Latency of acid hydrolases in rat kidney cortex

1. Some lysosomal populations in the rat kidney cortex appear to be mechanically weak and are readily disrupted by gentle homogenization, while other populations remain intact even after repeated homogenization. 2. Lysosomes in the rat kidney cortex appear to be resistant to hypertonic media but are readily disrupted under hypotonic conditions. 3. Lysosomes in rat kidney cortex are readily disrupted when incubated in isotonic sucrose at 37 degrees C. 4. Measurement of total and free activity of three acid hydrolases: N-acetyl-beta-D-glucosaminidase (NAG), acid beta-galactosidase and acid beta-glycerophosphatase, indicates that the latency of these enzymes is relatively low in the homogenate (10-29%) and the ML-fraction (14-42%), but high (60-95%) in the purified large lysosomes (protein droplets). 5. The latency of purified small lysosomes is relatively lower (30-60%) than that of large lysosomes, suggesting that small lysosome populations are relatively permeable to the acid hydrolase substrates. 6. Latency variations of acid hydrolases amongst subcellular fractions appear to reflect the heterogeneity of lysosomal populations present in the kidney cortical homogenate.     

Phenylalanine hydroxylation in isolated rat kidney tubules

1. Phenylalanine hydroxylation has been demonstrated to occur in isolated rat kidney tubules under physiological conditions. 2. The hydroxylation flux response is hyperbolic with apparent Km and Vmax values of ca 85 microM phenylalanine and 49 nmol tyrosine formed/mg dry wt per hr respectively. 3. Hydroxylation in kidney tubules is substantially less sensitive to effectors of cyclic AMP turnover and Ca2+ mobilization than phenylalanine hydroxylation in isolated liver cells.     

Ring hydroxylation of 25-hydroxycholecalciferol by rat renal microsomes

Two metabolites have been isolated from rat renal microsomes incubated with 25-hydroxycholecalciferol. Postmitochondrial supernatant fractions from kidneys of thyroidectomized and parathyroidectomized rats were incubated with magnesium acetate, potassium acetate, an NADPH generating system, and 25-hydroxycholecalciferol at a level of 20 micrograms/ml postmitochondrial supernatant for 60 min at 30 degrees C. Lipid extracts of the incubation mixtures were purified by silica gel TLC and HPLC. Two peaks were obtained. Metabolite chi 2 eluted at 18 min and metabolite chi 1 at 23 min when chromatographed on a silica column developed with hexane-isopropanol. Metabolites chi 1 and chi 2 were found to have maximal absorbance at 265 nm. Both metabolites were periodate sensitive, indicating vicinal hydroxyl groups. Mass spectral analysis of metabolite chi 2, which was isolated in greater quantity than metabolite chi 1, indicates that metabolite chi 2 had resulted from hydroxylation of the A ring. Results indicate that 25-hydroxycholecalciferol is hydroxylated on carbon 2 or carbon 4 by renal microsomes. Metabolites chi 1 and chi 2, because of similarity in chromatographic migration and periodate sensitivity, are, perhaps, isomers or 2- and 4-hydroxylated metabolites.     

Oxidative decarboxylation of retinoic acid in microsomes of rat liver and kidney

Liver and kidney microsomes have been found to catalyze a rapid decarboxylation of retinoic acid in vitro. The reaction requires NADPH and Fe(2+), and is further stimulated by the presence of pyrophosphate. Thiamine pyrophosphate contained sufficient iron as an impurity to provide strong enhancement of the reaction in the absence of added iron. The decarboxylation could also be shown to occur nonenzymatically in the presence of ascorbate, Fe(2+), and boiled microsomes, but there was little autoxidation resulting in decarboxylation. The reaction was strongly inhibited by chelating agents, N,N'-diphenyl-p-phenylene diamine, phenazine methosulfate, and ferricyanide, and resembled lipid peroxidation in both its cofactor requirements and response to inhibitors. The product of the reaction appeared to lack only the C-15 of the original retinoic acid molecule. It was not retained by diethylaminoethyl cellulose, was more polar than retinoic acid upon silicic acid chromatography, had a lower UV absorption maximum (295 m micro ) than the starting product, and seemed to have an aldehyde group at C-14. The physiological significance of the decarboxylation remains to be assessed, but its rapidity makes it important to in vitro work on retinoic acid.     

Polyamines and amino acid incorporation in vitro into microsomes of rat cerebral cortex

1. Polyamines were found to be associated with microsomes of rat cerebral cortex, the amount of spermine being about four times that of spermidine. Cell sap contained more spermidine than spermine. 2. Both polyamines were able to stimulate the incorporation of [(14)C]valine into microsomes in vitro with a maximum rate equal to 250% of the control. Polyamines stimulated at concentrations close to the amount of spermine and spermidine naturally present in the system. 3. Spermine (0.05mm) was used to study the mechanism of action of polyamines. The increasing of microsome and cell-sap concentration facilitated the action of spermine, but the same process was inhibited by increasing pH5-enzyme concentration. 4. Spermine did not affect the association of [(14)C]valine with tRNA in cell sap, but increased the rate of aminoacyl-tRNA formation in pH5 enzyme preparations. However, this process was not affected in any case when incorporating microsomes were present. 5. It is suggested that microsomes are the main site of action of polyamines.     

