A Labile CO2-Fixing Enzyme Complex in Spinach Chloroplasts

1972 ◽  
Vol 27 (8) ◽  
pp. 925-932 ◽  
Author(s):  
Bruno Müller
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Phosphoglycerate Kinase ◽  
Gel Chromatography ◽  
Enzyme Complex ◽  
Molecular Weights ◽  
Spinach Chloroplasts ◽  
Reversible Dissociation ◽  
Glyceraldehyde Phosphate Dehydrogenase ◽  
Ribulose Diphosphate Carboxylase

Activity and activation of ribulose 5-phosphate kinase, ribulose diphosphate carboxylase, phosphoglycerate kinase, and the NADP- and NAD-dependent glyceraldehyde phosphate dehydrogenases from isolated diloroplasts were traced in the course of differential ultracentrifugation. Activation of some of these enzymes was carried out by incubation with NADPH2 or ATP. Activity of ribulose diphosphate carboxylase could be increased 2,4-fold by incubation with NADPH2 while phosphoglycerate kinase could not be activated.The degree of activation of the NADP-specific glyceraldehyde phosphate dehydrogenase and ribulose phosphate kinase was correlated with the velocity of sedimentation. Enzymes, which could be activated manifold, sedimented more quickly. Two fractions could be obtained after ultracentri fugation: the sedimented fraction could be highly activated while the fraction in the supernatant could not.It was shown by gel chromatography with Sephadex for the NADP-specific glyceraldehyde phosphate dehydrogenase and ribulose phosphate kinase that the enzyme fractions which could be greatly activated had molecular weights of at least 400 000. In contrast, the enzyme fraction which could not be activated had a molecular weight of 50 000 for the ribulose phosphate kinase and of 240 000 for the glyceraldehyde phosphate dehydrogenases.Results are discussed in the way that a reversible dissociation of a labile enzyme complex takes place between ribulose phosphate kinase, glyceraldehyde phosphate dehydrogenases, and ribulose diphosphate carboxylase. Enzyme activity changes with the state of aggregation. The dissociated enzymes have higher activity than the complexed ones. Dissociation can be performed by ATP and NADPH2, which are assumed to be the physiological regulators, and by unphysiological means as dilution and high salt concentrations.

Studies on Soluble Fibrin Complexes as a Function of pH

1975 ◽  
Author(s):  
Y. Benabid ◽  
E. Concord ◽  
M. Suscillon
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Gel Electrophoresis ◽  
Gel Chromatography ◽  
Agarose Gel ◽  
Monomer Mixture ◽  
Soluble Fibrin ◽  
Molecular Weights ◽  
Fibrin Monomer

Purified fibrinogen solutions, incubated with thrombin. CNBr. Sepharose, were subjected to agarose gel chromatography and eluted at different pH (6.5; 7.5; 8.5). Among high molecular weight derivatives formed by thrombin, the major component was a dimer. Gel chromatography at pH 8.5 showed a complexes peak distinct of that from fibrinogen, whereas at pH 6.5, only the fibrinogen peak appeared: fibrin monomer was eluted with fibrinogen as demonstrated by polyacrylamid gel electrophoresis 3.75% pH 8.9. SDS urea electrophoresis after reduction indicated that complexes peak contained two α-chains (α and α′). When fibrinogen was incubated with thrombin in the presence of FSF and calcium, several derivatives with higher and higher molecular weights were formed besides the dimer, and elution profiles of chromatography were identical at pH 6.5 and 8.5, thus indicating stable complexes formation. If fibrinogen-fibrin monomer mixture was subjected to FSF action at different pH, no complexes were formed at pH 6.5. These results confirm that at pH 6.5, any association was prevented.

scholarly journals Gelatinases in Boar Seminal Plasma and Their Relation to Semen Indicators

