scholarly journals The octameric structure of β-glucosidase from Botryodiplodia theobromae Pat

1991 ◽  
Vol 275 (3) ◽  
pp. 721-725 ◽  
Author(s):  
G M Umezurike
Keyword(s):  
Substrate Inhibition ◽  
Hydrolysis Product ◽  
Gel Filtration ◽  
Molecular Forms ◽  
Botryodiplodia Theobromae ◽  
Complete Inactivation ◽  
Alkaline Conditions ◽  
Michaelis Constants ◽  
Beta Glucosidase ◽  
Inactive Protein

1. Whereas only beta-glucosidase A (beta-D-glucoside glucohydrolase, EC 3.2.1.21) was produced by the tropical fungus Botryodiplodia theobromae Pat. (I.M.I. 115626; A.T.C.C. 26123) in young cultures containing D-cellobiose as carbon source, lower-Mr forms (B, C and D) were found in older cultures when the pH had drifted from the initial value of pH 6.2 to pH 7.9. 2. The Michaelis constants (Km) of the various molecular forms of the enzyme were 0.30 +/- 0.03 mM-, 0.26 +/- 0.01 mM-, 0.20 +/- 0.02 mM- and 0.16 +/- 0.01 mM-o-nitrophenyl beta-D-glucopyranoside for beta-glucosidase forms A (Mr 320,000), B (Mr 160,000), C (Mr 80,000) and D (Mr 40,000) respectively. 3. Only beta-glucosidase D showed substrate inhibition. 4. Only L-arginine was found as the N-terminal residue, and beta-glucosidase A contained 31.7 +/- 0.6 mol of N-terminal L-arginine/mol of the enzyme. 5. Storage of purified beta-glucosidase A under mildly alkaline conditions caused its dissociation into the lower-Mr forms, whereas adjustment of the pH of a solution of beta-glucosidase A to pH 12.0 with 1 M-NaOH led to complete inactivation on incubation at 40 degrees C for 1 h and to the release of 25.2 +/- 1.5 mol of inorganic phosphate/mol of the enzyme. 6. O-Phospho-L-serine was isolated from the acid-hydrolysis product of beta-glucosidase A but not from that of beta-glucosidase D. 7. Reduction and carboxamidomethylation of the various forms of beta-glucosidase gave only one enzymically inactive protein with an Mr of 10,000-11,000. 8. After partial succinylation (3-carboxypropionylation) of beta-glucosidase D at pH 5.0 and removal of the precipitated protein formed, the supernatant solution contained beta-glucosidase components similar to the other molecular forms (A, B and C) and an aggregate (beta-glucosidase Xs) that gave a positive result in the alkaline hydroxylamine test, whereas N-succinylated beta-glucosidase D, an aggregate (form Xp) that behaved like beta-glucosidase Xs and traces of forms A, B and C were found by gel filtration of the solution of the precipitate solubilized at neutral pH (7.0-7.7). 9. These observations are discussed in terms of the proposed octameric structure of beta-glucosidase A based on the result of electron microscopy [Umezurike (1975) Biochem. J. 145, 361-368].

scholarly journals The subunit structure of β-glucosidase from Botryodiplodia theobromae Pat

1975 ◽  
Vol 145 (2) ◽  
pp. 361-368 ◽  
Author(s):  
G M Umezurike
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Substrate Inhibition ◽  
Gel Filtration ◽  
Heat Stability ◽  
Reduced Form ◽  
Subunit Structure ◽  
Botryodiplodia Theobromae ◽  
Low Concentrations ◽  
Michaelis Constants

