Ontogeny of hepatocyte proliferation inhibitor activity during human liver development and its effect on cell proliferation in in vivo and in vitro studies

1993 ◽  
Vol 71 (5-6) ◽  
pp. 241-247 ◽  
Author(s):  
B. Geetha Devi ◽  
C. M. Habeebullah ◽  
P. D. Gupta
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Hepg2 Cells ◽  
Human Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Hepatocyte Proliferation ◽  
Liver Development ◽  
Dodecyl Sulfate

Ontogeny of hepatocyte proliferation inhibitory (HPI) activity was studied during human liver development. HPI activity was first noticed in the cytosolic fraction of 20-week-old fetal liver and thereafter it began to increase with the liver maturation. An inverse correlation between the ontogeny of HPI activity and mitotic counting of the hepatocytes during human liver development was established. The crude HPI fraction from fetal and adult human liver inhibited the incorporation of [3H]thymidine into the DNA of 2-day-old rat liver and HepG2 cells and the inhibitory effect was directly proportional to the age of the HPI source. Prolonged exposure of HepG2 cells to the HPI fraction was also found to be cytotoxic. Preliminary characterization of the crude HPI fraction revealed an age-dependent increase in proteins of 18, 28, and 60 kilodaltons following silver chloride staining of sodium dodecyl sulfate - polyacrylamide gel electrophoresis and densitometric scanning.Key words: human liver, hepatocyte proliferation inhibitor, ontogeny, sodium dodecyl sulfate - polyacrylamide gel electrophoresis, HepG2 cells, cytotoxic.

Covalent guanylyl intermediate formed by HeLa cell mRNA capping enzyme

1982 ◽  
Vol 2 (8) ◽  
pp. 993-1001
Author(s):  
D Wang ◽  
Y Furuichi ◽  
A J Shatkin
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Hela Cell ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Enzyme Complex ◽  
Cell Nuclei ◽  
Dodecyl Sulfate ◽  
Enzyme Intermediate

Guanylyltransferase, an enzyme that catalyzes formation of mRNA 5'-terminal caps, was isolated from HeLa cell nuclei. The partially purified preparation, after incubation with [alpha-32P]GTP, yielded a single radiolabeled polypeptide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The guanylylated product was stable at neutral and alkaline pHs and had a pI of 4 by isoelectric focusing. An apparent molecular weight of approximately 68,000 was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. The formation of a covalently linked, radiolabeled GMP-protein complex and the associated release of PPi required the presence of [alpha-32P]GTP and divalent cations and incubation between pH 7 and 9. Reaction with [beta-32P]GTP, [alpha-32P]CTP, [alpha-32P]UTP, or [alpha-32P]ATP did not label the approximately 68,000-dalton polypeptide. Phosphoamide linkage of the GMP-enzyme complex was indicated by its sensitivity to cleavage by acidic hydroxylamine or HCl and not by NaOH or alkaline phosphatase. Both formation of the GMP-enzyme intermediate and synthesis of cap structures of type GpppApG from GTP and ppApG were remarkably temperature independent; the rates of enzyme activity at 0 to 4 degrees C were 30% or more of those obtained at 37 degrees C. Radiolabeled GMP-enzyme complex, isolated by heparin-Sepharose chromatography from reaction mixtures, functioned effectively as a GMP donor for cap synthesis with 5'-diphosphorylated oligo- and polynucleotide acceptors. Alternatively, protein-bound GMP could be transferred to PPi to form GTP. The formation of a guanylylated enzyme intermediate appears to be characteristic of viral and cellular guanylyltransferases that modify eucaryotic mRNA 5' termini.

Isolation, purification, and characterization of a peptide that contains the β-ketoacyl reductase, enoyl reductase, and β-hydroxyacyl dehydrase activities of the pigeon liver fatty acid synthetase

1985 ◽  
Vol 63 (1) ◽  
pp. 50-56
Author(s):  
Rajinder N. Puri ◽  
John W. Porter
Keyword(s):  
Fatty Acid ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fatty Acid Synthetase ◽  
Polyacrylamide Gel ◽  
Liver Fatty Acid ◽  
Dodecyl Sulfate ◽  
Pigeon Liver

Controlled proteolytic cleavage of pigeon liver fatty acid synthetase with elastase (4% w/w) for 5 h yields two peptides that are designated II and IV. After 5 h of proteolysis the incubation mixture containing these peptides retains all of the component enzyme activities of the fatty acid synthetase complex. The two peptides are then separated by chromatography on an Affi-Gel Blue column. Gel filtration of the fraction containing peptide II yields a homogeneous peptide as shown by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The molecular weight of this peptide has been estimated to be 130 000 by sodium dodecyl sulfate – polyacrylamide gel electrophoresis, size exclusion chromatography, and amino acid analysis. The sedimentation coefficient for peptide II is approximately 7.4S. Peptide II contains the domains for the β-ketoacyl and enoyl reductases and β-hydroxyacyl dehydrase activities of the fatty acid synthetase complex.

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