glycerol cyclic phosphate as substrate is 7.2 mM, and the pH optimum broad (6.9–7.5). The molecular weight (by gel filtration) of the enzyme is approx. 35500. 4. The phosphodiesterase has no requirement for Ca2+ or Mg2+, but is stimulated by reducing agents (cysteine, dithiothreitol) and Fe2+. 5. The purified phosphodiesterase preparation also hydrolysed 3′:5′-cyclic AMP, producing 5′-AMP exclusively, and 2′:3′-cyclic AMP, forming 3′-AMP and 2′-AMP in the ratio 7:3. Bis-(p-nitrophenyl) phosphate was slowly hydrolysed, but other phosphodiesters tested were not attacked. 6. The phosphodiesterase is inhibited by theophylline and o-phenanthroline. It is inhibited by Pi and by a variety of phosphomonoesters, of which certain aromatic primary phosphates are particularly effective.

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.

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.

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.

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.

scholarly journals PREPARATION AND CHARACTERIZATION OF AN IMMUNOELECTRON MICROSCOPE TRACER CONSISTING OF A HEME-OCTAPEPTIDE COUPLED TO Fab

1974 ◽  
Vol 139 (1) ◽  
pp. 208-223 ◽  
Author(s):  
J. P. Kraehenbuhl ◽  
R. E. Galardy ◽  
J. D. Jamieson
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Antibody Fragments ◽  
Amino Groups ◽  
Electron Microscope Level ◽  
Ph Optimum ◽  
Peroxidatic Activity ◽  
Stokes Radius ◽  
Trypsin Hydrolysis

A heme-octapeptide (mol wt 1,550) has been obtained from cytochrome c by successive pepsin and trypsin hydrolysis and purified by gel filtration and countercurrent distribution. It possesses peroxidatic activity characterized by an apparent Km of 0.2 M, an apparent vmax of 4 mmol/min per mg of peptide, and a pH optimum of 7.0. Using a novel two-step conjugation procedure, the heme-octapeptide was coupled to rabbit Fab antibody fragments by first derivatizing it with the N-hydroxysuccinimide ester of p-formylbenzoic acid and subsequently allowing it to form a Schiff base with the amino groups of Fab. Stable covalent linkages were then obtained by reduction of the Schiff bases with sodium borohydride. The conjugate consists of ∼2 heme-octapeptides attached to each Fab molecule. The molecular weight is 45,000 daltons when coupled to sheep Fab and 50,000 daltons with a Stokes radius of 32 Å, when conjugated to rabbit Fab. Its peroxidatic activity is characterized by an apparent Km of 0.4 M, an apparent vmax of 0.4 mmol/min and per mg of attached heme-octapeptide and a pH optimum of 7.0. The conjugate has been used for the localization at the electron microscope level of secretory immunoglobulins in the mammary gland of lactating rabbits.

scholarly journals Factors affecting the activity and stability of the palmitoyl-coenzyme A hydrolase of rat brain

1979 ◽  
Vol 179 (3) ◽  
pp. 515-523 ◽  
Author(s):  
Thomas E. Knauer
Keyword(s):  
Molecular Weight ◽  
Rat Brain ◽  
Gel Filtration ◽  
Lower Molecular Weight ◽  
Supernatant Fraction ◽  
Ph Optimum ◽  
Factors Affecting ◽  
Chain Acyl ◽  
Coa Thioesters ◽  
Molecular Weight Form