1. A homologous series of beta-glcosidase (beta-D-glcoside glcohydrolase, EC 3.2.1.21), which varied in relative amounts in different preparations from cultures of similar and different age, was observed in cultures od Botryodiplodia theobromae Pat grown for 4-8 week on cotton flock (cellulose) as carbon source. 2. Aging of the purified high-molecular-weight species led to some amount of siddociation into a homolous series of lower-molecular-weight speices. 3. Rough molecular-weight estimates, by gel filtration, of the various species derived from the purifeid high-molecular-weight enzyme were 350000-3800000, 170000, 180000, 83000-87000 and 45000-47000. 4. Electron micrographs of the negatively stained 350000-380000-molecular-weight enzyme showed that the molecule is an octamer in which each roughly spherical monomer occupies a corner of a cube with each side about 7.14nm long. 5. Carboxamidomethylation of the reduced form of each molecular-weight species of the enzyme led to irreversible dissociation of the molecules into electrophoretically identical polypeptides with a moleclar weight of 10000-12000. 6. These results suggest a slow association-dissociation of the type (8n)in equilibrium 2 (4n) in equilibrium 4(2n) in equilibrium 8(n), where n is defined as the monomer. The monomer is in turn made up of four polypeptide a subunits whi-ch are non-catalytic. 7. The Michaelis constants (Km) and heat stability of the four wnzymically active molecular species derived from the purified enzyme increased with molecular complexity, whereas all four species were inhibited by glycerol (100nM) at low concentrations of substrate (o-nitrophenyl beta-D-glucopyranoside) but activated at high substrat concentrations. 8. Only the lowest-molecular-weight species (45species (45,000-47000 mol. wt.) showed substrate inhibition.

scholarly journals The β-glucosidase in the gut contents of the snail Achatina achatina

1976 ◽  
Vol 157 (2) ◽  
pp. 381-387 ◽  
Author(s):  
G M Umezurike
Keyword(s):  
Molecular Weight ◽  
Heat Stability ◽  
Molecular Forms ◽  
Gut Contents ◽  
Ph Optimum ◽  
Optimum Ph ◽  
Michaelis Constants ◽  
Molecular Complexity ◽  
Beta Glucosidase ◽  
Digestive Glands

1. The enzyme beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) from the gut contents of active Achatina achatina exists in two molecular forms, beta-glucosidase C (mol.wt. about 82000) and D (mol.wt. about 41000). 2. Only the lower-molecular-weight species was found in the gut contents of aestivating snails or in extracts from their digestive glands and washed gut walls. 3. On re-activation of some aestivating snails, betion of ATP and Mg2+ to the isolated gut contents or to extracts from washed gut walls led to the formation of higher-molecular-weight forms of the enzyme, beta-glucosidase A (mol.wt. about 329000) and beta-glucosidase B (mol.wt. about 165000). 5. All these forms of the enzyme have similar pH optimum (pH 5.0-5.6). 6. The Michaelis constants (Km) and heat stability of the enzyme increased with increasing molecular complexity.

Differential neural regulation of circulating somatostatin-14 and somatostatin-28 in conscious dogs

1993 ◽  
Vol 264 (5) ◽  
pp. G902-G909 ◽  
Author(s):  
G. R. Greenberg
Keyword(s):  
Plasma Concentrations ◽  
Gel Filtration ◽  
Conscious Dogs ◽  
Molecular Forms ◽  
Neural Pathways ◽  
Vagus Nerves ◽  
Solid Meal ◽  
Liquid Meal ◽  
Basal Plasma ◽  
Somatostatin 14

