scholarly journals Formation of complexes between 125I-labelled human or bovine somatotropins and binding proteins in vivo in rat liver and kidney

1983 ◽  
Vol 214 (1) ◽  
pp. 121-132 ◽  
Author(s):  
J S Bonifacino ◽  
L P Roguin ◽  
A C Paladini
Keyword(s):  
Rat Liver ◽  
Gel Filtration ◽  
Radioactive Material ◽  
Bovine Somatotropin ◽  
Liver Uptake ◽  
Liver And Kidney ◽  
Specific Proteins ◽  
Triton X ◽  
Formation Of Complexes

At 5 min after intravenous injection, both 125I-labelled human somatotropin and 125I-labelled bovine somatotropin were concentrated in rat liver and kidney. When the labelled hormones were administered along with an excess of the corresponding unlabelled hormone, a significant decrease of the uptake was observed in the liver, but not in the kidney. Study of the subcellular distribution of radioiodinated somatotropins in liver revealed that most of the radioactivity was specifically concentrated in the microsomal fraction. In contrast, the kidney fraction that accounted for most of the radioactivity was the 100 000 g supernatant. After solubilization, with 1% (w/v) Triton X-100, of the microsomal fractions obtained from both organs, the radioactive material was analysed by gel filtration on Sepharose CL-6B. By using this approach, it was demonstrated that both 125I-labelled human somatotropin and 125I-labelled bovine somatotropin bind in vivo to proteins present in liver. A small proportion of 125I-labelled human somatotropin was also shown to form complexes with proteins present in kidney. The present results demonstrate that the liver uptake is mainly due to binding of somatotropins to specific proteins, in contrast with the kidney, in which binding to specific sites contributes minimally to the overall uptake.

scholarly journals Properties of subunits of the multicatalytic proteinase complex revealed by the use of subunit-specific antibodies

1991 ◽  
Vol 278 (1) ◽  
pp. 171-177 ◽  
Author(s):  
A J Rivett ◽  
S T Sweeney
Keyword(s):  
Molecular Mass ◽  
Rat Liver ◽  
Gel Filtration ◽  
Immunoblot Analysis ◽  
Rat Tissues ◽  
Specific Antibodies ◽  
Multicatalytic Proteinase ◽  
Cell Extracts ◽  
Liver And Kidney ◽  
Lysosomal Proteinase

The multicatalytic proteinase (MCP) is a high-molecular-mass non-lysosomal proteinase that gives rise to a characteristic pattern of bands of molecular mass 22-34 kDa on SDS/PAGE gels. Isoelectric-focusing gels of the enzyme purified from rat liver show 16 bands with isoelectric points in the range of pH 5-8.5. Two-dimensional PAGE gels reveal that there are more than the previously reported 13 polypeptides associated with the MCP from rat liver and show a pattern of 15-20 major spots and several minor ones, similar to that of MCP isolated from some other sources. Possible relationships between the different polypeptides were investigated by immunoblot analysis of electrophoretically purified proteinase subunits with affinity-purified subunit-specific antibodies as well as antibodies raised against individual denatured subunits of the complex. The results demonstrate that many of the major polypeptide components of the MCP complex are antigenically distinct. Moreover comparison of immunoreactive material in crude cell extracts with that in purified MCP preparations has shown that the polypeptides are not derived from a smaller number of higher-molecular-mass subunits. Also, individual subunits have the same apparent molecular mass in a variety of rat tissues, suggesting close similarity between MCPs of different tissues. The highest concentrations of MCP subunits occur in liver and kidney. Gel-filtration analysis of crude extracts has demonstrated that MCP polypeptides are also associated with a higher-molecular-mass complex, which may be the 26 S proteinase that has been implicated in the degradation of ubiquitin-protein conjugates.

