scholarly journals Some properties of cytochrome b5 from liver microsomes of man, monkey, pig and chicken

1969 ◽  
Vol 115 (4) ◽  
pp. 849-856 ◽  
Author(s):  
F. G. Nóbrega ◽  
P. S. Araujo ◽  
M. Pasetto ◽  
I. Raw
Keyword(s):  
Amino Acids ◽  
Spectral Properties ◽  
Cytochrome B5 ◽  
Liver Microsomes ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Gradient Elution ◽  
Closely Related Species ◽  
Ammonium Sulphate Fractionation ◽  
Terminal Amino

1. Cytochrome b5 was released from liver microsomes of man, monkey, pig and chicken by incubation with a crude lipase preparation. 2. By using DEAE-cellulose chromatography, ammonium sulphate fractionation, Sephadex-gel filtration and a final gradient elution on DEAE-Sephadex A-50, cytochromes b5 were obtained from the four species studied, all possessing similar spectral properties. 3. Stokes radii of the cytochromes were measured by gel filtration. 4. N-Terminal amino acids for the different cytochromes were serine for man and monkey, alanine for pig and glycine for chicken. 5. Amino acid analyses of the cytochromes are presented. 6. Peptide ‘fingerprint’ patterns of tryptic digests of the different cytochromes are discussed and clearly show increasing similarity for more closely related species.

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 Rabbit Tamm–Horsfall urinary glycoprotein. Chemical composition and subunit structure

1971 ◽  
Vol 122 (5) ◽  
pp. 623-631 ◽  
Author(s):  
Anne M. S. Marr ◽  
A. Neuberger ◽  
Wendy A. Ratcliffe
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Chemical Composition ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Cross Reactivity ◽  
Subunit Structure ◽  
Terminal Amino

1. Tamm–Horsfall glycoprotein from rabbit urine has been isolated and characterized. The homogeneity of the preparation has been established by a variety of procedures including disc gel electrophoresis and ultracentrifugation in aqueous solution, sodium dodecyl sulphate and formic acid. 2. The chemical composition has been determined and a carbohydrate content of approx. 31% was obtained. The relative contents of the amino acids were shown to be very similar to those in human Tamm–Horsfall glycoprotein. A trace of lipid was also detected. 3. Leucine was identified as the only N-terminal amino acid. 4. The subunit structure was investigated in the presence of sodium dodecyl sulphate by gel filtration and disc gel electrophoresis. These studies indicated that the subunit possessed a molecular weight of approx. 84000±6000. A similar value was obtained after reduction and S-alkylation of the glycoprotein indicating that the disulphide bonds were all intrachain. 5. A minimum value for the chemical molecular weight of 85000±6000 was obtained from the number of N-terminal amino acids released by cyanogen bromide cleavage of the glycoprotein. 6. The immunological properties of the glycoprotein were studied. Cross reactivity was demonstrated between human Tamm–Horsfall glycoprotein and a guinea-pig anti-rabbit Tamm–Horsfall antiserum.

scholarly journals Characterization and biosynthesis of cytochrome b5 in rat liver microsomes

1968 ◽  
Vol 107 (6) ◽  
pp. 839-849 ◽  
Author(s):  
J. R. Sargent ◽  
B. P. Vadlamudi
Keyword(s):  
Amino Acid ◽  
Rat Liver ◽  
Spectral Properties ◽  
Proteolytic Enzymes ◽  
Cytochrome B5 ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Isolated Rat Liver ◽  
Peptide Linkage ◽  
Isolated Rat

1. Cytochrome b5 is released from rat liver microsomes by both proteolytic enzymes and by treatments that disrupt phospholipids. Cytochrome P-420 is only released to a marked extent by treatments that disrupt phospholipids. 2. Cytochrome b5 was isolated in a pure state from both the rough and smooth fractions of rat liver microsomes after treatment with trypsin, and was shown to contain two cytochrome components with identical spectral properties. 3. Amino acid analyses of the two components are presented, together with peptide ‘fingerprint’ patterns of tryptic digests of the two components. 4. Studies based on the direct isolation of cytochrome b5 after administration of a single dose of radioactive amino acid to rats demonstrate that the cytochrome is synthesized initially in the rough fraction of microsomes and only subsequently appears in the smooth fraction. 5. Isolated rat liver microsomes are capable of incorporating radioactive amino acids into cytochrome b5 under standard conditions. 6. Under these conditions the amino acid is incorporated into peptide linkage in the cytochrome.