Somatostatin-like immunoreactivity (SLI) released into the circulation after nutrients or secretagogues is heterogeneous. To determine whether similar neural pathways regulate secretion of SLI molecular forms, circulating somatostatin-28 (S-28) and somatostatin-14 (S-14) responses to ingestion of a solid meal, intraduodenal perfusion of a liquid defined formula meal, and intravenous infusion of cholecystokinin octapeptide (CCK-OP, 250 pmol.kg-1.h-1) were measured in four conscious dogs with and without cryogenic blockade of the cervical vagus nerves. SLI was separated by gel-filtration chromatography of extracted, acidified plasma and quantified by radioimmunoassay. Basal plasma concentrations of S-28 were 4.1 +/- 0.6 fmol/ml and of S-14 were 3.8 +/- 0.4 fmol/ml. Ingestion of the solid meal increased plasma SLI threefold, and elevations of S-28 and S-14 were equivalent. After the intraduodenal liquid meal or infusion of CCK-OP, plasma SLI rose twofold, but increments of S-28 exceeded S-14, comprising approximately 70% of SLI released. Vagal blockade by cooling reversibly inhibited both the S-28 and S-14 responses to the solid meal, intraduodenal liquid meal, and CCK-OP. In contrast, atropine (50 micrograms/kg iv), given after solid food, intraduodenal nutrients, and CCK-OP, suppressed S-28 but further increased S-14 responses. Atropine did not, however, alter the suppression of S-14 and S-28 by vagal cooling.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Purification and characterization of two human erythrocyte arylamidases preferentially hydrolysing N-terminal arginine or lysine residues

1978 ◽  
Vol 175 (3) ◽  
pp. 1051-1067 ◽  
Author(s):  
K K Mäkinen ◽  
P L Mäkinen
Keyword(s):  
Substrate Inhibition ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Gel Permeation Chromatography ◽  
Ph Optimum ◽  
Preparative Scale ◽  
Molecular Weights ◽  
Gel Permeation ◽  
Lysine Residues ◽  
Enzyme I

Two arylamidases (I and II) were purified from human erythrocytes by a procedure that comprised removal of haemoglobin from disrupted cells with CM-Sephadex D-50, followed by treatment of the haemoglobin-free preparation subsequently with DEAE-cellulose, gel-permeation chromatography on Sephadex G-200, gradient solubilization on Celite, isoelectric focusing in a pH gradient from 4 to 6, gel-permeation chromatography on Sephadex G-100 (superfine), and finally affinity chromatography on Sepharose 4B covalently coupled to L-arginine. In preparative-scale purifications, enzymes I and II were separated at the second gel-permeation chromatography. Enzyme II was obtained as a homogeneous protein, as shown by several criteria. Enzyme I hydrolysed, with decreasing rates, the L-amino acid 2-naphtylamides of lysine, arginine, alanine, methionine, phenylalanine and leucine, and the reactions were slightly inhibited by 0.2 M-NaCl. Enzyme II hydrolysed most rapidly the corresponding derivatives of arginine, leucine, valine, methionine, proline and alanine, in that order, and the hydrolyses were strongly dependent on Cl-. The hydrolysis of these substrates proceeded rapidly at physiological Cl- concentration (0.15 M). The molecular weights (by gel filtration) of enzymes I and II were 85 000 and 52 500 respectively. The pH optimum was approx. 7.2 for both enzymes. The isoelectric point of enzyme II was approx. 4.8. Enzyme I was activated by Co2+, which did not affect enzyme II to any noticeable extent. The kinetics of reactions catalysed by enzyme I were characterized by strong substrate inhibition, but enzyme II was not inhibited by high substrate concentrations. The Cl- activated enzyme II also showed endopeptidase activity in hydrolysing bradykinin.

scholarly journals Bioelectrochemical Enhancement of Biogenic Methane Conversion of Coal

Energies ◽  
2018 ◽  
Vol 11 (10) ◽  
pp. 2577 ◽  
Author(s):  
Dong-Mei Piao ◽  
Young-Chae Song ◽  
Dong-Hoon Kim
Keyword(s):  
Methane Production ◽  
Methane Conversion ◽  
Substrate Inhibition ◽  
Hydrolysis Product ◽  
Oxygen Demand ◽  
Methane Yield ◽  
Anaerobic Reactor ◽  
Adaptation Time ◽  
Biogenic Methane ◽  
Inhibition Effect

