Charles D. Kochakian

Definition of androgens and protein anabolic steroids

Abstract The protein anabolic effect of testosterone and related steroids is expressed by the synthesis of protein in many extragenital tissues in addition to the accessory sex organs. The main site of extragenital actions is the skeletal muscles. The degree of response of the muscles, however, varies widely among the individual muscles and also among species. The response of the ‘levator ani’ (properly the dorsal bulbocavernosus) has been used as an assay method. Comparison of the response of this muscle with that of the seminal vesicles and/or the prostate of the immature castrated rat has suggested a large number of steroids with preferential ‘levator ani’ activity. The use of this muscle as representative of other skeletal muscles has been severely criticized. Furthermore, the apparent separation of the two activities in the animal experiments has not been supported by clinical trials. Consequently, nitrogen balance assays have been made in the castrated rat, in female ovariectomized monkey and in man. Many steroids were found to be more active than testosterone propionate or methyltestosterone. A separation of the action of a steroid on extragenital tissue from that of the accessory sex organs will probably not be achieved until the mechanism of action in the two types of tissue is elucidated.

Androgen regulation of RNA polymerase activity in isolated mouse kidney nuclei

Abstract 1. 1. Isolated mouse kidney nuclei have RNA polymerase activity. The reaction is dependent on DNA, divalent cations (Mg 2+ , Mn 2+ ) and the four ribonucleoside triphosphates. The amount of RNA obtained was decreased by the addition of actinomycin D, ribonuclease or deoxyribonuclease to the enzyme system. The addition of Mn 2+ and (NH 4 ) 2 SO 4 greatly enhanced the activity. The optimum was 8.1 at the high and 8.8 at the low ionic concentration. 2. 2. Castration of adult male mice produced a sharp decrease in the RNA polymerase activity of the isolated nuclei of the kidney. Testosterone propionate reversed the effect of castration. The addition of Mn 2+ and (NH 4 ) 2 SO 4 to the incubation system enhanced the enzyme activity but decreased the percent differences. The stimulating effect of the androgen was evident within 2 h after subcutaneous injection and was dose dependent. 3. 3. Adrenalectomy further decreased the RNA polymerase activity, which was not only restored but also increased by testosterone propionate to the same level as in non-adrenalectomized castrated mice. 4. 4. The suspension of the kidney nuclei in a solution of three of the nucleotides and the addition of the fourth nucleotide to the enzyme system in varying amounts resulted in a higher maximal polymerase activity for the castrated mice when CTP was varied and a lower value with GTP. Thus, the effects of castration and androgen were much less with the former and greater with the latter nucleotide. 5. 5. The incorporation of all the nucleotides in RNA was decreased by castration and restored to normal by testosterone propionate. The base composition of the newly synthesized RNA was not markedly changed. The A+U G+C ratio was approx. 0.50 for the three groups. The androgen caused a slight increase in the ratio. The addition of Micrococcus lysodeikticus RNA polymerase to the enzyme system did not produce any significant changes. The addition of (NH 4 ) 2 SO 4 produced an increase in the incorporation of the nucleotides and an increase in the A+U G+C ratio to approx. 1.0.

Intracellular regulation of nucleic acids of mouse kidney by androgens.

Abstract The RNAs of the respective intracellular parts of the mouse kidney decreased in direct proportion to the decrease in kidney weight after castration, but the microsomal RNA decreased at a faster rate. The DNA also decreased but to a lesser degree than the RNAs. The administration of testosterone propionate or androstan-17β-o1,3-one for intervals up to 6 weeks increased the kidney weight above normal and produced a much greater percentage increase in the microsomal RNA than the weight. The soluble, nuclear and mitochondrial RNA increased essentially in proportion to the increase in weight of the kidney.

