Kinetic study of the interaction between rat haptoglobin and rat liver cathepsin B

1980 ◽  
Vol 58 (5) ◽  
pp. 410-417 ◽  
Author(s):  
M. Pagano ◽  
R. Engler ◽  
M. Gelin ◽  
M. F. Jayle
Keyword(s):  
Molecular Weight ◽  
Kinetic Study ◽  
Rat Liver ◽  
Cathepsin B ◽  
Michaelis Constant ◽  
Rat Serum ◽  
Acute Phase Reactants ◽  
Serum Haptoglobin ◽  
Lysosomal Proteinase

One of the roles of the acute phase reactants (APR), according to Koj, is to regulate the action of tissue proteinases released during the inflammatory reaction. To study this phenomenon in vitro, rat liver cathepsin B (EC 3.4.22.1), a lysosomal proteinase, and a typical APR, rat serum haptoglobin, were purified. By kinetic study, haptoglobin was found to inhibit cathepsin B and the inhibitory activity of a competitive type was increased with the molecular weight of the substrate. When 125I-labelled denatured bovine serum albumin (BSA) was used as a substrate, the apparent Michaelis constant (Km app.) for cathepsin B was 5 ± 0.4 × 10−6 M. When haptoglobin was added, the apparent inhibition constant (Ki app.) was 3.5 ± 1.5 × 10−8 M. Native haptoglobin was not catabolized by cathepsin B while under the same conditions denatured haptoglobin was degraded by the enzyme.

Inhibition of cathepsin L and B by haptoglobin, the haptoglobin–hemoglobin complex, and asialohaptoglobin. "In vitro" studies in the rat

1982 ◽  
Vol 60 (6) ◽  
pp. 631-637 ◽  
Author(s):  
M. Pagano ◽  
M. A. Nicola ◽  
R. Engler
Keyword(s):  
Rat Liver ◽  
Inflammatory Reaction ◽  
Cathepsin B ◽  
Cathepsin L ◽  
In Vitro Studies ◽  
The Other ◽  
Michaelis Constant ◽  
Thiol Proteinases ◽  
Thiol Proteinase

In broadening our research on the inhibition of cathepsin B (EC 3.4.22.1) by rat haptoglobin, we have used the haptoglobin–hemoglobin complex and asialohaptoglobin. The inhibition of cathepsin L (EC 3.4.22.15), another lysosomal thiol proteinase, by haptoglobin and its related molecules has also been investigated.With azocasein as substrate, both enzymes were inhibited by both haptoglobin and its related molecules. When azocasein was used as a substrate, the apparent Michaelis constant (Km, app.) for cathepsin L was 1 × 10−5 ± 0.4 × 10−5 M. When haptoglobin was added, the apparent inhibition constant (Ki, app.) was 3 × 10−8 ± 2.5 × 10−8 M.The results suggest that rat haptoglobin specifically inhibits lysosomal thiol proteinases and that it has a regulatory role in tissue proteolysis associated with the inflammatory reaction. On the other hand, these properties would seem to be peculiar to the systems rat haptoglobin – rat liver cathepsin B or L.

scholarly journals Novel Role for Albumin in Innate Immunity: Serum Albumin Inhibits the Growth of Blastomyces dermatitidis Yeast Form In Vitro

2003 ◽  
Vol 71 (11) ◽  
pp. 6648-6652 ◽  
Author(s):  
Steven Giles ◽  
Charles Czuprynski
Keyword(s):  
Molecular Weight ◽  
Inhibitory Activity ◽  
Mammalian Species ◽  
Blastomyces Dermatitidis ◽  
Low Molecular Weight ◽  
Rat Serum ◽  
Yeast Form ◽  
Domain Iii

ABSTRACT In this study we found that serum inhibitory activity against Blastomyces dermatitidis was principally mediated by albumin. This was confirmed in experiments using albumin from several mammalian species. Analbuminemic rat serum did not inhibit B. dermatitidis growth in vivo; however, the addition of albumin restored inhibitory activity. Inhibitory activity does not require albumin domain III and appears to involve binding of a low-molecular-weight yeast-derived growth factor.