This study demonstrated the enhancement of biogenic coal conversion to methane in a bioelectrochemical anaerobic reactor with polarized electrodes. The electrode with 1.0 V polarization increased the methane yield of coal to 52.5 mL/g lignite, which is the highest value reported to the best of our knowledge. The electrode with 2.0 V polarization shortened the adaptation time for methane production from coal, although the methane yield was slightly less than that of the 1.0 V electrode. After the methane production from coal in the bioelectrochemical reactor, the hydrolysis product, soluble organic residue, was still above 3600 mg chemical oxygen demand (COD)/L. The hydrolysis product has a substrate inhibition effect and inhibited further conversion of coal to methane. The dilution of the hydrolysis product mitigates the substrate inhibition to methane production, and a 5.7-fold dilution inhibited the methane conversion rate by 50%. An additional methane yield of 55.3 mL/g lignite was obtained when the hydrolysis product was diluted 10-fold in the anaerobic toxicity test. The biogenic conversion of coal to methane was significantly improved by the polarization of the electrode in the bioelectrochemical anaerobic reactor, and the dilution of the hydrolysis product further improved the methane yield.

scholarly journals Molecular Forms of Prostate-specific Antigen in Malignant and Benign Prostatic Tissue: Biochemical and Diagnostic Implications

2000 ◽  
Vol 46 (1) ◽  
pp. 47-54 ◽  
Author(s):  
Klaus Jung ◽  
Brigitte Brux ◽  
Michael Lein ◽  
Birgit Rudolph ◽  
Glen Kristiansen ◽  
...  
Keyword(s):  
Prostate Specific Antigen ◽  
Specific Antigen ◽  
Gel Filtration ◽  
Tumor Stage ◽  
Prostatic Hyperplasia ◽  
Prostatic Tissue ◽  
Molecular Forms ◽  
Tissue Samples ◽  
Total Psa ◽  
Lower Ratio

Abstract Background: Patients with prostate cancer (PCa) show a lower ratio of free prostate-specific antigen (fPSA) to total PSA (tPSA) in serum than patients with benign prostatic hyperplasia (BPH). The patterns of the intracellular PSA isoforms in malignant and benign prostatic tissue have been studied as potential molecular reasons for this phenomenon. Methods: Prostatic tissue samples were obtained after cystoprostatectomy from patients with bladder cancer (n = 10), from BPH patients (transurethral resection of the prostate, n = 10; adenomectomy, n = 10), and from the cancerous and noncancerous parts of the same prostates removed surgically by prostatectomy because of PCa (n = 20). PSA pattern was characterized by gel filtration, immunoblotting, and immunoassays for tPSA, fPSA, α1-antichymotrypsin-PSA (ACT-PSA), and complexed PSA (Bayer Immuno 1 assay). Comparisons were made with the PSA concentrations in serum. Results: The major portion of tPSA in all tissue samples was fPSA; complexed PSA forms were <2%. Samples from cystoprostatectomy patients had the lowest and those from adenomectomy patients the highest values of tPSA and fPSA. PSA concentrations were lower in cancerous than in the noncancerous parts of the prostate. No significant correlations were found between tumor stage or grade and the amounts of tPSA, fPSA, and ACT-PSA in tissue. Tissue PSA values were not correlated with the serum PSA concentrations nor with the ratios fPSA/tPSA and ACT-PSA/tPSA in sera. Conclusions: The amounts of tPSA and the PSA isoforms in prostatic tissue explain neither the concentrations of tPSA and PSA isoforms in serum nor the behavior of the ratio fPSA/tPSA in patients with BPH and PCa.

scholarly journals Purification and kinetic properties of ox brain histamine N-methyltransferase

1986 ◽  
Vol 233 (3) ◽  
pp. 669-676 ◽  
Author(s):  
W L Gitomer ◽  
K F Tipton
Keyword(s):  
Acetic Acid ◽  
Steady State ◽  
Mouse Brain ◽  
Initial Rate ◽  
Substrate Inhibition ◽  
Gel Filtration ◽  
Product Inhibition ◽  
Kinetic Properties ◽  
Brain Histamine ◽  
Inhibition Studies