2010 ◽  
Vol 79 (3) ◽  
pp. 491-496 ◽  
Author(s):  
Maja Zakošek Pipan ◽  
Marjan Kosec ◽  
Janko Mrkun ◽  
Petra Zrimšek
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Seminal Plasma ◽  
Animal Species ◽  
Sperm Concentration ◽  
Gelatin Zymography ◽  
Motile Sperm ◽  
Molecular Weights ◽  
Simultaneous Identification ◽  
First Time

Matrix metalloproteinases were detected in reproductive tissues and seminal plasma of various animal species. The aim of this study was to determine for the first time the presence of gelatinases and metalloproteases in boar seminal plasma and to correlate the results with semen indicators. Gelatin zymography was used for simultaneous identification and measurement of gelatinase enzyme activity associated with their molecular weights. Several gelatinase forms were identified in seminal plasma of boars. Those that were stimulated by CaCl2 and inhibited by EDTA and phenanthroline were considered as metalloproteases. Negative correlation between semen indicators (sperm index, sperm concentration and concentration of progressive motile sperm) and the concentrations of metalloprotease at 78 kDa and 66 kDa means that higher values of semen indicators correlate with lower concentrations of these metaloproteases in seminal plasma. Gelatinases with molecular weight of 225, 78 and 66 kDa correlated with higher levels of acrosome damage. Samples with sperm index above 110 M/ml contained gelatinases of significantly lower band intensities at 78 and 66 kDa compared to samples with SI less than 110 M/ml. Bands with 225, 78 and 66 kDa are suggested to belong to a dimer of MMP-9, proMMP-2 and mature MMP-2.

Separation of Sub-Units of Antihemophilic Factor (AHF) by Agarose Gel Chromatography

1972 ◽  
Vol 27 (02) ◽  
pp. 212-219 ◽  
Author(s):  
H. J Weiss ◽  
Louise L. Phillips ◽  
W. Rosner
Keyword(s):  
Molecular Weight ◽  
Molecular Size ◽  
Void Volume ◽  
Gel Chromatography ◽  
Agarose Gel ◽  
Major Peak ◽  
Molecular Weights ◽  
Congenital Afibrinogenemia ◽  
Weight Substance ◽  
Antihemophilic Factor

SummaryThe molecular weight of antihemophilic factor (AHF) in plasma and cryoprecipitate was studied by chromatography on agarose gel (Bio-Gel A, 1.5 M). At a pH of 7.4 and the ionic strength of plasma, AHF appeared in the void volume as a sharp, symmetrical peak, indicating a molecular weight of 1.5 million or greater. Similar findings were obtained in a patient with congenital afibrinogenemia. At a pH of 7.7, the major peak of AHF-activity was again found in the void volume, but a spreading of activity into higher elution volumes was also observed. In 1 M NaCl, pH 7.4, AHF dissociated into active sub-units of varying molecular size. The molecular weights of the smallest subunits were estimated to be 169,000-194,000. These studies provide further evidence that AHF is a high molecular weight substance, not associated with fibrinogen, whose quarternary structure may be disrupted to produce active sub-units of varying sizes.

Cellulolytic enzyme system of Acetivibrio cellulolyticus

1981 ◽  
Vol 27 (3) ◽  
pp. 288-294 ◽  
Author(s):  
J. N. Saddler ◽  
A. W. Khan
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cellulolytic Enzymes ◽  
Cellulolytic Enzyme ◽  
Endoglucanase Activity ◽  
Molecular Weights ◽  
Reversible Dissociation ◽  
Electrophoretic Mobilities ◽  
Novel Method ◽  
Culture Supernate

