70~ SHORT COMMUNICATIONS

Inhibition of 3~-hydroxysteroid-mediated transhydrogenase of rat liver by various quinones and flavonoids

~,fl-Unsaturated 3-oxosteroids were reported to inhibit 3~-hydroxysteroid-mediated transhydrogenase obtained from the soluble fraction of rat liver 1. The influence of naphthoquinones, steroidal quinones and flavonoids on the transhydrogenase system was studied to elucidate further the structural specificity of the compounds which block the reaction.

The preparation and assay of 3a-hydroxysteroid-mediated transhydrogenase were previously described 1, 2. The per cent inhibition of 3a-hydroxysteroid-mediated transhydrogenase by various compounds tested is presented in ~Fable I. The following compoufids at a concentration of lO -5 M were non-inhibitory: Vitamin K1, p-benzo- quinone, DL-~-tocopherylquinone, coenzyme Q10, 9,Io-anthraq uinone, io-hydroxy- estrane-3,I7-dione , genistein, hesperidin, hesperidin methyl chalcone, hydroquinone and 1,4-naphthalenediol.

Various increments of androstane-3,I7-dione were added to the assay system to reverse the inhibition effected by 3-hydroxy-2,5(Io)-estradiene-I,4,I7-trione (Table II). Androstane-3,I7-dione at concentrations greater than 6.5" Io -6 M had an inhibitory infuence (Table II). Concentrations between 9.7" 1°-7 and 6. 5 • lO -6 M of androstane-3,I7-dione were considered in the graphical evaluation which indicated a non-competitive inhibition. Km and Ki for androstane-3,I7-dione and 3-hydroxy- 2,5(io)-estradiene-i,4,i7-trione derived from Lineweaver-Burk plots of Michaelis-

TABLE I

I N H I B I T I O N O1~ 3~X-HYDROXYSTEROID-MEDIATED TRANSHYDROGENASE BY VARIOUS COMPOUNDS

The sys tem contained 7.5 mg of enzyme protein (o.I ml), ~o/*moles of KCN, I / ,mole of DPN +, o ,o2/ tmole of T P N +, io /~moles of glucose-6-P, 4/~moles of MgClz, 0.6 Kornberg units of glucose- 6-P dehydrogenase, 2oo/ tmoles of Tris buffer (pH 7.5), and wate r to a final volume of 3.o ml. After io min of equilibration, io /~g of androsterone dissolved in 0.0o5 ml of dioxane were added to initiate the reaction and o.oo5 ml of dioxane was added to the control cuvette. Appropr ia te amoun t s of inhibi tory compounds in O.OLO ml of solvent were added to both cuvettes after io min.

The reaction was followed by measur ing changes in absorbancy at 34 ° m # .

Comt, o u * ~ Inhibition ( % )

Conventrat ions ( M ) z o -s r o -g z o -7

1,2-Naphthoquinone ioo 23 o i ,4 -Naphthoquinone 54 6 o Menadione i oo 48 o Vi tamin K s ioo 2 o Lapachol 64 20 o Lomatiol 66 2 o Juglone ioo 57 o 2 ,5(Io)-Estradiene-I ,4 , I7- t r ione 40 o o 3-Hydroxy-2,5 ( io)-estradiene- 1,4,17-trione ioo 68 14 Quercitin 67 25 2 Quercitr in 38 5 o Ru t in I oo 47 22 17-Deoxytestosterone 72 2 o o 2 -Hydroxyes t rone 18 o o Aldactone ioo 57 14 Bis hydroxycoumar in 16 o o

B i o c h i n * . B i o p h y s . A c t a , 59 (1962) 7o8-71o

71o SHORT COMMUNICATIONS

In studying the rates of DPNH and TPNH oxidation by quinones, 1.5 mg of enzyme protein was utilized. I t was noted that when DPNH and TP N H were in- cubated with 7.5 mg of enzyme protein (o.I ml), they were oxidized at a rate of 9.6 and lO.4 m~moles/min, respectively. The addition of KCN and sodium isoamyl- ethylbarbiturate (Amytal) at a concentration of lO -4 M did not influence the oxi- dation. This spontaneous oxidation was prevented, however, when the enzyme protein was reduced to 1.5 mg (0.02 ml) and in the presence of lO -4 M KCN.

The non-enzymic and enzymic rates of oxidation of DPNH and TP N H by various quinones are presented in Table III. Significant oxidation of reduced pyridine nucle- otides occurred non-enzymically with p-benzoquinone, 1,2-naphthoquinone and 1, 4- naphthoquinone. The rates of oxidation with or without added albumin in the assay system were the same. The enzymic rates were corrected for the non-enzymic oxidation.

It can be concluded that the 3a-hydroxysteroid-mediated transhydrogenase is inhibited by naphthoquinones with short side chains, steroidal quinones, and flavonoids in addition to a,fl-unsaturated 3-oxosteroids. The 3-hydroxy or oxo group on the steroid nucleus appears to be important in the mediation and inhibition of hydrogen transfer.

The author is indebted to Professor L. FIESER, Harvard University, for lomatiol (2-hydroxy-3-(3-methyl-2-butenyl)-I,4-naphthoquinone); Dr. C. KAGAWA, Searle & Co., for Aldactone (3-(3-oxo-7a-acetylthio-I7fl-hydroxy-4-androsten-I7a-yl)-pro- pionic acid lactone); Dr. K. FOLKERS, Merck, Sharp & Dohme Research Lab., for coenzyme Qz0; Dr. J. FISHMAN, Sloan-Kettering Institute, for 2-hydroxyestrone; and Dr. E. SCHWENK, Worcester Foundation for Experimental Biology, for 2,5(10)- estradiene-I,4,i7-trione, 3-hydroxy-2,5(Io)-estradiene-I,4,I7-trione and Io-hydroxy- estrane-3,I7-dione; Dr. A. SEGALOFF, Alton Ochsner Medical Foundation, for 5a- androstane and I7-deoxytestosterone.

This investigation was supported in part by a research grant (C-38o9) from the National Cancer Institute, Public Health Service, and by a grant-in-aid from the U.S. Atomic Energy Commission, Grant No. AT (3o-1)-91o.

Division of Clinical Investigation, Sloan-Kettering Institute, New York, N.Y . (U.S.A.)

SAMUEL S. KOIDE

i S. S. KOIDE, C. CHEN AND S. FREEMAN, Biochim. Biophys. Acta, 38 (196o) 374. 2 B. HURLOCK AND P. TALALAY, J. Biol. Chem., 288 (1958) 886.

Received January 3oth, 1962 Biochim. Biophys. Acta, 59 (1962) 7o8-71o

Acid phosphatase activity of some algae and its inhibition by molybdenum

Acid phosphatases (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2) are reported to be present in a wide variety of organisms. The enzyme system was shown to be located on the surface of the cells as reported by ROTHSTEIN 1 in yeast and by SPENCER 2 in tomato roots. Molybdenum was shown to inhibit the activity of the enzyme in vivo, although HEWITT 3 could find no inhibition in cauliflower leaves

Biochim. Biophys. ,4eta, 59 (1962) 71o-712