Palmitoyl-CoA hydrolase (EC 3.1.2.2) catalyses the irreversible hydrolysis of long-chain acyl-CoA thioesters. This enzyme is found primarily in the postmicrosomal supernatant fraction prepared from homogenates of rat brain. Either of two forms of the hydrolase, a lower-molecular-weight species of approx. 70000 or a higher-molecular-weight species of approx. 130000 can be isolated by gel filtration. The higher-molecular-weight form is obtained from columns of Sephadex G-200 eluted with buffer containing 10μm-palmitoyl-CoA or 20% (v/v) glycerol, whereas the lower-molecular-weight form is obtained when the eluting buffer does not contain palmitoyl-CoA or glycerol. The two forms of the hydrolase have the same pH optimum of 7.5, are equally sensitive to the thiol-blocking reagents p-hydroxymercuribenzoate, HgCl2, and 5,5′-dithiobis-(2-nitrobenzoic acid), and exhibit the same Km (1.8μm) with palmitoyl-CoA as substrate. The two forms differ in the availability or reactivity of certain external thiol groups, as determined by covalent chromatography with activated thiol Sepharose. Dilute solutions of the lower-molecular-weight form of the hydrolase rapidly lose activity (50% in 60min at 0°C), but there is no change in the Km with palmitoyl-CoA as substrate during this progressive inactivation. Dilutions of the hydrolase in buffer containing 10μm-palmitoyl-CoA retain full activity. However, addition of palmitoyl-CoA to solutions of the lower-molecular-weight form will not restore previously lost hydrolase activity. The evidence supports the conclusion that the substrate palmitoyl-CoA promotes the formation of a relatively stable dimer from two unstable subunits. This process may not be reversible, since the removal of palmitoyl-CoA or glycerol from solutions of the higher-molecular-weight form does not result in the appearance of the lower-molecular-weight form of the hydrolase.

scholarly journals Purification and properties of molecular-weight variants of human placental alkaline phosphatase

1968 ◽  
Vol 108 (5) ◽  
pp. 779-792 ◽  
Author(s):  
Nimai K. Ghosh ◽  
William H. Fishman
Keyword(s):  
Molecular Weight ◽  
Alkaline Phosphatase ◽  
Large Scale ◽  
Equilibrium Dialysis ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Exchange Chromatography ◽  
Placental Alkaline Phosphatase ◽  
Ph Optimum ◽  
Starch Gel

1. Alkaline phosphatase of human placenta was purified by a procedure involving homogenization with tris buffer, pH8·6, extraction with butanol, ammonium sulphate fractionation, exposure to heat, ethanol fractionation, gel filtration, triethylaminoethylcellulose anion-exchange chromatography, continuous curtain electrophoresis on paper and equilibrium dialysis. Methods for both laboratory-scale and large-scale preparation were devised. 2. Two major molecular-weight variants designated A and B were separated by molecular sieving with Sephadex G-200 and variant A was purified 4000-fold. 3. Variant B, which comes off the Sephadex G-200 column before variant A, is the electrophoretically slower-moving species on starch gel and is quite heterogeneous. 4. Purified variant A was fairly homogeneous on the basis of electrophoretic studies on starch gel and Sephadex gel, ultracentrifugation and immunodiffusion. 5. The respective molecular weights for variants A and B were 70000 and over 200000 on the basis of sucrose-density-gradient ultracentrifugation. Variant A exhibited a sedimentation coefficient of 4·2s. 6. Crystalline variant B could be converted into fast-moving variant A and vice versa. 7. Kinetic studies indicated no difference between the two variants. These include linear rates of hydrolysis, pH optimum, Michaelis constants and uncompetitive stereospecific l-phenylalanine inhibition. 8. The amino acid compositions of variants A and B and of placental albumin were determined.

Purification and characterization of an acid phosphatase from peanut (Arachis hypogaea) seed

1984 ◽  
Vol 62 (2) ◽  
pp. 385-391 ◽  
Author(s):  
Sheikh Mehboob Basha
Keyword(s):  
Molecular Weight ◽  
Acid Phosphatase ◽  
Arachis Hypogaea ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Exchange Chromatography ◽  
Arachis Hypogaea L ◽  
Nitrophenyl Phosphate ◽  
Phosphorylated Sugars

An acid phosphatase (EC 3.1.3.2) from peanut (Arachis hypogaea L.) seed has been purified 433-fold by ammonium sulfate fractionation, gel filtration, and ion-exchange chromatography. The purified preparation was found to be homogeneous by electrophoresis and gel filtration. The molecular weight of the enzyme was estimated to be approximately 240 000 and it was found to be composed of six identical subunits, each with an apparent molecular weight of 42 500. Following isoelectric focusing, the isolectric point (pI) of the enzyme was found to be around pH 5.6. The apparent Km value with p-nitrophenyl phosphate as substrate was 2 ? 10−1 μM. The enzyme was inhibited by Hg2+, Fe2+, Cu2+, Zn2+, Pb2+, and F−. Higher concentrations (2–50 μM) and long incubation periods (60–90 min) with Ca2+ and Mg2+ ions were shown to activate the enzyme. This enzyme showed no effect toward phosphorylated sugars but appear to hydrolyze ATP, ADP, AMP, and β-glycerophosphate.

