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.

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 α-Crystallin. The isolation and characterization of distinct macromolecular fractions

1971 ◽  
Vol 124 (2) ◽  
pp. 337-343 ◽  
Author(s):  
Abraham Spector ◽  
Lu-Ku Li ◽  
Robert C. Augusteyn ◽  
Arthur Schneider ◽  
Thomas Freund
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Chemical Difference ◽  
Molecular Weights ◽  
Isolation And Characterization ◽  
Different Populations ◽  
Molecular Weight Fractions

α-Crystallin was isolated from calf lens periphery by chromatography on DEAE-cellulose and gel filtration. Three distinct populations of macromolecules have been isolated with molecular weights in the ranges approx. 6×105−9×105, 0.9×106−4×106and greater than 10×106. The concentration of macromolecules at the molecular-weight limits of a population are very low. The members of the different populations do not appear to be in equilibrium with each other. Further, in those molecular-weight fractions investigated, no equilibrium between members of the same population was observed. The population of lowest molecular weight comprises 65–75% of the total material. The amino acid and subunit composition of the different-sized fractions appear very similar, if not identical. The only chemical difference observed between the fractions is the presence of significant amounts of sugar in the higher-molecular-weight fractions. Subunit molecular weights of approx. 19.5×103and 22.5×103were observed for all α-crystallin fractions.

scholarly journals Characterization of amino acids and low-molecular-weight peptides bound to cytoplasmic granules from the posterior pituitary

1970 ◽  
Vol 48 (4) ◽  
pp. 455-462 ◽  
Author(s):  
Seon Shin ◽  
Frank LaBella ◽  
Gary Queen
Keyword(s):  
Amino Acids ◽  
Gel Filtration ◽  
Cation Exchange Resin ◽  
Exchange Chromatography ◽  
Posterior Pituitary ◽  
Total Acid ◽  
Cytoplasmic Granules ◽  
Molecular Weights ◽  
Positive Material ◽  
Fraction I

A number of peptides and amino acids, representing 30–40% of the total acid-extractable, ninhydrin-positive material of the tissue, were associated with cytoplasmic granules (sedimenting at 3 000 000 g-min after preliminary removal of "nuclei and debris") isolated from bovine posterior pituitary glands. Acetic acid (0.2 N) extracts of a purified neurosecretory granule fraction showed only slight differences in the pattern of peptides and amino acids from extracts of the total cell particulate fraction. Gel filtration of extracts on Sephadex G-25 yielded three major fractions: fraction I consisting of peptide material of molecular weights > 4000, fraction II of molecular weights averaging about 3000, and fraction III of molecular weights < 2000. Fraction III was further resolved by anion-exchange chromatography into 12 subfractions. Vasopressin and oxytocin were contained in subfractions 2 and 3, respectively. Each of these subfractions was in turn chromatographed on a cation-exchange resin and resolved into a total for fraction III of 22 major components: lysine, arginine, phenylalanine, ammonia, and 18 peptides. Three of the peptides contained only aspartic and glutamic acids in the ratios 8:1, 5:1, and 4:1. The sequences of four dipeptides were ascertained. Another peptide was not retarded by Dowex 50 and yielded glutamic acid upon acid hydrolysis. Still another peptide yielded tyrosine plus an unknown ninhydrin-positive component after hydrolysis. The amino acid compositions were determined for nine other peptides containing three to nine residues. Additional peptides in fraction III were detected in lesser or trace amounts. Isolated granule fractions from both bovine posterior pituitary and rat liver were dialyzed against isotonic sucrose or distilled water. The rate of loss of ninhydrin-positive material from the sample dialyzed against water indicated that a large proportion of the "free" amino acids and peptides of these tissues were contained within intracellular organelles.

scholarly journals Purification and characterization of two human erythrocyte arylamidases preferentially hydrolysing N-terminal arginine or lysine residues

1978 ◽  
Vol 175 (3) ◽  
pp. 1051-1067 ◽  
Author(s):  
K K Mäkinen ◽  
P L Mäkinen
Keyword(s):  
Substrate Inhibition ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Gel Permeation Chromatography ◽  
Ph Optimum ◽  
Preparative Scale ◽  
Molecular Weights ◽  
Gel Permeation ◽  
Lysine Residues ◽  
Enzyme I

