scholarly journals Human plasma uridine diphosphate galactose-glycoprotein galactosyltransfertase. Purification, properties and kinetics of the enzyme-catalysed reaction

1977 ◽  
Vol 167 (3) ◽  
pp. 621-628 ◽  
Author(s):  
A Bella ◽  
J S Whitehead ◽  
Y S Kim
Keyword(s):  
Molecular Weight ◽  
Human Plasma ◽  
Gel Filtration ◽  
Amino Sugar ◽  
Enzymic Activity ◽  
Transferase Activity ◽  
Uridine Diphosphate ◽  
Absolute Requirement ◽  
Km Value ◽  
Alpha Lactalbumin

The soluble galactosyltransferase of human plasma catalysed the transfer of galactose from UDP-galactose to high- and low-molecular-weight derivatives of N-acetylglucosamine, forming a beta-1-4 linkage. The enzyme was purified by using (NH4)2SO4 precipitation and affinity chromatography on an alpha-lactalbumin-Sepharose column. The galactosyltransferase was maximally bound to this column in the presence of N-acetylglucosamine, and the enzyme was eluted by omitting the amino sugar from the developing buffer. The molecular weight of the enzyme was estimated to be 85000 by gel filtration. The assay conditions for optimum enzymic activity was 30 degrees C and pH7.5. Mn2+ ion was found to be an absolute requirement for transferase activity. The Km for Mn2+ was 0.4 mM and that for the substrate, UDP-galactose, was 0.024 mM. The Km for the acceptors was 0.21 mM for alpha1-acid glycoprotein and 3.9 mM for N-acetylglucosamine. In the presence of alpha-lactalbumin, glucose became a good acceptor for the enzyme and had a Km value of 2.9 mM. Results of the kinetic study indicated that the free enzyme reacts with Mn2+ under conditions of thermodynamic equilibrium, and the other substrates are added sequentially.

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.

The Nature of Inactive Renin in Human Plasma and Amniotic Fluid

1978 ◽  
Vol 55 (1) ◽  
pp. 41-50 ◽  
Author(s):  
A. A. Shulkes ◽  
R. R. Gibson ◽  
S. L. Skinner
Keyword(s):  
Molecular Weight ◽  
Amniotic Fluid ◽  
Human Plasma ◽  
Gel Filtration ◽  
Dilution Effect ◽  
Exchange Chromatography ◽  
Acid Activation ◽  
Temperature Dependent ◽  
Inactive Renin ◽  
Full Activation

1. The properties of inactive and active renin in human plasma and amniotic fluid were studied chromatographically. Activation was achieved at pH 3.3 with and without added pepsin. 2. Acid activation of renin was time- and temperature-dependent but was inhibited by dilution of the sample. The dilution effect was corrected by adding pepsin. Such characteristics indicate that activation at low pH is catalysed by intrinsic enzymes. 3. Separation and/or dilution of the activating enzyme during ion-exchange chromatography concealed the eluted position of inactive renin and reduced the amount recovered. Only after full activation of the eluted renin was achieved with added pepsin was a distinct peak of inactive renin exposed. 4. At pH 7.5 inactive renin carried a lower negative charge than the active enzyme. This charge difference was lost after activation. 5. No molecular-weight differences between active, inactive renin or the International Renin Standard were detected by gel filtration. No renin of larger molecular weight was present. 6. These findings will be helpful in purification studies of human inactive renin.

Two Different Types of Inactive Renin in Human Plasma: Molecular and Kinetic Properties

1984 ◽  
Vol 67 (4) ◽  
pp. 421-425
Author(s):  
Yoichi Imamura ◽  
Kunio Hiwada ◽  
Tatsuo Kokubu
Keyword(s):  
Molecular Weight ◽  
Human Plasma ◽  
Kinetic Properties ◽  
Carbohydrate Composition ◽  
Ph Profile ◽  
Isoelectric Points ◽  
Different Types ◽  
Km Value ◽  
Inactive Renin ◽  
Fresh Plasma

1. We separated inactive renin in human plasma into two types, adsorbed and non-adsorbed, by chromatography on a concanavalin A-Sepharose column. About 75% of fresh plasma inactive renin was adsorbed to the column, and the rest passed through it. Non-adsorbed and adsorbed inactive renins were partially purified. 2. Non-adsorbed inactive renin had a molecular weight of 48000 and an isoelectric point of 5.44. Adsorbed inactive renin had a molecular weight of 46000 and isoelectric points of 5.56 and 5.80. 3. After activation with trypsin, both activated inactive renins were similar with respect to molecular weight (45000), thermostability, Km value (0.56 μmol/l) and pH profile. But pI values of both activated inactive renins differed. 4. These results indicate that there exist in human plasma two different types of inactive renin which differ in carbohydrate composition.