Polyacrylamide gel electrophoresis of the cellulolytic system from culture supernates of Acetivibrio cellulolyticus showed the presence of four major enzymes: a β-glucosidase, an exoglucanase, and two endoglucanases. The relative proportions of these enzymes in the culture supernate were affected by the nature of the cellulosic substrate and by the length of the incubation period. The molecular weights of the cellulolytic enzymes were β-glucosidase, 81 000; exoglucanase, 38 000; endoglucanase C2, 33 000; and endoglucanase C3, 10 400, as estimated by their electrophoretic mobilities relative to proteins of known molecular weight. Treatment of the high molecular weight endoglucanase with SDS–mercaptoethanol led to reversible dissociation of the enzyme into polypeptide subunits similar to the low molecular weight endoglucanase. Endoglucanase activity could be assayed for directly using a novel method of incorporating carboxymethyl cellulose in the polyacrylamide gels. The molecular weights and functions of these enzymes are compared with those detected in culture filtrates of various fungi.

scholarly journals On the Relationship Between Molecular Mass and Anticoagulant Activity in a Low Molecular Weight Heparin (Enoxaparin)

1992 ◽  
Vol 67 (05) ◽  
pp. 556-562 ◽  
Author(s):  
Ana-Victoria Bendetowicz ◽  
Elisabeth Pacaud ◽  
Suzette Béguin ◽  
André Uzan ◽  
H Coenraad Hemker
Keyword(s):  
Molecular Weight ◽  
Catalytic Activity ◽  
Low Molecular Weight Heparin ◽  
Anticoagulant Activity ◽  
Low Molecular Weight ◽  
Gel Chromatography ◽  
Molecular Weights ◽  
Thrombin Inhibition ◽  
Specific Catalytic Activity ◽  
The Relationship

SummaryA low molecular weight heparin (enoxaparin, mean molecular weight ~ 4,400) was separated by gel chromatography into eight different fractions with a narrow distribution around the following mean molecular weights: 1,800, 2,400, 2,900, 4,200, 6,200, 8,600, 9,800 and 11,000. We compared the influence of enoxaparin on the generation of thrombin in plasma to that of the eight fractions.We determined: a) the % of material with high affinity to antithrombin III (HAM) and the % of HAM above the critical chainlength necessary to allow for thrombin inhibition (ACLM), b) the specific catalytic activity on the decay of endogenous thrombin, and c) the inhibition of over-all thrombin formation in the extrinsic and the intrinsic pathway. From b and c we calculated the inhibition of prothrombin conversion in these pathways.We found that a) there is a gradual decrease of the HAM fraction with decreasing molecular weight; b) the specific catalytic activity for the inactivation of thrombin does not vary significantly between the fractions when expressed in terms of ACLM; c) the potency to inhibit prothrombin conversion does not vary significantly between the fractions when expressed in terms of HAM.

scholarly journals The Molecular Weight At the Air-Water Interface of Some Keratin Derivatives Extracted From Wool

1955 ◽  
Vol 8 (1) ◽  
pp. 122 ◽  
Author(s):  
BS Harrap
Keyword(s):  
Molecular Weight ◽  
Cell Walls ◽  
Average Molecular Weight ◽  
Cortical Cell ◽  
Number Average Molecular Weight ◽  
Molecular Weights ◽  
Reversible Dissociation ◽  
Electrophoretic Patterns ◽  
Air Water Interface ◽  
Electrophoretic Component

Using the monolayer technique, number-average molecular weights have been determined for a series of extracts of wool prepared by successive treatments with alkaline sodium thioglycollate. The molecular weights of these extracts have been discussed in relation to their electrophoretic patterns. The change in the number-average molecular weight in the successive extracts has been correlated with the presence of certain electrophoretic components. The possibility of extraction of lipoidal or other non-protein material from the cortical cell walls is discussed. A reversible dissociation of the major electrophoretic component at high pH was observed.

scholarly journals Binding to antithrombin of heparin fractions with different molecular weights

1981 ◽  
Vol 193 (2) ◽  
pp. 427-433 ◽  
Author(s):  
Ȧke Danielsson ◽  
Ingemar Björk
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Binding Constants ◽  
Gel Chromatography ◽  
Molecular Weights ◽  
High Affinity ◽  
Heparin Fractions ◽  
Fluorescence Titrations ◽  
In The Beginning ◽  
Difference Absorption

