scholarly journals Properties of phosphoenolpyruvate carboxykinase (guanosine triphosphate) synthesized in hepatoma cells in the presence of amino acid analogues

1975 ◽  
Vol 146 (3) ◽  
pp. 585-593 ◽  
Author(s):  
S E Knowles ◽  
J M Gunn ◽  
L Reshef ◽  
R W Hanson ◽  
F J Ballard
Keyword(s):  
Amino Acid ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Hepatoma Cells ◽  
Dibutyryl Cyclic Amp ◽  
Enzyme Degradation ◽  
Amino Acid Analogues ◽  
Enzyme Molecules ◽  
Natural Amino Acids

1. Phosphoenolpyruvate carboxykinase (GTP) was induced by a combination of dibutyryl cyclic AMP, theophyline and dexamethasone in Reuber H35 hepatoma cells under conditions where an amino acid in the medium was replaced by an appropriate analogue. 2. With canavanine replacing arginine or with 5-fluorotryptophan or 6-fluorotryptophan replacing tryptophan the induced enzyme had a lower catalytic activity-relative to antibody reactivity. 3. These aberrant enzyme molecules were heat-labile in vitro. 4. Measurements of enzyme degradation in vivo indicated that the canavanine-containing enzyme and the 6-fluorotryptophan-containing enzyme were degraded more rapidly than the enzyme containing all natural amino acids.

scholarly journals Increased degradation rates of protein synthesized in hepatoma cells in the presence of amino acid analogues

1975 ◽  
Vol 146 (3) ◽  
pp. 595-600 ◽  
Author(s):  
S E Knowles ◽  
J M Gunn ◽  
R W Hanson ◽  
F J Ballard
Keyword(s):  
Amino Acid ◽  
Cell Cultures ◽  
Incubation Medium ◽  
Hepatoma Cells ◽  
Cell Protein ◽  
Chase Period ◽  
Separate Cell ◽  
Degradation Rates ◽  
Amino Acid Analogues

1. Reuber H35 hepatoma cells incorporate the arginine analogue canavanine into cell protein when arginine is omitted from the incubation medium. 2. By labelling arginine-containing proteins with (14-C)leucine and then canavanine-containing proteins with (3-H)leucine in the same cells, it is possible to measure the degradation of both types of protein during a subsequent ‘chase’ period. With this technique it has been shown that canavanine-containing proteins are degraded at a rate severalfold greater than normal proteins. Comparable results were found when 6-fluorotryptophan was used as an analogue to tryptophan. 3. Control experiments in which the labelling order was reversed or where the animo acid and its analogue were incubated in separate cell cultures support the conclusion that abberrant proteins are rapidly degraded in vivo.

scholarly journals Glucagon resistance of hepatoma cells. Evidence for receptor and post-receptor defects

1983 ◽  
Vol 214 (3) ◽  
pp. 845-850 ◽  
Author(s):  
M Fehlmann ◽  
M Crettaz ◽  
C R Kahn
Keyword(s):  
Amino Acid ◽  
Cyclic Amp ◽  
Amino Acid Transport ◽  
Hepatoma Cell ◽  
Insulin Binding ◽  
Hepatoma Cells ◽  
Hepatoma Cell Line ◽  
Acid Transport ◽  
Dibutyryl Cyclic Amp

Of all available liver cells in culture, only primary cultured hepatocytes are known to respond to glucagon in vitro. In the present study we investigated whether glucagon could stimulate amino acid transport and tyrosine aminotransferase (TAT;EC 2.6.1.5) activity (two well-characterized glucagon effects in the liver) in Fao cells, a highly differentiated rat hepatoma cell line. We found that glucagon had no effect on transport of alpha-aminoisobutyric acid (AIB; a non-metabolizable alanine analogue) nor on TAT activity, even though both activities could be fully induced by insulin [2-fold and 3-fold effects for AIB transport and TAT activity, respectively, after 6h; EC50 (median effective concentration) = 0.3 nM], or by dexamethasone (5-8-fold effects after 20 h; EC50 = 2 nM). Analysis of [125I]iodoglucagon binding revealed that Fao cells bind less than 1% as much glucagon as do hepatocytes, whereas insulin binding in Fao cells was 50% higher than in hepatocytes. The addition of dibutyryl cyclic AMP, which fully mimics the glucagon stimulation of both AIB transport and TAT activity in hepatocytes, induced TAT activity in Fao cells (a 2-fold effect at 0.1 mM-dibutyryl cyclic AMP) but had no effect on AIB transport. Cholera toxin stimulated TAT activity to the same extent as did dibutyryl cyclic AMP. These results indicate that the lack of glucagon responsiveness in cultured hepatoma cells results from both a receptor defect and, for amino acid transport, an additional post-receptor defect. Moreover, the results show that amino acid transport and TAT activity, which appeared to be co-induced by insulin or by dexamethasone in these cells, respond differently to cyclic AMP. This suggests that different mechanisms are involved in the induction of these activities by glucagon in liver.

