Studies on Microsomal Nucleoside Diphosphatase of Rat Hepatocytes

Kuriyama, Yoshiaki

doi:10.1016/s0021-9258(19)45202-2

Cited by 80 publications

(6 citation statements)

References 33 publications

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“…Similar criteria have been used to localize nucleosidediphosphatase on the luminal face of the endoplasmic reticulum in rat liver (Kuriyama, 1972). It has been argued that the latency of the nucleosidediphosphatase (or inosine-S'-diphosphatase) of rat liver endoplasmic reticulum may be due (A) Freshly prepared Golgi (900 Mg of protein) was preincubated at 22 °C for 45 min in 1 mL of 0.14 M sucrose containing 900 µg of concanavalin A, 55 Mmol of mercaptoethanol, 10 Mmol of MnCl2, 10 Mmol of CaCl2, and 20 Mmol of A-acetylglucosamine.…”

Section: Discussionmentioning

confidence: 99%

Orientation and role of nucleoside diphosphatase and 5'-nucleotidase in Golgi vesicles from rat liver

Brandan¹,

Fleischer²

1982

Biochemistry

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The fate of UDP formed during the galactosylation of added N-acetylglucosamine in Golgi vesicles isolated from rat liver using D2O-sucrose gradients has been determined. UDP-Gal labeled with [14C]uracil was used, and the products of the reaction were separated and quantitated by using high-pressure liquid chromatography. [14C]Uridine rather than [14C]UDP or [14C]UMP was found to accumulate, indicating the presence of both UDPase and UMPase activities in the Golgi. Golgi vesicles were shown to contain a nucleosidediphosphatase activity that is membrane bound. It appears to be located on the luminal face of the Golgi since it is activated 3-5-fold by detergents and 4-fold by treatment of the vesicles with Filipin. We have shown previously that Filipin disrupts the Golgi but does not solubilize membrane-bound enzymes. The nucleosidediphosphatase of the Golgi differs from that present in rough endoplasmic reticulum in its absolute requirement for Ca2+ for activity and in its substrate specificity that is higher for UDP than for IDP. Golgi vesicles also contain UMPase activity that is stimulated only 2-fold by detergents or Filipin. Concanavalin A inhibits this activity about 80% in both intact and detergent-treated vesicles. The Golgi UMPase is thus probably identical with 5'-nucleotidase. These results are consistent with histochemical evidence from other laboratories that indicate that 5'-nucleotidase is present on both sides of liver Golgi membranes. In the presence of concanavalin A and N-acetylglucosamine, intact Golgi vesicles were found to convert UDP-Gal to UMP. These findings indicate that UDP formed by galactosyltransferase in the lumen of the vesicles is rapidly converted to UMP by UDPase in the lumen but that UMP moves rapidly out of the lumen of the Golgi and is broken down to uridine by 5'-nucleotidase on the cytoplasmic side of the vesicles.

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Section: Discussionmentioning

confidence: 99%

Orientation and role of nucleoside diphosphatase and 5'-nucleotidase in Golgi vesicles from rat liver

Brandan¹,

Fleischer²

1982

Biochemistry

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“…The microsomes (40 to 50 mg/mL) were frozen (-85 °C) until needed. The first step in delipidation of microsomes was adopted from the method of Kuriyama (1972) for solubilization of nucleotide diphosphatase. Microsomes were suspended in 10 mM Tris-HCl, 0.25 M sucrose, pH 7.5, to a concentration of 12 to 15 mg of protein per mL.…”

Section: Methodsmentioning

confidence: 99%

Preparation and properties of a phospholipid-free form of microsomal UDP-glucuronyltransferase

