scholarly journals Dolichyl monophosphate and its sugar derivatives in plants

1977 ◽  
Vol 161 (1) ◽  
pp. 93-101 ◽  
Author(s):  
C T Brett ◽  
L F Leloir
Keyword(s):  
Enzyme Preparation ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Sodium Deoxycholate ◽  
Soya Bean ◽  
Pea Seedlings ◽  
Lipid Moiety ◽  
Sugar Derivatives ◽  
Chloroform Methanol ◽  
Cellulose Column

A glucose acceptor was isolated from soya beans by extraction with chloroform/methanol (2:1, v/v), followed by DEAE-cellulose column chromatography of the extract. This acceptor could not be distinguished from liver dolichyl monophosphate by t.l.c. It could replace dolichyl monophosphate as a mannose acceptor with a liver enzyme and its glucosylated derivative could replace dolichyl monophosphate glucose as a glucose donor in the same system. These results, together with those already reported [Pont Lezica, Brett, Romero Martinez & Dankert (1975) Biochem, Biophys. Res. Commun. 66, 980-987], indicate that the acceptor from soya bean is a dolichyl monophosphate. Gel filtration of its glucosylated derivative on Sephadex G-75 in the presence of sodium deoxycholate indicated that the acceptor contained 17 or 18 isoprene units. An enzyme preparation from pea seedlings was shown to use endogenous acceptors to form lipid phosphate sugars containing mannose and N-acetylglucosamine from GDP-mannose and UDP-N-acetylglucosamine. Chromatographic and degradative techniques indicated that the compounds formed were lipid monophosphate mannose, lipid pyrophosphate N-acetylglucosamine, lipid pyrophosphate chitobiose and a series of lipid pyrophosphate oligosaccharides containing both mannose and N-acetylglucosamine. None of these compounds was degraded by catalytic hydrogenation, and so the lipid moiety in each case was probably an alpha-saturated polyprenol. The endogenous acceptors for mannose and N-acetylglucosamine in peas may therefore be dolichyl monophosphate, as has been found in mammalian systems.

scholarly journals Recovery of dolichyl diphosphate oligosaccharide in methanolic aqueous phase prepared from rat liver microsomal fractions

1986 ◽  
Vol 236 (3) ◽  
pp. 913-916
Author(s):  
M Sarkar ◽  
S Mookerjea
Keyword(s):  
Aqueous Phase ◽  
Rat Liver ◽  
Liver Microsomes ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Rat Liver Microsomes ◽  
Endogenous Protein ◽  
Phase Map ◽  
Chloroform Methanol ◽  
Cellulose Column

The synthesis of dolichyl diphosphate oligosaccharide was studied by incubating rat liver microsomes (microsomal fractions) with GDP-[14C]mannose, UDP-glucose, UDP-N-acetylglucosamine and [3H]dolichol phosphate. The labelled products obtained by the first step of extraction of the microsomes in methanolic aqueous phase (MAP fraction in chloroform/methanol/water; 3:2:1, by vol.) and in CMW fraction (chloroform/methanol/water; 10:10:3, by vol.) obtained by extraction of the interphase after the first step of extraction were analysed on a DEAE-cellulose column. With the progress of incubation, the radioactivity in unchanged GDP-mannose decreased, whereas the labelled dol-P-P-oligo in the MAP fraction increased about 5-6-fold. The lipid oligosaccharide in this fraction accounted for about 50-60% of the GDP-mannose used, whereas the recovery of the labelled lipid oligosaccharide in the CMW fraction was about 10%. The lipid oligosaccharide from both reactions after mild acid hydrolysis were analysed by gel filtration on Bio-Gel P-4. The oligosaccharide from the MAP fraction gave a peak of higher Mr distinctly separate from the lower-Mr peak obtained from the CMW fraction. Microsomes incubated with labelled lipid oligosaccharide from the MAP fraction showed incorporation of the label into endogenous protein.