Two arylamidases (I and II) were purified from human erythrocytes by a procedure that comprised removal of haemoglobin from disrupted cells with CM-Sephadex D-50, followed by treatment of the haemoglobin-free preparation subsequently with DEAE-cellulose, gel-permeation chromatography on Sephadex G-200, gradient solubilization on Celite, isoelectric focusing in a pH gradient from 4 to 6, gel-permeation chromatography on Sephadex G-100 (superfine), and finally affinity chromatography on Sepharose 4B covalently coupled to L-arginine. In preparative-scale purifications, enzymes I and II were separated at the second gel-permeation chromatography. Enzyme II was obtained as a homogeneous protein, as shown by several criteria. Enzyme I hydrolysed, with decreasing rates, the L-amino acid 2-naphtylamides of lysine, arginine, alanine, methionine, phenylalanine and leucine, and the reactions were slightly inhibited by 0.2 M-NaCl. Enzyme II hydrolysed most rapidly the corresponding derivatives of arginine, leucine, valine, methionine, proline and alanine, in that order, and the hydrolyses were strongly dependent on Cl-. The hydrolysis of these substrates proceeded rapidly at physiological Cl- concentration (0.15 M). The molecular weights (by gel filtration) of enzymes I and II were 85 000 and 52 500 respectively. The pH optimum was approx. 7.2 for both enzymes. The isoelectric point of enzyme II was approx. 4.8. Enzyme I was activated by Co2+, which did not affect enzyme II to any noticeable extent. The kinetics of reactions catalysed by enzyme I were characterized by strong substrate inhibition, but enzyme II was not inhibited by high substrate concentrations. The Cl- activated enzyme II also showed endopeptidase activity in hydrolysing bradykinin.

scholarly journals Isolation of palmitoyl-CoA hydrolases from human blood platelets

1981 ◽  
Vol 199 (3) ◽  
pp. 639-647 ◽  
Author(s):  
R K Berge ◽  
L E Hagen ◽  
M Farstad
Keyword(s):  
Human Blood ◽  
Blood Platelets ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Apparent Molecular Weight ◽  
Cytosol Fraction ◽  
Molecular Weights ◽  
Apparent Homogeneity ◽  
Coa Hydrolase ◽  
Human Blood Platelets

The palmitoyl-CoA hydrolase activity, which in human blood platelets is mainly localized in the cytosol fraction [Berge, Vollset & Farstad (1980) Scand. J. Clin. Lab. Invest. 40, 271--279], was found to be extremely labile. Inclusion of glycerol or palmitoyl-CoA stabilized the activity during preparation. Gel-filtration studies revealed multiple forms of the enzyme with molecular weights corresponding to about 70 000, 40 000 and 24 000. The relative recovery of the mol.wt.-70 000 form was increased by the presence of 20% (v/v) glycerol or 10 microM-palmitoyl-CoA. The three enzyme forms are probably unrelated, since they were not interconvertible. The three different species of palmitoyl-CoA hydrolase were purified by DEAE-cellulose and hydroxyapatite chromatography, isoelectric focusing and high-pressure liquid chromatography (h.p.l.c.) to apparent homogeneity. The three enzymes had isoelectric points (pI) of 7.0, 6.1 and 4.9. The corresponding molecular weights were 27 000--33 000, 66 000--72 000 and 45 000--49 000, calculated from h.p.l.c. and Ultrogel AcA-44 chromatography. The apparently purified enzymes were unstable, as most of the activity was lost during purification. The enzyme with an apparent molecular weight of 45 000--49 000 was split into fractions with molecular weights of less than 10 000 by re-chromatography on h.p.l.c. concomitantly with a loss of activity. The stimulation of the activity by the presence of serum albumin seems to depend on the availability of palmitoyl-CoA, as has been reported for other palmitoyl-CoA hydrolases. [Berge & Farstad (1979) Eur. J. Biochem. 96, 393--401].

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.

Comparison of some properties of soil humic acids and synthetic phenolic polymers incorporating amino derivaties

Soil Research ◽  
1966 ◽  
Vol 4 (1) ◽  
pp. 41 ◽  
Author(s):  
JN Ladd ◽  
JHA Butler
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Acid Hydrolysis ◽  
Humic Acids ◽  
Gel Filtration ◽  
Quinone Imine ◽  
Peptide Bonds ◽  
Molecular Weights ◽  
Soil Humic Acids ◽  
Properties Of Soil

Twenty-three model phenolic polymers, either nitrogen-free or incorporating amino acids, peptides, or proteins, have been prepared from p-benzoquinone and catechol under mild oxidative conditions. Two lines of experimentation have demonstrated properties of soil humic acids closely similar to those of polymers incorporating proteins, but different from those of polymers incorporating amino acids: (1) fractionation of humic acids and synthetic polymers by 'Sephadex' gel filtration showed that the percentage of components of molecular weights nominally greater than 100 000 ranged from 52-76 % for eight humic acids tested, 53-59 % for benzoquinone-protein polymers (excluding polymers containing protamine), but less than 20% for all other polymers; (2) acid hydrolysis with 6M HCl resulted in a partial release of polymer nitrogen. Amino acid nitrogen in the hydrolysates accounted for 32.4-51.9 % of humic acid nitrogen, 31.2-56.3 % of the nitrogen of polymers incorporating protein, but less than 10.8% of the nitrogen of polymers incorporating individual amino acids. Experiments with model monomeric N-phenylglycine derivatives and with polymers incorporating simple peptides showed that the bond between the carbon atom of an aromatic ring and the nitrogen atom of an a-amino acid is far more stable to acid hydrolysis than peptide bonds or bonds linking amino acids in humic acids. Glycine is, however, readily released from N-phenylglycine derivatives when conditions favour their oxidation to a quinone-imine intermediate. Incorporation of proteins into phenolic polymers prevented the detection of peptide bonds by the Folin reagent.

