Transcortin in rat kidney tissue: distribution in microsomal fractions

During chromatography of renal tissue cytosolic proteins on DEAE-cellulose the protein specifically binding [3H]corticosterone is eluted within the potassium phosphate concentration range of 0.08-0.10 M. Analysis of kidney slices revealed the synthesis of [3H]transcortin whose electrophoretic mobility was close to that of the blood plasma protein. Using radioimmunochemical methods, it has been found that transcortin-specific [125I]IgG antibodies interact with growing polypeptide chains of membrane-bound polyribosomes. Free polyribosomes do not bind antibodies against transcortin.     

Effect of gold thioglucose on subcellular selenium distribution in rat liver and kidney

Biological Trace Element Research - Reported are the subcellular distributions of selenium (Se), gold, glutathione peroxidase, and enzyme markers for nuclei, mitochondria, lysosomes, and soluble...     

Isolation of ribonuclease-free intact polyribosomes from rat kidney.

High levels of RNAase present in rat kidney have prevented isolation of intact polyribosomes from this tissue. This problem has been circumvented by a thorough in situ arterial perfusion of rat kidney, coupled with homogenization of the perfused rat kidney in heparin and detergents-fortified high-speed supernatant prepared from rat liver. This procedure reduced RNAase activity in the homogenate by as much as 70%. Sedimentation of the polyribosomes from this homogenate through a layer of 2.0 M sucrose resulted in a 78--80% yield of polyribosomes from the rat kidney. The resulting polyribosomal pellet contained less than 8% of the RNAase activity present in polyribosomes from non-perfused rat kidney. The remaining RNAase activity was separated from the larger polyribomes by sucrose density gradient centrifugation. The majority of the polyribosomes were larger than tetramers. This procedure also incrased both the yield and size of polyribosomes from rat and mouse liver.     

Purification of rat kidney arginases A1 and A4 and their subcellular distribution

Arginase A1 and arginase A4 were isolated from rat kidney. Arginase A4, which is the main form of arginase in rat kidney, was obtained at a highly purified preparation; its specific activity was 1057 mumoles ornithine . min-1 . mg-1 protein. The two forms differed in subcellular localization. Form A1 was restricted to the cytosol while form A4 occurred mainly in the mitochondrial matrix. Kidney arginases A1 and A4 were found to differ in immunological properties. Kidney arginase A1, in contrast to arginase A4, precipitated with antibodies against arginase A1 from rat liver. Arginase A1 from kidney was shown to differ from arginase A1 from the liver. The two enzymes could be distinguished by double diffusion test and immunoelectrophoresis.     

Protein kinase C in mouse kidney: subcellular distribution and endogenous substrates

The subcellular distribution, kinetic properties, and endogenous substrates of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) were examined in mouse kidney cortex. Protein kinase C associated with the particulate, mitochondrial, and brush border membrane fractions was assayed after solubilization in 0.2% Triton X-100 under conditions shown to be noninhibitory to catalytic activity. Of recovered activity, 52% was associated with the cytosolic fraction; mitochondrial and brush border membrane associated protein kinase C constituted 12 and 3%, respectively, of the activity recovered in the particulate fraction. Protein kinase C associated with brush border membranes exhibited a high affinity for ATP (apparent Km = 62 +/- 10 microM) and the highest apparent maximal velocity (1146 +/- 116 pmol P/(mg protein.min] of the renal fractions examined. Maximal stimulation of protein kinase C by diacylglycerol (in the presence of phosphatidylserine) was achieved at both 25 and 300 microM calcium in all renal fractions. These results are consistent with previous reports demonstrating that diacylglycerol increases the apparent affinity of protein kinase C for calcium. Phorbol 12-myristate 13-acetate, but not 4 alpha-phorbol, was able to substitute for diacylglycerol and stimulate cytosolic and particulate renal protein kinase C. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, a specific inhibitor of protein kinase C, led to significant inhibition of catalytic activity in all renal subcellular fractions. Endogenous substrates for protein kinase C were demonstrated in renal cytosolic (26, 45, 63, and 105 kilodaltons (kDa], particulate (26, 33, 68, and 105 kDa), mitochondrial (43 kDa), and brush border membrane (26, 41, 52, 88, and 105 kDa) fractions. The possible physiological significance of protein kinase C in mammalian kidney is discussed.     

The subcellular distribution of poly-A-degrading activity in mouse kidney.

