DOCSLIB.ORG

Y-Glutamyltransferase in Putative Premalignant Liver Cell Populations During Hepatocarcinogenesis1

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Y-Glutamyltransferase in Putative Premalignant Liver Cell Populations During Hepatocarcinogenesis1 [CANCER RESEARCH 38, 823-829, March 1978] y-Glutamyltransferase in Putative Premalignant Liver Cell Populations During Hepatocarcinogenesis1 Ross Cameron,2 John Kellen, Arnost Kolin, Aaron Malkin, and Emmanuel Farber Department of Pathology. University of Toronto. Toronto U5G 1L5 ¡R.C., A. K., E. F.], and Department of Clinical Biochemistry, Sunnybrook Medical Centre, Toronto M4N 3U5 [J. K., A. M.¡,Ontario Canada ABSTRACT the fetus but very low activity in the adult. In primary tumors induced by aflatoxin B,, 3'-Me-DAB, N-OH-2-AAF, and 2- The activity of y-glutamyltransferase, as measured AAF and in transplantable hepatomas of rats, a 30- to 50- quantitatively and by histochemical staining, was studied fold increase in GGT activity has been observed (8, 9, 11, in different cell populations during the induction of liver 34). GGT activity of whole liver showed a 5-fold increase as cancer with 2-acetylaminofluorene (2-AAF) or diethylni- early as 4 weeks after 2-AAF treatment, by 6 weeks after 3'- trosamine and compared with findings in fetal and in Me-DAB, and by 8 weeks after N-OH-2-AAF (9, 11). Since intact and regenerating adult liver. The enzyme activity is duct epithelial cells show the highest concentration of GGT 20-fold higher in 12-week nodules than in control livers in normal liver as judged by histochemistry and since and 30-fold higher in 20-week nodules than in controls. A proliferation of these cells ("oval cells"), often to striking similar 30-fold increase in activity relative to control is degrees, is a common reaction in the liver during carcino- present in hepatomas, induced by either 2-AAF or dieth- genesis with many chemicals, the interpretation of the ylnitrosamine, and in fetal hepatocytes. The enzyme increase in GGT activity in homogenates of whole liver is shows increases in activity in foci of very early putative complicated. Histochemically, GGT is demonstrable in pro preneoplastic hepatocytes induced by a single dose of liferating bile duct cells [oval cells (5)] after 4 weeks of N- diethylnitrosamine and selected by low doses of 2-AAF hydroxy-A/-2-fluorenylacetamide treatment (11) and in hy- plus partial hepatectomy. By 7 days, the foci show a 4- perplastic nodules induced by aflatoxin B, (15) and N-OH-2- fold increase in enzyme activity, and by 3 weeks they are AAF (11). These observations suggested that GGT may be a 40-fold higher than in the control liver. Histochemically, marker not only for neoplastic hepatocytes but also for the foci are strongly positive for y-glutamyltransferase, preneoplastic hepatocytes (15). especially in the bile canaliculi. By 21 days, the ductular A number of hepatocyte populations have been deline (oval) cells induced by 2-AAF have disappeared. When ated as possible precursor lesions for liver cancer induced stained for the enzyme activity, the foci stand out clearly by chemical carcinogens (7). Recently, in this laboratory, a against the negative background of the liver, allowing putative premalignant population of hepatocytes, "the fo easy quantitation. It appears that y-glutamyltransferase cus," has been identified morphologically within 1 week is a useful marker for preneoplastic hepatocytes. after carcinogen treatment (29). These proliferating foci of hepatocytes emerge as early as 30 hr after partial hepatec INTRODUCTION tomy in the livers of rats treated with a single dose of diethylnitrosamine i.p. to "initiate" and with a diet contain Tissue antigens and enzymes that will serve as pheno- ing a low level of 2-AAF to "select" the initiated 2-AAF- typic markers for malignant cells are becoming increasingly resistant hepatocytes during the regeneration period after important as diagnostic aids (2, 20, 27). Some of these hepatectomy (29, 30). These foci have been produced with markers may appear in the blood and other body fluids