Erickson¹,

Zakim²,

Vessey³

1978

Biochemistry

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“…On the other hand, various types of investigations on the localization of a number of enzymes have resulted in conclusions similar to those reached in this paper. The distributions of NADH-and NAI2PHcytochrome c reductase, cytochrome bs, nucleo-side pyrophosphatase, cytochrome P-450, G6Pase, acetanilid esterase, IDPase, and GDPmannosyltransferase were studied by using proteolytic treatment (1,13,31,34,47); the localization of P-450 was investigated with 125I-labeling (63); the localization of cytochrome b~, NADHand NADPH-cytochrome c reductases, IDPase, and acetanilide esterase was investigated by using antibodies (1,34,35,46,48,51,(59)(60)(61); the site of various electron-transport enzymes, IDPase, G6Pase, and UDPGA-transferase, was analyzed by studying substrate permeability and latency (2,21,23,27,34); the asymmetry of esterases was studied by using charged and uncharged substrates and inhibitors (28); the site of G6Pase was investigated by histochemical methods (18,36,37); and, finally, the distribution of a number of electrontransport enzymes was studied by following the product released both in vitro and in vivo (25,43). The data in the above references are generally in agreement with the findings described in this paper, which therefore confirms previous findings on the localization of some enzymes attained by using a different approach.…”

Section: Nilsson and Dallner Enzyme And Phospholipid Asymmetry In Liver Membranesmentioning

confidence: 99%

“…On the other hand, a number of studies regarding the topology of proteins in this membrane have appeared. The distribution of a number of enzymes was studied by using approaches such as substrate permeability and latency (2,21,23,27,34), use of charged and uncharged substrates and inhibitors (28), antibodies (1,34,35,46,48,51,(59)(60)(61), proteolysis (1,13,31,34,47), and 125I-labeling (63). Isolated microsomal vesicles are impermeable to various charged substances, which may be the reason why many microsomal enzymes display latency (43).…”

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confidence: 99%

Enzyme and phospholipid asymmetry in liver microsomal membranes.

Nilsson¹,

Dallner²

1977

The Journal of Cell Biology

159

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The transverse distribution of enzyme proteins and phospholipids within microsomal membranes was studied by analyzing membrane composition after treatment with proteases and phospholipases. Upon trypsin treatment of closed microsomal vesicles, NADH-and NADPH-cytochrome c reductases as well as cytochrome b~ were solubilized or inactivated, while cytochrome P-450 was partially inactivated. When microsomes were exposed to a concentration of deoxycholate which makes them permeable to macromolecules but does not disrupt the membrane, the detergent alone was sufficient to release four enzymes: nucleoside diphosphatase, esterase, /3-glucuronidase, and a portion of the DT-diaphorase. Introduction of trypsin into the vesicle lumen inactivated glucose-6-phosphatase completely and cytochrome P-450 partially. The rest of this cytochrome, ATPase, AMPase, UDP-glucuronyltransferase, and the remaining 50% of DT-diaphorase activity were not affected by proteolysis from either side of the membrane. Phospholipase A treatment of intact microsomes in the presence of albumin hydrolyzed all of the phosphatidylethanolamine, phosphatidylserine, and 55% of the phosphatidylcholine. From this observaton, it was concluded that these lipids are localized in the outer half of the bilayer of the microsomal membrane. Phosphatidylinositol, 45% of the phosphatidylcholine, and sphingomyelin are tentatively assigned to the inner half of this bilayer. It appears that the various enzyme proteins and phospholipids of the microsomal membrane display an asymmetric distribution in the transverse plane.It has been established that several enzymes and glycoproteins of a number of biological membranes such as erythrocytes (56), reticulocytes (38), plasma membranes (17), and inner mitochondrial membranes (29) are not randomly distributed in the transverse plane but are concentrated at the outer or inner surface of these membranes. Bretscher (7) carried out studies with nonpenetrating probes and Zwaal and his coworkers (64) used phospholipases to come to the conclusion that the phospholipids in the erythrocyte membrane are also asymmetrically distributed in the transverse plane.The membrane of the endoplasmic reticulum (ER) contains a large number of enzyme systems, some of them involved in metabolic reactions closely associated with the cytoplasm and some of them participating in the biosynthesis of secretory macromolecules completed and transported within the lumen of the ER and Golgi system (53). Therefore, it is to be expected that, like several other biological membranes, the ER mem-

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Studies on Microsomal Nucleoside Diphosphatase of Rat Hepatocytes

Cited by 80 publications

References 33 publications

Orientation and role of nucleoside diphosphatase and 5'-nucleotidase in Golgi vesicles from rat liver

Orientation and role of nucleoside diphosphatase and 5'-nucleotidase in Golgi vesicles from rat liver

Preparation and properties of a phospholipid-free form of microsomal UDP-glucuronyltransferase

Enzyme and phospholipid asymmetry in liver microsomal membranes.

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