Purification of Ristocetin Willebrand Factor (RWF)

1975 ◽  
Author(s):  
J. D. Olson ◽  
D. N. Fass ◽  
W. J. Brockway ◽  
E. J. W. Bowie ◽  
K. G. Mann
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Apparent Molecular Weight ◽  
Amino Sugar ◽  
Procoagulant Activity ◽  
Factor Viii Inhibitor ◽  
Induced Aggregation

A component required for the ristocetin-induced aggregation of platelets was isolated in yields of 20-30% from pooled human and porcine plasma by cryoethanol concentration of the RWF, after removal of the vitamin K dependent coagulation factors on barium citrate. The concentrate was further purified using gel filtration (4% agarose) and ion exchange (DEAE-cellulose) chromatography. Sodium dodecyl sulfate polyacrylamide gels of the isolated factor indicated an apparent molecular weight of greater than 500,000. After reduction of RWF with mercaptoethanol, a single band is resolved with an apparent molecular weight of 230,000. The purified component had no factor VIII procoagulant activity and did not compete for the activity of naturally occurring factor VIII inhibitor (human). Antisera raised in rabbits directed against the purified component inhibited the RWF activity but not the factor VIII procoagulant activity of plasma. Amino acid analysis indicated the presence of all normal amino acids and failed to detect any amino sugar. Analysis of lipid revealed a significant amount of lipid composed of mono, di and triglycerides, cholesterol, cholesterol esters and free fatty acid, with small portions of phospholipids.

Characterization Of Soluble DES-A And DES-AB Fibrin In Human Plasma At 37°C By Agarose-Gel Filtration

1981 ◽  
Author(s):  
G Müller-Berghaus ◽  
J-C Bernhard ◽  
I Mahn
Keyword(s):  
Molecular Weight ◽  
Human Plasma ◽  
High Molecular Weight ◽  
Gel Filtration ◽  
Void Volume ◽  
Agarose Gel ◽  
High Molecular Weight Material ◽  
Different Temperatures ◽  
Lower Temperature

In two consecutive steps, thrombin cleaves the fibrinopeptides A and B from fibrinogen producing des-A fibrin and des-AB fibrin. Labeled des-A fibrin was prepared by batroxobin and labeled des-AB fibrin by clotting of 125I-fibrinogen with thrombin. Fibrin solubilized in buffered urea was mixed with plasma containing 131I-fibrinogen (fibrin:fibrinogen ratio = 1:20). These fibrinfibrinogen mixtures were applied to sepharose CL- 6B columns eq ui librated with buffered plasma (0.0025 M EDTA, 0.1 M NaCl, 0.05 M tris, 0.005 M EACA, 2 AT U hirudin/ml, 500 KIU a protinin/ml, 0.003 M NaN3, pH 7.4). Plasma was used as an equilibration and elution medium to prevent precipitation of fibrin in the columns. At 20°C, labeled des-A fibrin as well as des-AB fibrin were eluted in the void volume as high-molecular weight aggregates (peak A) and separated from m onomeric labeled fibrinogen (peak B). At 37°C, however, des- A fibrin was eluted at the same position as monomeric fibrinogen (peak B), whereas des-AB fibrin was eluted in the void volume as at 20°C. Rechromatography of isolated fractions of peak A and peak B at different temperatures showed that monomeric fibrin isolated at 37°C formed high molecular weight material at 20°C, and high-molecular weight fibrin isolated at 20°C dissociated at 37 ° C. The results suggest that des-A fibrin solubilized in plasma in the absence of calcium ismonomeric at 37°C but forms high-molecular weight aggregates at lower temperature, whereas des-AB fibrin is oligomeric at 20°C as well as at 37°C.