and a number of hepatocarcinogens as initiators, namely, dieth allow for early identification of the presence of neoplastic ylnitrosamine, A/-hydroxy-/V-2-fluorenylacetamide, dimeth- cells (11, 27). In the liver, a-fetoprotein, preneoplastic ylnitrosamine, and aflatoxin B,. The foci show a functional antigen, and GGT3 have been identified as possible positive resistance to the cytotoxic effects of carcinogens, a prop markers for preneoplastic hepatocytes (4). GGT has been erty found in hyperplastic nodules and hepatomas (1, 6, studied both biochemically and histochemically in many 10). In order to confirm the possibility that GGT could be a tissues. GGT is found in a number of epithelial cells includ marker for premalignant hepatocytes, putative premalig ing those located in jejunal villi, pancreatic acini, bile ducts, nant hepatocyte populations including early hyperplastic seminal vesicles, and proximal convoluted tubules (23, 25), nodules, late hyperplastic nodules, and foci were examined as well as in lymphocytes, the retina, and choroid plexus for GGT activity, both biochemically and histochemically. (19, 22). In the liver, hepatocytes show high GGT activity in The striking increase in activity of this enzyme in such hepatocytes in comparison with those in normal and regen 1 This research was supported in part by grants from the National Cancer erating liver is the subject of this communication. Institute of Canada and Medical Research Council of Canada and the Connaught Development Fund of the University of Toronto. 2 Research Fellow of the National Cancer Institute of Canada (1974 to MATERIALS AND METHODS 1976). 3 The abbreviations used are: GGT, -y-glutamyltransferase (EC 2.3.2.2); 3'- Me-DAB, 3'-methyldimethylaminoazobenzene; N-OH-2-AAF. N-hydroxy-2- Animals and Carcinogenic Diet Regimens. For produc acetylaminofluorene; 2-AAF, 2-acetylaminofluorene. tion of foci, male Fischer 344 rats (Charles River, Wilming Received September 6. 1977; accepted December 13, 1977. ton, Mass.) weighing 100 to 200 g were given 1 dose of MARCH 1978 823 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1978 American Association for Cancer Research. R. Cameron et al. diethylnitrosamine, 100 or 200 mg/kg i.p. Two weeks later, to 10 min. Protein was determined according to the method the rats were fed a standard basal diet (24% protein) of Lowry ef al. (18). containing 0.02% 2-AAF (Bioserv, Frenchtown, N. J.) for 1 •y-Glutamyl-p-nitroanilide, glycylglycine, and bovine se week and were then subjected to two-thirds partial hepatec- rum albumin were obtained from Sigma Chemical Co., St. tomy (12). 2-AAF was continued 1 week after hepatectomy Louis, Mo. All other chemicals were from other commercial (Fig. 1), at which time multiple foci are visible grossly in the sources except as specified. liver as small, grayish white nodules about 1 mm in diameter GGT Histochemistry. The GGT was detected according (29). Essential controls were treated as shown in Chart 1. to the method described by Rutenberg ef al. (26). Slices of For chronic feeding experiments, the basal diet containing liver with foci, nodules, hepatomas, or control liver were 0.02% 2-AAF was fed to rats for 20 weeks continuously, and serially cut in a cryostat (5 nm), air dried, fixed in ice-cold then the rats were given Purina rat chow. For diethylnitros- acetone for 30 min, and incubated in freshly prepared amine-induced hepatomas, diethylnitrosamine was given in medium containing y-glutamyl-4-methoxy-2-naphthylamide the drinking water continuously (50 ppm) for 36 weeks. (Vega-Fox Biochemicals, Tucson, Ariz.) as substrate and Biochemistry. All animals were sacrificed by cervical Fast Blue BB salt (GURR-Searle Diagnostic, High Wy- fracture after a 24-hr period of fasting. Grossly visible combe, Buckinghamshire, England) as coupling agent (26). nodules and foci were shelled out. Where sufficient tissue Spare sections were stored at 4°and retained their enzy (about 0.5 g) could be obtained, biochemical analysis was matic activity unchanged for at least 6 months. Control performed on tissue from each