Characterization of an isozyme of S-adenosylmethionine synthetase from rat liver

1984 ◽  
Vol 62 (5) ◽  
pp. 276-279 ◽  
Author(s):  
C. H. Lin ◽  
W. Chung ◽  
K. P. Strickland ◽  
A. J. Hudson
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Enzymatic Synthesis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Aromatic Amino Acids ◽  
Adenosylmethionine Synthetase ◽  
Cellulose Column

An isozyme of S-adenosylmethionine synthetase has been purified to homogeneity by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and gel filtration on a Sephadex G-200 column. The purified enzyme is very unstable and has a molecular weight of 120 000 consisting of two identical subunits. Amino acid analysis on the purified enzyme showed glycine, glutamate, and aspartate to be the most abundant and the aromatic amino acids to be the least abundant. It possesses tripolyphosphatase activity which can be stimulated five to six times by S-adenosylmethionine (20–40 μM). The findings support the conclusion that an enzyme-bound tripolyphosphate is an obligatory intermediate in the enzymatic synthesis of S-adenosylmethionine from ATP and methionine.

scholarly journals Purification and subunit structure of argininosuccinate lyase from Chlamydomonas reinhardi

1977 ◽  
Vol 167 (1) ◽  
pp. 71-75 ◽  
Author(s):  
R F Matagne ◽  
J P Schlösser
Keyword(s):  
Enzyme Activity ◽  
Crude Extract ◽  
Enzyme Preparation ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Activity Analysis ◽  
Disc Electrophoresis ◽  
Subunit Structure ◽  
Argininosuccinate Lyase

Argininosuccinate lyase (EC 4.3.2.1) was purified by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and gel filtration on Sephadex G-200. The final enzyme preparation was purified 46-fold compared with the crude extract. Electrophoresis of this preparation revealed three bands, the major one having the enzyme activity. Analysis of the enzyme by gel filtration and by disc electrophoresis (in two different concentrations of acrylamide) gave mol.wts. of 200000 (+/- 15000) and 190000 (+/- 20000) respectively. Treatment with sodium dodecyl sulphate and mercaptoethanol dissociated the enzyme into subunits of mol.wt. 39000 (+/-2000). The results are indicative of the multimeric structure of the enzyme, which is composed of five (perhaps four or six) identical subunits.

Effect of Streptococcal Extracellular Nuclease on the Carrier Activity of RNA for Streptolysin S

1983 ◽  
Vol 38 (1-2) ◽  
pp. 107-111 ◽  
Author(s):  
Akira Taketo ◽  
Yoriko Taketo
Keyword(s):  
Hemolytic Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Nucleotide Composition ◽  
Active Fraction ◽  
Streptolysin S ◽  
The Core ◽  
Extracellular Nuclease ◽  
Effective Component ◽  
Cellulose Column

Upon digestion with a streptococcal extracellular nuclease, yeast RNA yielded acid-insoluble core having increased carrier activity for streptolysin S. The carrier activity was found in minor fractions of the core which were eluted from a DEAE-cellulose column at higher salt concentrations. Upon gel filtration through a Sephadex G-75 column, the effective component (Fr. I) was eluted earlier than bulk oligonucleotides (Fr. II). Nucleotide composition (in mol %) of Fr. I was AMP: 21.8; GMP: 55.1; CMP: 8.2; UMP: 14.9, whereas that of Fr. II was AMP: 38.0; GMP: 33.1; CMP: 8.0; UMP: 20.9. Chromatographic patterns of SLS complex induced by Fr. I were similar to those of the toxin formed in the presence of active fraction prepared from RNase I core. Hemolytic activity of the latter complex was, like the former, unaffected by streptococcal nuclease treatment. The carrier activity of DNA digested with the nuclease was also investigated.

A proteolytic pseudomonad from skin lesions of rainbow trout (Salmo gairdnerii). II. Some properties of the proteinase

1968 ◽  
Vol 14 (8) ◽  
pp. 875-880 ◽  
Author(s):  
M. F. Li ◽  
Carol Jordan
Keyword(s):  
Rainbow Trout ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Skin Lesions ◽  
Complexing Agents ◽  
Bivalent Cations ◽  
Wide Ph Range ◽  
Ph Range ◽  
Trout Muscle ◽  
Cellulose Column

