Lewis L. Engel

CLINICAL, MORPHOLOGICAL AND BIOCHEMICAL STUDIES OF A VIRILIZING TUMOR IN THE TESTIS*

(IProm the Worcester Foundation for Experimental Biology, Shrewsbury, Mass.; The John Collins Warren Laboratories, Collis P. Huntington Memorial Hospital of Harvard University at Massachusetts General Hospital, and the Department of Biological Chemistry, Harvard Medical School, Boston, Mass.; and the Departments of Medicine, Obstetrics and Gynecology, and Pathology, Division of Endocrinology, Duke University Medical Center, Durham, N. C.)

The estimation of urinary metabolites of administered estradiol-17 β-16-C14

Abstract A procedure for the quantitative estimation of five identified urinary metabolites and two groups of urinary metabolites of estradiol-C 14 is described. It is based upon ( a ) hydrolysis of glucosiduronates with bacterial β-glucuronidase, ( b ) separation of neutral from phenolic substances with an anion-exchange resin, and ( c ) the separation by gradientelution partition chromatography of 2-methoxyestrone, estrone, estradiol-17β, 16-epiestriol, and estriol. In addition, a group of metabolites eluted between estradiol-17β and 16-epiestriol and a group of metabolites more polar than estriol may be separated and estimated.

The interaction of steroid hormones and coenzyme components

Abstract 1. 1. The distribution-equilibrium method has been refined to permit the measurement of equilibrium constants for complex formation between several steroids and coenzyme components in aqueous solution. 2. 2. From the relation of the structure of the steroid and the coenzyme component to the equilibrium constant, it is concluded that complexes result from interactions between the purine nucleus of coenzyme components which contain purines, and the α-side of rings, C, D, and part of ring B of the steroid. The flatness of this region of the steroid is of primary importance for complex formation. Interactions found to occur with riboflavine phosphate are presumably of a similar type. 3. 3. The nature of the forces leading to complex formation are discussed. The interaction appears to be of a non-polawr character, most of the energy being derived from the recombination of water molecules which are displaced from the interacting surfaces.

The twin ion technique for detection of metabolites by gas chromatography-mass spectrometry: Intermediates in estrogen biosynthesis☆

Abstract [4- 14 C + 7-D 0.44 ]Androstenedione and [4- 14 C + 7β-D 0.42 ]testosterone were prepared. When they were examined by mass spectrometry, the above proportion of deuterium and protium forms resulted in mass spectra in which the molecular ion (M + ) and (M + + 1) were of equal intensity. Fragment ions that contained deuterium were also twins. When doubly-labeled androstenedione and testosterone were used as substrates for the aromatizing enzymes of human placenta, the mass spectra of metabolites were characteristically labeled and thus readily distinguished from unlabeled material. Metabolites were quantitated by counting 14 C. 17β,19-Dihydroxyandrost-4-en-3-one, 19-hydroxyandrost-4-ene-3,17-dione, 17β-hydroxy-3-oxoandrost-4-en-19-al, 3,17-dioxoandrost-4-en-19-al, estradiol-17β, and estrone were isolated, identified by their mass spectra, and quantitated following incubation of doubly-labeled androstenedione and testosterone with human placental microsomes.

The subunit structure of human placental 17β-estradiol dehydrogenase☆☆☆

Abstract Human placental 17β-estradiol dehydrogenase gave a single band by sodium dodecyl sulfate - polyacrylamide gel electrophoresis, corresponding to a molecular weight of approximately 33,500 daltons. Alanine was the only amino acid detected by N-terminal analysis. Ultracentrifugation gave a molecular weight of 67,700 daltons which suggests that in solution the enzyme exists as a dimer.