scholarly journals Cytochrome P-450 induction by clofibrate. Purification and properties of a hepatic cytochrome P-450 relatively specific for the 12- and 11-hydroxylation of dodecanoic acid (lauric acid)

1982 ◽  
Vol 203 (1) ◽  
pp. 161-168 ◽  
Author(s):  
G. Gordon Gibson ◽  
Terry C. Orton ◽  
Paul P. Tamburini
Keyword(s):  
Endoplasmic Reticulum ◽  
Lauric Acid ◽  
Dodecanoic Acid ◽  
Specific Content ◽  
Molecular Weights ◽  
Purification And Properties ◽  
Mixed Function Oxidase System ◽  
Cytochrome P 450 ◽  
Hepatic Cytochrome

Hypolipidaemic drugs induce peroxisomal proliferation in the liver and many induce the formation of the hepatic endoplasmic reticulum in general and the formation of cytochrome P-450 in particular. We have induced the formation of rat liver microsomal cytochrome P-450 by the administration of the hypolipidaemic drug clofibrate, isolated the endoplasmic reticulum, solubilized the cytochrome P-450 from these membranes and subdivided the cytochrome P-450 into four fractions by the use of hydrophobic, anionic, cationic and adsorption chromatography. One of these fractions (cytochrome P-450 fraction 1) was highly purified to a specific content of 17nmol of cytochrome P-450/mg of protein and the protein was active in a reconstituted enzyme system towards the 12- and 11-hydroxylation of the fatty acid, dodecanoic (lauric) acid, with preferential activity towards the 12-hydroxy metabolite. This reconstituted activity was absolutely dependent on NADPH, NADPH-cytochrome P-450 reductase and cytochrome P-450, indicating the role of the mixed-function oxidase system in the metabolism of lauric acid. Another fraction of the haemoprotein (cytochrome P-450 fraction 2) preferentially formed 11-hydroxylauric acid, whereas a third fraction (cytochrome P-450 fraction 3) exhibited only trace laurate oxidase activity and was similar to the phenobarbitone form of the haemoprotein in that these last two cytochromes rapidly turned-over the drug benzphetamine. The molecular weights and spectral properties of these cytochrome P-450 fractions are reported, along with the phenobarbitone-induced form of the enzyme and the nature of the cytochrome(s) induced by clofibrate pretreatment are discussed in the terms of possible haemoprotein heterogeneity.

ISOLATION OF LH, FSH AND TSH FROM HUMAN PITUITARIES AFTER THE REMOVAL OF HGH

1974 ◽  
Vol 77 (3) ◽  
pp. 485-497 ◽  
Author(s):  
P. A. Torjesen ◽  
T. Sand ◽  
N. Norman ◽  
O. Trygstad ◽  
I. Foss
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Disc Electrophoresis ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Molecular Weights ◽  
Pituitary Glands

ABSTRACT Highly purified human LH, FSH and TSH were isolated from batches of 300 frozen pituitary glands (200 g) by pH, acetone and ethanol fractionation, Sephadex gel filtration, ion-exchange chromatography on DEAE-cellulose and CM-Sephadex, and preparative polyacrylamide-gel electrophoresis. Sodium dodecyl-sulphate (SDS) polyacrylamide gel electrophoresis was used in order to check the purity, the identity and the molecular weight of the purified LH, FSH and TSH. This procedure showed that the hormone preparations consisted of two subunits with molecular weights of: LH: 21 300 and 17 900, FSH: 22 100 and 18 300 and TSH: 20 800 and 16 400. The purity of the hormone preparations was also evaluated by analytical disc electrophoresis at pH 8.9. The purified hormone preparations had radioimmunological activity as follows: LH: 20 000 IU/mg, FSH: 16 500 IU/mg and TSH: 5 IU/mg. All preparations had high biological potency.

Changes in proteins of Domiati cheese during pickling

1972 ◽  
Vol 39 (2) ◽  
pp. 219-225 ◽  
Author(s):  
M. H. Abd El-Salam ◽  
Safinaz El-Shibiny
Keyword(s):  
Amino Acids ◽  
Room Temperature ◽  
Gel Filtration ◽  
Electrophoretic Pattern ◽  
Pasteurized Milk ◽  
Molecular Weights ◽  
Electrophoretic Patterns ◽  
Related Substances ◽  
Nitrogenous Substances

SummaryDomiati cheese from homogenized and unhomogenized pasteurized milk was pickled for 4 months at room temperature. During pickling, the soluble tyrosine and tryptophan contents of the cheese gradually increased, indicating progressive proteolysis.Gel filtration of cheese extract on Sephadex G-25 revealed the presence of nitrogenous substances of various molecular weights. The maximum formation of amino acids and related substances was observed after 15 days storage.The changes in the electrophoretic patterns of proteins in the cheese during pickling indicated that both αs - and β-caseins were attacked. β-Casein, however, was much less affected.Homogenization was found to affect the formation of soluble nitrogenous substances, but was without effect on the electrophoretic pattern of the protein in the cheese.

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