Distribution of Dipeptidase Activity between Lysosomes and Soluble Fraction of Rat Liver

1975 ◽  
Vol 53 (5) ◽  
pp. 502-508 ◽  
Author(s):  
Steve L. Taylor ◽  
Al L. Tappel
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Soluble Fraction ◽  
Bimodal Distribution ◽  
Sulfhydryl Reagents ◽  
Ph Optimum ◽  
Triton Wr 1339 ◽  
Dipeptidase Activity ◽  
Soluble Fractions

Dipeptidase activity toward Arg-Phe, Arg-Gly, and Trp-Leu exhibited bimodal distribution in the lysosomal and soluble fractions of rat liver. The majority (50–70%) of the dipeptidase activity was present in the soluble fraction. Some evidence for a plasma membrane dipeptidase, which hydrolyzes Trp-Leu but not Arg-Phe or Arg-Gly, also was found. The lysosomal dipeptidase activity had a pH optimum of 6.0–7.0, and was activated by sulfhydryl reagents. Lysosomal localization for some of the dipeptidase activity was established with Triton WR-1339 fractionation and latency experiments.

Acute effects of ethanol on hepatic endocytosis and processing of insulin in perfused rat liver

1993 ◽  
Vol 264 (3) ◽  
pp. E420-E427 ◽  
Author(s):  
J. Fawcett ◽  
B. Hammond ◽  
G. D. Smith
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Rate Constant ◽  
Binding Constants ◽  
Compartmental Analysis ◽  
Insulin Binding ◽  
Bimodal Distribution ◽  
Subcellular Fractionation ◽  
Perfused Rat Liver ◽  
Binding Studies

This study utilizes the perfused rat liver combined with subcellular fractionation and compartmental analysis to investigate the effects of ethanol on hepatic uptake, endocytosis, and processing of insulin. At 4 min after the start of a 2-min pulse of radiolabeled insulin, increasing ethanol concentrations progressively inhibited insulin uptake by the liver (57% at 50 mM ethanol). Subcellular fractionation of the perfused livers showed a progressive shift in distribution from a predominantly endosomal location (control) to a bimodal distribution between endosomes and plasma membrane. This could be largely accounted for by a specific reduction in the endosome-associated insulin. Binding studies showed no changes in the binding properties of the plasma membrane insulin receptor. Compartmental analysis of the perfusate efflux curves confirmed the lack of effect of ethanol on the binding constants but showed a significant decrease in the endocytic rate constant (50%) together with an increase in the retroendocytic rate constant (33%). Simulation studies with the compartmental model showed that these changes could account for the observed decrease in uptake by the liver. No changes were found in the subsequent endocytic degradation of insulin.

scholarly journals Alkaline ribonuclease and phosphodiesterase activity in rat liver plasma membranes

1973 ◽  
Vol 132 (3) ◽  
pp. 449-458 ◽  
Author(s):  
Terence D. Prospero ◽  
Malcolm L. E. Burge ◽  
Kenneth A. Norris ◽  
Richard H. Hinton ◽  
Eric Reid
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Ribonuclease Activity ◽  
Nuclear Fraction ◽  
Phosphodiesterase Activity ◽  
Ph Optimum ◽  
Liver Plasma Membranes ◽  
Rat Liver Plasma Membranes

The ribonuclease and phosphodiesterase activities of rat liver plasma membranes, purified from the crude nuclear fraction by centrifugation in an A-XII zonal rotor and flotation, were examined and compared. The plasma membrane is responsible for between 65 and 90% of the phosphodiesterase activity of the cell and between 25 and 30% of the particulate ribonuclease activity measured at pH8.7 in the presence of 7.5mm-MgCl2. Both enzymes were most active between pH8.5 and 8.9. Close to the pH optimum, both enzymes were more active in Tris buffer than in Bicine or glycine buffer. Both plasma-membrane phosphodiesterase and ribonuclease were strongly activated by Mg2+, there being at least a 12-fold difference between the activity in the presence of Mg2+ and of EDTA. There is, however, a difference in the response of the enzymes to Mg2+ and EDTA in that the phosphodiesterase is fully activated by 1.0mm-MgCl2 and fully inhibited by 1.0mm-EDTA, whereas the ribonuclease requires 7.5mm-MgCl2 for full activation and 5mm-EDTA for full inhibition. Density-gradient centrifugation has indicated that on solubilization in Triton X-100 most of the ribonuclease activity is released into a small fragment of the same size as that containing the phosphodiesterase activity. The relationship between the two activities is discussed in view of these results.