Fatty acid composition of phospholipids in mitochondria and microsomes during diethylnitrosamine carcinogenesis in rat liver

Changes in lipid composition and function of subcellular organelles have been described in transplanted and primary tumours. We examine here the fatty acid composition of individual phospholipids (PL) in hyperplastic nodules and primary hepatoma induced by diethylnitrosamine (DEN), compared to that of normal liver and of transplantable Yoshida AH-130 hepatoma. Phosphatidylcholine and phosphatidylethanolamine fatty acid composition in mitochondria and microsomes from primary hepatoma were markedly different from normal liver; C18:0/C18:1 ratio was lower and the ratio between monosaturated and polyunsaturated fatty acids was higher. Linoleic acid content of mitochondrial cardiolipin, usually very high in normal rat liver, was notably lower in primary hepatoma. Cholesterol/phospholipid ratio in both microsomes and mitochondria from DEN-induced hepatoma was higher than in normal liver. Hyperplastic nodules showed no changes in cholesterol content whereas modifications in fatty acid composition were already observable. These modifications of membrane structure may be related to the functional changes found in nodular cells. Changes in fatty acid composition of membrane phospholipids, occurring in both primary hepatoma and preneoplastic nodules, might be one of the causes for decreased rate of lipid peroxidation peculiar to these tissues.     

Peroxisomal oxidation of L-2-hydroxyphytanic acid in rat kidney cortex

A previously unreported metabolite of mammalian phytanic acid catabolism, 2-oxophytanic acid, was identified by gas chromatography/mass spectrometry analysis. The formation of 2-oxophytanic acid was demonstrated to result from the oxidation of L-2-hydroxyphytanic acid, a reaction catalysed by a rat-kidney-cortex H2O2-generating oxidase. The pH optimum for the L-2-hydroxyphytanate oxidase activity was 8.5 and its apparent Km and Vm were about 0.15 mM and 0.35 mumol min-1 (g tissue)-1, respectively. L-2-Hydroxyisocaproate, a substrate of rat kidney L-alpha-hydroxyacid oxidase type B, inhibited the formation of 2-oxophytanate from L-2-hydroxyphytanic acid. Fractionation studies have indicated that 40% of L-2-hydroxyphytanate oxidase was associated with a particulate fraction and that the activity distribution of the oxidase closely paralleled that of catalase, a well known peroxisomal marker enzyme.     

Fatty acid remodelling of phosphatidylinositol under conditions of de novo synthesis in rat liver microsomes

Phosphatidylinositol (PI) is initially synthesized in mammalian cells with a fatty acid composition similar to that of its precursor, primarily monounsaturated forms of cytidine diphosphodiglyceride (CDP-DAG). However, at the steady state, over 80% of PI exists in the 1-stearoyl, 2-arachidonoyl form. The fatty acid remodelling of PI is due to a number of deacylation/reacylation mechanisms. In the preceding paper we demonstrated that de novo synthesized PI is rapidly deacylated and subsequently reacylated. In this report we present further evidence that cycles of deacylation and reacylation are involved in the remodelling of PI. Incubation of microsomes with CDP-DAG of different fatty acid composition results in quantitative and qualitative differences in lysoPI formation. Additionally, analyses of the resulting lysoPI and PI species reveal that multiple species of fatty acids are incorporated into the 1-position of both PI and lysoPI. Addition of acylation cofactors (fatty acyl CoAs or ATP plus CoA) potentiate reacylation in this system. The addition of stearoyl or myristoyl CoA during de novo synthesis of PI results in the incorporation of these added fatty acids into the I-positive of PI. In addition, some evidence is presented that multiple mechanisms for remodelling of the 1-position of PI may be active in the microsomes, including ATP- and CoA-dependent acylation, ATP-independent, CoA-dependent acylation and CoA-independent mechanisms. Finally, the disappearance of only a subset of lysoPI species upon the addition of acylation cofactors suggests that the reacylation step exhibits some substrate specificity.     

Purification and characterization of two forms of cytochrome P-450 from rat kidney cortex microsomes

Two forms of cytochrome P-450 (P-450), designated P-450 k-1 and P-450 k-2, have been purified about 100-fold from rat kidney cortex microsomes. P-450 k-1 and P-450 k-2 have monomeric molecular weights of 51,500 and 52,000, respectively, on sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis. Absolute spectra of the oxidized forms indicate that P-450 k-1 is largely in the low-spin state and partly in the high-spin state, and that P-450 k-2 is essentially all in the former. The absorption maxima in reduced carbon monoxide difference spectra are at 450.5 and 451 nm with P-450 k-1 and P-450 k-2, respectively. The two P-450s catalyze the omega- and (omega-1)-hydroxylation of fatty acids such as caprate, laurate, myristate, and palmitate, although P-450 k-1 exhibits a higher specific activity with all fatty acids tested. In addition, P-450 k-1 is capable of hydroxylating prostaglandin (PG) A1 and A2 at the omega-position, whereas P-450 k-2 has no activity toward PGs. These activities are all stimulated by addition of cytochrome b5. The two P-450s give different peptide map patterns when partially digested with Staphylococcus aureus V8 protease or papain.