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J H Shim

Publications and source records attributed to J H Shim.

43 records · Page 3Linked to original sources
PubMed1995

Trichodiene synthase. Identification of active site residues by site-directed mutagenesis.

Derivatization of 5,5'-dithiobis(2-nitrobenzoic acid)-treated trichodiene synthase with [methyl-14C]methyl methanethiosulfonate and analysis of the derived tryptic peptides suggested the presence of two cysteine residues at the active site. The corresponding C146A and C190A mutants were constructed by site-directed mutagenesis. The C190A mutant displayed partial but significantly reduced activity, with a reduction in kcat/Km of 3000 compared to the wild-type trichodiene synthase, while the C146A mutant was essentially inactive. A hybrid trichodiene synthase, constructed from amino acids 1-309 of the Fusarium sporotrichioides enzyme and amino acids 310-383 of the Gibberella pulicaris cyclase, had steady state kinetic parameters nearly identical to those of the wild-type F. sporotrichioides enzyme. From this parent hybrid, a series of mutants was constructed by site-directed mutagenesis in which the amino acids in the base-rich region, 302-306 (DRRYR), were systematically modified. Three of these mutants were overexpressed and purified to homogeneity. The importance of Arg304 for catalysis was established by the observation that the R304K mutant showed a more than 25-fold increase in Km, as well as a 200-fold reduction in kcat. In addition, analysis of the incubation products of the R304K mutant by gas chromatography-mass spectrometry (GC-MS) indicated that farnesyl diphosphate was converted not only to trichodiene but to at least two additional C15H24 hydrocarbons, mle 204. Replacement of the Tyr305 residue of trichodiene synthase with Phe had little effect on kcat, while increasing the Km by a factor of ca. 7-8.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Cycloserine-induced lichenoid drug eruption.

A 65-year-old male patient who has taken antituberculous drugs including cycloserine for 8 months showed multiple pruritic, violaceous, flat-topped papules and plaques on his back which had developed 5 months into therapy. Histopathologic findings were consistent with lichenoid drug eruption. Patch test with the drugs revealed a strongly positive reaction to cycloserine only. Four months after discontinuation of oral cycloserine, pruritus resolved and the lesions subsided without any new lesions. Cycloserine is one of the antituberculous drugs. Skin manifestations resulting from its administration are reported to be rare, but to our knowledge, this is the first case report of lichenoid drug eruption from its use.

Aged↗
PubMed1993

Irreversible inactivation of monoterpene cyclases by a mechanism-based inhibitor.

Monoterpene synthases (cyclases) catalyze the divalent metal ion-dependent transformation of geranyl pyrophosphate to representative of the various monocyclic and bicyclic skeletal types by an electrophilic reaction mechanism involving coupled isomerization and cyclization steps. An analogue of the geranyl substrate, in which the terminal gem-dimethyl groups were joined to form a cyclopropyl function (6-cyclopropylidene-3E-methyl-hex-2-en-l-yl pyrophosphate) was shown to be a potent inhibitor of (-)-4S-limonene synthase from Mentha spicata and of several other monoterpene cyclases from diverse plant species. Inhibition was concentration and time dependent (pseudo-first-order kinetics), as well as absolutely contingent on the presence of the divalent metal ion cofactor. A double reciprocal plot of kinactivation versus inhibitor concentration gave an apparent Ki of approximately 0.3 microM and a maximum rate of inactivation of about 0.3 min-1 with limonene synthase. As expected for an active-site-directed process, the natural substrate, geranyl pyrophosphate, afforded protection against inactivation by the cyclopropylidene analogue. Selectivity of the inhibition was demonstrated with [1-3H]6-cyclopropylidene-3E-methyl-hex-2-en-1-yl pyrophosphate by specific labeling of limonene synthase in crude enzyme extracts as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, radio-fluorography, and immunoblotting. The radioactive cyclase-inactivator complex was formed with 1:1 stoichiometry and was stable to extended dialysis and boiling in 2% sodium dodecyl sulfate, suggesting irreversible covalent modification of the enzyme involving a chemical reaction between cyclase and inhibitor. Thermally denatured limonene synthase and synthase that had been inactivated with the histidine-directed reagent diethylpyrocarbonate or the cysteine-directed reagent p-hydroxymercuribenzoate (two reagents known to modify the active site of the enzyme and inhibit catalysis) were not labeled when treated with the [1-3H]-analogue, indicating that the functional enzyme was necessary to effect complex formation. All of the evidence is consistent with the analogue serving as a mechanism-based inactivator that must undergo both ionization-dependent isomerization and cyclization steps to reveal an allylic cation which alkylates the protein. In addition to furnishing supporting evidence for the electrophilic reaction sequence, this mechanism-based inactivator provides a powerful new approach for the examination of cyclase active sites.

Intramolecular Lyases↗
PubMed1985

Aromatase activity in the perinatal rat forebrain: effects of age, sex and intrauterine position.