Occurrence and Biosynthesis of Ceramide Phosphorylethanolamine in Chicken and Rat Liver

1972 ◽  
Vol 50 (2) ◽  
pp. 166-173 ◽  
Author(s):  
Bernd A. Muehlenberg ◽  
Michael Sribney ◽  
Marilyn Kemp Duffe
Keyword(s):  
Rat Liver ◽  
Reaction Rate ◽  
Hydroxyl Group ◽  
Transferase Activity ◽  
Manganese Ions ◽  
Sulfhydryl Reagents ◽  
Ph Optimum ◽  
Optimal Activity ◽  
Threo Isomer ◽  
Do So

Ceramide phosphorylethanolamine has been found to occur in chicken and rat liver. An enzyme (CDP-ethanolamine:ceramide ethanolaminephosphotransferase) has been found in a number of tissues which catalyzes the biosynthesis of this lipid. The enzyme catalyzes the transfer of the phosphorylethanolamine moiety of CDP-ethanolamine to the free primary hydroxyl group of a ceramide (N-acylsphingosine). The chicken liver enzyme requires 0.010 M manganese ions for optimal activity and has a pH optimum of 7.7. The Km for the substrate N-octanoyl-threo-sphingosine was found to be 2.5 × 10−4 M. A study of the effect of increasing CDP-ethanolamine concentration on the reaction rate indicates from sigmoid kinetics that the coenzyme modulates and possibly regulates PE-ceramide transferase activity. The enzyme differs from sphingomyelin synthetase (CDP-choline:ceramide cholinephosphotransferase) in that it will only utilize the unnatural threo isomer of N-acylsphingosines (threo-ceramides) as acceptors for the phosphorylethanolamine moiety of CDP-ethanolamine. Sphingomyelin synthetase has been shown to utilize erythro-ceramides the presence of sulfhydryl reagents (Sribney, M.: Can. J. Biochem. 49, 306 (1971)); the enzyme catalyzing the biosynthesis of ceramide phosphorylethanolamine, however, does not do so, even in the presence of a variety of sulfhydryl reagents tested.

scholarly journals Identification and subcellular localization of sphinganine-phosphatases in rat liver

1995 ◽  
Vol 311 (1) ◽  
pp. 139-146 ◽  
Author(s):  
P De Ceuster ◽  
G P Mannaerts ◽  
P P Van Veldhoven
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Water Soluble ◽  
Nuclear Fraction ◽  
Cell Fractionation ◽  
Acidic Ph ◽  
Ph Optimum ◽  
High Affinity ◽  
Neuronal Tissues

One of the primary products of [4,5-3H]sphinganine phosphate, added to fibroblast cultures, is sphinganine [Van Veldhoven and Mannaerts (1994) Biochem. J. 299, 597-601], implicating the physiological action of (a) hitherto unknown phosphatase(s). We have now further characterized this activity in rat liver. In homogenates, the dephosphorylation appeared to be catalysed by multiple enzymes. A low-affinity system was active at acidic pH, whereas at physiological pH values hydrolysis was carried out by a high-affinity enzyme. The latter was sensitive to Zn2+ and detergents and possessed a pH optimum of 7.5. Upon cell fractionation the major portion of the high-affinity activity was recovered in the nuclear and microsomal fractions. Further separation of the microsomal fraction showed an association predominantly with vesicles derived from the plasma membrane. Likewise, when plasma membranes were prepared from the nuclear fraction, the high-affinity phosphatase co-purified with the plasma membrane markers. From the differential effects of bivalent cations, chelators, water-soluble and amphiphilic phosphate esters, detergents and other compounds, it could be concluded that the plasma membrane-associated sphinganine-phosphatase activity is not due to alkaline phosphatase, dolichol-phosphatase, the N-ethylmaleimide-insensitive phosphatidate phosphatase or ceramide-phosphatase. The dephosphorylation observed at acidic pH in homogenates appeared also to be enriched in purified plasma membranes and might represent a side-activity of ceramide-phosphatase. We speculate that the high-affinity phosphatase, which is especially active in neuronal tissues, plays a role in the attenuation of bioactive phosphorylated sphingoid bases such as sphingenine phosphate, and propose to name it sphingosine-phosphatase.

scholarly journals Phosphodiesterase II in epithelial cells from guinea-pig and rat small intestine

1974 ◽  
Vol 142 (3) ◽  
pp. 545-553 ◽  
Author(s):  
Peter R. Flanagan ◽  
S. H. Zbarsky
Keyword(s):  
Epithelial Cells ◽  
Guinea Pig ◽  
Specific Activity ◽  
Soluble Fraction ◽  
Gel Filtration ◽  
Equilibrium Density ◽  
Small Intestinal Mucosa ◽  
Ph Optimum ◽  
Triton Wr 1339 ◽  
Small Intestinal

