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Translation Series No. 784 CANADA

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Translation Series No. 784 CANADA 1 `s.L-f0 „ N t•PM ",•..)11,0 ■.S s. T. I. NATLN."._ FISHERIES RESEARCH BOARD OF CANADA' OTTAWA Translation Series No. 784 CANADA Studies on steroid hydroxylases--with particular emphasis on their electron transports By Tokuji Kimura Original title: Suteroido Suisankakosokei no Kenkyu--Toku ni Sono Denshidentatsukei ni tsuite. From: Seikagaku (Journal of the Japanese Biochemical Society), Vol. 38, pp. 209-223. 1966. Translated by Translation Bureau (N.T.) Foreign Languages Division Department of the Secretary of State of Canada Fisheries Research Board of Canada Technological Research Laboratory Halifax, N. S. 1966 te7V2, ele •‘: DEPARTMENT OF THE SECRETARY OF STATE SECRÉTARIAT D'ÉTAT é BUREAU FOR TRANSLATIONS BUREAU DES TRADUCTIONS FOREIGN LANGUAGES DIVISION DES LANGUES DIVISION CANADA ÉTRANGÈRES TRANSLATED FROM •• TRADUCTION DE INTO •••• Japanese English SUBJECT SUJET Biochemistry AUTHOR AUTEUR Tokuji KIMURA TITLE IN ENGLISH ••• TITRE ANGLAIS Studies on Steroid Hydroxylases --with Particelar Emphasis on Their Electron Transports-- TITLE IN FOREIGN LANGUAGE •• TITRE EN LANGUE ETRANGERE SUTEROIDO SUISANKAKOSOKEI NO KENKYU --Toku ni Sono Denshidentatsukei ni tsuite--- REFERENCE -• RgFÉRENCE (NAME or BOOK OR PUBLICATION •- NOM bU LIVRE OU PUBLICATION ) SEIKAGAKU (NJournal of Biochechemical Society of Japan) Vol.38, PUBLISHER .•• ÉDITEUR CITY ••• VILLE DATE PAGES May 1966 pp.209 -223 Mr. Paul Larose, Chief Translator, REQUEST RECEIVED FROM OUR NUMBER Department of Fisheries REQUIS PAR NOTRE DOSSIER N° 3227 DEPARTMENT Fisheries TRANSLATOR MINISTRE TRADUCTEUR LT. YOUR NUMBER DATE COMPLETED Oct. 20 VOTRE DOSSIER N° 5-3'7 REMPLIE LE 1 66 DATE RECEIVED nEçu LE - Sept. 14, 1966 -7 i-71 /11 7fi1t p209 Studies on Steroid Hydroxylases --with Particular Emphasis on Tâeir Electron Transports-- By Tokuji KIMURA,(Laboratory of Biochemistry, College of Science, St. Paul's University, Tokyo) Introduction Many excellent papers have been published on a variety of studies made on the synthesieof steroid hormones in animal organs and on bacteria. The present report describes the steroid hydro- xylases identified in mitochondria by centrifugation after the break down of cells in the process of snythesis of steroid hor- mones with cholesterol as starting material. Hydroxylases known to be falling under this category are as follows: 1) ChoUstero1-20hydroxylase l) 2) 2) Cholesterol-22-hydroxylase 3-5) 3) Cholesterol desmolase 4) Steroid-11,e-hydroxylase (E0 1.14.1. 63 8) 9) 5) Steroid-18-hydroxylasen) 11) Besides these hydroxylases, steroid-21-hydroxylase[EC 1.14.1.81 steroid-17-hydroxylase 1.14.1.7pare known to be present in microsomes. In adrenal cortex, the synthesis of corticoids from cholesterol are enhanced by le-OH steroid dgâydrogePase(EC 1.1.1. and steroid-A-isomeraseEC 5.3.3.1)together with these hydroxylases. Their activities indicate inclusion of hydrogyl group into setroid molecules irrespective of kyokuzalsei(polarity ) 2 of cell granules using NADPH as reducing agent and requiring 12) molecular oxygen. According to the classification of oxygenases, these belong to monooxygenases and the entire process of the chemical reaction may be shown in the following formula: AH NADPH 4 Q2 ileir-;7---+ A-OH + IL H20 Similarly to the chemical reaction of monomikanasF ..An general, one atom of molecular oxygen is included in steroid particles and the other to form water. However, as discussed later in this report, the stoichiometry of this reaction is not yet established because these hydroxylases contain autoxirdation system of NADPH. The reason for the author's particular interesl in hydroxylases found in mitochondria may be attributed to the following: 1) Since strong electron transïisstpintsysthickhich conjugate with „,ceiGh.tlyè tkbNis -found in mitochondria, how would this electron transmission system functionally effect eeetenia transmission system of hydroxylaqpand what would be the microstructual relation of the former with the latter in the granule. 2) Comparison with mechanism of non-blIlbs hydroxylation primarily in connection with detoxication in physeological terms,(for example,hydroxylation of aniline,etc.,) which are identified in microsomes of liver etc. › 5) Comparison of electron transmission mecnanism of steroid hydroxylases which are considered to be granular com- plex and that of ferroflavoproteins also considered as granule but which required a special procedure for solublization. Among these ferroflavoproteins are, for instance, succinic dehydro- genase IED 1.3.99.11 13) containing four atoms of iron (non-hemin) and a mole of FAD(flavin adenino dinucledide), 14) NADH dehydrogenase, choline dehydrogenase EC 1.1.99.1115) 1.)1,,p.Ephatg) 16) and p(-glyceroi. -... -dénydrogenase 1 EC 1.1.2.1) 4) Enzymes that participate in cholesterol decompostion are found separately in mitochondria and microsomes. The source or site of formation of these enzymes within the cells and sig- , nificance of tneir kydkuzaisei( polarity ). 