An Endogalactosaminidase from Streptomyces griseus

1975 ◽  
Vol 53 (12) ◽  
pp. 1237-1249 ◽  
Author(s):  
Jose L. Reissig ◽  
Wei-Hsien Lai ◽  
James E. Glasgow
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Culture Filtrate ◽  
Deae Cellulose ◽  
Streptomyces Griseus ◽  
Glass Surfaces

An endogalactosaminidase has been purified 34-fold from the culture filtrate of Streptomyces griseus. This enzyme cleaves GalN–GalN linkages in oligogalactosaminoglycan, a galactosamine-rich oligosaccharide isolated from the culture filtrate of a Neurospora mutant. Since some or all of the GalN–GalN bonds in this molecule link positions 1 and 4, and are in the α-configuration, we are probably dealing with an endo-α-(1 → 4)-galactosaminidase, but this characterization is only tentative because the few bonds cleaved by the enzyme could have a different structure. The enzyme is inactive towards N-acetyl-oligogalactosaminoglycan and chitosan.The endogalactosaminidase preparations also cleave high molecular weight galactosaminoglycan (obtained from Neurospora) into fragments [Formula: see text] daltons in molecular weight, and catalyze the release of Neurospora sporelings from the glass surfaces to which they are anchored. Galactosaminoglycan-cleaving and sporeling-releasing activities elute jointly from DEAE-cellulose columns. This observation provides further support for an earlier proposal that the sporelings are anchored to the glass by means of galactosaminoglycan molecules.

scholarly journals Deoxyribonucleic acid polymerases of Euglena gracilis. Purification and properties of two distinct deoxyribonucleic acid polymerases of high molecular weight

1975 ◽  
Vol 151 (2) ◽  
pp. 227-238 ◽  
Author(s):  
A G McLennan ◽  
H M Keir
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Euglena Gracilis ◽  
Deoxyribonucleic Acid ◽  
Deae Cellulose ◽  
Low Molecular Weight ◽  
Optimum Conditions ◽  
Electrophoretic Mobilities ◽  
Substrate Activation ◽  
Purification And Properties

Two DNA polymerases of high molecular weight, pol A (mol.wt. 190 000) and pol B (mol.wt. 240 ooo), have been purified 6300-fold and 1600-fold respectively from an extramitochondrial supernatant of a bleached strain of Euglena gracilis. They have very similar requirements when assayed with an ‘activated’-DNA primer-template [the optimum conditions of pH and ionic (K+ and Mn2+) composition being 7.2, 25 mM and 0.2 mM respectively]. 0.2 mM-Mn2+ was about 1.5-2-fold as effective as 2 mM-Mg2+, owing to substrate activation by deoxyribonucleoside 5′-triphosphates in the presence of Mn2+. Km values for the triphosphates in the absence of activation were about 10(-6)M with Mn2+ and 8 × 10(-6) M with Mg2+ for both enzymes. They were inhibited to the same extent by N-ethylmaleimide, novobiocin and o-phenanthroline, but differed in their chromatographic behaviour on DEAE-cellulose and in their electrophoretic mobilities on polyacrylamide gel. No evidence was found for the existence in these cells of a DNA polymerase of low molecular weight, but there were indications that a third enzyme of high molecular weight might exist.

Subfractions of Fibrinogen Isolated from Human Plasma by Affinity Chromatography

1975 ◽  
Author(s):  
H. Hörmann ◽  
A. Stemberger ◽  
W. Jilg
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Human Plasma ◽  
High Molecular Weight ◽  
Deae Cellulose ◽  
Lag Phase ◽  
High Solubility ◽  
Stable Conformation ◽  
Associated Proteins ◽  
Deutsche Forschungsgemeinschaft

Fibrinogen was isolated from human plasma by affinity chromatography using thrombin activated immobilized fibrinogen. Elution with 1.0 M KBr yielded a preparation of 90–95% clottability. It contained, in addition to fibrinogen, polymers and proteins of lower molecular weight. Most of the associated proteins were removed by adsorption of fibrinogen on DEAE-Cellulose followed by stepwise elution. Under these conditions fibrinogen was also separated into several fractions including high solubility fibrinogen, high molecular weight derivatives and a fibrinogen fraction which was associated with another protein. High solubility fibrinogen was investigated in more detail. Its plasmic digestion showed a lag phase which was not recognized, if normal fibrinogen was degraded. This indicates a relatively stable conformation of high solubility fibrinogen.(Supported by Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 51.)