Site Specific Incorporation of Amino Acid Analogues into Proteins In Vivo

2000 ◽  
Author(s):  
Anne K. Kowal ◽  
Caroline Kohrer ◽  
Uttam L. RajBhandary
Keyword(s):  
Amino Acid ◽  
Site Specific ◽  
Amino Acid Analogues

Oxytocin analogues with non-coded amino acid residues in position 8: [8-Neopentylglycine]oxytocin and [8-cycloleucine]oxytocin

1987 ◽  
Vol 52 (9) ◽  
pp. 2317-2325 ◽  
Author(s):  
Jan Hlaváček ◽  
Jan Pospíšek ◽  
Jiřina Slaninová ◽  
Walter Y. Chan ◽  
Victor J. Hruby
Keyword(s):  
Amino Acid ◽  
Solid Phase Synthesis ◽  
Solid Phase ◽  
The Other ◽  
Amino Acid Residues ◽  
Phase Synthesis ◽  
Other Hand ◽  
Fragment Condensation

[8-Neopentylglycine]oxytocin (II) and [8-cycloleucine]oxytocin (III) were prepared by a combination of solid-phase synthesis and fragment condensation. Both analogues exhibited decreased uterotonic potency in vitro, each being about 15-30% that of oxytocin. Analogue II also displayed similarly decreased uterotonic potency in vivo and galactogogic potency. On the other hand, analogue III exhibited almost the same potency as oxytocin in the uterotonic assay in vivo and in the galactogogic assay.

Synthesis of Fragments of the Vasoactive Intestinal Peptide (VIP) and Analysis of Their Residual Biological Activities

1995 ◽  
Vol 60 (7) ◽  
pp. 1229-1235 ◽  
Author(s):  
Ivana Zoulíková ◽  
Ivan Svoboda ◽  
Jiří Velek ◽  
Václav Kašička ◽  
Jiřina Slaninová ◽  
...  
Keyword(s):  
Amino Acid ◽  
Biological Activities ◽  
Residual Activity ◽  
Detailed Examination ◽  
Amino Acid Residues ◽  
Linear Peptide ◽  
Zone Electrophoresis ◽  
Capillary Zone

The vasoactive intestinal (poly)peptide (VIP) is a linear peptide containing 28 amino acid residues, whose primary structure indicates a low metabolic stability. The following VIP fragments, as potential metabolites, and their analogues were prepared by synthesis on a solid: [His(Dnp)1]VIP(1-10), VIP(11-14), [D-Arg12]VIP(11-14), [Lys(Pac)15,21,Arg20]VIP(15-22), and VIP(23-28). After purification, the peptides were characterized by amino acid analysis, mass spectrometry, RP HPLC, and capillary zone electrophoresis. In some tests, detailed examination of the biological activity of the substances in vivo and in vitro gave evidence of a low, residual activity of some fragments, viz. a depressoric activity in vivo for [His(Dnp)1]VIP(1-10) and a stimulating activity for the release of α-amylase in vitro and in vivo for [Lys(Pac)15,21,Arg20]VIP(15-22) and VIP(23-28).

scholarly journals Expression of laminin γ 1 cultured hepatocytes involves repeated CTC and GC elements in the LAMC1 promoter

1996 ◽  
Vol 313 (3) ◽  
pp. 745-752 ◽  
Author(s):  
Françoise LEVAVASSEUR ◽  
Jocelyne LIÉTARD ◽  
Kohei OGAWA ◽  
Nathalie THÉRET ◽  
Peter D. BURBELO ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Hepatoma Cell ◽  
Primary Cultures ◽  
Regulatory Elements ◽  
Hepatoma Cells ◽  
Basement Membranes ◽  
Major Protein ◽  
Cultured Hepatocytes