17α and 17β-oxidoreductases of adult female hamster liver and kidney☆

Abstract The liver and kidney cytosols of adult female hamsters converted androstenedione to epitestosterone and testosterone and small quantities of 5β-androstanes. The kidney and liver cytosols were more effective in the production of epitestosterone with NADH than NADPH as coenzyme. The coenzymes had a reverse effect in the production of testosterone. Maximum production of epitestosterone occurred at pH 6.0 then gradually decreased with increase to pH 7.5. Testosterone production did not show a decrease until about pH 7.5. Liver homogenate stimulated the production of primarily highly polar metabolites. NADPH was more effective than NADH. This activity was localized in the microsomal fraction. Liver homogenate and microsomes also produced testosterone, epitestosterone and androstane-3α, 17β-diol. NADH was more effective than NADPH in the production of testosterone and epitestosterone but the reverse occurred in the production of 5β-androstane-3α,17β-diol. NADPH also effected the production of a small quantity of 3α-hydroxy-5β-androstan-17-one by both the homogenate and microsomes. The cytosol of the kidney converted testosterone only to traces of 5β-androstanes while the liver cytosol yielded appreciable quantities of 5β-androstane-3α,17β-diol and smaller quantities of the 3β-diol; NADPH was more effective than NADH. The NADPH system also produced androstenedione. Both the liver and kidney also exhibited strong 17β-hydroxy dehydrogenase activity. The activity in the cytosol of both tissues was stronger with NAD + than NADP + while it was much stronger with NAD + in the liver microsomes and with NADP + in the kidney microsomes. Spectrophotometric assay of the liver cytosol with NADP + revealed not only 17β-hydroxy dehydrogenase activity but also almost equal 17α-hydroxy and very high 3α-hydroxy dehydrogenase activity. The pH optimum for the 17β- and 17α-hydroxy activities was essentially the same, about 9.8, while that for the 3α-hydroxy activity was slightly higher, 10.5.

Influence of Castration and Testosterone Propionate on Cardiac Output, Renal Blood Flow, and Blood Volume in Mice

Summary The cardiac output and renal blood flow has been determined in mice by the adaptation of the 86Rb method of Sapir-stein. Cardiac output also was determined with [131I] albumin. Castration decreased the cardiac output, renal 86Rb uptake and renal blood flow. Testosterone propionate was ineffective after 2 days but within 7 days had restored these values to normal. Extension of the androgen treatment did not produce any further changes. The blood volume of the mice was decreased approximately 10% by castration and restored to normal by testosterone propionate.

The induction, purification and characterization of 17β-hydroxy-C19-steroid dehydrogenase of the female guinea pig kidney☆

The female guinea pig kidney cytosol and microsomes contain a trace or no detectable NADP+ or NAD+-linked dehydrogenase activity for several hydroxy-C19-steroids. The administration of testosterone induced a gradual increase in the 17β-C19-steroid dehydrogenase activity of the cytosol, but not of the microsomes, to the level in the male after 50 days. The induced enzyme was purified by (NH4)2SO4 precipitation, Sephadex G-75 nitration and DEAE-Sephadex A-50 and CM-Sephadex C-50 chromatography to give a protein which gave a molecular weight of 32,000 and a single protein and enzyme staining band on gel electrophoresis. The pure enzyme dissociated into three gel electrophoretic bands on removal of 2-mercaptoethanol from the solution and was restored by replacement of the mercaptoethanol. The enzyme in the cytosol, however, dissociated on storage at 4°C for 48 h or longer in the presence or absence of 2-mercaptoethanol. The KM value, steroid specificity, gel electrophoretic pattern, isoelectric value, pH optimum and NADP+ and NAD+ requirement were practically identical with those of the purified major 17β-hydroxy-C19-steroid dehydrogenase of the male guinea pig kidney. The several hydroxy-C19-steroid dehydrogenase activities of the liver were not influenced by testosterone administration. The highest enzyme activity was exhibited with 5β-dihydrotestosterone as substrate.

C19-steroid 5β-reductase and 3- and 17-oxidoreductases of adult male hamster kidney