The interaction between bovine antithrombin, a plasma proteinase inhibitor, and heparin species of different molecular weights was studied. A commercial heparin preparation was divided by gel chromatography into a number of fractions with average molecular weights ranging from 6000 to 34700. Each of these fractions was further fractionated by affinity chromatography on matrix-bound antithrombin. In the latter procedure, those heparin fractions that had molecular weights lower than about 14000 were separated into three peaks. The material in the first of these was not adsorbed on the column, and the other two peaks corresponded to the low-affinity and high-affinity peaks described previously. In contrast, high-molecular-weight heparin samples gave only the low-affinity and high-affinity fractions. U.v. difference absorption studies showed that the non-adsorbed heparin fraction bound to antithrombin in solution with a binding constant at physiological ionic strength only slightly lower than that of low-affinity heparin. The division between the two fractions thus is arbitrary and only dependent on the conditions selected for the affinity-chromatography experiment. Stoicheiometries and binding constants for the binding of several high-affinity heparin species to antithrombin were determined by fluorescence titrations. High-affinity heparin fractions of equal elution positions in the beginning of the peaks of the affinity chromatographies, but with different molecular weights, showed stoicheiometries that were not experimentally distinguishable from 1:1 and also had no appreciable differences in binding constants. However, the anticoagulant activities, calculated on a molar basis, of these fractions increased markedly with molecular weight, a behaviour that thus cannot be explained by differences in the binding of the fractions to antithrombin. In contrast, high-affinity samples of similar molecular weights, which were eluted at increasing ionic strengths from matrix-linked antithrombin, were found to have an increasing proportion of chains with two binding sites for antithrombin and also to have progressively higher binding constants. These binding properties at least partly explain the increasing anticoagulant activities that were observed for these fractions.

scholarly journals Self-association of dermatan sulphate proteoglycans from bovine sclera

1981 ◽  
Vol 197 (2) ◽  
pp. 483-490 ◽  
Author(s):  
L Cöster ◽  
L A Fransson ◽  
J Sheehan ◽  
I A Nieduszynski ◽  
C F Phelps
Keyword(s):  
Molecular Weight ◽  
Light Scattering ◽  
Guanidine Hydrochloride ◽  
Apparent Molecular Weight ◽  
Sedimentation Equilibrium ◽  
Gel Chromatography ◽  
Side Chains ◽  
Dermatan Sulphate ◽  
Molecular Weights ◽  
High Particle

1. Two proteodermatan sulphate fractions (I and II) from bovine sclera were studied by gel chromatography, light-scattering and ultracentrifugation under various conditions. 2. Gel chromatography of proteoglycans in the absence or presence of hyaluronate was performed under associative conditions. No effect on the elution profile was noted. 3. Ultracentrifugation experiments (sedimentation-velocity and sedimentation-equilibrium) with proteoglycan I and II in 6 M-guanidine hydrochloride gave molecular weights (Mw) of 160000-220000 and 70000-100000 respectively. As the protein contents were 45% and 60% respectively, it may be calculated that proteoglycan I contained four to five side chains, whereas proteoglycan II contained one or two. Sedimentation-equilibrium runs performed in 0.15 M-NaCl gave an apparent molecular weight (Mw) of 500000-800000 for proteoglycan I and 90000-110000 for proteoglycan II. 4. In light-scattering experiments both proteoglycans I and II yielded high particle weights in 0.15 M-NaCl (3.1 × 10(6) and 3.4 × 10(6) daltons respectively). In the presence of 6 M-guanidine hydrochloride the molecular weights decreased to 410000 and 130000 respectively. The particle weights in 0.15 M-NaCl were not altered by the addition of hyaluronate or hyaluronate oligosaccharides. 5. The dermatan sulphate side chains of scleral proteoglycans (L-iduronate/D-glucuronate ratio 7:13) gave a particle weight of 100000 daltons in 0.15 M-NaCl. In 1.00 M-KCl/0.02M-EDTA the molecular weight was 24000. Addition of free scleral dermatan sulphate chains to a solution of proteoglycan II promoted further multimerization of the macromolecule.