An extracellular proteinase from a pseudomonad pathogenic to rainbow trout was purified 33-fold by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and Sephadex G 75 gel filtration. The purified enzyme was active over a wide pH range, from pH 5.0 to 10.0. Heating at 98 °C for 1 h did not completely inactivate the enzyme. Its observed temperature optimum was 45 °C. Michaelis–Menten constants were found to be 0.625% for casein and 0.263% for rainbow trout muscle albumin. Activation energies calculated for these substrates were 8.1 kcal and 11.4 kcal per mole, respectively. The involvement of bivalent cations and free sulfhydryl groups in the enzymatic activity was demonstrated by the inhibition caused by metal-complexing agents and p-chloromercuribenzoate, respectively.

scholarly journals Glucosylation of phosphorylpolyisoprenol and sterol at the plasma membrane of soya-bean (Glycine max) protoplasts

1980 ◽  
Vol 186 (2) ◽  
pp. 411-421 ◽  
Author(s):  
C M Chadwick ◽  
D H Northcote
Keyword(s):  
Plasma Membrane ◽  
Glycine Max ◽  
Neutral Lipids ◽  
Deae Cellulose ◽  
Soya Bean ◽  
Radioactive Material ◽  
Optimum Conditions ◽  
Radioactive Label ◽  
Acidic Fraction ◽  
Chloroform Methanol

Protoplasts were prepared from cells of soya-bean (Glycine max) suspension cultures and the plasma membrane was labelled with diazotized [G-3H]sulphanilic acid. Homogenates were fractionated by differential and isopycnic centrifugation, and membrane fractions in a density gradient were characterized by enzymic markers and the radioactive label. When fractions containing a large amount of protein were incubated with UDP-[U-14C]glucose, radioactive material soluble in chloroform/methanol was formed and this separated into acidic and neutral fractions on ion-exchange chromatograms of DEAE-cellulose. The acidic fraction was shown to consist of dolichol phosphate glucose, and the neutral fraction sterol glucosides and acylsterol glucosides. Optimum conditions for glucosylation of dolichol phosphate were established as 5 mM-MgCl2, pH 6.0, and the enzyme had a Michaelis constant of 1.5 × 10(-5) m-UDP-glucose. Optimum conditions for glucosylation of sterol were 5 mM-MgCl2, pH 8.0 GDP-[U-14C]glucose was a poor substrate for the synthesis of both acidic and neutral lipids. Although the synthesis of dolichol phosphate glucose and sterol glucosides occurred throughout the sucrose gradient, the specific activities of both glucosyltransferases were greatest in a fraction coincident with the radioactively labelled plasma membrane. Results are discussed in relation to the likely role fo these transglucosylase activities.

Aldehyde Dehydrogenases in Rat Liver

1972 ◽  
Vol 50 (7) ◽  
pp. 741-748 ◽  
Author(s):  
G. T. Shum ◽  
A. H. Blair
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Supernatant Fraction ◽  
Aldehyde Dehydrogenases ◽  
Low Concentrations ◽  
Enzyme I ◽  
Cellulose Column ◽  
Aldehyde Dehydrogenase Activity

Two enzymes (I and II) with NAD+-dependent aldehyde dehydrogenase activity have been separated and partially purified from the supernatant fraction of rat liver. Resolution was effected by DEAE-cellulose column chromatography. In addition to the differences in charge properties, these two proteins differ in substrate specificity, that of enzyme II being comparatively restricted. Enzyme I has a relatively sharp optimum in activity at pH 8 whereas enzyme II exhibits an optimal range between pH 8 and 9.5. Both enzymes are strongly inhibited by low concentrations of p-chloromercuribenzene sulfonic acid and this inhibition can be reversed by dithiothreitol. Both enzymes are also inhibited by arsenite; inhibition of enzyme I is enhanced by mercaptoethanol but inhibition of enzyme II is not so affected. Molecular weight estimation by gel filtration indicates each protein has a molecular weight of approximately 180 000.

scholarly journals Purification and chemical characterization of human hexosaminidases A and B

1976 ◽  
Vol 159 (3) ◽  
pp. 535-539 ◽  
Author(s):  
J E S. Lee ◽  
A Yoshida
Keyword(s):  
Column Chromatography ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Gel Chromatography ◽  
Deae Cellulose Column ◽  
Hexosaminidase A ◽  
Cellulose Column