Histamine N-methyltransferase (EC 2.1.1.8) was purified 1100-fold from ox brain. The native enzyme has an Mr of 34800 +/- 2400 as measured by gel filtration on Sephadex G-100. The enzyme is highly specific for histamine. It does not methylate noradrenaline, adrenaline, DL-3,4-dihydroxymandelic acid, 3,4-dihydroxyphenylacetic acid, 3-hydroxytyramine or imidazole-4-acetic acid. Unlike the enzyme from rat and mouse brain, ox brain histamine N-methyltransferase did not exhibit substrate inhibition by histamine. Initial rate and product inhibition studies were consistent with an ordered steady-state mechanism with S-adenosylmethionine being the first substrate to bind to the enzyme and N-methylhistamine being the first product to dissociate.

scholarly journals Different molecular forms of plasminogen and plasmin produced by urokinase in human plasma and their relation to protease inhibitors and lysis of fibrinogen and fibrin

1974 ◽  
Vol 143 (2) ◽  
pp. 273-283 ◽  
Author(s):  
Sten Müllertz
Keyword(s):  
Human Plasma ◽  
Protease Inhibitors ◽  
Gel Electrophoresis ◽  
Protease Activity ◽  
Gel Filtration ◽  
Molecular Forms ◽  
Crossed Immunoelectrophoresis ◽  
Low Concentrations ◽  
Α2 Macroglobulin ◽  
Esterase Inhibitor

Urokinase-activated human plasma was studied by gel electrophoresis, gel filtration, crossed immunoelectrophoresis and electroimmunoassay with specific antibodies and by assay of esterase and protease activity of isolated fractions. Urokinase induced the formation of different components with plasminogen+plasmin antigenicity. At low concentrations of urokinase, a component with a KD value of 0.18 by gel filtration and post β1 mobility by gel electrophoresis was detected. The isolated component had no enzyme or plasminogen activity. In this plasma sample fibrinogen was not degraded for 10h, but when fibrin was formed, by addition of thrombin, fibrin was quickly lysed, and simultaneously a component with a KD value of 0 and α2 mobility appeared, which was probably plasmin in a complex with α2 macroglobulin. This complex showed both esterase and protease activity. After gel filtration with lysine buffer of the clotted and lysed plasma another two components were observed with about the same KD value by gel filtration as plasminogen (0.35), but β1 and γ mobilities by gel electrophoresis. They appeared to be modified plasminogen molecules, and possibly plasmin with γ mobility. Similar processes occurred without fibrin at higher urokinase concentrations. Here a relatively slow degradation of fibrinogen was correlated to the appearance of the plasmin–α2 macroglobulin complex. The fibrin surface appeared to catalyse the ultimate production of active plasmin with a subsequent preferential degradation of fibrin and the formation of a plasmin–α2 macroglobulin complex. The gel filtration and electrophoresis of the plasma protease inhibitors, α1 antitrypsin, inter-α-inhibitor, antithrombin III, and C1-esterase inhibitor indicated that any complex between plasmin and these inhibitors was completely dissociated. The β1 and post β1 components appear to lack correlates among components occurring in purified preparations of plasminogen and plasmin.