Mechanisms of conversion of xanthine dehydrogenase to xanthine oxidase in ischemic rat liver and kidney

1988 ◽  
Vol 254 (5) ◽  
pp. G753-G760 ◽  
Author(s):  
T. G. McKelvey ◽  
M. E. Hollwarth ◽  
D. N. Granger ◽  
T. D. Engerson ◽  
U. Landler ◽  
...  
Keyword(s):  
Xanthine Oxidase ◽  
Rat Liver ◽  
Ischemia Reperfusion ◽  
Xanthine Dehydrogenase ◽  
Serum Glutamic Oxaloacetic Transaminase ◽  
Liver Model ◽  
Damage Process ◽  
Liver And Kidney ◽  
Sulfhydryl Modification

Previous studies have proposed and supported a role for the proteolytic, irreversible conversion of xanthine dehydrogenase to xanthine oxidase (XO) in postischemic injury in a wide variety of organs. A second mechanism of conversion, due to sulfhydryl modification and reversible with dithiothreitol (DTT), is potentially important but has not been well investigated. In this study rat liver and kidney were found to produce significant amounts of DTT-reversible XO during normothermic global ischemia. Formation of reversible XO precedes that of irreversible XO by approximately 0.5 h with a strong correlation (r = 0.92) existing between the rate of irreversible XO formation and the concentration of reversible XO. The formation of reversible XO is preceded by a depletion of glutathione with concentrations of glutathione during ischemia correlating (r = 0.85) with the observed concentration of reversible XO. While a large increase in the extent of liver damage occurs concurrently with conversion in an in vivo liver model of liver ischemia, an ischemia-reperfusion regimen (1 h of ischemia plus 0.5 h of reperfusion) that resulted in no conversion caused significant elevations in serum glutamic pyruvic transaminase and serum glutamic-oxaloacetic transaminase. Rats depleted of XO by tungsten dieting release 65% less enzyme after the same insult, suggesting that endogenous XO may also participate in the damage process independent of any conversion.

scholarly journals Purification and characterization of N-ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP2) from rat liver

1995 ◽  
Vol 308 (3) ◽  
pp. 983-989 ◽  
Author(s):  
I N Fleming ◽  
S J Yeaman
Keyword(s):  
Phosphatidic Acid ◽  
Rat Liver ◽  
Lysophosphatidic Acid ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Sds Page ◽  
Chromatography Columns ◽  
Triton X ◽  
Second Peak

N-Ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP; EC 3.1.3.4) was purified 5900-fold from rat liver. The enzyme was solubilized from membranes with octylglucoside, fractionated with (NH4)2SO4, and purified in the presence of Triton X-100 by chromatography on Sephacryl S300, hydroxyapatite, heparin-Sepharose and Affi-Gel Blue. Silver-stained SDS/PAGE indicated that the enzyme was an 83 kDa polypeptide. Sephacryl S-300 gel filtration also produced a second peak of enzyme activity, which was eluted from all of the chromatography columns at a different position from the purified enzyme. SDS/PAGE indicated that it contained three polypeptides (83 kDa, 54 kDa and 34 kDa), and gel filtration suggested that it was not an aggregate of the purified enzyme. Both forms were sensitive to inhibition by amphiphilic amines, Mn2+ and Zn2+, but not by N-ethylmaleimide. Purified PAP required detergent for activity, but was not activated by Mg2+, fatty acids or phospholipids. The enzyme was able to dephosphorylate lysophosphatidic acid or phosphatidic acid, and was inhibited by diacylglycerol and monoacylglycerol. No evidence was obtained for regulation of PAP by reversible phosphorylation.

Comparison of somatostatin-28 and somatostatin-14 clearance by the perfused rat liver

1985 ◽  
Vol 63 (1) ◽  
pp. 62-67 ◽  
Author(s):  
M. Seno ◽  
Y. Seino ◽  
Y. Takemura ◽  
S. Nishi ◽  
H. Ishida ◽  
...  
Keyword(s):  
Rat Liver ◽  
Gel Filtration ◽  
Perfused Rat Liver ◽  
Half Time ◽  
Hepatic Extraction Ratio ◽  
Plasma Extract ◽  
Second Peak ◽  
Somatostatin 14