Most (95%) of the poly-A-degrading activity of the mouse kidney was found in the cytoplasmic fraction and only 5% was found in the nuclear fraction; 43% of the poly-A-degrading activity of the cytoplasm was found in the mitochondria, 22% in the microsomes, and 30% in the soluble fraction. Differences in activity and specificity indicate that poly A is degraded in the nucleus by enzymes that are separate and distinct from the enzymes in the cytoplasm that degrade poly A. The nuclear poly-A-degrading activity can be separated into an endonuclease with a general specificity and exonuclease, similar to one found in Ehrlich ascites tumor cells, which shows some specificity for poly A.     

gamma-Endorphin generating endopeptidase in rat brain: subcellular and regional distribution

beta-Endorphin is converted into the biologically active fragment gamma-endorphin by an endopeptidase which we term "gamma-endorphin generating endopeptidase". Subcellular and regional distributions of this endopeptidase activity in rat brain were studied by a newly developed assay. After subcellular fractionation of rat brain tissue gamma-endorphin generating endopeptidase activity was predominantly recovered in the cytosolic fraction. A 10 to 15 fold lower activity was present in synaptosomes, mitochondria and synaptic membranes. Hardly any endopeptidase activity was detected in nuclei and myelin. The endopeptidase activity in cytosolic and particulate fraction was found throughout brain, pituitary and spinal cord in a rather homogeneous fashion. Cytosolic activity in all brain parts was 10 to 15 fold higher than the activity in the particulate fraction. It is suggested that rather the beta-endorphin distribution than the endopeptidase is restricting for gamma-endorphin production in certain brain parts.     

Purification, characterization, molecular cloning, and subcellular distribution of neutral ceramidase of rat kidney

Previously, we reported two types of neutral ceramidase in mice, one solubilized by freeze-thawing and one not. The former was purified as a 94-kDa protein from mouse liver, and cloned (Tani, M., Okino, N., Mori, K., Tanigawa, T., Izu, H., and Ito, M. (2000) J. Biol. Chem. 275, 11229--11234). In this paper, we describe the purification, molecular cloning, and subcellular distribution of a 112-kDa membrane-bound neutral ceramidase of rat kidney, which was completely insoluble by freeze-thawing. The open reading frame of the enzyme encoded a polypeptide of 761 amino acids having nine putative N-glycosylation sites and one possible transmembrane domain. In the ceramidase overexpressing HEK293 cells, 133-kDa (Golgi-form) and 113-kDa (endoplasmic reticulum-form) Myc-tagged ceramidases were detected, whereas these two proteins were converted to a 87-kDa protein concomitantly with loss of activity when expressed in the presence of tunicamycin, indicating that the N-glycosylation process is indispensable for the expression of the enzyme activity. Immunohistochemical analysis clearly showed that the ceramidase was mainly localized at the apical membrane of proximal tubules, distal tubules, and collecting ducts in rat kidney, while in liver the enzyme was distributed with endosome-like organelles in hepatocytes. Interestingly, the kidney ceramidase was found to be enriched in the raft microdomains with cholesterol and GM1 ganglioside.     

The subcellular location in rat kidney of the peripheral benzodiazepine acceptor

In rat kidney high-affinity binding sites for [3H]Ro-5-4864 and [3H]PK-11195 with the properties of the peripheral-type acceptor were found enriched in mitochondrial (M) and light-mitochondrial-lysosomal (L) fractions on differential centrifugation. When the combined M and L fractions were subjected to sucrose density gradient centrifugation, these binding sites were found enriched at a density of 1.155 g/ml coincident with a population of light mitochondria, whereas a population of heavier mitochondria (rho = 1.175 g/ml) had few or no binding sites. Transmission electron microscopy showed that whereas the heavier mitochondria appeared highly pure and intact, the lighter mitochondria appeared less intact and to be contaminated with vesicular structures. After fractionation of the light mitochondria and vesicles by centrifugation, both fractions showed the same ratio of [3H]Ro-4864 binding sites to monoamine oxidase activity consistent with the vesicles being of mitochondrial outer-membrane origin. Digitonin pre-treatment had no effect on the density of acceptor-rich fractions on sucrose density gradient centrifugation. However, pretreatment with succinate/iodophenylnitrophenylphenyltetrazolium (INT) perturbed equally the density of acceptor-rich fractions and mitochondrial marker enzymes. When mitochondrial fractions were subjected to sonication prior to density gradient centrifugation the binding sites were now found highly enriched in a much lighter fraction coincident with the monoamine oxidase activity and thus consistent with being outer-membrane vesicles. When a mitochondrial fraction was subjected to hypotonic treatment before assay no evidence for activation/unmasking of binding sites was found. The hypotonic treatment did not release any inhibitor of the binding sites. These results are consistent with the peripheral benzodiazepine acceptor having an outer-membrane location on a sub-population of rat kidney mitochondria. Those mitochondria showing high levels of the acceptor are either light mitochondria or appear more susceptible to osmotic damage than those mitochondria in which the acceptor is absent or at low levels.     