Plasma Met-enkephalin and catecholamine responses to insulin-induced hypoglycaemia in greyhounds

1987 ◽  
Vol 114 (1) ◽  
pp. 81-87 ◽  
Author(s):  
S. Medbak ◽  
D. F. J. Mason ◽  
L. H. Rees
Keyword(s):  
Blood Glucose ◽  
Plasma Concentrations ◽  
Gel Filtration ◽  
Opioid Antagonist ◽  
Molecular Forms ◽  
Endogenous Opioid ◽  
Gel Filtration Chromatography ◽  
Plasma Adrenaline ◽  
Molecular Weights ◽  
Blood Glucose Concentrations

ABSTRACT The involvement of endogenous opioid peptides in the stress response was investigated by measuring plasma concentrations of Met-enkephalin-like immunoreactivity (MLI), adrenaline and noradrenaline during insulin-induced hypoglycaemia in conscious greyhounds. Moreover, the molecular forms of circulating MLI were characterized using gel filtration chromatography. In the first group of animals, i.v. administration of insulin (0·3 units/kg) provoked marked hypoglycaemia (blood glucose concentrations fell from 4·4 ± 0·1 to 1·5 ±0·2 mmol/l; mean ± s.e.m.) which was associated with significant (P< 0·001) rises in plasma MLI concentrations from a basal concentration of 45 ± 8 to a peak of 189 ±39 ng/l. A within-subject study comparing five different insulin doses ranging from 0·004 to 0·3 units/kg showed dose-related effects on blood glucose with nadir concentrations of 4·1 ± 0·6 mmol/l (after the smallest dose of insulin) and 0·8 ± 0·1 mmol/l (after the largest dose of insulin). This was associated with dose-related rises in plasma MLI with peak concentrations of 56±17 and 558 ± 35 ng/l, plasma adrenaline with peak concentrations of 0·45± 0·06 and 15·76±1·33 nmol/l and plasma noradrenaline with peak concentrations of 0·49 ± 0·07 and 2·27 ± 0·45 nmol/l following the smallest and largest doses of insulin respectively. These results are the first demonstration of raised plasma MLI concentrations following hypoglycaemia. Moreover, they show that the hormonal responses vary with the degree of hypoglycaemia achieved. Together with reports by other investigators these findings might suggest opioid modulation of the responses of the sympathoadrenal system to hypoglycaemia. These responses were, however, not modified by the opioid antagonist naloxone. Gel filtration chromatography of neat (unextracted) plasma revealed the predominance of large molecular weight enkephalin-containing peptides, with approximate molecular weights of 18 000 and 8000, both basally and following stimulation by hypoglycaemia. J. Endocr. (1987) 114,81–87

Somatostatin stimulates acetylcholine release in the canine heart

1989 ◽  
Vol 257 (2) ◽  
pp. H483-H487
Author(s):  
J. W. Wiley ◽  
L. Uccioli ◽  
C. Owyang ◽  
T. Yamada
Keyword(s):  
Acetylcholine Release ◽  
Gel Filtration ◽  
Negative Inotropic Effect ◽  
Molecular Forms ◽  
Dependent Manner ◽  
Canine Heart ◽  
Release Of Acetylcholine ◽  
The Right ◽  
A Minor ◽  
Somatostatin 14

Previous reports of somatostatin's atropine-sensitive negative inotropic effect on cardiac function prompted the present studies to characterize the molecular forms and actions of somatostatin in the canine heart. Radioimmunoassay of cardiac extracts revealed concentrations of somatostatin-like immunoreactivity ranging from 0.50 +/- 0.13 pmol/g tissue (means +/- SE, n = 6) in the pulmonary artery to 0.78 +/- 0.23 pmol/g tissue in the right ventricle. On gel filtration of the extracts, two major molecular forms of somatostatin-like immunoreactivity were elicited, the predominant one coeluting with somatostatin-14 and a minor peak corresponding to somatostatin-28. Experiments performed with slices of atrial septum indicated that somatostatin-14 (10(-10) to 10(-7) M) stimulated the release of acetylcholine in a dose-dependent manner. This action of somatostatin-14 was additive with K+-evoked acetylcholine release, unaffected by hexamethonium, and blocked by tetrodotoxin, suggesting mediation via a nonnicotinic postganglionic neural pathway. Our studies lead us to conclude that somatostatin may function as a neurotransmitter in the heart, which exerts its negative inotropic action by promoting the release of acetylcholine.

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