Harold Werbin

Utilization of adrenal gland cholesterol for synthesis of cortisol by the intact normal and the ACTH-treated guinea pig.

Guinea pigs were fed a diet containing cholesterol-4-C14 for several weeks in order to establish isotopic equilibrium with respect to (a) labeled cholesterol in the adrenal glands and other tissues and (b) labeled cortisol in the urine. In the guinea pig, about 60% of adrenal gland cholesterol was derived from plasma. In this respect the guinea pig differs from the rat, in which practically all adrenal gland cholesterol is derived from plasma. Only 13% of testicular cholesterol was derived from plasma. Evidence compatible with the view that adrenal gland cholesterol is the principal precursor of urinary cortisol in the guinea pig is presented. ACTH treatments that increased the daily excretion of cortisol in urine about threefold failed to alter the values for (a) the ratio of the specific activitiy of adrenal cholesterol to that of blood cholesterol and (b) the ratio of the specific activity of urinary cortisol to that of adrenal cholesterol. These findings are consistent with the view that ACTH administration does not induce a new pathway of synthesis of urinary cortisol but, rather, speeds up an existing pathway from adrenal gland cholesterol.

Rapid Sensitive Method for Determining H3-Water in Body Fluids by Liquid Scintillation Spectrometry.

Summary 1) Mesenteric arteries of rats with acute renal hypertension were analyzed for electrolyte and water content. A technic is described for obtaining these vessels. 2) Arteries showed a significant rise in sodium, potassium, and chloride, as well as expansion of total water. However, total cation concentration in tissue water was not significantly altered. 3) Electrolyte changes may have resulted from renal dysfunction associated with retention of salt and water. Whether they were incidental to the hypertensive state or played a role in its genesis is not established.

PHOTOREACTIVATION OF UV-IRRADIATED BLUE--GREEN ALGAL VIRUS LPP-1.

Abstract UV damage to LPP-1, a DNA virus that infects the blue-green alga Plectonema boryanum , can be partially reversed by illuminating the alga subsequent to infection. Photoreactivation is induced by white or blue light but not by red or “black light” (maximum intensity at 356 mg). Since red light supports photosynthesis, there is apparently no interdependence between photosynthesis and photoreactivation. The UV-irradiated alga was photoreactivated by “black light” as well as by white or blue light. The finding that. “black light” restores algal but not viral replication suggests that the mechanisms respectively involved in the photorepair of viral and algal nucleic acids are dissimilar.

Partial separation of beef adrenal Δ5-3-ketosteroid isomerases: Androst-5-ene-3,17-dione isomerase and pregn-5-ene-3,20-dione isomerase

Two distinct Δ5-3-ketosteroid isomerases, one acting on the androst-5-ene-3,17-dione and the other on pregn-5-ene-3,20-dione, were demonstrated in beef adrenal cortex homogenates and were partly separated by ammonium sulfate fractionation from a solubilized preparation of the glands. In one enzyme fraction, androst-5-ene-3,17-dione isomerase was almost free of pregn-5-ene-3,20-dione isomerase activity. In a second fraction, pregn-5-ene-3,20-dione isomerase was twice as active as androst-5-ene-3,17-dione isomerase, whereas in the solubilized preparation androst-5-ene-3,17-dione isomerase was 5–8 times as active as pregn-5-ene-3,20-dione isomerase.

Δ5-3-ketosteroid isomerase: Solubilization and stabilization in beef adrenal gland preparations

Abstract The presence in beef adrenal gland of Δ5-3-ketosteroid isomerase that converts pregn-5-ene-3,20-dione to progesterone is demonstrated. The enzyme is unstable, difficult to solubilize, and appears to be associated with the particulate material of the cell. A procedure for solubilizing and concentrating Δ5-3-ketosteroid isomerase from beef adrenal cortex is described. This involved extraction of the tissue with hypertonic buffer, and resolubilization of the enzyme activity. A tenfold enriched isomerase preparation was obtained that was stable for three weeks. It is concluded that, in the adrenal cortex, two enzymes, a Δ5-3β-hydroxysteroid dehydrogenase and a Δ5-3-ketosteroid isomerase, are required for the conversion of Δ5-3β-hydroxysteroids to Δ4-3-ketosteroids.

Comparative photoreactivation of ultraviolet light-inactivated tobacco mosaic virus ribonucleic acid on Chenopodium, pinto bean, and tobacco plants☆

Tobacco mosaic virus nucleic acid (TMV-RNA), inactivated by ultraviolet light, 2537 A, was photoreactivated on leaves of Chenopodium, Pinto bean, and tobacco. There was greater photoreactivation on tobacco leaves than on the leaves of the other two plants. The quantum yield for the inactivation of the nucleic acid was about the same regardless of which plant was used as host when leaves were infected and left in the dark. This finding indicates the lack of dark recovery mechanisms in plant leaves for repair of TMV-RNA inactivated at 2537 A. Illuminated leaves had higher temperatures than leaves left in the dark. This temperature difference did not influence the extent of photoreactivation, nor did the presence of bentonite in the inoculum.