animal separately; other sections incubated in a similar medium without substrate wise, nodules or foci and surrounding nonnodular liver were negative. tissue were pooled. Careful histological and histochemical studies were performed on all tissues used for biochemical assay. The tissue was fixed in Carnoy's solution for histo RESULTS logical examination and stained with hematoxylin and eosin. Histology. Animals fed the continuous 0.02% 2-AAF regi Gray-white hyperplastic nodules were identified as early men develop grossly visible gray-white nodules within 6 to as 6 to 8 weeks in livers of animals fed the 0.02% 2-AAF diet 8 weeks after beginning the diet. Such nodules, microscop continuously, and at 12 weeks they were sufficiently large ically, are typical hyperplastic liver nodules (3). Seven for gross isolation for discrete biochemical and morpholog months after the beginning of 2-AAF feeding, a few rats ical study. Late liver-colored hyperplastic nodules develop develop hepatocellular carcinomas and by 1 year most rats by 16 to 20 weeks on a continuous 2-AAF diet (3, 6). have hepatomas. With diethylnitrosamine given continu Assay for GGT. Tissue was homogenized in 10 volumes ously in the drinking water, rats first develop hepatomas at of ice-cold 0.25 M sucrose with 10 strokes of a Thomas about 10 months following the beginning of carcinogen homogenizer. The homogenate was centrifuged at 1,000 x treatment, and by 1.5 years most rats have hepatomas. With g for 10 min, and the pellet containing nuclei and debris a single dose of diethylnitrosamine (250 to 300 mg/kg ¡.p.), was discarded. The supernatant was further centrifuged at some rats develop hepatomas by 12 to 15 months. All the 100,000 x g for 60 min (International IEC B60 centrifuge hepatomas were typical trabecular hepatocellular carcino with Rotor A169) to pellet the nonnuclear particulate frac mas. tion. This pellet was resuspended in 5 volumes of 0.1 M Foci appear very early following initiation with a single Tris-HCI (pH 7.6) containing 0.01 M MgCL and used for dose of diethylnitrosamine and selection with 2-AAF com assay.
Load more

Recommended publications
  • NADPH-Dependent A-Oxidation of Unsaturated Fatty Acids With
    Proc. Natl. Acad. Sci. USA Vol. 89, pp. 6673-6677, August 1992 Biochemistry NADPH-dependent a-oxidation of unsaturated fatty acids with double bonds extending from odd-numbered carbon atoms (5-enoyl-CoA/A3,A2-enoyl-CoA isomerase/A3',52'4-dienoyl-CoA isomerase/2,4-dienoyl-CoA reductase) TOR E. SMELAND*, MOHAMED NADA*, DEAN CUEBASt, AND HORST SCHULZ* *Department of Chemistry, City College of the City University of New York, New York, NY 10031; and tJoined Departments of Chemistry, Manhattan College/College of Mount Saint Vincent, Riverdale, NY 10471 Communicated by Salih J. Wakil, April 13, 1992 ABSTRACT The mitochondrial metabolism of 5-enoyl- a recent observation of Tserng and Jin (5) who reported that CoAs, which are formed during the (3-oxidation of unsaturated the mitochondrial -oxidation of 5-enoyl-CoAs is dependent fatty acids with double bonds extending from odd-numbered on NADPH. Their analysis of metabolites by gas chroma- carbon atoms, was studied with mitochondrial extracts and tography/mass spectrometry led them to propose that the purified enzymes of (3-oxidation. Metabolites were identified double bond of 5-enoyl-CoAs is reduced by NADPH to yield spectrophotometrically and by high performance liquid chro- the corresponding saturated fatty acyl-CoAs, which are then matography. 5-cis-Octenoyl-CoA, a putative metabolite of further degraded by P-oxidation. linolenic acid, was efficiently dehydrogenated by medium- This report addresses the question of how 5-enoyl-CoAs chain acyl-CoA dehydrogenase (EC 1.3.99.3) to 2-trans-5-cis- are chain-shortened by P-oxidation. We demonstrate that octadienoyl-CoA, which was isomerized to 3,5-octadienoyl- 5-enoyl-CoAs, after dehydrogenation to 2,5-dienoyl-CoAs, CoA either by mitochondrial A3,A2-enoyl-CoA isomerase (EC can be isomerized to 2,4-dienoyl-CoAs, which are reduced by 5.3.3.8) or by peroxisomal trifunctional enzyme.