Complexing of Low Mr-Weight Human Urokinase In Rat Serum And Its Degradation Into Fragments Of Very Small Molecular Weight In Vivo But Not In Vitro

1981 ◽  
Author(s):  
Ph Schneider ◽  
M Ruegg ◽  
F Bachmann
Keyword(s):  
Molecular Weight ◽  
Inferior Vena ◽  
Vena Cava ◽  
Albino Rats ◽  
Small Molecular Weight ◽  
Rat Serum ◽  
Blood Samples ◽  
Sds Page

Highly purified lew molecular weight urokinase (LMR-UK), moving on SDS-PAGE (reduced and nan-reduced) as a single band of 32 kdalton, was labelled with 125I by the chlora- mine-T method. 106 cpn of this 125I-LMr-UK (94% TCA preci- pitable) were injected into the inferior vena cava of la- par atomized albino rats, which were maintained at 37°C. Blood samples were collected by cardiac puncture 5, 30 and 90 min respectively after the injection. Serun, obtained from these samples, was fractionated on a Sephadex G-100 column, calibrated with proteins of known Mr. Radioactivity was measured in the collected fractions.In the 5 min sample, the radioactivity was distributed in 2 peaks, corresponding to 32 kdalton and to < 70 kdalton respectively. In the 30 min sample, the distribution was characterized by a diminution of the 32 kdalton peak and the appearance of a third peak corresponding to a Mr of < 4 kdalton. In the 90 min sample, the LMr-UK peak had disappeared almost completely. About 40% of the 125I-activity was present in a skewed high Mr peak with a broad maximum in the 85-100 kdalton region; ≥ 60% of the 125I-activity was recovered in late fractions corresponding to < 4 kdalton. In control experiments, pooled rat serum was incubated in vitro with 125I-LMr-UK for 5, 30 and 90 min respectively and samples were fractionated on the same column. The radioactivity distribution shewed only the 32 and > 70 kdalton peaks, but no < 4 kdalton peak.These results suggest that LMr-UK is complexed to a carrier protein, both in vivo and in vitro, but that it is degraded into small fragments in vivo only. Attempts to characterize the nature of these complexes are in progress.

scholarly journals Molecular hybridization between rat liver deoxyribonucleic acid and complementary ribonucleic acid

1970 ◽  
Vol 120 (2) ◽  
pp. 225-235 ◽  
Author(s):  
Marialuisa Melli ◽  
J. O. Bishop
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Sodium Citrate ◽  
Filter Method ◽  
Dna Molecules ◽  
Membrane Filters ◽  
Diminished Capacity ◽  
Cross Linkage ◽  
Total Dna

RNA (cRNA) was synthesized in vitro on a template of rat liver DNA and its hybridization with rat liver DNA was studied by using the nitrocellulose-filter method. Sonication of the DNA diminished its apparent capacity to hybridize with RNA by about 50%. This is not due to cross-linkage of DNA molecules, because it could be shown that less than 2% of the sonicated DNA was cross-linked. The effect is due instead to the small size of the sonicated DNA molecules. Below a single-stranded molecular weight of 5×105 the DNA showed a progressive loss of capacity to hybridize with decrease in molecular weight. Evidence is presented suggesting that the apparently diminished capacity of the DNA to hybridize is due to loss of hybridized DNA from the membrane filters. When cRNA at concentrations of up to 25μg/ml is annealed with sonicated total DNA, an apparent hybridization saturation value is found at which about 2.5% of the DNA is hybridized with RNA. Increasing the cRNA concentration tenfold brought about the hybridization of a second component of the DNA approximately equal in amount to the first. The renaturation of rat liver DNA was studied by measuring the fall in the extinction at 260nm and two different components of renaturation were observed within the reiterated fraction of DNA. By hybridizing cRNA with different fractions of rat DNA the two components of the hybridization curve are shown to correspond to the two components of the renaturation curve. The conclusion is drawn that at a cRNA concentration of 250μg/ml most of the reiterated fraction of rat liver DNA is hybridized after annealing for 16h under standard conditions (0.30m-sodium chloride–30mm-sodium citrate at 65°C). Even with such a high cRNA concentration little or no hybridization of the slowly renaturing DNA fraction occurs. It is suggested that the most highly reiterated DNA component is poorly transcribed in vitro.