scholarly journals The potential dolichol recognition sequence of β-1,4-mannosyltransferase is not required for enzymic activity using phytanyl-pyrophosphoryl-α-N,N'- diacetylchitobioside as acceptor

1994 ◽  
Vol 299 (1) ◽  
pp. 23-27 ◽  
Author(s):  
L Revers ◽  
I B H Wilson ◽  
M C Webberley ◽  
S L Flitsch
Keyword(s):  
Amino Acid ◽  
Transmembrane Domain ◽  
Consensus Sequence ◽  
Gel Filtration ◽  
Enzymic Activity ◽  
Recognition Sequence ◽  
Mannose Residue ◽  
Before And After ◽  
Km Value ◽  
Good Agreement

The ALG1 gene of Saccharomyces cerevisiae encodes beta-1,4-mannosyltransferase, an essential membrane-associated enzyme involved in the assembly of dolichyl-linked oligosaccharide precursors for N-glycosylation [Albright and Robbins (1990) J. Biol. Chem. 265, 7042-7049], which catalyses the transfer of a mannose residue from GDP-mannose to dolichyl-pyrophosphoryl-alpha-N,N'- diacetylchitobioside; it also possesses a putative transmembrane domain, bearing an 11-amino-acid consensus sequence, which has been proposed to mediate dolichol recognition. Here we report the construction and bacterial expression of a mutant beta-1,4-mannosyltransferase derived from ALG1, which carries a 34-amino-acid deletion resulting in the absence of the entire N-terminal transmembrane domain. This truncated enzyme has an apparent Km value of 17 microM for phytanyl-pyrophosphoryl-alpha-N,N'-diacetylchitobioside, a known acceptor for beta-1,4-mannosyltransferase [Flitsch, Pinches, Taylor and Turner (1992) J. Chem. Soc., Perkin Trans. 1, 2087-2093]. The intact enzyme, expressed in the same system, has an apparent Km value of 25 microM. These figures are in good agreement with previously reported values for wild-type beta-1,4-mannosyl-transferase incubated with the natural dolichyl-linked substrate. Gel-filtration chromatography (before and after beta-mannosidase digestion) of the products of both forms of the enzyme verifies the formation of Man beta 1->4GlcNAc beta 1->4GlcNAc. We therefore conclude that the putative dolichol recognition sequence is not necessary for recognition of the phytanyl analogue of its natural dolichol substrate and suggest it probably also is not needed for its natural substrate.

Purification and Properties of Phosphoglyceromutase from Sheep Muscle

1971 ◽  
Vol 49 (11) ◽  
pp. 1183-1194 ◽  
Author(s):  
Eric James ◽  
R. O. Hurst ◽  
T. G. Flynn
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Diffusion Constant ◽  
Sedimentation Coefficient ◽  
Enzymic Activity ◽  
Sulfhydryl Groups ◽  
Cellulose Acetate Electrophoresis ◽  
Absolute Requirement ◽  
Protein Components ◽  
Sheep Muscle

Phosphoglyceromutase (2,3-diphospho-D-glycerate phosphotransferase, EC 2.7.5.3) has been purified from sheep muscle. The enzyme has a specific activity of 828 units/mg and is stable for several months at 0–2 °C in 0.1 μ phosphate buffer, pH 7.0. The Km for 2,3-diphosphoglycerate (DPGA) is 0.003 mM; the Km for 3-phosphoglycerate is 9.0 mM. A small amount of DPGA-phosphatase activity was associated with the enzyme.At pH 5.4 and 7.0 sheep phosphoglyceromutase was shown to be homogeneous by sedimenting as a single sharp peak in the ultracentrifuge and by the appearance of a single band on both disc gel and cellulose acetate electrophoresis. The sedimentation coefficient of the enzyme at pH 7.0 was 4.1 S, the diffusion constant 7.21 × 10−7 cm2/s, and the molecular weight calculated from both the-sedimentation and gel filtration data was of the order of 51 000.Disc gel electrophoresis of the enzyme at pH 9.3 revealed the presence of three protein components which were shown to be charge isomers.Titration of the enzyme with p-chloromercuribenzoate indicated that 4.0 sulfhydryl groups were present per mole. Reaction with 5,5′-dithio-bis-(2-nitrobenzoate) showed that one of the sulfhydryl groups may be an absolute requirement for enzymic activity.