PHOTOBIOLOGY OF RNA BACTERIOPHAGES—I. ULTRAVIOLET INACTIVATION AND PHOTOREACTIVATION STUDIES*

Abstract— Biologically active f2‐RNA, Obtained from bacteriophage f2, was inactivated by ultraviolet (u.v) light (2537 Å) with a quantum yield of 3.3 ± 0.3 times 10‐3 when assayed in the dark with protoplasts of an F‐ strain of E. coli k12. Assay under “black light” gave a quantum yield of 2.7 ± 0.5 times 10‐3 which was just enough lower to suggest that 17 per cent photorecovery of the u.v. lesions has taken place.

PHOTOBIOLOGY OF RNA BACTERIOPHAGES–II U.V.–IRRADIATION OF f2: EFFECTS ON EXTRACELLULAR STAGES OF INFECTION AND ON EARLY REPLICATION*

Abstract— The effect of u.v. irradiation (2537 Å) on the RNA bacteriophage f2 has been studied with respect to the adsorption of f2 to E. coli K12 (male strain), the penetration of f2‐RN A into the host cell and the conversion of the phage nucleic acid to the double‐stranded replicative intermediate. The biological parameter most sensitive to u.v. was the plaque‐forming ability of the phage. Its loss could be attributed to several factors. (1). A binding of capsid protein to phage nucleic acid interfering with host penetration by the f2‐RNA. (2). Desorption of some irradiated phage at 37° from their attachment sites on the host. (3). Molecular alterations in the RNA preventing formation of the replicative intermediate within the host.

The effect of high pressure on the rates of proteolytic hydrolysis. I. Chymotrypsin

Abstract A convenient method which requires only small amounts of enzyme is described for the measurement of the relative rate of hydrolysis of casein by crystalline chymotrypsin. This method is applicable to other proteolytic enzyme-substrate systems provided split products are liberated which absorb light in the ultraviolet region. From the change of the specific rate constant of an enzymatic reaction with pressure it is possible to calculate the volume of activation, Δ V . If this rate constant refers to the rate of disappearance of [ES], then the calculated Δ V may be interpreted as the difference in volume between [ES] and the activated complex [ES]. A relative value of −13.8 ml. for Δ V has been experimentally estimated for the chymotrypsin hydrolysis of casein at 14.8 °C. and pH 7.60. The rate of hydrolysis of 0.02 M l -tyrosine ethyl ester at 25.1 °C. and pH 7.80 has been found to follow zero-order kinetics. A volume of activation of −13.5 ml. was calculated for this hydrolysis.

Evidence for the presence of 17-hydroxypregnenedione isomerase in beef adrenal cortex

Abstract 1. 1. Since 17α-hydroxypregnenolone is a precursor in the biosynthesis of corticosteroids, an attempt was made to determine whether 17α-hydroxypregn-5-ene-3,20-dione was isomerized by the same enzyme as was pregn-5-ene-3,20-dione or whether a separate enzyme, 17α-hydroxypregn-5-ene-3,20-dione Δ 5(6) −Δ 4(5) isomerase, also existed in the adrenal gland. 2. 2. Several fractions prepared from bovine adrenal cortex were assayed for 17α-hydroxypregn-5-ene-3,20-dione Delta; 5(6) −Δ 4(5) -isomerase and pregn-5-ene-3,20-dione gD 5(6) −Δ 4(5) -isomerase activities. Some fractions were free of 17α-hydroxypregn-5-ene-3,20-dione Δ 5(6) −Δ 4(5) -isomerase activity, yet contained pregn-5-ene-3,20-dione Δ 5(6) −Δ 4(5) -isomerase activity. From this and other findings it is inferred that 17α-hydroxypregn-5-ene-3,20-dione and pregn-5-ene-3,20-dione were isomerized by two distinct enzymes in the adrenal cortex. 3. 3. A scheme is presented indicating the different roles of 17α-hydroxypregn-5-ene-3,20-dione Δ 5(6) −Δ 4(5) -isomerase, pregn-5-ene-3,20-dione Δ 5(6) −Δ 4(5) -isomerase, and androst-5-ene-3,17-dione Δ 5(6) −Δ 4(5) -isomerase in the biosynthesis of cortisol and aldosterone and in the metabolism of adrenal androgens.