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.

scholarly journals The α macroglobulins of rat serum

1976 ◽  
Vol 159 (3) ◽  
pp. 643-650 ◽  
Author(s):  
A H Gordon
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Complex Mixture ◽  
Male Rats ◽  
Normal Male ◽  
Rat Serum ◽  
Α2 Macroglobulin ◽  
Total Amino Acids

A three-stage method for isolation of α1 macroglobulin and α2 macroglobulin from the serum of normal and injured rats is described. The methods successively used, namely gel filtration, ultracentrifugation and chromatography on DEAE-cellulose, were chosen to minimize loss of tryptic esterase-protecting activity. The two proteins differed slightly with respect to the following properties: mol.wt., α1 macroglobulin 7.46 × 10(5), α2 macroglobulin 7.16 × 10(5); isoelectric focusing, α1, macroglobulin pI 4.4, α2 macroglobulin pI4.5. Amino acid analyses were identical, except with respect to tyrosine: α1 macroglobulin 3.96 ± 0.24, α2 macroglobulin 3.16 ± 0.32 mol/100 mol of total amino acids. When isolated from the serum of uninjured rats, α1 macroglobulin retained the capacity to bind 1.05 mol of trypsin/mol. However, if isolated from serum 2 days after injury only 0.78 mol of trypsin/mol of α1 macroglobulin was bound. α2 macroglobulin isolated from this latter serum bound on average 0.97 mol of trypsin/mol. When reduced with N-acetylcysteine, both molecules formed subunits of size corresponding to that expected for quarter molecules. When α2 macroglobulin was reduced with dithiothreitol, quarter molecules were again produced. α1 macroglobulin, however, when thus treated gave a more complex mixture, containing a component having a mol.wt. of less than 6 × 10(4). Antisera raised against the two proteins permitted estimation of the concentration of each protein in the plasmas or sera of normal and injured rats. Plasma from normal male rats contained 3.76 ± 0.56 mg of α1 macroglobulin/ml (n = 33) and 0.016 ± 0.001 mg of α2 macroglobulin/ml (n=33). After injury by injection of turpentine and cortisone, the concentrations in plasma were at 3 days 5.19 ± 0.81 mg of α1 macroglobulin/ml (n = 12) and at 2 days 1.38 ± 0.35 mg of α2 macroglobulin/ml (n = 12). Antisera to the two proteins did not cross-react with one another. The quarter molecules formed by reduction of both proteins showed increased antigenicity.

Purification of a base-specific ribonuclease Ru from Rhizopus niveus

1980 ◽  
Vol 58 (6) ◽  
pp. 489-493 ◽  
Author(s):  
Hiroyuki Horitsu ◽  
Noriyuki Takihi ◽  
Masahisa Sugiura ◽  
Mikio Tomoyeda
Keyword(s):  
Amino Acids ◽  
Aspartic Acid ◽  
Column Chromatography ◽  
Deae Cellulose ◽  
Disc Electrophoresis ◽  
Acetone Precipitation ◽  
Base Specificity ◽  
Terminal Amino ◽  
Carboxyl Terminal ◽  
Rhizopus Niveus

A base-specific ribonuclease (RNase) Ru (EC 3.1.27.5) was isolated and purified from Rhizopus niveus in a yield of 17% by the procedures of acetone precipitation, column chromatography on Duolite A-2, DEAE-cellulose, CM-cellulose, and 2′(3′)-aminohexyl-5′-UMP-agarose. The enzyme was shown to be homogeneous by polyacrylamide disc electrophoresis. The amino- and carboxyl-terminal amino acids of the enzyme were determined to be an arginine and an aspartic acid, respectively. The enzyme has a base specificity: it released only 3′-UMP from yeast RNA or poly(U) and, in addition, small amounts of 3′-CMP from poly(C).

Purification of the delta toxin of Staphylococcus aureus

1970 ◽  
Vol 16 (8) ◽  
pp. 703-708 ◽  
Author(s):  
J. D. Caird ◽  
G. M. Wiseman
Keyword(s):  
Amino Acids ◽  
Staphylococcus Aureus ◽  
Amino Acid ◽  
Ammonium Sulphate ◽  
Deae Cellulose ◽  
Aureus Strain ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Two Peaks

An improved procedure for the purification of the delta toxin of Staphylococcus aureus strain E-delta has been devised which relies upon precipitation at pH 4.0 and further treatment with ammonium sulphate. A final step consists of passage twice through a column of DEAE-cellulose. Toxin obtained by this method appeared to be homogeneous on the basis of immunodiffusion and electrophoresis studies. However, two peaks with sedimentation coefficients of 2.8 S and 9.8 S were obtained when toxin was examined in the ultracentrifuge. Proline was identified as the N-terminal amino acid. No other N-terminal amino acids were detected in the purified toxin.

