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Effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP) on the dimerization of lipoprotein lipase
No full textLipoprotein lipase (LPL), an enzyme playing the central role in triglyceride metabolism, is a glycoprotein and a homodimer of identical subunits. Dimerization and proper processing of oligosaccharide chains are important maturation steps in post-translational regulation of enzyme activity. Indirect evidences suggest that dimerization of LPL occurs in endoplasmic reticulum (ER) or Golgi. In this study, we investigated the dimerization status of LPL in 3T3-L1 adipocytes, using sucrose density gradient ultracentrifugation and carbonyl cyanide m-chlorophenylhydrazone (CCCP), an inhibitor of ER-Golgi protein transport. In the presence of CCCP, no increase of cellular LPL activity was detected during 2 h of recovery period after the depletion of LPL with heparin and cycloheximide. Only endoglycosidase H (endo H)-sensitive subunits were found in CCCP-treated cells after endo H digestion, suggesting that inactive LPL was retained in ER. In the presence of castanospermine, an inhibitor of ER glucosidase I, LPL subunits of both control and CCCP-treated cells had same molecular weight, indicating that complete oligosaccharides were transferred to LPL subunits in the presence of CCCP. In sucrose density gradient ultracentrifugation, all the LPL protein synthesized in the presence of CCCP was found at the dimeric fractions as in control cells. Most of LPL protein in control cells showed high affinity for heparin, and there was no difference between the control and CCCP-treated cells. These results suggest that dimerization and acquisition of high affinity for heparin of LPL can occur in ER of CCCP-treated cells without acquisition of catalytic activity.
ASSAY OF ORNITHINE AMINOTRANSFERASE WITH NINHYDRIN
No full textWe developed an assay system for ornithine amino-transferase (EC 2.6.1.13) using ninhydrin. Pyrroline 5-carboxylate, a product of enzymatic transamination, reacts with ninhydrin under hot acidic conditions to form a reddish pigment soluble in ethanol. The millimolar extinction coefficient of reaction product dissolved in ethanol was 16.5 at 510 nm. Acidification with perchloric acid effectively abolished the interfering color development by L-ornithine and L-glutamate. The paired activity measurement in mouse tissues by ninhydrin and o-aminobenzaldehyde methods showed a good correlation (gamma = 0.985). In our ninhydrin method, stable ninhydrin replaced unstable o-aminobenzaldehyde, and sensitivity was much higher than that with the conventional o-aminobenzaldehyde method. (C) 1994 Academic Press, Inc.
INHIBITORY MECHANISM OF CA2+ ON THE HEMOLYSIS CAUSED BY VIBRIO-VULNIFICUS CYTOLYSIN
No full textCalcium in millimolar concentrations protected mouse erythrocytes from hemolysis caused by Vibrio vulnificus cytolysin without affecting the release of intracellular K+ from the cells. This effect was maximal at 25 mM CaCl2. The protection was not absolute and could be partially overcome by increased concentrations of cytolysin. Calcium failed to block both the binding and oligomer formation of cytolysins on the erythrocyte membrane. After pore formation, the continued presence of calcium is required for the prevention of hemolysis. There was hardly any inflow of calcium into the erythrocytes through pores as measured by Ca-45(2+) uptake. The presence of calcium after the abolition of Ca2+ gradient by ionomycin cannot inhibit the hemolysis caused by cytolysin. These results suggest that calcium exerts its major inhibitory effect on V. vulnificus cytolysin-induced hemolysis as an osmotic protectant, and that cytolysin may become an useful tool for permeabilizing cells selectively for small ions such as potassium or sodium while preventing the Ca2+ flow.
ROLE OF CA2+ IN ALLOXAN-INDUCED PANCREATIC BETA-CELL DAMAGE
No full textPretreatment of rats with verapamil, a Ca2+-antagonist, completely prevented alloxan-induced hyperglycemia. Verapamil also abolished the inhibition of insulin secretion by alloxan and H2O2 in isolated rat pancreatic islets. H2O2 generation from alloxan was not affected by verapamil, but alloxan- and H2O2-induced DNA strand breaks were completely prevented. Treatment of beta-cells with alloxan and H2O2 caused elevation of cytosolic free Ca2+, and this increase of Ca2+ was also abolished by verapamil. These results suggest that alloxan-derived oxygen radicals may disturb intracellular Ca2+ homeostasis by increasing Ca2+ influx, which results in secondary reactions ultimately leading to DNA strand breaks and cytotoxicity of beta-cells.