Purification and some properties of uridine diphosphate N-acetylglucosamine pyrophosphorylase from Neurospora crassa

1979 ◽  
Vol 25 (12) ◽  
pp. 1381-1386 ◽  
Author(s):  
Kenji Yamamoto ◽  
Mitsuaki Moriguchi ◽  
Hiroyasu Kawai ◽  
Tatsurokuro Tochikura
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Neurospora Crassa ◽  
Isoelectric Point ◽  
Enzyme Preparation ◽  
Divalent Cations ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Uridine Diphosphate ◽  
Chromatographic Techniques

Uridine diphosphate N-acetylglucosamine pyrophosphorylase (EC. 2.7.7.23) of Neurospora crassa has been purified approximately 210-fold with dithiothreitol as the stabilizing agent by use of chromatographic techniques. The enzyme preparation appeared to be homogeneous when subjected to electrophoresis. The molecular weight was estimated as approximately 37 000 by gel filtration. The enzyme had an isoelectric point around pH 4.4.Maximum activity of the enzyme was observed at pH7.5. The enzyme required Mg2+, which may be replaced by other divalent cations such as Mn2+ and Co2+ for lesser degrees of effectiveness. The enzyme was strictly specific for UDP-N-acetylglucosamine as the substrate. The estimated values of Km were 2.2 mM for UDP-N-acetylglucosamine and 5.4 mM for inorganic pyrophosphate.The enzyme activity was highly stimulated by the addition of dithiothreitol or dithioerythritol but was lost by sulfhydryl inhibitory reagents.

On a Plasminogen Activator from Human Plasma

1979 ◽  
Vol 42 (05) ◽  
pp. 1607-1614 ◽  
Author(s):  
A Diaz Batista ◽  
G Hernandez Solana ◽  
J F Corral Almonte
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Human Plasma ◽  
Plasminogen Activator ◽  
Electrophoretic Mobility ◽  
Esterase Activity ◽  
Fibrinolytic Activity ◽  
Gel Filtration

SummaryA plasminogen activating substance was purified from the dialysates of the eluates of glass adsorbed kallikrein from fresh human plasma, by chromatography on QAE-Sephadex A-50 and gel filtration in Sephadex G-25. The preparation was concentrated by lyophilization. Its electrophoretic mobility was found to be similar to that of prealbumin. Its molecular weight appeared to be 15000–18000 daltons. The analysis of aminoacids of this activator showed that it contains a high proportion of acid amino acids. The purified activator showed esterase activity, fibrinolytic activity and kininogenase activity on heated human plasma. These activities were respectively equivalent to 150 μM BAEe/mg protein, 19 × 103 units of streptokinase/μg protein and 250 μg bradykinin/mg protein.

scholarly journals The multiple forms of α-D-mannosidase in human plasma

1979 ◽  
Vol 179 (3) ◽  
pp. 583-592 ◽  
Author(s):  
S Hirani ◽  
B Winchester
Keyword(s):  
Molecular Weight ◽  
Human Plasma ◽  
Concanavalin A ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
The Other ◽  
Multiple Forms ◽  
Simple Correlation ◽  
Multiple Components

The acidic alpha-D-mannosidase in human plasma closely resembles liver acidic alpha-D-mannosidase in its affinity for concanavalin A-Sepharose, molecular weight and resolution into multiple components on DEAE-cellulose. A combination of chromatography on concanavalin A-Sepharose and gel filtration on Sephadex G-200 and Sepharose 6B suggests that four forms of intermediate alpha-D-mannosidase, which differ either in their molecular weight of affinity for concanavalin A, exist in human plasma. A practical classification and nomenclature for the multiple forms of intermediate alpha-D-mannosidase in plasma based on molecular weight and affinity for concanavalin A is proposed. Multiple forms of intermediate alpha-D-mannosidase were also observed by ion-exchange chromatography on DEAE-cellulose, but there was not a simple correlation between these forms and those obtained with the other separation procedures. The form of intermediate alpha-D-mannosidase least abundant in plasma, approx. 7% of the activity, has very similar properties to the neutral alpha-D-mannosidase in human liver. In contrast, the other three forms of intermediate alpha-D-mannosidase, which account for over 90% of the activity, do not appear to be present in liver, except perhaps in trace amounts.

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