Phosphodiesterase II activity was determined by using a synthetic substrate, the 2,4-dinitrophenyl ester of thymidine 3′-phosphate. The enzyme activity was determined in fractions obtained by differential centrifugation of homogenates of epithelial cells from the small intestinal mucosa of guinea pigs and rats. In guinea-pig preparations phosphodiesterase II occurred with highest specific activity in those fractions rich in succinate dehydrogenase and acid phosphatase. A lysosomal location for the guinea-pig enzyme was indicated by its structure-linked latency and by its association with particles that under-went a characteristic decrease in equilibrium density when Triton WR-1339 was injected into the animals. With rat preparations a much greater proportion of the phosphodiesterase II activity was found in the soluble fraction after ultracentrifugation. The rat enzyme exhibited a lower degree of latency and administration of Triton WR-1339 had no effect. The rat enzyme further differed from that of the guinea pig in other respects; it was more labile at 60°C, it exhibited a lower pH optimum and it had a higher molecular weight as determined by gel-filtration chromatography.

scholarly journals Heterogeneity of liver acid phosphatases in developing chick embryo

1969 ◽  
Vol 115 (2) ◽  
pp. 191-197 ◽  
Author(s):  
K.-M. Wang
Keyword(s):  
Acid Phosphatase ◽  
Phosphatase Activity ◽  
Acid Phosphatase Activity ◽  
Soluble Fraction ◽  
The Other ◽  
Acid Phosphatases ◽  
Peak Activity ◽  
Ph Optimum ◽  
Triton X ◽  
Soluble Fractions

1. The development, localization and heterogeneity of acid phosphatase and a Zn2+-activated acid phosphatase in cellular fractions of developing chick liver were studied. 2. Acid phosphatase is distributed abundantly in the particulate and soluble fractions. The soluble fraction is rich in Zn2+-activated acid phosphatase, which attains its peak activity at about 15 days of incubation. 3. The particulate acid phosphatase activity is inhibited by fluoride but not by sodium l(+)-tartrate or cysteine. On the other hand, the soluble Zn2+-activated acid phosphatase activity is inhibited by sodium l(+)-tartrate and cysteine but not by fluoride. 4. The pH optimum of these two enzymes is similar at about 5·6. 5. The soluble Zn2+-activated acid phosphatase activity appears to be thermally stabilized by the treatment with Triton X-100 or bovine serum albumin.

ACTIVATION OF SCHRADAN BY MAMMALIAN TISSUE HOMOGENATES

1956 ◽  
Vol 34 (6) ◽  
pp. 1131-1141 ◽  
Author(s):  
R. D. O'Brien
Keyword(s):  
Soluble Fraction ◽  
Mammalian Tissue ◽  
Ph Optimum ◽  
Tissue Homogenates ◽  
Extrahepatic Tissues ◽  
Mechanism Of Oxidation ◽  
Soluble Fractions

It has been confirmed that the combined microsomal and soluble fractions of liver, in the presence of magnesium, nicotinamide, and DPN, can convert schradan to a potent anticholinesterase. DPN can be replaced by TPN, but not by ATP. DPNH can replace the DPN and soluble fraction. Catalase enhances the conversion but only when DPNH or a source of it is provided. Acetone powders of microsomes, suitably fortified, are almost ineffective in converting schradan. The pH optimum of the whole schradan-converting system is 8.1. Other properties of the system are described. Certain extrahepatic tissues, especially lung, heart, and testis, can convert schradan. The livers of all species tested convert schradan. A peroxide-mediated mechanism of oxidation is proposed.

ACTIVATION OF SCHRADAN BY MAMMALIAN TISSUE HOMOGENATES

1956 ◽  
Vol 34 (1) ◽  
pp. 1131-1141 ◽  
Author(s):  
R. D. O'Brien
Keyword(s):  
Soluble Fraction ◽  
Mammalian Tissue ◽  
Ph Optimum ◽  
Tissue Homogenates ◽  
Extrahepatic Tissues ◽  
Mechanism Of Oxidation ◽  
Soluble Fractions

It has been confirmed that the combined microsomal and soluble fractions of liver, in the presence of magnesium, nicotinamide, and DPN, can convert schradan to a potent anticholinesterase. DPN can be replaced by TPN, but not by ATP. DPNH can replace the DPN and soluble fraction. Catalase enhances the conversion but only when DPNH or a source of it is provided. Acetone powders of microsomes, suitably fortified, are almost ineffective in converting schradan. The pH optimum of the whole schradan-converting system is 8.1. Other properties of the system are described. Certain extrahepatic tissues, especially lung, heart, and testis, can convert schradan. The livers of all species tested convert schradan. A peroxide-mediated mechanism of oxidation is proposed.

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