Sex and regional differences in aromatase activity were characterized in brains of rats sacrificed on embryonic days 18 (E18) and 20 (E20) and on postnatal days 2, 4 and 13 (D2, D4, D13). Aromatase activity was measured in vitro in homogenates of the hypothalamus/preoptic area (HPOA) and temporal lobe (TL) from individual rats, using [3H]19-hydroxyandrostenedione as substrate in the presence of NADPH. The apparent Km of aromatase for 19-hydroxyandrostenedione in TL at D4 was similar in males (34 nM) and females (22 nM,). Aromatase activity in the HPOA was highest prenatally (E18) and gradually declined to low levels by D13. Aromatase activity in the TL remained constant from E18 to D2, increased significantly on D4, and subsequently declined to low levels by D13. The level of aromatase activity was significantly greater in males than in females on E18 and D4 in the HPOA and on D4 in the TL. Differences in aromatase activity within regions of the HPOA were studied at E20 and D4. At both ages, the activity was highest in the preoptic area, lower in the anterior hypothalamus (AH), and lowest in the posterior hypothalamus. Aromatase activity was significantly higher in males than in females in the AH, but only on D4. The contiguity of males in utero was not correlated with aromatase activity in brain regions of adjacent female fetuses. The present results indicate that significant sex differences in aromatase activity exist in specific brain regions only at discrete times during perinatal development.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗
PubMed1985

Androgen aromatization and 5 alpha-reduction in ferret brain during perinatal development: effects of sex and testosterone manipulation.

Ferrets of both sexes were killed 8 or 5 days before expected parturition as well as 7, 15, 30, or 51 days after birth, and the activities of aromatase (using 19-[3H]hydroxyandrostenedione as substrate) and of 5 alpha-reductase (using [3H] testosterone as substrate) were assayed in whole homogenates of preoptic area plus anterior hypothalamus (POA), mediobasal hypothalamus (MBH), temporal lobe (TL), and cerebral cortex. Aromatase and testosterone 5 alpha-reductase activities were also measured in these regions in adult gonadectomized male and female ferrets. Compared with adults of both sexes in which aromatase activity was low in all brain regions studied, fetal ferrets had high levels of aromatase activity in POA plus MBH and in TL. At these prenatal ages, aromatase activity in POA plus MBH was significantly higher in males than in females. Aromatase activity in POA, MBH, and TL remained high in both sexes on postnatal days 7, 15, and 30, before declining by postnatal day 51. Cortical aromatase activity was uniformly low across all perinatal ages. The existence of a sex difference in aromatase activity in fetal POA plus MBH cannot be explained by a concurrent sex difference in circulating testosterone. Administration of testosterone to pregnant female ferrets over days 30-41 of gestation caused 150- to 350-fold increases in maternal plasma concentrations of testosterone and 2- to 5-fold increases in fetal plasma testosterone. However, aromatase activity was not affected in the POA and MBH of fetuses or mothers, although activity was significantly increased in the TL of mothers given testosterone. Furthermore, castration of neonatal or adult breeding males decreased plasma androgen levels by factors of 8 and 480, respectively, but resulted in only modest reductions in POA, MBH, and TL aromatase activity (a significant reduction occurred only in the adult male TL). Relatively high levels of testosterone 5 alpha-reductase activity were found in all brain regions across all perinatal ages, as well as in gonadectomized adult ferrets; there was no sex differences at any postnatal age studied. Prenatally, males had higher levels of 5 alpha-reductase activity than females only on day -8 in the POA plus MBH. The results show that estrogen and 5 alpha-reduced androgens can be synthesized in the brains of ferrets of both sexes during the perinatal period of sexual differentiation. A functional role for this neural metabolism of androgen remains to be demonstrated in this carnivorous species.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1983

Normal differentiation of masculine sexual behavior in male ferrets despite neonatal inhibition of brain aromatase or 5-alpha-reductase activity.

Male ferrets born in the laboratory received subcutaneous Silastic capsules containing either the aromatase inhibitor, androst-1,4,6-triene-3, 17-dione (ATD), the 5 alpha-reductase inhibitor, testosterone-17 beta-carboxylic acid (17 beta C), or no hormone, for 15 days beginning on the day of birth; an additional group of females received empty Silastic capsules. All ferrets were gonadectomized when 11 weeks of age and were subsequently tested for masculine sexual behavior after a latin-square sequence of treatments with subcutaneous Silastic capsules containing testosterone (T), estradiol (E), or dihydrotestosterone (DHT). After T, control males displayed significantly more neck gripping, mounting and pelvic thrusting than control females, and males treated neonatally with ATD or 17 beta C were no less responsive than control males. After DHT, little masculine sexual behavior was shown by any group. After E, the duration of mounting was significantly longer in control and ATD males than in control females or 17 beta C males. Subsequently, however, there were no differences between control and 17 beta C males on any parameter of masculine sexual performance, when they were retested sequentially after subcutaneous implantation of E followed by E + DHT. Additional groups of newborn male and female ferrets received subcutaneous capsules containing either ATD, 17 beta C, or no hormone and were killed on postnatal day 7. Administration of ATD, but not 17 beta C, strongly inhibited aromatase activity in the hypothalamus + preoptic area. In all groups, the formation of significantly inhibited cortical 5 alpha-reductase activity. Plasma concentrations of T were equivalent on postnatal day 7 in males given each of the neonatal treatments. These results suggest that behavioral masculinization in the male ferret results primarily from the neonatal action in brain of T itself, and not from its estrogenic or 5 alpha-reduced androgenic metabolites.

5-alpha Reductase Inhibitors↗