    [Show full text]
  • Regulation of the Tyrosine Kinase Itk by the Peptidyl-Prolyl Isomerase Cyclophilin A
    Regulation of the tyrosine kinase Itk by the peptidyl-prolyl isomerase cyclophilin A Kristine N. Brazin, Robert J. Mallis, D. Bruce Fulton, and Amy H. Andreotti* Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011 Edited by Owen N. Witte, University of California, Los Angeles, CA, and approved December 14, 2001 (received for review October 5, 2001) Interleukin-2 tyrosine kinase (Itk) is a nonreceptor protein tyrosine ulation of the cis and trans conformers. The majority of folded kinase of the Tec family that participates in the intracellular proteins for which three-dimensional structural information has signaling events leading to T cell activation. Tec family members been gathered contain trans prolyl imide bonds. The cis con- contain the conserved SH3, SH2, and catalytic domains common to formation occurs at a frequency of Ϸ6% in folded proteins (17), many kinase families, but they are distinguished by unique se- and a small subset of proteins are conformationally heteroge- quences outside of this region. The mechanism by which Itk and neous with respect to cis͞trans isomerization (18–21). Further- related Tec kinases are regulated is not well understood. Our more, the activation energy for interconversion between cis and studies indicate that Itk catalytic activity is inhibited by the peptidyl trans proline is high (Ϸ20 kcal͞mol) leading to slow intercon- prolyl isomerase activity of cyclophilin A (CypA). NMR structural version rates (22). This barrier is a rate-limiting step in protein studies combined with mutational analysis show that a proline- folding and may serve to kinetically isolate two functionally and dependent conformational switch within the Itk SH2 domain reg- conformationally distinct molecules.
    [Show full text]
  • Phosphoglucose Isomerase (Pgi)
    NIPRO ENZYMES PHOSPHOGLUCOSE ISOMERASE (PGI) [EC 5. 3. 1. 9] from Bacillus stearothermophilus D-Glucose 6-phosphate ↔ D-Fructose 6-phosphate SPECIFICATION State : Lyophilized Specific activity : more than 400 U/mg protein Contaminants : (as PGI activity = 100 %) Phosphofructokinase < 0.01 % 6-Phosphogluconate dehydrogenase < 0.01 % Phosphoglucomutase < 0.01 % NADPH oxidase < 0.01 % Glutathione reductase < 0.01 % PROPERTIES Molecular weight : ca. 200,000 Subunit molecular weight : ca. 54,000 Optimum pH : 9.0 - 10.0 (Fig. 1) pH stability : 6.0 - 10.5 (Fig. 2) Isoelectric point : 4.2 Thermal stability : No detectable decrease in activity up to 60 °C. (Fig. 3, 4) Michaelis constants : (95mM Tris-HCI buffer, pH 9.0, at 30 °C) Fructose 6-phospate 0.27 mM STORAGE Stable at -20 °C for at least one year NIPRO ENZYMES ASSAY Principle The change in absorbance is measured at 340nm according to the following reactions. Fructose 6-phosphate PGI Glucose 6-phosphate + G6PDH + Glucose 6-phosphate + NADP Gluconolactone 6-phosphate + NADPH + H Unit Definition One unit of activity is defined as the amount of PGI that forms 1 μmol of glucose 6-phosphate per minute at 30 °C. Solutions Ⅰ Buffer solution ; 100 mM Tris-HCl, pH 9.0 Ⅱ Fructose 6-phosphate (F6P) solution ; 100 mM (0.310 g F6P disodium salt/10 mL distilled water) + + Ⅲ NADP solution ; 22.5 mM (0.188 g NADP sodium salt∙4H2O/10 mL distilled water) Ⅳ Glucose-6-phosphate dehydrogenase (G6PDH) ; (from yeast, Roche Diagnostics K.K., No. 127 671) suspension in 3.2 M (NH4)2SO4 solution (10 mg/2 mL) approx.