Uptake and subcellular distribution of injected transferrin in rat liver

Radioactively labelled transferrin was injected into rats intravenously and its uptake and subcellular distribution in the liver was investigated. The amount of radioactivity in the liver remained constant from 10 min after injection. It was not influenced by asialoglycoproteins. The radioactive label was identified as asialotransferrin. After subcellular fractionation by differential and zonal sucrose density-gradient centrifugation the label was enriched in a low-density endocytic compartment showing fluorescence quenching of acridine orange and N-ethylmaleimide-sensitive ATPase activity. The data fit into a model of continuous transferrin-receptor-mediated recycling through the hepatocyte's endocytic compartment.     

The subcellular distribution of rat liver serine-pyruvate aminotransferase.

1. The subcellular distribution of L-serine-pyruvate aminotransferase activity in rat liver was investigated. About 80% was recovered from cell-free homogenates in a 'total-particles' fraction and the remainder in the cytosol. 2. Subfractionation of the particles by differential sedimentation and on sucrose density gradients showed a distribution for serine-pyruvate aminotransferase activity closely matching that observed for mitochondrial marker enzymes. 3. A study of the solubilization of enzymes from combined subcellular particles by digitonin at various concentrations also indicated a common subcellular location for serine-pyruvate aminotransferase and established mitochondrial enzymes. 4. The increase in liver serine-pyruvate amino-transferase activity induced by glucagon injection was accounted for as an increased mitochondrial activity.     

Methionyl aminopeptidase from rat liver: distribution of the membrane-bound subcellular enzyme

The selective distribution of methionyl aminopeptidase (MAP) among rat liver mitochondria (heavy and light) and microsomes is reported. Several properties of MAP from the three subcellular fractions showed that the enzyme is a typical aminopeptidase able to remove N-terminal methionine from oligopeptides and methionyl-2-naphthylamide but not from Met-Ala-Ser. MAP is a membrane-bound enzyme sensitive to SH-group oxidants and inhibitable by L-methionine but not by usual arylaminopeptidase inhibitors. It is suggested that, MAP may play an important role during protein synthesis in rat liver.Abbreviations AANA Aminoacyl-2-Naphthylamides (MetNA, AlaNA, etc...) - AApNA Aminoacyl-pNitroanilides (MetpNA, AlapNA, etc...) - AANH₂ L-Aminoacylamides (MetNH₂, AlaNH₂, etc...) - APase Acid Phosphatase - BSA Bovine Serum Albumin - DEAF Diethylaminoethyl - EDTA Ethylenediaminetetraacetic Acid - GDH Glutamate Dehydrogenase - MLBK Methionyl-Lysyl-Bradykinin (Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) - MAP Methionyl Aminopeptidase - pOHMB Sodium p-Hydroxymercuribenzoate - SDS Sodium Dodecyl Sulfate - SRA Specific Relative Activity     

Kinetics of the early subcellular distribution of cadmium in rat hepatocytes

The kinetics of the early subcellular distribution of cadmium (Cd) was characterized in primary cultures of rat hepatocytes exposed to 10, 50 and 100 M Cd in a serum-free WME medium for 10, 30 or 60 min. Our results demonstrate a time- and concentration-dependent increase in Cd content with the highest metal concentration measured in the cytosol, whereas the lowest was observed in the mitochondria. With the exception of early localization in the plasma membrane, Cd concentrations in fractions were characterized by the following decreasing order of magnitude: cytosol > low density molecules nuclei > lysosomes mitochondria. We also found evidence for: (i) a two-step process for Cd distribution in the nuclei and mitochondria; and (ii) a time-dependent slow process of transfer from the plasma membrane to the cytosol. Saturation in Cd uptake was observed at 50 M in most cell fractions at 10 and 30 min, except for the plasma membrane. The lack of apparent saturation for Cd accumulation at 60 min was not related to an increase in metallothionein synthesis. Altogether, our data provide insights into the dynamics of transfer between intracellular compartments, and allow a better identification of the organelles that are the most subjected to Cd toxicity for early exposure conditions.Deceased March 25, 2004.     

Phosphatidylinositol kinase in rat pancreatic islets: subcellular distribution and sensitivity to calcium

Plasma membrane of pancreatic islets contains a calcium sensitive phosphatidylinositol kinase. This enzyme catalyzes the first reaction in the pathway leading to the production of inositol trisphosphate, which is believed to cause a redistribution of intracellular calcium. Since the activity of this enzyme is inhibited by calcium (K0.5 = 10 microM), a loss of calcium from plasma membrane (the site of PI kinase) may be necessary for activation of the enzyme in vivo.