Regulation of rat liver maturation in vitro by glucocorticoids

1988 ◽  
Vol 8 (1) ◽  
pp. 203-209
Author(s):  
J Y Chou ◽  
Y J Wan ◽  
T Sakiyama
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Fetal Liver ◽  
Tyrosine Aminotransferase ◽  
Temperature Sensitive ◽  
Adult Rat ◽  
Normal Hepatocyte ◽  
Fetal Rat ◽  
Afp Mrna

The biochemistry of liver maturation was studied by using the RLA209-15 fetal rat hepatocyte line that is temperature sensitive for maintenance of the differentiated fetal liver phenotype. At 33 degrees C these cells were dedifferentiated; but at 40 degrees C they were phenotypically differentiated and, like normal fetal hepatocytes, synthesized moderate levels of albumin and transferrin, high levels of authentic (69,000 and 73,000 molecular weight) rat fetal alpha-fetoprotein (AFP), and low levels of a 65,000-molecular-weight variant AFP. Our results indicated that administration of glucocorticoid hormones to RLA209-15 cells at 40 degrees C induced a series of events associated with normal hepatocyte maturation; synthesis of fetal AFP was inhibited, whereas the synthesis of variant AFP, albumin, transferrin, tyrosine aminotransferase, and alpha 1-acid glycoprotein was induced. The variant AFP was produced by RLA209-15 cells at both temperatures and was encoded by an mRNA of 1.7 kilobases (kb). The fetal AFP was encoded by an mRNA of 2.2 kb. Normal adult rat liver contained three AFP mRNAs of 2.2 (minor), 1.7, and 1.5 kb. The 1.7-kb adult liver AFP mRNA comigrated with the RNA found in RLA209-15 cells, and both directed the synthesis of a 50,000-molecular-weight precursor polypeptide of the variant AFP. Administration of glucocorticoids to RLA209-15 cells grown at 33 degrees C stimulated synthesis of both the fetal and variant AFPs, but the levels of the 2.2-kb AFP mRNA were preferentially increased. RLA209-15 cells contained two glucocorticoid receptor mRNAs of 6.8 and 4.5 kb. The glucocorticoid-mediated maturation described above was blocked by the antiglucocorticoid RU486.

scholarly journals Regulation of rat liver maturation in vitro by glucocorticoids.

1988 ◽  
Vol 8 (1) ◽  
pp. 203-209 ◽  
Author(s):  
J Y Chou ◽  
Y J Wan ◽  
T Sakiyama
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Fetal Liver ◽  
Tyrosine Aminotransferase ◽  
Temperature Sensitive ◽  
Adult Rat ◽  
Normal Hepatocyte ◽  
Fetal Rat ◽  
Afp Mrna

The biochemistry of liver maturation was studied by using the RLA209-15 fetal rat hepatocyte line that is temperature sensitive for maintenance of the differentiated fetal liver phenotype. At 33 degrees C these cells were dedifferentiated; but at 40 degrees C they were phenotypically differentiated and, like normal fetal hepatocytes, synthesized moderate levels of albumin and transferrin, high levels of authentic (69,000 and 73,000 molecular weight) rat fetal alpha-fetoprotein (AFP), and low levels of a 65,000-molecular-weight variant AFP. Our results indicated that administration of glucocorticoid hormones to RLA209-15 cells at 40 degrees C induced a series of events associated with normal hepatocyte maturation; synthesis of fetal AFP was inhibited, whereas the synthesis of variant AFP, albumin, transferrin, tyrosine aminotransferase, and alpha 1-acid glycoprotein was induced. The variant AFP was produced by RLA209-15 cells at both temperatures and was encoded by an mRNA of 1.7 kilobases (kb). The fetal AFP was encoded by an mRNA of 2.2 kb. Normal adult rat liver contained three AFP mRNAs of 2.2 (minor), 1.7, and 1.5 kb. The 1.7-kb adult liver AFP mRNA comigrated with the RNA found in RLA209-15 cells, and both directed the synthesis of a 50,000-molecular-weight precursor polypeptide of the variant AFP. Administration of glucocorticoids to RLA209-15 cells grown at 33 degrees C stimulated synthesis of both the fetal and variant AFPs, but the levels of the 2.2-kb AFP mRNA were preferentially increased. RLA209-15 cells contained two glucocorticoid receptor mRNAs of 6.8 and 4.5 kb. The glucocorticoid-mediated maturation described above was blocked by the antiglucocorticoid RU486.