    [Show full text]
  • Inhibitor for Protein Disulfide‑Isomerase Family a Member 3
    ONCOLOGY LETTERS 21: 28, 2021 Inhibitor for protein disulfide‑isomerase family A member 3 enhances the antiproliferative effect of inhibitor for mechanistic target of rapamycin in liver cancer: An in vitro study on combination treatment with everolimus and 16F16 YOHEI KANEYA1,2, HIDEYUKI TAKATA2, RYUICHI WADA1,3, SHOKO KURE1,3, KOUSUKE ISHINO1, MITSUHIRO KUDO1, RYOTA KONDO2, NOBUHIKO TANIAI4, RYUJI OHASHI1,3, HIROSHI YOSHIDA2 and ZENYA NAITO1,3 Departments of 1Integrated Diagnostic Pathology, and 2Gastrointestinal and Hepato‑Biliary‑Pancreatic Surgery, Nippon Medical School; 3Department of Diagnostic Pathology, Nippon Medical School Hospital, Tokyo 113‑8602; 4Department of Gastrointestinal and Hepato‑Biliary‑Pancreatic Surgery, Nippon Medical School Musashi Kosugi Hospital, Tokyo 211‑8533, Japan Received April 16, 2020; Accepted October 28, 2020 DOI: 10.3892/ol.2020.12289 Abstract. mTOR is involved in the proliferation of liver 90.2±10.8% by 16F16 but to 62.3±12.2% by combination treat‑ cancer. However, the clinical benefit of treatment with mTOR ment with Ev and 16F16. HuH‑6 cells were resistant to Ev, and inhibitors for liver cancer is controversial. Protein disulfide proliferation was reduced to 86.7±6.1% by Ev and 86.6±4.8% isomerase A member 3 (PDIA3) is a chaperone protein, and by 16F16. However, combination treatment suppressed prolif‑ it supports the assembly of mTOR complex 1 (mTORC1) and eration to 57.7±4.0%. Phosphorylation of S6K was reduced by stabilizes signaling. Inhibition of PDIA3 function by a small Ev in both Li‑7 and HuH‑6 cells. Phosphorylation of 4E‑BP1 molecule known as 16F16 may destabilize mTORC1 and was reduced by combination treatment in both Li‑7 and HuH‑6 enhance the effect of the mTOR inhibitor everolimus (Ev).
    [Show full text]
  • Deamidated Human Triosephosphate Isomerase Is a Promising Druggable Target
    biomolecules Article Deamidated Human Triosephosphate Isomerase Is a Promising Druggable Target Sergio Enríquez-Flores 1,*, Luis Antonio Flores-López 1,2, Itzhel García-Torres 1, Ignacio de la Mora-de la Mora 1 , Nallely Cabrera 3, Pedro Gutiérrez-Castrellón 4 , Yoalli Martínez-Pérez 5 and Gabriel López-Velázquez 1,* 1 Grupo de Investigación en Biomoléculas y Salud Infantil, Laboratorio de EIMyT, Instituto Nacional de Pediatría, Secretaría de Salud, Mexico City 04530, Mexico; [email protected] (L.A.F.-L.); [email protected] (I.G.-T.); [email protected] (I.d.l.M.-d.l.M.) 2 CONACYT-Instituto Nacional de Pediatría, Secretaría de Salud, Mexico City 04530, Mexico 3 Departamento de Bioquímica y Biología Estructural, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City 04510, Mexico; [email protected] 4 Hospital General Dr. Manuel Gea González, Mexico City 14080, Mexico; [email protected] 5 Unidad de Investigación en Medicina Experimental, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico City 04510, Mexico; [email protected] * Correspondence: [email protected] (S.E.-F.); [email protected] (G.L.-V.); Tel.: +52-55-10840900 (G.L.-V.) Received: 9 June 2020; Accepted: 10 July 2020; Published: 15 July 2020 Abstract: Therapeutic strategies for the treatment of any severe disease are based on the discovery and validation of druggable targets. The human genome encodes only 600–1500 targets for small-molecule drugs, but posttranslational modifications lead to a considerably larger druggable proteome. The spontaneous conversion of asparagine (Asn) residues to aspartic acid or isoaspartic acid is a frequent modification in proteins as part of the process called deamidation.