Laminin γ1 chain is present in all basement membranes and is expressed at high levels in various diseases, such as hepatic fibrosis. We have identified cis- and trans-acting elements involved in the regulation of this gene in normal rat liver, as well as in hepatocyte primary cultures and hepatoma cell lines. Northern-blot analyses showed that laminin γ1 mRNA was barely detectable in freshly isolated hepatocytes and expressed at high levels in hepatocyte primary cultures, as early as 4 h after liver dissociation. Actinomycin D and cycloheximide treatment in vivo and in vitro indicated that laminin γ1 overexpression in cultured hepatocytes was under the control of transcriptional mechanisms. Transfection of deletion mutants of the 5´ flanking region of murine LAMC1 gene in hepatoma cells that constitutively express laminin γ1 indicated that regulatory elements were located between -594 bp and -94 bp. This segment included GC- and CTC-containing motifs. Gel-shift analyses showed that two complexes were resolved with different affinity for the CTC sequence depending on the location of the GC box. The pattern of complex formation with nuclear factors from freshly isolated and cultured hepatocytes was different from that obtained with total liver and similar to that with hepatoma cells. Southwestern analysis indicated that several polypeptides bound the CTC-rich sequence. Affinity chromatography demonstrated that a Mr 60000 polypeptide was a major protein binding to the CTC motif. This polypeptide is probably involved in the transcriptional activation of various proto-oncogenes and extracellular matrix genes that are expressed at high levels in both hepatoma cells and early hepatocyte cultures.

A novel polymorphism of human gene has an amino acid substitution (V365M) that decreases enzymatic activity in vitro and in vivo

2004 ◽  
Vol 76 (6) ◽  
pp. 519-527 ◽  
Author(s):  
T FUKAMI ◽  
M NAKAJIMA ◽  
R YOSHIDA ◽  
Y TSUCHIYA ◽  
Y FUJIKI ◽  
...  
Keyword(s):  
Amino Acid ◽  
Enzymatic Activity ◽  
Amino Acid Substitution ◽  
Human Gene

Reduced and S-carboxymethylated human growth hormone: a probe for diabetogenic action

1984 ◽  
Vol 247 (5) ◽  
pp. E639-E644
Author(s):  
C. M. Cameron ◽  
J. L. Kostyo ◽  
J. A. Rillema ◽  
S. E. Gennick
Keyword(s):  
Growth Hormone ◽  
Amino Acid ◽  
Human Growth Hormone ◽  
Human Growth ◽  
Mouse Mammary Gland ◽  
Amino Acid Incorporation ◽  
Acid Incorporation ◽  
Hypophysectomized Rats

The biological activity profile of reduced and S-carboxymethylated human growth hormone (RCM-hGH) was determined to establish its suitability for study of the diabetogenic property of hGH. RCM-hGH was found to have greatly attenuated in vivo growth-promoting activity in the 9-day weight-gain test in hypophysectomized rats (approximately 1%) and to have a similar low order of in vitro activity in stimulating amino acid incorporation into the protein of the isolated rat diaphragm. RCM-hGH also only had approximately 1% of the in vitro insulin-like activity of the native hormone on isolated adipose tissue from hypophysectomized rats. In contrast, RCM-hGH retained substantial in vivo diabetogenic activity in the ob/ob mouse, appearing to have approximately 50% of the activity of the native hormone. RCM-hGH was also found to retain significant, although attenuated (25%), in vitro lactogenic activity when tested for the ability to stimulate amino acid incorporation into a casein-rich protein fraction in mouse mammary gland explants. Because RCM-hGH exhibits a high degree of diabetogenic activity, although lacking significant anabolic or insulin-like activities, it will be useful as a "monovalent" probe for the study of the molecular mechanism of the diabetogenic action of GH.

“In vivo” amplification of biological activity of tetragastrin by amino acid hydroxamates

1984 ◽  
Vol 4 (12) ◽  
pp. 1009-1015 ◽  
Author(s):  
J. P. Bali ◽  
H. Mattras ◽  
A. Previero ◽  
M. A. Coletti-Previero
Keyword(s):  
Biological Activity ◽  
Amino Acid ◽  
Aromatic Amino Acid ◽  
Aminopeptidase Activity ◽  
Proteolytic Degradation ◽  
Short Peptides ◽  
Rat Blood ◽  
Active Peptides

Rat blood was shown to contain an aminopeptidase which rapidly hydrolyses short peptides containing an aromatic amino acid as N-terminal residue. Using tetragastrin (Trp-Met-Asp-PheNH 2) as substrate, we showed that some amino acid hydroxamates inhibit rat aminopeptidase activity ‘in vitro’ in the following order: HTrpNHOH > HPheNHOH ≫ HAIaNHOH. The same hydroxamates markedly enhanced the biological activity of tetragastrin ‘in vivo’. The amplification of the secretory effect, correlated with the amount of the hydroxamate used, strongly suggests that these compounds can stabilize a number of active peptides in vivo by inhibiting their proteolytic degradation.

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