Cytotoxic mechanism of Vibrio vulnificus cytolysin in CPAE cells
No full textVibrio vulnificus is an estuarian bacterium that causes septicemia and serious wound infection. The cytolysin, one of the important virulence determinants in V. vulnificus infection, has been reported to have lethal activity primarily by increasing pulmonary vascular permeability. In the present study, we investigated the cytotoxic mechanism of V. vulnificus cytolysin in cultured pulmonary artery endothelial (CPAE) cells, which are possible target cells of cytolysin in vivo. V. vulnificus cytolysin caused the CPAE cell damages with elevation of the cytosolic free Ca2+, DNA fragmentation, and decrease of the cellular NAD(+) and ATP level. These cytotoxic effects of V. vulnificus cytolysin were prevented by EGTA and aminobenzamide, but were not affected by verapamil or catalase. These results indicate that the elevation of cytosolic free Ca2+ induced by V. vulnificus cytolysin causes the increase of DNA fragmentation and the damaged DNA activates nuclear poly(ADP-ribose) synthetase, which depletes the cellular NAD(+) and ATP, resulting in cell death. (C) 2002 Elsevier Science Inc. All rights reserved.
HEPARIN-RELEASABLE PROATRIAL NATRIURETIC PEPTIDE PROCESSING ENZYME FROM BOVINE ATRIA
No full textThe proatrial natriuretic peptide (pro-ANP) processing enzyme, which catalyzes the conversion of pro-ANP to atrial natriuretic peptide (ANP), the active circulating form, was partially purified from bovine atria and characterized. The enzyme, which selectively cleaves the Arg(98)-Ser(99) peptide bond of pro-ANP, was released from the membrane fraction of atrial cells by heparin, suggesting that the enzyme is located at the outer surface of plasma membrane. The enzyme showed optimal pH of 8.0 and was strongly inhibited by serine protease inhibitors. The molecular weight was 560 kDa on gel filtration.
Protective effect of C-reactive protein against the lethality induced by Vibrio vulnificus lipopolysaccharide
No full textVibrio vulnificus infection has attracted special interest because of its high mortality. A strong clinical association exists between hepatic dysfunction and increased morbidity and mortality from V. vulnificus infection. In this study, the effect of C-reactive protein (CRP), a typical hepatogenic acute phase protein, on the lethality induced by V. vulnificus lipopolysaccharide (LPS) was investigated in galactosamine-sensitized mice. The pretreatment of CRP, in a dose of at least 2 mg/kg, 2 hr before the challenge of LPS completely protected mice against the lethality by V. vulnificus LPS, The elevation of serum tumor necrosis factor-alpha (TNF-alpha) induced by LPS administration was not affected by CRP pretreatment, However, the LPS- or TNF-alpha-induced hepatotoxicity was completely prevented by CRP, These results indicate that CRP does not prevent the synthesis, but prevents the hepatotoxic action of TNF-alpha. The possibility that impaired production of acute phase proteins in patients with pre-existing hepatic dysfunction may predispose the higher risk of V. vulnificus infection needs to be evaluated further.
Glycosylation of lipoprotein lipase in human subcutaneous lipomas
No full textGlycosylation of lipoprotein lipase (LPL) was studied in human subcutaneous lipomas. Heparin-releasable LPL activities were higher in lipomas than those in adjacent normal adipose tissues, and showed good correlation with cellular LPL protein mass. Molecular weight of LPL subunit was 57 kDa in both tissues. After endoglycosidase H-digestion, two types of LPL subunits were found in normal adipose tissues: partially sensitive (55 kDa) and totally sensitive (52 kDa) form. In lipoma tissues, the fraction of partially sensitive form (55 kDa) was increased comparing with control adipose tissues. These results suggest that partially sensitive subunits constitute the major secretable form of LPL in human subcutaneous lipomas.
HEMOLYTIC MECHANISM OF CYTOLYSIN PRODUCED FROM V-VULNIFICUS
No full textThe characteristics of hemolytic action of cytolysin produced from V. vulnificus were investigated in mouse erythrocytes. The cytolysin bound erythrocyte membranes in temperature-independent manner and then lysed cells temperature-dependently. Hemoglobin release by the cytolysin was completely inhibited by the presence of raffinose or melezitose, but K+ release was not affected. The cytolysin-induced hemolysis was always accompanied with the conversion of membrane-bound cytolysin into an oligomer of 210 kDa, corresponding to a tetramer of native cytolysins. Nonesterified cholesterol inactivated the cytolysin by converting active monomeric cytolysin into inactive oligomer. The results suggest that the cytolysin lyses erythrocytes due to the formation of small pores on erythrocyte membrane by cholesterol-mediated oligomerization of the cytolysin.
PROTECTIVE MECHANISM OF GLUCOSE AGAINST ALLOXAN-INDUCED PANCREATIC BETA-CELL DAMAGE
No full textGlucose prevented the alloxan- or H2O2-induced inhibition of insulin secretion in rat pancreatic islets. Hydrogen peroxide was detected during the incubation of islets with alloxan, and this generation of hydrogen peroxide was not affected by glucose. Treatment of beta-cells with alloxan or H2O2 caused elevation of cytosolic free Ca2+ and decrease of cellular NAD(+). Glucose blocked the decrease of cellular NAD(+) level, but did not abolish the increase of cytosolic Ca2+. These results indicate that glucose protected pancreatic beta-cell damage after the H2O2 generation and Ca2+ influx on a chain of reactions in the diabetogenesis of alloxan. (C) 1995 Academic Press, Inc.
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