N-Acetyl-β-hexosaminidases A and B were purified to homogeneity from human placenta. In the initial step of purification, the enzymes were adsorbed on concanavalin A-Sepharose 4B and eluted from the column with α-methyl D-mannosides. Subsequent purification steps included DEAE-cellulose column chromatography, QAE-Sephadex [diethyl-(2-hydroxypropyl)aminoethyl-Sephadex] column chromatography, Sephadex G-200 gel filtration and preparative disc polyacrylamide-gel electrophoresis, followed by another QAE-Sephadex chromatography for the hexosaminidase A preparation, and DEAE-cellulose column chromatography, calcium phosphate gel chromatography, Sephadex G-200 gel filtration, QAE-Sephadex chromatography and CM-cellulose chromatography for the hexosaminidase B preparation. The purified preparations, particularly hexosaminidase A, had significantly higher specific enzyme activities than previously reported. The preparations moved on polyacrylamide-gel electrophoresis as single protein bands, which also stained for enzyme activity. Sedimentation-equilibrium centrifugation indicated homogenous dispersion of the enzymes, and the molecular weight was estimated as about 110000 for both enzymes. Complete amino acid and carbohydrate compositions of the two isoenzymes were determined, and, in contrast with previous suggestions, no sialic acid was found in the enzymes.

Milk-clotting activity of proteinases produced by Rhizopus

1969 ◽  
Vol 15 (1) ◽  
pp. 99-104 ◽  
Author(s):  
Hwa L. Wang ◽  
Doris I. Ruttle ◽  
C. W. Hesseltine
Keyword(s):  
Sodium Chloride ◽  
Culture Filtrate ◽  
Enzyme Preparation ◽  
Deae Cellulose ◽  
Active Components ◽  
Milk Clotting ◽  
Salt Gradient ◽  
High Yields ◽  
Cellulose Column ◽  
Milk Clotting Activity

Rhizopus oligosporus NRRL 3271 produces an enzyme having high milk-clotting activity. High yields of the enzyme were noted in the culture filtrates of milk, wheat flour, or wheat bran. The enzyme was stable at 40 °C, or below, but its activity was destroyed rapidly by heating at 60 °C. The enzyme was fairly stable between pH 3 and 6 and very unstable at a pH below 2 or above 7. The enzyme was recovered from the culture filtrate by ammonium sulfate fractionation (30–75% saturation). When the enzyme preparation so obtained was chromatographed on a DEAE-cellulose column and eluted by salt gradient, four active components were noted, two of which accounted for most of the activity. The NRRL 3271 enzyme and animal rennin behave similarly toward the pH of milk, added calcium chloride, and sodium chloride. Although the NRRL 3271 enzyme caused a higher degree of proteolysis than did rennin, the proteolysis apparently was not high enough to interfere with curd formation. Therefore, the NRRL 3271 enzyme merits further study as a potential replacement for rennin or as an adjunct to be incorporated into rennin for the manufacture of cheese.

scholarly journals The complete amino acid sequence of a mouse ϰ light chain

1972 ◽  
Vol 128 (2) ◽  
pp. 427-444 ◽  
Author(s):  
J. Svasti ◽  
C. Milstein
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Paper Electrophoresis ◽  
Tryptic Peptides ◽  
Myeloma Protein ◽  
Complete Amino Acid Sequence ◽  
Cellulose Column ◽  
Mouse Myeloma

The complete amino acid sequence of the κ-chain of the mouse myeloma protein MOPC 21 was established. The protein was reduced and alkylated with iodo[2-14C]acetic acid, and 21 tryptic peptides were isolated, mainly by paper electrophoresis and paper chromatography. Three large tryptic peptides (of 35, 36 and 42 residues), which were difficult to isolate in this manner, were obtained pure and in excellent yields by a combination of Sephadex G-50 gel filtration in 1% (w/v) NH4HCO3 and chromatography on a DEAE-cellulose column in ammonium acetate buffer, pH8.1. Peptides overlapping the tryptic peptides were isolated from a chymotryptic digest. The chain is 214 residues long. Microheterogeneity of two peptides was observed and is believed to be due to deamidation. It was not excluded that such deamidation could occur in serum from which the protein was isolated. The sequence is compared with the sequences of two other mouse κ-chains, and with the human κ-chain basic sequences.

Sign in / Sign up

Export Citation Format

Share Document