Subunit components of casein micelles from bovine, ovine, caprine and equine milks

1989 ◽  
Vol 56 (1) ◽  
pp. 61-68 ◽  
Author(s):  
Tomotada Ono ◽  
Hideaki Kohno ◽  
Satoshi Odagiri ◽  
Toshio Takagi
Keyword(s):  
Molecular Weight ◽  
Light Scattering ◽  
High Performance ◽  
Laser Light ◽  
Laser Light Scattering ◽  
The Other ◽  
Gel Chromatography ◽  
Casein Micelles ◽  
Molecular Weights ◽  
Combined Use

SummarySubunit components of ovine, caprine and equine casein micelles were separated by gel chromatography using a TSK-G4000SW high-performance column and the subunit components of the fractions analysed and compared with bovine casein. Molecular weights of the casein complexes were determined by the combined use of high-performance gel chromatography and low-angle laser light scattering. The caprine and ovine caseins were separated into three peaks (F2, F3 and F4) which were similar to those of bovine casein with respect to composition and molecular weight (500, 100 and 23 K). These F2, F3 and F4 peaks consisted mainly of αs- and κ-casein, αs- and β-casein and β-casein respectively. The equine casein was separated into two components corresponding to F3 and F4 of bovine casein. These F3 and F4 peaks consisted mainly of αs- and β-casein and β-casein respectively. The molecular weight of equine F3 (850 K) was different from that of the other three species. The contents of F2 and F4 in these caseins were dependent on the contents of κ-casein and β-casein respectively.

Purification and some properties of five endo-1,4-β-D-xylanases and β-D-xylosidase produced by a strain of Aspergillus niger

1979 ◽  
Vol 57 (2) ◽  
pp. 125-134 ◽  
Author(s):  
Michael John ◽  
Bernd Schmidt ◽  
Jurgen Schmidt
Keyword(s):  
Molecular Weight ◽  
Aspergillus Niger ◽  
Substrate Specificity ◽  
Culture Medium ◽  
The Other ◽  
Crystalline Cellulose ◽  
Gel Chromatography ◽  
Molecular Weights ◽  
Optimal Activity ◽  
Equatorial Forest

Five different xylanases and a β-D-xylosidase in the culture medium of Aspergillus niger have been purified to homogeneity from 13- to 52-fold by a procedure of gel and hydroxylapatite chromatography. The strain was isolated from soil of the African equatorial forest. Gel chromatography of the purified enzymes indicated that three of the xylanases have molecular weights of 31 000 and the other two xylanases have molecular weights of 50 000.β-D-Xylosidase has a molecular weight of 78 000. The pH curves of the xylanases were quite diverse and showed pH optima ranging from 4.0 to 6.5. Characteristic action patterns were obtained for each of the purified xylanases by gel chromatography of the xylan digests on Bio-Gel P-2. The enzymes degraded arabinoxylan by an endomechanism, producing L-arabinose, D-xylose, xylobiose, and a mixture of branched arabinose–xylose and D-xylose oligosaccharides. All xylanases seemed to be capable of liberating L-arabinose from either arabinoxylan or the arabinose–xylose oligosaccharides. Branched arabinose-containing D-xylose oligosaccharides were slowly hydrolyzed, so that these sugars accumulate in the digest. Two xylanases showed relatively broad substrate specificity and were able to degrade also crystalline cellulose. β-D-Xylosidase showed optimal activity at pH 6.7 to 7.0 and at 42 °C. The Km for o-nitrophenyl-β-D-xylopyranoside was 0.22 mM and xylotriose was hydrolyzed more rapidly than xylobiose.

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