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PubMed1997

The role of cyclic AMP production, calcium channel activation and enzyme activities in the inhibition of testosterone secretion by amphetamine.

1. The aim of this study was to investigate the mechanism by which amphetamine exerts its inhibitory effect on testicular interstitial cells of male rats. 2. Administration of amphetamine (10(-12)-10(-6) M) in vitro resulted in a dose-dependent inhibition of both basal and human chorionic gonadotropin (hCG, 0.05 iu ml(-1))-stimulated release of testosterone. 3. Amphetamine (10(-9) M) enhanced the basal and hCG-increased levels of adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation in vitro (P<0.05) in rat testicular interstitial cells. 4. Administration of SQ22536, an adenylyl cyclase inhibitor, decreased the basal release (P<0.05) of testosterone in vitro and abolished the inhibitory effect of amphetamine. 5. Nifedipine (10(-6) M) alone decreased the secretion of testosterone (P<0.01) but it failed to modify the inhibitory action of amphetamine (10(-10)-10(-6) M). 6. Amphetamine (10(-10)-10(-6) M) significantly (P<0.05 or P<0.01) decreased the activities of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), P450c17, and 17-ketosteroid reductase (17-KSR) as indicated by thin-layer chromatography. (t.l.c.). 7. These results suggest that increased cyclic AMP production, decreased Ca2+ channel activity and decreased activities of 3beta-HSD, P450c17, and 17-KSR are involved in the inhibition of testosterone production induced by the administration of amphetamine.

17-Hydroxysteroid Dehydrogenases↗
PubMed1988

Kinetics of a model for zymogen activation: the case of high activating enzyme concentrations.

Transient phase and steady state equations have been derived for the following enzyme activation mechanism: (formula; see text) in which the concentrations of activating enzyme, E, and substrate, A, are greatly in excess of that of zymogen, Ei. EEi, EEa, EaA and EaY are four intermediates; W is a peptide related from Ei during EEa formation and X and Y are the products of Ea reaction on A. From the general equations, approximate solutions under certain simplifying conditions have been derived. Finally, some formal particular cases of the above mechanism are considered.

Enzyme Activation↗
PubMed2000

GM1 synthase depends on N-glycosylation for enzyme activity and trafficking to the Golgi complex.

Glycosyltransferase cDNAs contain a variable number of potential N-glycosylation sites. Here we examined the occupancy and relevance for the activity and intracellular trafficking of the only potential N-glycosylation site of the mouse beta1,3galactosyltransferase (Gal-T2 or GA1/GM1/GD1b synthase) in Gal-T2 cDNA transfected CHO-K1 cells. Transfected cells synthesize a Golgi located active enzyme of 43 kDa whose N-glycan was metabolically labeled from [3H]mannose and was Endo-H sensitive. Inhibition of N-glycosylation by Tunicamycin or by point mutation of the N-glycosylation site resulted in the synthesis of a polypeptide of 40 kDa which lacked enzyme activity and was concentrated in the endoplasmic reticulum (ER). Inhibition of ER glucosidases by Castanospermine impaired the exit of a form of Gal-T2 having reduced enzyme activity from the ER. The N-terminal Gal-T2 domain (aa 1-52) was able to direct and to retain the green fluorescence protein in the Golgi complex. Taken together, these results indicate that Gal-T2 depends on N-glycosylation for its activity and for proper trafficking to, but not its retention in, the Golgi complex.

Animals↗
PubMed1994

Human beta 1,4 galactosyltransferase and alpha 2,6 sialyltransferase expressed in Saccharomyces cerevisiae are retained as active enzymes in the endoplasmic reticulum.

Biosynthesis and intracellular transport of recombinant human full-length beta 1,4 galactosyltransferase (GT) and full-length alpha 2,6 sialyltransferase (ST) were investigated in Saccharomyces cerevisiae. Recently, enzymic activity of recombinant GT (rGT) in crude homogenates of S. cerevisiae could successfully be demonstrated [Krezdorn, C., Watzele, G., Kleene, R. B., Ivanov, S. X. & Berger, E. G. (1993) Eur. J. Biochem. 212, 113-120]. In the present work, we show that, in yeast strains transformed with plasmid pDPSIA containing the cDNA coding for human ST, rST enzymic activity using asialo-fetuin or N-acetyllactosamine as acceptor substrates could readily be detected. Analysis by 1H-NMR spectroscopy of the disaccharide product of rGT, as recently reported, and the trisaccharide product of rST demonstrated that only the expected glycosidic linkages were formed. Following mechanical disruption of yeast cells, both enzymes sedimented with a fraction enriched in membranes of the endoplasmic reticulum (ER) and were activated by Triton X-100 3-5-fold. rGT and rST could be immunoprecipitated from their [35S]Met-labelled transformed yeast extracts using polyclonal antibodies raised against fusion proteins consisting of beta-galactosidase-GT or beta-galactosidase-ST, respectively, expressed in Escherichia coli. For rGT a single glycosylated form of apparent molecular mass 48 kDa was reported, but for rST two main bands corresponding to apparent molecular masses of 48 kDa and 44 kDa, respectively, were detected. Immunoprecipitation from either tunicamycin-treated [35S]Met-labelled transformed yeast cells or labelling with radio-active sugars both indicated that the 44-kDa form of rST was non-glycosylated and that the 48-kDa form of rST was core N-glycosylated. In addition, core glycosylation of both recombinant enzymes demonstrated that they were competent for translocation across the ER membranes. However, the 44-kDa form of rST was converted to the 48-kDa glycosylated form only slowly, suggesting a mechanism of posttranslational translocation. Absence of hyperglycosylation of rST and rGT in wild type and lack of the Golgi-specific man-alpha 1,6-man epitope suggest that the recombinant enzymes did not enter the yeast Golgi apparatus. These results indicated that both rGT and rST are retained as enzymically active enzymes in the ER of yeast and suggest a ribonucleoprotein-independent import of rST into the ER.