Hydroxysteroid dehydrogenase of Pseudomonas testosteroni: Separation of a 17β-hydroxysteroid dehydrogenase from the 3(17)β-hydroxysteroid dehydrogenase and comparison of the two enzymes

When a crude extract of Pseudomonas testosteroni induced with testosterone was subjected to polyacrylamide gel electrophoresis, six bands that stained for 17 beta-hydroxysteroid dehydrogenase activity was observed. A protein fraction containing the enzyme corresponding to the fastest migrating band and devoid of the other hydroxysteroid dehydrogenase activities has been obtained. This preparation appears to be distinct from the previously isolated 3(17) beta-hydroxysteroid dehydrogenase (EC 1.1.1.51) in its chromatography properties on DEAE-cellulose, substrate and cofactor specificity, immunological properties and heat stability. The preparation appears devoid of 3alpha-, 3beta-, 11beta-, 17alpha-, 20alpha-, and 20beta-hydroxysteroid dehydrogenase activities. The enzyme transfers th 4-pro-S-hydrogen of NADH from estradiol-17beta (1,3,5(10)estratriene-3,17beta-diol) to estrone (3-hydroxy-1,3,5(10)-estratriene-17-one).

Quantitative gas chromatography of steroid methoxime-trimethylsilyl ethers

Abstract A method of preparing the 0-methyloxime trimethylsilyl ethers of nanomole quantitites of C 19 -and C 21 -steroids is described. The derivatives, identified by their mass spectra, can be separated and quantitated by gas chromatography using either isothermal or temperature programmed conditions. Thermal instability renders many free steroids unsuitable for gas-liquid chromatography. Derivative formation, such as the conversion of hydroxyl groups to trimethylsilyl ethers and of ketones to methoximes (0-methyloximes) overcomes this problem (3–9). In earlier investigations, large amounts of steroids were employed and quantitation was not investigated. We describe a gas chromatographic method of separation and quantitation of small amounts of C 19 — and C 21 -steroids as their methoxime-trimethylsilyl ethers. The derivatives were identified by gas chromatography-mass spectrometry. This technique is rapid and has been applied to the analysis of steroids in tissues and biological fluids (9).

Soluble guinea pig liver TPN dependent 17β-hydroxysteroid (testosterone) dehydrogenase: Partial purification and substrate specificity

Ammonium sulfate fractionation, DEAE-cellulose and hydroxylapatite chromatography have been employed to effect 200–230 fold purification of the soluble TPN dependent 17β-hydroxysteroid (testosterone) dehydrogenase of guinea pig liver. The purified preparations also oxidize saturated C19-3β-hydroxysteroids of the 5α series and saturated C19-17β-hydroxysteroids. Studies on substrate specificity indicate that the planarity of the substrate influences the reactivity.

[6] Catalytic competence: A direct criterion for affinity labeling

Publisher Summary Affinity labels or active-site-directed irreversible enzyme inhibitors are designed by combining in the same molecule a moiety that resembles the substrate for an enzyme and a reactive group that will attack some amino acid residue in the protein with the formation of a covalent bond. The success of affinity labeling depends upon the fortuitous presence of a reactive amino acid residue at such a distance from the substrate binding site that the covalently bound substrate like moiety can rest either in the active site or very close to it. This chapter explains the four criteria that have been established for the definition of affinity labels. It examines and provides evidence that the reaction of potential affinity label, 3-bromoacetoxyestrone, with human placental estradiol dehydrogenase and have found that it satisfies the first four criteria for an affinity label. Furthermore, the 17-keto group of this compound offers an opportunity to determine whether, after reaction with the enzyme, the covalently bound steroid is suitably positioned to serve as a substrate in the dehydrogenase reaction.

Mechanisms of Action of Estrogens1

Publisher Summary At the biochemical level there is a small but growing body of information concerning effects of estrogenic hormones on biochemical processes. Any complete concept of mechanism of action of estrogens must ultimately bridge the gap between these phenomena and the changes noted above. It is clear from the outset that at the present time no such bridge can be built. Experimental attack from both directions should serve to narrow the gap and ultimately establish a logical connection between the events at the molecular level and those occurring in the whole animals that are seen as morphologic changes in the target tissue. Knowledge of mechanisms of hormone action is certainly less well developed than the understanding of the biochemical functions of certain vitamins. However, it is no more possible to explain the functional and anatomical changes induced by estrogen administration to the susceptible animal in terms of the presently known effects of estrogenic hormones on biochemical systems than it is possible to explain the syndrome of pellagra in terms of defects in the functioning of the known biochemical systems in which niacinamide plays a part.