Human hemopexin. Preparation of the heme-rich protein

1982 ◽  
Vol 47 (2) ◽  
pp. 535-542 ◽  
Author(s):  
Ladislav Morávek ◽  
Josef Borvák ◽  
Karel Grüner ◽  
Bedřich Meloun ◽  
Petr Štrop ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Amino Acid Analysis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Simplified Procedure ◽  
Pooled Samples ◽  
Terminal Amino Acid Sequence

A simplified procedure was developed for the preparation of hemopexin from Cohn fraction IV obtained from partially hemolyzed pooled samples of serum. The method is based on precipitation with rivanol, chromatography on DEAE-cellulose, and gel filtration; it permits large quantities of the material to be treated on a laboratory scale. The preparation of heme-rich hemopexin obtained was characterized by amino acid analysis and the following N-terminal amino acid sequence: Thr-Pro-Leu-Pro-Arg-Gly-Ser-Ala-His-Gly-Asn-Val-Ala-Glu-Gly-Glu-Thr(Thr)Thr-Asn-Pro-Asp-Val-(Gly)(Leu).

scholarly journals Three Glycoproteins with Antimutagenic Activity Identified in Lactobacillus plantarum KLAB21

2001 ◽  
Vol 67 (8) ◽  
pp. 3445-3449 ◽  
Author(s):  
Chang-Ho Rhee ◽  
Heui-Dong Park
Keyword(s):  
Amino Acids ◽  
Lactobacillus Plantarum ◽  
Culture Supernatant ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Total Sugar ◽  
Antimutagenic Activity ◽  
Serovar Typhimurium ◽  
Molecular Masses ◽  
Coomassie Brilliant

ABSTRACT Antimutagenic substances were purified from a culture supernatant of Lactobacillus plantarum KLAB21 cells isolated from kimchi, a Korean traditional fermented vegetable, and their characteristics were investigated. The antimutagenic substances were separated into two fractions by DEAE-cellulose ion-exchange column chromatography, which were designated the R1 and R2 fractions. The R1 fraction was then divided into two fractions again by Sephadex G200 gel filtration chromatography, and the fractions were designated R1-1 and R1-2. All three fractions were further purified using a Sepharose CL-6B gel filtration column. All the purified fractions were successfully stained with fuchsin as well as Coomassie brilliant blue, suggesting that they are glycoproteins. The purified fractions were confirmed to possess antimutagenic activity againstN-methyl-N′-nitro-N-nitrosoguanidine on Salmonella enterica serovar Typhimurium TA100 cells. Their molecular masses were determined to be 16 (R1-1), 11 (R1-2), and 14 (R2) kDa on the Sepharose CL-6B column. Total sugar contents were 8.4% (R1-1), 7.3% (R1-2), and 9.4% (R2). The amino acid compositions of the fractions were different from each other; the major amino acids were glutamic acid (21.5%) and phenylalanine (17.1%) in the R1-1 fraction and glycine (41.3%) in the R1-2 fraction, but valine (31%) and phenylalanine (22.6%) were the major amino acids in the R2 fraction.

Inhibition of the synthesis of hepatic cytochrome P-450 by the interferon-inducing agent poly rI.rC

1984 ◽  
Vol 62 (4) ◽  
pp. 379-383 ◽  
Author(s):  
Gurmit Singh ◽  
Kenneth W. Renton
Keyword(s):  
Amino Acids ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Microsomal Protein ◽  
Interferon Inducer ◽  
Molecular Weights ◽  
Drug Biotransformation ◽  
Mixed Function Oxidase System ◽  
Pyrene Hydroxylase ◽  
Cytochrome P 450

The levels of cytochrome P-450, cytochrome b5, aminopyrine N-demethylase, and benzo[a]pyrene hydroxylase were depressed in hepatic microsomes following treatment of mice with the interferon inducer poly rI.rC. The decrease in the hepatic mixed function oxidase system was accompanied by an increase in the incorporation of amino acids into total microsomal protein. Fractionation of solubilized microsomes using a Sephacryl S-200 gel filtration column demonstrated that the increase in amino acid incorporation tended to be associated with proteins with molecular weights under 67 000. The fractions which contained cytochrome P-450 were further separated using a DEAE cellulose column. The amount of labelled amino acids associated with the cytochrome P-450 fractions was uniformly depressed in preparations from poly rI.rC treated animals compared with saline-treated controls. These results suggest that poly rI.rC causes a depression in the rate of synthesis of the apoprotein of cytochrome P-450 while increasing the incorporation of amino acids into other hepatic proteins. The decrease in apocytochrome P-450 synthesis explains the marked loss of drug biotransformation which occurs following induction of interferon.

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