scholarly journals Characterization of a high-molecular-weight form of human acrosin. Comparison with human pancreatic trypsin

1981 ◽  
Vol 199 (2) ◽  
pp. 307-316 ◽  
Author(s):  
R A Anderson ◽  
S A Beyler ◽  
S R Mack ◽  
L J D Zaneveld
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Deae Cellulose ◽  
High Molecular Weight Form ◽  
Rate Limiting Step ◽  
Frozen Semen ◽  
Bean Trypsin Inhibitor ◽  
Hydrolysis Of Esters ◽  
Hydrolysis Of ◽  
Molecular Weight Form

A high-molecular-weight form of acrosin (alpha-acrosin, EC 3.4.21.10) was extracted from spermatozoa obtained from frozen semen and purified over 300-fold. Purification was effected by sequential use of Sephadex G-150, CM-cellulose and DEAE-cellulose chromatography. Properties of human acrosin were compared with those of human pancreatic trypsin. The molecular weight (Mr) of acrosin (70000) was greater than that of trypsin (Mr 21000). Isoelectric points for acrosin (pI = 9.0) and trypsin (pI = 8.2) were also different. alpha-N-Benzoyl-L-arginine ethyl ester was hydrolysed 50% more rapidly by acrosin than by trypsin. Acrosin had similar kcat. values for the hydrolysis of esters with different acylating groups (i.e. benzoyl-L-arginine and p-tosyl-L-arginine esters). In contrast, trypsin had dissimilar kcat. values for the hydrolysis of esters with different acylating groups. Kinetic data argue against deacylation as the rate-limiting step in ester hydrolysis by acrosin. Acrosin was less sensitive than trypsin to inhibition by 7-amino-1-chloro-3-L-tosylamidoheptan-2-one (‘TLCK’), di-isopropyl fluorophosphate and soya-bean trypsin inhibitor. D-Fructose and D-arabinose inhibited acrosin, but had no effect on trypsin. The data indicate that definite differences exist between human acrosin and trypsin.

scholarly journals Identification and properties of renal mineralocorticoid receptors in relation to glucocorticoid binders in rat liver and kidney

1976 ◽  
Vol 154 (3) ◽  
pp. 567-575 ◽  
Author(s):  
M K Agarwal
Keyword(s):  
Molecular Weight ◽  
Blood Serum ◽  
High Molecular Weight ◽  
Rat Liver ◽  
Rat Kidney ◽  
Deae Cellulose ◽  
Competitive Binding ◽  
Mineralocorticoid Receptors ◽  
Liver And Kidney

The binding of the natural mineralocorticoid aldosterone and the glucocorticoid corticosterone to macromolecules in rat liver and kidney cytoplasmic fractions was compared by various chromatographic procedures. Equilibration of kidney cytosol with 10nM-aldosterone, either alone or in the presence of a competing steroid, was ideal for ionexchange chromatography of DEAE-cellulose DE-52, and revealed the presence of four sorts of binding components. One of these, eluted in the 0.001M-phosphate pre-wash, and another, less abundant, forming a peak at 0.006M-phosphate, did not bind corticosterone at equimolar concentrations, and appear to constitute the mineralocorticoid-specific ‘MR‘ receptor in rat kidney. They could not be detected in the liver. Radioactivity eluted in the 0.02 and 0.06M-phosphate regions on DEAE-cellulose DE-52 appears to be due to [3H]aldosterone binding to glucocorticoid-specific ‘GR’ receptors and to transcortin respectively, since labelling was greater with corticosterone even at 10 nM than with the mineralocorticoid at 100nM and since [14C]corticosterone bound to blood serum transcortin was always co-chromatographed in the 0.06M-phosphate region. These two components appear to be identical with those in the liver and could be labelled maximally only by 100nM-corticosterone. The separation between specific mineralo- and glucocorticoid-binding species was less clear when chromatography was attempted on DEAE-Sephadex A-50 columns, possibly because of disaggregation into subunits in the presence of the high KC1 concentrations required for elution. Competitive binding followed by filtration through Sephadex G-200 gel indicated that cellular MR binders, unlike GR receptors, exist mostly as high-molecular-weight aggregates, although both appear to exhibit a comparable monomeric molecular weight of approx. 67000.