    [Show full text]
  • Protein Disulfide-Isomerase A3 Significantly Reduces Ischemia
    Neurochemistry International 122 (2019) 19–30 Contents lists available at ScienceDirect Neurochemistry International journal homepage: www.elsevier.com/locate/neuint Protein disulfide-isomerase A3 significantly reduces ischemia-induced damage by reducing oxidative and endoplasmic reticulum stress T Dae Young Yooa,b,1, Su Bin Choc,1, Hyo Young Junga, Woosuk Kima, Kwon Young Leed, Jong Whi Kima, Seung Myung Moone,f, Moo-Ho Wong, Jung Hoon Choid, Yeo Sung Yoona, ∗ ∗∗ Dae Won Kimh, Soo Young Choic, , In Koo Hwanga, a Department of Anatomy and Cell Biology, College of Veterinary Medicine, Research Institute for Veterinary Science, Seoul National University, Seoul, 08826, South Korea b Department of Anatomy, College of Medicine, Soonchunhyang University, Cheonan, Chungcheongnam, 31151, South Korea c Department of Biomedical Sciences, Research Institute for Bioscience and Biotechnology, Hallym University, Chuncheon, 24252, South Korea d Department of Anatomy, College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, Chuncheon, 24341, South Korea e Department of Neurosurgery, Dongtan Sacred Heart Hospital, College of Medicine, Hallym University, Hwaseong, 18450, South Korea f Research Institute for Complementary & Alternative Medicine, Hallym University, Chuncheon, 24253, South Korea g Department of Neurobiology, School of Medicine, Kangwon National University, Chuncheon, 24341, South Korea h Department of Biochemistry and Molecular Biology, Research Institute of Oral Sciences, College of Dentistry, Gangneung-Wonju
    [Show full text]
  • Scaffoldless Engineered Enzyme Assembly for Enhanced Methanol Utilization
    Scaffoldless engineered enzyme assembly for enhanced methanol utilization J. Vincent Pricea, Long Chena, W. Brian Whitakera,b, Eleftherios Papoutsakisa,b, and Wilfred Chena,1 aDepartment of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716; and bThe Delaware Biotechnology Institute, University of Delaware, Newark, DE 19711 Edited by Arnold L. Demain, Drew University, Madison, NJ, and approved September 27, 2016 (received for review February 4, 2016) Methanol is an important feedstock derived from natural gas and organisms requires electrons in the form of NADH and culture can be chemically converted into commodity and specialty chem- conditions largely microaerobic or anaerobic (7, 10). Thus, or- icals at high pressure and temperature. Although biological ganisms that can grow anaerobically or microaerobically and NAD- conversion of methanol can proceed at ambient conditions, there dependent Mdhs are essential for effective conversion of methanol + is a dearth of engineered microorganisms that use methanol to to desirable metabolites (7). Although NAD(P) -dependent Mdhs produce metabolites. In nature, methanol dehydrogenase (Mdh), are the best candidates for engineering synthetic methylotrophs which converts methanol to formaldehyde, highly favors the reverse like Escherichia coli, these enzymes have poor predicted thermo- reaction. Thus, efficient coupling with the irreversible sequestration dynamic properties and are thus dependent on maintaining low of formaldehyde by 3-hexulose-6-phosphate synthase (Hps)
    [Show full text]
  • Properties of D-Xylose Isomerase from Streptomyces Albus SERGIO SANCHEZ' and KARL L
    APPLin MICoOBIOLGY, June 1975, p. 745-750 Vol. 29, No. 6 Copyright 0 1975 American Society for Microbiology Printed in U.S.A. Properties of D-Xylose Isomerase from Streptomyces albus SERGIO SANCHEZ' AND KARL L. SMILEY* Northern Regional Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois 61604 Received for publication 6 January 1975 A partially purified D-xylose isomerase has been isolated from cells of Streptomyces albus NRRL 5778 and some of its properties have been deter- mined. D-Glucose, D-xylose, D-ribose, L-arabinose, and L-rhamnose served as substrates for the enzyme with respective Km values of 86, 93, 350, 153, and 312 mM and Vmax values measuring 1.23, 2.9, 2.63, 0.153, and 0.048 tmol/min per mg of protein. The hexose D-allose was also isomerized. The enzyme was strongly activated by 1.0 mM Mg2+ but only partially activated by 1.0 mM Co2+. The respective Km values for Mg2+ and Co2+ were 0.3 and 0.003 mM. Mg2+ and Co2+ appear to have separate binding sites on the isomerase. These cations also protect the enzyme from thermal denaturation and from D-sorbitol inhibition. The optimum temperature for ketose formation was 70 to 80 C at pH values ranging from 7 to 9. D-Sorbitol acts as a competitive inhibitor with a Ki of 5.5 mM against D-glucose, D-xylose, and D-ribose. Induction experiments, Mg2+ activation, and D-sorbitol inhibition indicated that a single enzyme (D-xylose isomerase) was responsible for the isomerization of the pentoses, methyl pentose, and glucose.