The hepatic clearances of somatostatin (SS)-28 and SS-14 by the perfused rat liver were compared, using a recirculating, plasma-free, erythrocyte-containing perfusion system. The disappearance rate constant, half time, clearance, and hepatic extraction ratio when 1.2 nM SS-28 was added to the perfusate were 0.0221 ± 0.0051 min−1, 36.6 ± 7.6 min, 0.34 ± 0.08 mL/min, and 17.2 ± 3.9%, respectively. The corresponding values obtained when SS-14 was added to the perfusate were 0.0405 ± 0.0022 min−1, 17.3 ± 1.0 min, 0.71 ± 0.05 mL/min, and 35.4 ± 2.6%, respectively. The differences between the SS-28 and SS-14 indices were all statistically significant. In addition, the perfusates with SS-28 added were eluted on Sephadex G-25 fine columns and somatostatinlike immunoreactivity (SLI) was determined. No SS-14 was found in perfusate containing SS-28 at both 5 and 30 min after the beginning of the perfusion. To investigate whether or not the liver plays an important role in the clearance of SS-28 or the conversion of SS-28 to SS-14 in vivo, the plasma disappearance of 2 μg SS-28 was compared in the whole rat and the functionally hepatectomized model. The half time of plasma SS-28 was 1.43 ± 0.12 min in the whole rat, significantly shorter than the 2.20 ± 0.14 min in the hepatectomized model. Gel filtration of plasma extract samples at 0.5 min after the SS-28 injection showed two major peaks of SLI: a first peak corresponding to SS-28 and a second peak coeluted in the position of SS-14 in both the whole rat and the hepatectomized model. At 4 min after the SS-28 injection, the first peak disappeared and only a small second peak was observed. These results suggest that SS-28 is cleared by the rat liver in vivo and in vitro and that it is cleared more slowly than SS-14. Furthermore, we find that little, if any, conversion of SS-28 to SS-14 occurs in the liver.

Studies on the Biosynthesis of Flavin Nucleotides from 2-14C-Riboflavin by Rat Liver and Kidney

1973 ◽  
Vol 51 (6) ◽  
pp. 772-782 ◽  
Author(s):  
A. G. Fazekas ◽  
T. Sandor
Keyword(s):  
Rat Liver ◽  
Flavin Adenine Dinucleotide ◽  
Total Radioactivity ◽  
Flavin Mononucleotide ◽  
Time Intervals ◽  
Adult Rats ◽  
Liver And Kidney ◽  
Rat Liver Cytosol

2-14C-Riboflavin was injected subcutaneously into young adult rats to study the biosynthesis of flavin mononucleotide (FMN) and flavin–adenine dinucleotide (FAD) in the liver and kidneys. Animals were sacrificed at different time intervals following the administration of labelled riboflavin (RF), and radioactive flavins were determined in their tissues by a newly devised method. Both tissues accumulated radioactive riboflavin rapidly and peak levels were obtained at 90 min after the injection, when over 80% of the total radioactivity of the liver was present in FAD. At this time the liver contained 17% of the injected dose of 2-14C-RF. The kidneys contained relatively high quantities of free RF due to the concentration and urinary excretion of the vitamin.Analysis of subcellular fractions of the liver of animals injected with 2-14C-RF revealed that most of the radioactivity was present in mitochondria and nuclei. The flavin nucleotides of rat liver cytosol became progressively associated with macromolecules in vivo. However, there was no significant binding of free RF by macromolecules in blood plasma or liver cytosol.Kinetic studies and incubations with liver slices indicated that RF freely diffuses into the liver cells, is rapidly converted into FAD, and becomes attached to apoenzymes. The tissue uptake of RF and FMN formation is considerably influenced by the concentration of RF present in the system, both in vivo and in vitro.

scholarly journals Preparation and properties of a complex from rat liver of polyribosomes with components of the cytoskeleton

1983 ◽  
Vol 216 (1) ◽  
pp. 215-226 ◽  
Author(s):  
A Adams ◽  
E G Fey ◽  
S F Pike ◽  
C J Taylorson ◽  
H A White ◽  
...  
Keyword(s):  
Rat Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Subcellular Fractionation ◽  
Cytoskeletal Proteins ◽  
Deoxyribonuclease I ◽  
Microsomal Membranes ◽  
Cytoskeletal Elements ◽  
Triton X

Gel filtration with 1% agarose (Bio-Gel A-150m) separates polyribosomes bound to microsomal membranes from ‘free’ polyribosomes when these fractions are prepared by standard centrifugal techniques. However, when polyribosomes contained in an unfractionated postmitochondrial supernatant are run on an identical column, over 90% of the total polyribosomes are present as aggregates, designated ‘membrane-cytomatrix’, which are eluted in the column void volume. Polyribosomes are not released from these aggregates on removal of microsomal phospholipids by treatment of postmitochondrial supernatant with 1% Triton X-100, a neutral detergent. The aggregates are disrupted by the usual ultracentrifugation techniques used in subcellular fractionation. After treatment of membrane-cytomatrix with Triton X-100 to remove phospholipids and membrane proteins, 58% of the polyribosomes still remain associated with protein-containing complexes in the form of a cytomatrix and are not ‘free’. Preparations of both membrane-cytomatrix and cytomatrix are capable of sustained protein synthesis. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed that the cytoskeletal proteins actin and myosin are present in the cytomatrix. Incubation of cytomatrix preparations with the actin-depolymerizing agent deoxyribonuclease I caused release of the polyribosomes. Polyribosome release by deoxyribonuclease I was prevented by prior incubation with phalloidin, which is known to stabilize F-actin. Thus polyribosomes are associated with cytoskeletal elements in rat liver, and this association is dependent on polymeric forms of actin.