scholarly journals The xylanase system of Coniophora cerebella

1968 ◽  
Vol 108 (4) ◽  
pp. 571-576 ◽  
Author(s):  
N. J. King ◽  
D. B. Fuller
Keyword(s):  
Molecular Weight ◽  
Culture Filtrate ◽  
Uronic Acid ◽  
Enzyme System ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
The Other ◽  
Xylose Residue ◽  
Random Manner ◽  
Acidic Sugars

1. The culture filtrate of the fungus Coniophora cerebella grown on poplar 4-O-methylglucuronoxylan as carbon source and enzyme inducer contained an enzyme system that degraded the polysaccharide to xylose, acidic and neutral oligosaccharides and an enzyme-resistant polymer. Free uronic acid was not produced. 2. Cold ethanol fractionation of the culture filtrate yielded two active fractions, one of which had only xylanase (EC 3.2.1.8) and the other both xylanase and xylosidase (EC 3.2.1.37) activities. Further fractionation on DEAE-cellulose resolved the xylanase and xylosidase activities. 3. The xylanase degraded poplar 4-O-methylglucuronoxylan in an essentially random manner, producing oligosaccharides, but some xylose residues in the vicinity of uronic acid side groups were protected from hydrolysis, preventing a truly random attack. The xylosidase attacked the polysaccharide very slowly, releasing xylose, but the oligosaccharides produced by the action of the xylanase were much more susceptible to hydrolysis by the xylosidase. 4. The products of xylanase action were separated into neutral and acidic fractions. The neutral oligosaccharides were separated by chromatography on charcoal–Celite, and the major products were characterized as xylobiose, xylotriose, xylotetraose and xylopentaose. Some of the acidic sugars were branched, having the uronic acid residue attached to a xylose residue other than the terminal non-reducing one. 5. Gel filtration of various xylanase fractions gave values for the molecular weight of the enzyme from 34000 to 38000.

scholarly journals The Heterogeneity of the High Molecular Weight B12 Binder in Serum

Blood ◽  
1969 ◽  
Vol 33 (6) ◽  
pp. 899-908 ◽  
Author(s):  
CHRISTINE LAWRENCE
Keyword(s):  
Molecular Weight ◽  
Vitamin B12 ◽  
High Molecular Weight ◽  
Serum Proteins ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Weight Fraction ◽  
Low Molecular Weight ◽  
Transcobalamin I ◽  
Transcobalamin Ii

Abstract The binding of vitamin B12 by serum proteins was studied by separating Co57B12-enriched serum by Sephadex gel filtration, column chromatography with DEAE-cellulose, and paper electrophoresis. Each method of separation yielded two discrete B12-binding fractions. However, the analysis of each serum by all three separation technics indicated that one of the fractions was, in each case, bipartite. The "high" molecular weight B12-binding fraction defined by Sephadex gel filtration consisted of transcobalamin I and just part of the transcobalamin II fraction. The remaining portion of transcobalamin II was eluted from Sephadex gel in a "low" molecular weight fraction. Thus, transcobalamin II, equivalent to the β-globulin B12-binder, consisted of both "high" and "low" molecular weight components. This suggests that there are at least three serum proteins that can bind vitamin B12: two β-globulins, together comprising the transcobalamin II fraction and differing in molecular weight; and transcobalamin I.

scholarly journals Purification of a high molecular weight actin filament gelation protein from Acanthamoeba that shares antigenic determinants with vertebrate spectrins.

1984 ◽  
Vol 99 (6) ◽  
pp. 1970-1980 ◽  
Author(s):  
T D Pollard
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Actin Filament ◽  
Fluorescent Antibody ◽  
Deae Cellulose ◽  
Cross Reactivity ◽  
Actin Binding ◽  
Antigenic Determinants ◽  
Selective Precipitation ◽  
Pig Brain