    [Show full text]
  • Variants of Phosphohexose Isomerase in Gastrointestinal And
    [CANCER RESEARCH 48. 2998-3001, June, 1988] Variants of Phosphohexose Isomerase in Gastrointestinal and Mammary Carcinoma: Isoelectric Focusing Patterns of Normal and Tumor Tissues Derived from Surgical Specimens of the Same Patient1 Matthias Baumann, Damián Jezussek, Ralf-Torsten Richter, and Karl Brand2 Institute of Biochemistry, Medical Faculty, University of Erlangen-Nuremberg, Federal Republic of Germany ABSTRACT H2PO4 H2O, 5; MgSO4 7H2O,0.77; Krebs saline buffer: H2O, 1:4; pH 7.5). The occurrence of charge variants of phosphohexose isómeras? was Then the homogenate was sonicated by 6 pulses of 10 s each at 50 elucidated in cancerous and, for comparison, in noncancerous tissues W with cooling intervals of 1 min to make sure that the temperature of the preparation remained below 10'C. Subsequently, the extract was obtained from the same organ. Surgical specimens from 35 patients with gastrointestinal and mammary carcinoma were studied by means of the rehomogenized and centrifuged at 12,000 x g for 10 min. The super isoelectric focusing (IEF) technique. Differences in the IEF pattern could natant was dialyzed against 1% glycine buffer, pH 7.5, overnight at 4'C. LKB Multiplier 2117, LKB Power Supply Unit 2103, and Servalyt be demonstrated in 90% of the patients with gastric cancer. Correspond ing values for the patients with colorectal and mammary carcinoma were Precotes, pH 3-10, were used for the isoelectric focusing experiments. 50 and 73%, respectively. Almost all normal specimens proved to be A Julabo Paratherm FT20p electronic thermostat served to cool the plate to 4'C during the run. monomorphic, revealing only the major basic band with a pi of 9.1.
    [Show full text]
  • Phosphoglucose Isomerase1 (Ec 5.3.1.9)
    Enzymatic Assay of PHOSPHOGLUCOSE ISOMERASE1 (EC 5.3.1.9) PRINCIPLE: D-Fructose 6-Phosphate PGI> D-Glucose 6-Phosphate D-Glucose 6-Phosphate + ß-NADP G-6-PDH> 6-Phosphogluconate + ß-NADPH Abbreviations used: PGI = Phosphoglucose Isomerase ß-NADPH = ß-Nicotinamide Adenine Dinucleotide Phosphate, Reduced Form ß-NADP = ß-Nicotinamide Adenine Dinucleotide Phosphate, Oxidized Form G-6-PDH = Glucose-6-Phosphate Dehydrogenase CONDITIONS: T = 25°C, pH = 7.4, A340nm, Light path = 1 cm METHOD: Continuous Spectrophotometric Rate Determination REAGENTS: A. 250 mM Glycylglycine Buffer, pH 7.4 at 25° (Prepare 100 ml in deionized water using Glycylglycine, Free Base, Sigma Prod. No. G-1002. Adjust the pH to 7.4 with 1 M NaOH.) B. 100 mM D-Fructose 6-Phosphate Solution (F-6-P) (Prepare 1 ml in deionized water using D-Fructose 6-Phosphate, Disodium, Sigma Prod. No. F-3627.) C. 20 mM ß-Nicotinamide Adenine Dinucleotide Phosphate Solution (NADP) (Prepare 1 ml in deionized water using ß-Nicotinamide Adenine Dinucleotide Phosphate, Sodium Salt, Sigma Prod. No. N-0505. PREPARE FRESH.) D. 100 mM Magnesium Chloride Solution (MgCl2) (Prepare 10 ml in deionized water using Magnesium Chloride, Hexahydrate, Sigma Prod. No. M-0250.) Revised: 01/12/94 Page 1 of 4 Enzymatic Assay of PHOSPHOGLUCOSE ISOMERASE1 (EC 5.3.1.9) REAGENTS: (continued) E. Glucose-6-Phosphate Dehydrogenase Enzyme Solution (G-6-PDH) (Immediately before use, prepare a solution containing 50 units/ml of Glucose-6-Phosphate Dehydrogenase, Sigma Prod. No. G-6378, in cold deionized water.) F. Phosphoglucose Isomerase Enzyme Solution (PGI) (Immediately before use, prepare a solution containing 0.3 - 0.7 unit/ml in cold deionized water.) PROCEDURE: Pipette (in milliliters) the following reagents into suitable cuvettes: Test Blank Deionized Water 2.00 2.00 Reagent A (Buffer) 0.50 0.50 Reagent B (F-6-P) 0.10 0.10 Reagent C (NADP) 0.10 0.10 Reagent D (MgCl2) 0.10 0.10 Reagent E (G-6-PDH) 0.10 0.10 Mix by inversion and equilibrate to 25°C.