Abstract Male hamster kidney cytosol exhibited strong 5β-reductase activity. Incubation of cytosol with [4- 14 C]-testosterone at pH 6.7 yielded 5β-DHT with minor quantities of 5β-androstane-3α,17β-diol and 5β-androstane-3β,17β-diol. Incubation with [4- 14 C]-androstendione yielded 5β-androstanedione and smaller quantities of testosterone, 5β-DHT, 3α-hydroxy-5β-androstan-17-one, 3β-hydroxy-5β-androstan-17-one and 5β-androstane-3α,17β-diol. The two major metabolites were progressively increased with increase in the concentration of the respective substrates but the other metabolites showed very little change. The metabolism of the respective substrates was progressively decreased with changes in pH of the incubation mixture from 6.0–7.5 accompanied by a parallel decrease in the formation of the respective major metabolites. NADPH was much more effective than NADH as coenzyme. The microsomes exhibited a trace of 5β-reductase activity only with NADPH and androstenedione. The kidney homogenate at pH 10.1 effectively converted [4- 14 C]-testosterone to [4- 14 C]-androstenedione. The dehydrogenase activity was present in the cytosol and microsomes. NAD + was more effective than NADP + in the cytosol and the reverse was indicated for the microsomes. Spectrophotometric assay revealed not only NADP + -linked Hβ-dehydrogenase activity but also a lower 3α-dehydrogenase activity but no detectable 3β- or 17α-dehydrogenase activity. NAD + -linked activity was not explored because of the interference by the very high endogenous NAD + -reduetase activity.

Partial purification and some properties of guinea pig kidney 17 β-hydroxy-C19 steroid dehydrogenase

Abstract Guinea pig kidney 17β-hydroxy-C19-steroid dehydrogenase was partially purified by a combination of streptomycin sulfate and ammonium sulfate fractionation, Sephadex filtration, DEAE-cellulose chromatography and a second Sephadex filtration. The specific activity of both TPN- and DPN-linked activities was increased fifty-fold and thirty two-fold for the respective recovered enzyme activities. The purest fraction was increased two hundred thirty-fold in specific activity. The partially purified enzyme sedimented as one symmetrical peak on ultracentrifugation. The s020, w was 3.0S and the D020, w was 7.89×10−7 cm2 sec−1. On disc electrophoresis the TPN- and DPN-linked 17β-OH C19-steroid dehydrogenase activities were revealed in five prominent bands; three weakly stained protein bands showed no enzyme activity. The two cofactor linked activities were not separated throughout the purification steps and had a ratio of 1 to 8 in favor of the TPN-linked activity. The molecular weight was 35,100, 31,600 and 31,200 by ultracentrifugation, Sephadex filtration, and disc electrophoresis respectively. No bound cofactor was detected in the partially purified enzyme fraction. Testosterone produced the highest activity among the steroids tested; 17β-estradiol elicited only slight activity. A crude preparation of the enzyme in 0.25 M sucrose was stable but readily lost activity after purification. Addition of 7mM β-mercaptoethanol and 0.25 M sucrose or 20% glycerol prevented the loss in activity. The enzyme activity was lost on making cytosol 4 M in urea or on dialysis against 8 M urea. The partially purified enzyme was labile to heat but was stable at 4° or −20°. Thiol-blocking agents inhibited the enzyme activity. There were 2.8 moles−SH/mole protein.

Heterogeneity of guinea pig kidney 17β-hydroxy-C-19-steroid dehydrogenase activity observed by disc gel electrophoresis

Abstract Disc electrophoresis of guinea pig kidney cytosol gave five bands which possessed both TPN- and DPN-linked 17β-hydroxy-C19-steroid dehydrogenase activities. The intensity of the enzyme activities decreased from the slowest to the fastest migrating band. Electrophoresis at pH 7.9 shifted the intensity of the bands to the opposite direction. Re-electrophoresis of the individual bands gave the same migration as in the original preparation. The multiple bands persisted during the various purification procedures. Storage at −20 C produced a shift in the intensity of the enzyme activities which was prevented and restored by the addition of mercaptoethanol. Electrophoresis at a series of gel concentrations indicated that the enzymes in the different bands were charge isomers rather than size isomers and were best explained as conformational isomers.

Subcellular localization of the C19-steroid reductase activities of female rat liver

Abstract The role of DPNH and TPNH in the reduction of [4- 14 C]-testosterone or [4- 14 C] and rostenedione by female rat liver homogenate and its subcellular fractions was studied. The extracted metabolites were separated by thin layer and paper chromatography. 5α-Androstane-3α, 17β-diol was the major metabolite. Androsterone and 5α-dihydrotestosterone occurred in lesser quantities and 5α-androstane-3β, 17β-diol, 5α-androstanedione and epiandrosterone in small amounts. Androstenedione (or testosterone) was produced only in the presence of DPNH. The omission of the coenzymes or TPNH-generating system resulted in only a trace amount of the metabolites.