TSH binding proteins in rat and human serum

1987 ◽  
Vol 115 (4) ◽  
pp. 497-506 ◽  
Author(s):  
O. Spira ◽  
M. Gafni ◽  
C. Ben-David ◽  
J. Gross ◽  
A. Gordon
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Ammonium Sulphate ◽  
Serum Proteins ◽  
Biologically Active ◽  
Rat Serum ◽  
Ammonium Sulphate Precipitation ◽  
Human Sera ◽  
Front Running

Abstract. When serum of hypothyroid rats was fractionated on a Sephadex G-100 column, most of the immunoreactive TSH was found as a front running peak, together with the high molecular weight serum proteins. Similarly, a rTSH preparation (10 mU), chromatographed in the presence of 1 ml of normal rat serum also migrated at the front, however, when a high load of TSH (4.4 U) was added to 1 ml of serum, two immunoreactive peaks were found, suggesting the saturation of the front running fraction. Immunoelectrophoresis and autoradiography of rat or human sera containing the respective 125I-labelled TSHs showed binding of the labelled TSH to IgG, alpha-2-macroglobulin, and to a third unidentified protein, migrating near the albumin line. In order to determine if the bound TSH is biologically active, the high molecular weight protein fraction was separated from hypothyroid rat serum by 45% ammonium sulphate precipitation. Immunoreactivity was determined by RIA and the biological activity was determined, in vitro, by the stimulation of 99Tc uptake by FRTL-5 cells. The 45% ammonium sulphate precipitate contained almost all of the immunoreactivity and the bioactivity of the TSH of whole serum. These results indicate that: a) The endogenous circulating TSH in the hypothyroid rat exists mainly in a protein-bound form and this protein-bound TSH contains most of the hormonal bioactivity of the serum. b) Exogenous TSH binds to serum proteins in euthyroid and hypothyroid rats and in humans. There are three protein fractions in these sera that bind TSH, one of which is an immunoglobulin. The occurrence of TSH binding immunoglobulins may involve autoimmune mechanisms.

Cell-free translation of mitochondrial nicotinamide nucleotide transhydrogenase

1985 ◽  
Vol 5 (6) ◽  
pp. 483-490 ◽  
Author(s):  
Elisabeth Carlenor ◽  
Vigg Joste ◽  
B. Dean Nelson ◽  
Jan Rydström
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Liver Mitochondria ◽  
Cytochrome Bc1 Complex ◽  
Free System ◽  
Nicotinamide Nucleotide Transhydrogenase ◽  
Free Translation ◽  
A Cell ◽  
Matrix Fraction

Mammalian nicotinamide nucleotide transhydrogenase is translated as a 5000 daltons larger molecular weight precursor in a cell-free system programmed with rat liver polysomes. The mature rat liver enzyme had the same molecular weight as the purified beef heart enzyme, 115 000 daltons. The precursor was not processed in vitro by liver mitochondria or by a rat liver mitochondrial matrix fraction, nor did it appear to bind to mitochondria. In contrast, pre-FeS protein of the cytochrome bc1 complex was processed in the same samples by both mitochondria and matrix, suggesting an important difference in the processing mechanisms or in the efficiency of processing of the two precursors.

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