Philip A. Katzman

Studies on beta-glucuronidase from E. coli.

Summary Potent culture fluid preparations of β-glucuronidase are obtained when E. coli is grown at 25°C and pH 7.3 in a simple medium containing menthyl-β-glucuronide. The optimum incubation time is 8 days when continuous agitation is employed. The optimum pH for the hydrolysis of phenolphthalein mono-β-glucuronidei is 6.2, while that for estriol-4bT-glucuronide is 4.5-7.0. Bacterial β-glucuronidase is readily inhibited by ammonium ion. Dialysis markedly decreases the enzymatic activity of our preparations. The culture fluid contains phosphatase but no sulfatase activity.

Identification of estra-1,3,5 (10) ,16-tetraen-3-ol (estratetraenol) from the urine of pregnant women (1)

Abstract The neutral fraction obtained from enzymically hydrolyzed human late-pregnancy urine was subjected to thin layer chromatography in benzene. By means of Haennis iron-Kober reagent B; a zone corresponding to the mobility and color of estratetraenol was detected. Further purification was carried out by chromatography on silicic acid columns using ethyl acetate:cyclohexane (1:9). The fractions which contained the estratetraenol-like material were collected and methylated. The 3-methyl ether derivative of the urinary component and authentic estratetraenol exhibited identical chromatographic and chromogenic behavior. Analysis of the trimethylsilyl ether derivative of the urinary component in the LKB Gas Liquid Chromatograph-Mass Spectrometer showed a major fraction having a retention time and mass spectrum corresponding to those obtained with the authentic estrogen. Preliminary studies indicated that estratetraenol was present predominantly as the glucosiduronate, in a concentration of approximately 100 μg/liter of urine.

Hydrolysis of conjugated steroids by bacterial glucuronidase.

Summary Bacterial glucuronidase which hydrolyzes the glucuronides of estriol, pregnanediol, menthol and phenolphthalein liberates a large proportion of estrogen, ketosteroid and corticosteroid of human urine.

A Quantitative Procedure for Determining Normal Excretion of Prolan

Several methods have been developed for the preparation of active extracts of the gonad-stimulating substance of pregnancy urine. Although some of these procedures give good recoveries from pregnancy urine no one has reported a satisfactory procedure for the quantitative estimation of the small quantities of prolan that occur in normal urines. The excretion of prolan in conditions unassociated with pregnancy has been determined exclusively with untreated urine or by means of Zondeks 1 alcohol precipitation method which gives only a 5-fold concentration. Since alcohol causes an appreciable loss of activity (Wiesner and Marshall, 2 Katzman and Doisy, 3 ) this procedure is not suitable for use in normal excretion of the anterior pituitary-like substance. In attempting to purify pregnancy urine extracts by means of various protein precipitants we observed that precipitation with tungstic acid invariably gave remarkably complete recovery. Wiesner and Marshall 2 and Zondek, Scheibler and Krabbe 4 found that phosphotungstic acid quantitatively precipitates the active material of pregnancy urine but they have not reported studies on the recovery of the small amounts of prolan which occur in normal urine. We have not used phosphotungstic acid but it is evident from Table I that our tungstic acid method gives a surprisingly good recovery of minute quantities. The procedure is as follows: A 24-hour urine sample to which has been added 10 cc. of 10% sodium tungstate and 10 cc. of 0.5% casein is made faintly acid to congo red with dilute H2SO4. The precipitate is collected by centrifugation, washed with acetone, freed from acetone by reduced pressure, and dissolved by adding dilute NaOH until the mixture is faintly alkaline to phenolphthalein. BaCl2 and Ba(OH)2 are then added in the proportion necessary to prevent the solution from becoming acid, until precipitation is complete.

Separation of methylated bases of ribonucleic acid by two-dimensional thin-layer chromatography

Abstract The separation of fourteen methylated purine and pyrimidine bases that have been reported to occur in transfer RNA was investigated by thin-layer chromatography using two solvent systems and five adsorbants containing various percentages of Silica Gel GF and Avicel microcrystalline cellulose. By increasing the amount of cellulose in the adsorbant, disproportionate decreases in the R F values of the methylated bases were observed in both solvent systems. From these data a two-dimensional thin-layer chromatographic system has been developed that satisfactorily resolves a mixture of the fourteen methylated bases employed in this investigation.