5) Presumable action of Peptide hormones on mitochon- dria, direct or otherwise, may be inferred from the presence of the enzymes in mitochondria which are in sucn phase of metabo"- lism as to be receiving stimulus-_-: from AOTH(aarenocorticotropic hormone). I. Cellular Environment of Steroid HydroXeLazes It is necessary to investigate thephysiological environment of steroid hydroxylases, if we were to understand them under the inner celluar physiological conditions. Tlius, d'etermination of weightsof such cofactors as are pertinent to . hydroxylation, NADP and NADPH irpparticluar, was carried out. 4 Activities of enzymes in NADPH supplying system as well as activities of those in NADPH consuming system which are likely to be competitive with hydroxylases were also determined. Special attention was paid to physiological environment of hydroxylases on the basis of the statement made to the effect that ACTH acts to increase the concentration of NADPH by stimulating glycogen decomposition. 19 ) The cortex was hoemptbed and centrifuged as a whole without regard to the zonal difference. The quantitative analysis of enzymes and auxiliary enzymes in nucleus, mito- chondria, microsomes, and the solubles were made separately. Table-1 showes the distribution of bxldb_a-reductases in the adrenal cortical cells. As in other animal tissues, the major portion of glucose-6-phosphate dehydrogenaserEC 1.1.1. 493, and 6-phosphoglucon dehydrogenaseEE0 1.1.1. 43>ere present in the solubles of adrenal cortex and their extremely strong activities as compared with those in other organs are note- worthy, for example, the activities per tissue weight of these enzymes are as intense as 900 folds of those in heart musclature and 40 folds of those in liver, wkitle ,_ in adrenal medulla, they do not exceed 1 of the strength of those in 17) 5 the cortex. Further, the major portion of isocitrate dehydrogenase which reduces NADPrEC 1.1.1. 42'3 is also identified in the solubles. Although the total activities of enzymes in NADPH supply system which are found predominantly 1 (.?") 5 in the solubles of the cortex.---- ......- may be understood in connection with NADPH requirements of steroid hydroxylases, what physiological significance the fact that activities of those enzymes in NADPH supply system is virtually 100 times stronger than the total activities or hydroxylases bears is of interest to the author. Total activities of NADPH supply system is so much stronger even if we include activities of NADPH oxidases, which are known to have intense activities among NADPH reoxidases, into those of steroid hydroxylases. This is supported by the evidence on determination of NADP 18) and .NAD2â contents. Table-1. Distribution of Oxideze-Reductases in Adrenal Cortical Cells t 1•=r0"11")7 I *lc '.1---À • ‘11 tg it mi e • ye,=—.x -6= ii'iïOltrbiele* 60 70 3520 • -41:e..* Yet, = >Mete** ''' - 90 64 3040 V- -s:›Fit19.91(Meilt .(NADP) ' 120 2390 ›,,erare.**** (gum ,. .1270 , 'n litiel(ea*** ' 165 IS *... , 9,->rftx**** s . „ . ' , - • . - t- NX±I'Pli 0.**13$* ___-_ - i-• i NADPH - rgt '4t zit eqm '10.1. , - ..rytbs re**rs*— , • 7490 7 • Ali« - et or** 176 . -1-,.111' _ 43 F -.;21 -*Mite* ,. ï ,• - a , '.W -1IP -*fete* ' . x io-s: . *imigit.te-et , . 9 ,•tq-.3teet ***._ff-T.ext**.feeie*t *.*** g :6 1 14 .*R10, 6 p210 Table-1. Distribution of 0 xl61 ee —1/eductases in Adrenal Cortical Cells Enzymes Mitochondria Microsomes Solubles Glucose-6-phosphate 160 70 3520 dehydrogenase 6-phosphoglucon 90 64 3040 dehydrogenase Isocitrate dehydrogenase 90 120 2390 Malic acid dehydrogenase 360 100 1270 (decarbonate) Succinic dehydrogenase* 185 15 %pee) de,L-glycero In c ehydro- 990 genase* Cnoline dehydrogenase* 0 -- -- NADPH dehydrogenase** 846 71Y ' 5630 NADPH oxidase*** 35 14 23 NADH dehydrogenase** 6230 7490 7500 NADH oxidase*** 176 111 .38 Steroid-21-hydroxylase 4 5 0 Sterold-1370-hydroxylase 7 0 0 Cholesterol side chain 7x10 -2 0 0 breaking enzyme 6 )**** * take phenazine methosulfate as electron recepGors. ** take/ ferri'cyanide as electron receptors. *** take molecular oxygen as electron receptors. ****/Beef adrenal cortex; the figures indicate activity converted into unit of miamol/min per one g of poke adrenal cortex. (Kimura, Nakamura; unbublished data.) •' Table-2 Distribution of NADP and NADPH2 in the Adrenal Cells 18 ) Cell 2ractions NADP NADPH NADPH/NADP Mitochondria 3.0 3.5 1.1 Microsomes < 1 1.5 >1.5 Solubles 10.9 55.2 5.0 Figures indicate mewl of pyridine nucleotides/g of the adrenal cortex. Table-2 above showsAistribution
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