    [Show full text]
  • Triosephosphate Isomerase from Rabbit Muscle
    Triosephosphate Isomerase from rabbit muscle Catalog Number T2391 Storage Temperature 2–8 °C CAS RN 9023-78-3 This product is purified from rabbit muscle and is EC 5.3.1.1 supplied as a suspension in 3.2 M (NH4)2SO4, pH 6.0. Synonyms: TPI; D-Glyceraldehyde-3-phosphate ketolisomerase Specific Activity: ³4,000 units/mg protein Product Description Unit Definition: One unit will convert 1.0 mmole of Triosephosphate Isomerase (TPI) catalyzes the D-glyceraldehyde-3-phosphate to dihydroxyacetone interconversion of D-glyceraldehyde 3-phosphate (GAP) phosphate per minute at pH 7.6 at 25 °C. and dihydroxyacetone phosphate (DHAP). TPI plays a role in the glycolytic pathway and in gluconeogenesis. TPI is assayed spectrophotometrically in a 3.0 ml While the reaction is reversible, the formation of reaction mixture containing 0.5 mM Tris, pH 7.6, dihydroxyacetone phosphate is favored by a ratio of 1 280 mM triethanolamine, 0.132 mM b-NADH, 4.9 mM 20:1 over the reverse reaction. DL-glyceraldehyde 3-phosphate, 4 units of a-glycerophosphate dehydrogenase, and A deficiency in TPI is an autosomal recessive disorder 0.02–0.04 unit of triosephosphate isomerase. in children under five characterized by cardiomyopathy, congenital hemolytic anemia, and susceptibility to This product contains no detectable activity for the bacterial infection. Most children with this disorder do 1 following enzymes (detection limit: % of TPI activity): not survive beyond age five. 3-phosphoglyceric phosphokinase (0.01%) pyruvate kinase (0.001%) Molecular mass: 53.2 kDa (calculated) lactic dehydrogenase (0.001%) TPI is a homodimeric protein with two 25 kDa 2,3 phosphoglucose isomerase (0.001%) subunits.
    [Show full text]
  • Cyclophilins As Modulators of Viral Replication
    Viruses 2013, 5, 1684-1701; doi:10.3390/v5071684 OPEN ACCESS viruses ISSN 1999-4915 www.mdpi.com/journal/viruses Review Cyclophilins as Modulators of Viral Replication Stephen D. Frausto, Emily Lee and Hengli Tang * Department of Biological Science, The Florida State University, Tallahassee, FL 32306, USA; E-Mails: [email protected] (S.D.F); [email protected] (E.L.) * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +1-850-645-2402; Fax: +1-850-645-8447. Received: 22 May 2013; in revised form: 26 June 2013 / Accepted: 3 July 2013 / Published: 11 July 2013 Abstract: Cyclophilins are peptidyl‐prolyl cis/trans isomerases important in the proper folding of certain proteins. Mounting evidence supports varied roles of cyclophilins, either positive or negative, in the life cycles of diverse viruses, but the nature and mechanisms of these roles are yet to be defined. The potential for cyclophilins to serve as a drug target for antiviral therapy is evidenced by the success of non-immunosuppressive cyclophilin inhibitors (CPIs), including Alisporivir, in clinical trials targeting hepatitis C virus infection. In addition, as cyclophilins are implicated in the predisposition to, or severity of, various diseases, the ability to specifically and effectively modulate their function will prove increasingly useful for disease intervention. In this review, we will summarize the evidence of cyclophilins as key mediators of viral infection and prospective drug targets. Keywords: cyclosporin; HIV; HCV; cyclophilin 1. Introduction Unlike other infective agents, viruses do not encode a full complement of proteins that allow them to proliferate independent of the host.
    [Show full text]