Hydrolysis of Conjugates of Neutral Ketosteroids

Summary The hydrolysis of steroid conjugates has been studied by 5 methods. Continuous ether extraction of urine made 7.2 N with respect to HCl gave values appreciably higher than those obtained by boiling with 15 vol. % HC1 and these values approximated the sum of ketosteroids released by β-glucuronidase and by continuous extraction with ether of urines brought to pH 0.7 with HCl

Metabolism of progesterone in mouse vaginal tissue

Abstract The in vitro and in vivo metabolism of 1,2- 3H-progesterone was studied in estrogen-stimulated and control vaginae of ovariectomized mice. Employing two-dimensional thin-layer chromatography, gas-liquid chromatography and metabolite “trapping” techniques, the major and minor pathways for progesterone metabolism were determined in vitro and shown to involve saturation of the Δ4-double bond to yield 5α-pregnane compounds and reduction of the C20 and C3 ketone groups to form 20α- and 3α- and 3β-hydroxy derivatives, respectively. The quantities of 20β-hydroxy metabolites and 5β-epimers that were detected were considered not to be significant. The major metabolites formed by untreated tissues following in vitro incubation in the presence of both high (10−6M) and low (10−8M) progesterone concentrations were 3α-hydroxy-5α-pregnan-20-one and 5α-pregnane-3,20-dione. Although these two derivatives were also found in sizable quantities in estrogen-treated tissues, a marked increase (5-fold) in the rate of C20 ketone reduction at high progesterone concentrations (10−6M) to yield 20α-hydroxy-4-pregnen-3-one was demonstrated. Following intravaginal administration of 3H-progesterone in vivo , only progesterone and 3α-hydroxy-5α-pregnan-20-one were retained in appreciable quantities through 2 hr, suggesting rapid loss of 20α-hydroxy-4-pregnen-3-one and the 5α-pregnanediols from this tissue under in vivo conditions.

Hydrolysis of urinary estrogen and 17-ketosteroid glucosiduronides by bacterial and mammalian β-glucuronidases☆☆☆

Abstract The β-glucuronidase prepared from Escherichia coli was found to be much more effective than that obtained from beef liver in the hydrolysis of estrogen and 17-ketosteroid glucosiduronides of human pregnancy urine. Incubation at 50 ° improved the effectiveness of the mammalian enzyme, permitting maximal liberation of 17-ketosteroids but not of estrogens after 24 hr. of incubation. Excessively long periods of incubation with the bacterial enzyme lowered the yield of estrogen but not of 17-ketosteroid. The liberation of estriol from purified estriol glucosiduronides in buffered aqueous solution took place more rapidly with bacterial β-glucuronidase than with the beef-liver enzyme and was more rapid than the liberation of estrogen from pregnancy urine by these enzymes. From 50 to 70% of the activity of both the bacterial and mammalian enzyme preparations added to pregnancy urine was lost after 24 hr. of incubation at 37 ° as measured by the hydrolysis of phenolphthalein glucosiduronide. The inactivation of the bacterial enzyme was accelerated, rather than retarded, by the adition of CHCl3 and EDTA and reduced the yields of estrogen and 17-ketosteroids. Since the comparative activities of these β-glucuronidase preparations toward phenolphthalein glucosiduronide do not parallel their activities toward the steroid glucosiduronides in pregnancy urine, it is evident, that the ability to hydrolyze the former is not an adequate criterion for judging the effectiveness of these enzymes toward other substrates.

Methylation of the guanine bases of transfer RNA

Summary The extent of methylation of the guanine bases of tRNA was assessed by labeling KB cells with [2- 14 C]-guanosine under a variety of pulse and pulse-chase conditions. Approximately 95% of the radioactivity incorporated into tRNA was recovered and identified as [2- 14 C]-guanine and 1-, 7-, N 2 - and N 2 N 2 -methyl-[2- 14 C]-guanine. Analysis of the distribution of these constituents under long-pulse and pulse-chase conditions revealed that a maximum of 9.6% of the guanine bases of tRNA became methylated. As reflected by short pulses, the formation of N 2 -methylguanine occurred predominantly at a later stage of tRNA maturation than did 1-, 7- and N 2 N 2 -methylguanine.

The methylation of tRNA in KB cells after treatment with actinomycin D

Abstract Extensive shifts in the distribution of labeled methylated constituents of tRNA were observed in KB cells treated with actinomycin D for 30 min prior to a 90-min pulse with 3H-CH3-methionine. Although this treatment completely blocked the synthesis of tRNA, methylation continued to the extent of 12–15% of controls (pulsed without antibiotic). Under this condition the relative proportion of radioactivity incorporated into 3-methylcytosine, N2-methylguanine and 2′-O-methylribose was markedly increased (170–235% of control values), it was moderately reduced in 1-methyladenine, 5-methyl-cytosine and 5-methyluracil (35–70% of controls) and markedly reduced in 1-, 7- and N2N2-methylguanines (15–30% of controls). These data suggest that specific types of methylations occur at particular times during the processing of pre-tRNAs.