scholarly journals ISOLATION OF TWO DISTINCT CLASSES OF POLYSOMES FROM A NUCLEAR FRACTION OF RAT LIVER

1968 ◽  
Vol 37 (1) ◽  
pp. 163-181 ◽  
Author(s):  
Paul D. Sadowski ◽  
Janet Alcock Howden
Keyword(s):  
Outer Membrane ◽  
Rat Liver ◽  
Orotic Acid ◽  
Specific Activity ◽  
Nuclear Fraction ◽  
Differential Centrifugation ◽  
Rat Liver Nuclei ◽  
Liver Nuclei ◽  
Triton X

Isolated rat liver nuclei were washed with Triton-X-100 in the presence of liver cell sap. This treatment liberated a fraction of polysomes which were isolated by differential centrifugation and were designated "outer membrane polysomes." The outer membrane polysomes synthesized protein in vivo. Shortly after injection of orotic acid-14C, the RNA of outer membrane polysomes had a higher specific activity than that of cytoplasmic polysomes. It was postulated that outer membrane polysomes may be an intermediate in the transfer of newly synthesized RNA from the nucleus to the cytoplasm. In other experiments, Triton-washed rat liver nuclei were lysed in the presence of deoxycholate and deoxyribonuclease. A ribonucleoprotein fraction was isolated from the lysate by differential centrifugation. This fraction contained "intranuclear ribosomes," which sedimented like partially degraded polysomes in sucrose gradients. This degradation could be partially prevented if intranuclear ribosomes were purified by sedimentation through heavy sucrose. The resulting pellets were termed "intranuclear polysomes" because they contained some undergraded polysomes. Intranuclear polysomes were highly radioactive after a brief pulse with orotic acid-14C, but did not appear to synthesize protein rapidly in vivo. Intranuclear polysomes may represent the initial stage of assembly of polyribosomes in the nucleus.

scholarly journals Effect of a single dose of dimethylnitrosamine on biosynthesis of nucleic acid and protein in rat liver and kidney

1971 ◽  
Vol 125 (4) ◽  
pp. 943-952 ◽  
Author(s):  
B. W. Stewart ◽  
P. N. Magee
Keyword(s):  
Single Dose ◽  
Rat Liver ◽  
Sucrose Density Gradient ◽  
Polymerase Activity ◽  
Hepatic Necrosis ◽  
Deficient Diet ◽  
Morphological Studies ◽  
Liver And Kidney

1. Administration of a single dose of dimethylnitrosamine to rats temporarily fed on a protein-deficient diet causes a high incidence of kidney tumours. The effect of such a dose of dimethylnitrosamine (40mg/kg body wt.) on metabolism of nucleic acids and protein in rat liver and kidneys was examined during the week immediately after administration. 2. Incorporation of [14C]leucine and [14C]orotate into hepatic macromolecules was inhibited within 5h of injection of dimethylnitrosamine, and did not recover for at least 5 days. Interpretation of these results is complicated by the concomitant extensive hepatic necrosis. 3. Renal RNA synthesis was assayed by incorporation of [14C]orotate in vivo and measurement of DNA-dependent RNA polymerase activity in vitro. Both systems indicate biphasic inhibition; minimal activity was recorded 9h and 3 days after treatment. Changes in incorporation of [14C]leucine into renal protein were similar but less marked. 4. Sucrose-density-gradient analysis of renal cytoplasmic RNA indicated increased synthesis of rRNA 24h after injection of the nitrosamine. The rate of loss of radioactivity from kidney ribosomes pre-labelled with [14C]orotate was not modified by dimethylnitrosamine. 5. Dimethylnitrosamine increased incorporation of [3H]-thymidine into renal DNA. The three distinct periods of stimulated synthesis observed are discussed, with particular reference to recently published morphological studies of the sequential development of kidney tumours induced by dimethylnitrosamine in protein-depleted rats.

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