I have purified a high molecular weight actin filament gelation protein (GP-260) from Acanthamoeba castellanii, and found by immunological cross-reactivity that it is related to vertebrate spectrins, but not to two other high molecular weight actin-binding proteins, filamin or the microtubule-associated protein, MAP-2. GP-260 was purified by chromatography on DEAE-cellulose, selective precipitation with actin and myosin-II, chromatography on hydroxylapatite in 0.6 M Kl, and selective precipitation at low ionic strength. The yield was 1-2 micrograms/g cells. GP-260 had the same electrophoretic mobility in SDS as the 260,000-mol-wt alpha-chain of spectrin from pig erythrocytes and brain. Electron micrographs of GP-260 shadowed on mica showed slender rod-shaped particles 80-110 nm long. GP-260 raised the low shear apparent viscosity of solutions of Acanthamoeba actin filaments and, at 100 micrograms/ml, formed a gel with a 8 microM actin. Purified antibodies to GP-260 reacted with both 260,000- and 240,000-mol-wt polypeptides in samples of whole ameba proteins separated by gel electrophoresis in SDS, but only the 260,000-mol-wt polypeptide was extracted from the cell with 0.34 M sucrose and purified in this study. These antibodies to GP-260 also reacted with purified spectrin from pig brain and erythrocytes, and antibodies to human erythrocyte spectrin bound to GP-260 and the 240,000-mol-wt polypeptide present in the whole ameba. The antibodies to GP-260 did not bind to chicken gizzard filamin or pig brain MAP-2, but they did react with high molecular weight polypeptides from man, a marsupial, a fish, a clam, a myxomycete, and two other amebas. Fluorescent antibody staining with purified antibodies to GP-260 showed that it is concentrated near the plasma membrane in the ameba.

scholarly journals Glycoprotein biosynthesis in animal cells grown in suspension culture. Assembly of lipid-linked saccharides and formation of protein-bound ‘high-mannose’ oligosaccharides

1981 ◽  
Vol 195 (1) ◽  
pp. 139-151 ◽  
Author(s):  
D S Bailey ◽  
J Burke ◽  
R Sinclair ◽  
B B Mukherjee
Keyword(s):  
Molecular Weight ◽  
Suspension Culture ◽  
High Molecular Weight ◽  
Deae Cellulose ◽  
Glucose Deprivation ◽  
Animal Cells ◽  
Glycoprotein Biosynthesis ◽  
High Mannose

Glycoprotein biosynthesis was studied with mouse L-cells grown in suspension culture. Glucose-deprived cells incorporated [3H]mannose into ‘high-mannose’ protein-bound oligosaccharides and a few relatively high-molecular-weight lipid-linked oligosaccharides. The latter were retained by DEAE-cellulose and turned over quite slowly during pulse--chase experiments. Increased heterogeneity in size of lipid-linked oligosaccharides developed during prolonged glucose deprivation. Sequential elongation of lipid-linked oligosaccharides was also observed, and conditions that prevented the assembly of the higher lipid-linked oligosaccharides also prevented the formation of the larger protein-bound ‘high-mannose’ oligosaccharides. In parallel experiments, [3H]mannose was incorporated into a total polyribosome fraction, suggesting that mannose residues were transferred co-translationally to nascent protein. Membrane preparations from these cells catalysed the assembly from UDP-N-acetyl-D-[6-3H]glucosamine and GDP-D-[U-14C]mannose of polyisoprenyl diphosphate derivatives whose oligosaccharide moieties were heterogeneous in size. Elongation of the N-acetyl-D-[6-3H]glucosamine-initiated glycolipids with mannose residues produced several higher lipid-linked oligosaccharides similar to those seen during glucose deprivation in vivo. Glucosylation of these mannose-containing oligosaccharides from UDP-D-[6-3H]glucose was restricted to those of a relatively high molecular weight. Protein-bound saccharides formed in vitro were mainly smaller in size than those assembled on the lipid acceptors. These results support the involvement of lipid-linked saccharides in the synthesis of asparagine-linked glycoproteins, but show both in vivo and in vitro that protein-bound ‘high-mannose’ oligosaccharide formation can occur independently of higher lipid-linked oligosaccharide synthesis.

The role of hydration degree of cations and anions on the adsorption of high-molecular-weight nonionic polyacrylamide on glass surfaces

2012 ◽  
Vol 415 ◽  
pp. 91-97 ◽  
Author(s):  
A.R. Al-Hashmi ◽  
P.F. Luckham ◽  
R.S. Al-Maamari ◽  
A. Zaitoun ◽  
H.H. Al-Sharji
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Hydration Degree ◽  
Glass Surfaces
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