AMP metabolism by the marine bacterium Vibrio (Beneckea) natriegens: purification and properties of adenylate kinase

1981 ◽  
Vol 27 (10) ◽  
pp. 1053-1059 ◽  
Author(s):  
Karamchand Ramotar ◽  
Michael A. Pickard
Keyword(s):  
Molecular Weight ◽  
Adenylate Kinase ◽  
Gel Electrophoresis ◽  
Marine Bacterium ◽  
Specific Activity ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Atp Formation ◽  
Purification And Properties ◽  
Beneckea Natriegens

Adenylate kinase (EC 2.7.4.3) has been purified 484-fold from extracts of Vibrio natriegens to a specific activity of 1350 μmol ADP formed∙min−1∙mg protein−1. The preparation was 97% pure as judged by gel electrophoresis and exhibited molecular weight values of 29 000 by gel filtration and 32 000 by SDS–gel electrophoresis. The isoelectric point was at pH 4.7. Only ATP (Km 0.067 mM), ADP (Km 0.45 mM), and AMP (Km 0.12 mM) exhibited high activity as substrates, though dATP or dAMP could serve as cosubstrates with AMP or ATP, respectively, at reduced rates. The equilibrium constant in the direction of ATP formation was 1.09, and the pH optimum in both directions was broad, from pH 7.2 to pH 7.6. Enzyme activity was sensitive to the thiolalkylating agents iodacetamide and p-chloromercuriphenyl sulfonate.

scholarly journals The role of adenosine 3′:5′-cyclic monophosphate in the regulation of insulin release. Properties of islet-cell adenosine 3′:5′-cyclic monophosphate phosphodiesterase

1972 ◽  
Vol 129 (4) ◽  
pp. 945-952 ◽  
Author(s):  
D. J. Sams ◽  
W. Montague
Keyword(s):  
Cyclic Amp ◽  
Insulin Release ◽  
Islets Of Langerhans ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Supernatant Fraction ◽  
Phosphodiesterase Activity ◽  
Ph Optimum ◽  
Cyclic Monophosphate ◽  
Km Value

1. An assay has been developed with sufficient sensitivity for determination of the adenosine 3′:5′-cyclic monophosphate diesterase activity in islets of Langerhans, and has been used to investigate the response of the enzyme to various agents which are known to affect insulin release. 2. The subcellular distribution of the enzyme in islets of Langerhans prepared from guinea-pig pancreas was investigated and over 70% of the activity present in the original homogenate was recovered in the supernatant fraction. 3. Gel filtration of the activity present in the supernatant fraction on Sephadex G-200 gave a single peak of activity with an apparent molecular weight of 200000. The phosphodiesterase activity in the peak fraction showed two apparent Km values for adenosine 3′:5′-cyclic monophosphate (cyclic AMP) of 3μm and 30μm, suggesting the presence of two activities. The pH optimum of the activity with the low Km value was 8.7. 4. Theophylline, caffeine, 3-isobutyl-1-methylxanthine (SC-2964), glibenclamide, tolbutamide, xylitol and leucine were inhibitors of the activity with the low Km value; imidazole and arginine stimulated the activity, and glucose and diazoxide were without significant effect. 5. It is suggested that the agents theophylline, caffeine, SC-2964, glibenclamide, tolbutamide, leucine and imidazole may alter the intracellular concentration of cyclic AMP in islets of Langerhans by affecting the cyclic AMP phosphodiesterase activity in islet cells and in this way may affect insulin release.

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