Biological Transport↗
PubMed1978

Interferon-associated, dsRNA-dependent enzyme activities in a mutant 3T6 cell engaged in the semiconstitutive synthesis of interferon.

Cytoplasmic extracts of untreated cultures of a virus-resistant mutant of mouse 3T6 cells, designated 3T6-VrB2, contain two double-stranded, RNA-activated enzyme activities associated with interferon action. These are the synthesis of a low molecular weight oligonucleotide inhibitor of cell-free protein synthesis from ATP, and the phosphorylation of a 67,000 dalton polypeptide by transfer of the gamma phosphate of ATP. Basal levels of both enzyme activities are detectable in extracts of untreated parental 3T6 cells, and are greatly enhanced upon interferon pretreatment. A procedure was developed, using a nonionic detergent to effect cell lysis, which allowed the analysis of the protein kinase activity from as few as 2 x 10(7) cells. Using this procedure, direct proportionalities were demonstrated between the concentration of interferon to which 3T6 cells were exposed, and both the level of protein kinase activity and the magnitude of the antiviral state were established in these cells. Furthermore, untreated cultures of 3T6-VrB2 exhibited both an antiviral state and an intracellular protein kinase activity equal to that of cultures of the parental 3T6 cells pretreated with a single concentration of mouse interferon.

Cell Line↗
PubMed1995

Simultaneous determination of enzyme activity and enzyme quantity in single human erythrocytes.

An integrated laser-based fluorescence enzyme assay and particle-counting immunoassay system with zeptomole sensitivity has been developed for simultaneous determinations of activity and concentration of enzymes in single erythrocytes. The product NADPH of the reaction between glucose-6-phosphate and NADP+ catalyzed by glucose-6-phosphate dehydrogenase (G6PDH) is monitored. Simultaneously, the agglutination of antibody-coated particles in the presence of G6PDH produces large particles which are counted by light scattering. The correlation between the two parameters indicates that on average only about 35% of the enzyme in individual cells is active. There is more than a 10-fold cell-to-cell variation in the ratios of the two parameters. This indicates that there are multiple mechanisms for the degradation of intracellular enzymes as a function of cell age.

Adult↗
PubMed1989

Nitrogen Assimilating Enzyme Activities and Enzyme Protein during Development and Senescence of Effective and Plant Gene-Controlled Ineffective Alfalfa Nodules.

Effective (N(2)-fixing) alfalfa (Medicago sativa L.) and plant-controlled ineffective (non-N(2)-fixing) alfalfa recessive for the in(1) gene were compared to determine the effects of the in(1) gene on nodule development, acetylene reduction activity (ARA), and nodule enzymes associated with N assimilation and disease resistance. Effective nodule ARA reached a maximum before activities of glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AAT), asparagine synthetase (AS), and phosphoenolpyruvate carboxylase (PEPC) peaked. Ineffective nodule ARA was only 5% of effective nodule ARA. Developmental profiles of GS, GOGAT, AAT, and PEPC activities were similar for effective and ineffective nodules, but activities in ineffective nodules were lower and declined earlier. Little AS activity was detected in developing ineffective nodules. Changes in GS, GOGAT, AAT, and PEPC activities in developing and senescent effective and ineffective nodules generally paralleled amounts of immunologically detectable enzyme polypeptides. Effective nodule GS, GOGAT, AAT, AS, and PEPC activities declined after defoliation. Activities of glutamate dehydrogenase, malate dehydrogenase, phenylalanine ammonia lyase, and caffeic acid-o-methyltransferase were unrelated to nodule effectiveness. Maximum expression of nodule N-assimilating enzymes appeared to require the continued presence of a product associated with effective bacteroids that was lacking in in(1) effective nodules.

Journal Article↗
PubMed1999

Inhibition of aryl hydrocarbon-induced cytochrome P-450 1A1 enzyme activity and CYP1A1 expression by resveratrol.

We investigated the effect of resveratrol, a constituent of the human diet that has been shown to inhibit aryl hydrocarbon-induced carcinogenesis in animals, on the carcinogen activation pathway regulated by the aryl hydrocarbon receptor. Resveratrol inhibited the metabolism of the environmental aryl hydrocarbon benzo[a]pyrene (B[a]P) catalyzed by microsomes isolated from B[a]P-treated human hepatoma HepG2 cells. Resveratrol competitively inhibited, in a concentration-dependent manner, the activity of the carcinogen activating enzymes cytochrome P-450 (CYP)1A1/CYP1A2 in microsomes and intact HepG2 cells. Resveratrol inhibited the B[a]P-induced expression of the CYP1A1 gene, as measured at the mRNA and transcriptional levels. Resveratrol abolished the binding of B[a]P-activated nuclear aryl hydrocarbon receptor to the xenobiotic-responsive element of the CYP1A1 promoter but did not itself bind to the receptor. Resveratrol was also effective in inhibiting CYP1A1 transcription induced by the aryl hydrocarbon dimethylbenz[a]anthracene in human mammary carcinoma MCF-7 cells. These data demonstrate that resveratrol inhibits aryl hydrocarbon-induced CYP1A activity in vitro by directly inhibiting CYP1A1/1A2 enzyme activity and by inhibiting the signal transduction pathway that up-regulates the expression of carcinogen activating enzymes. These activities may be an important part of the chemopreventive activity of resveratrol in vivo.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1973

Activities of enzymes responsible for steroid biosynthesis and cholesterol ester metabolism in rabbit ovarian interstitial tissue and corpora lutea. A comparison of enzyme activities with flow rates.

A method involving the use of isolated cholesterol ester-storage granules as substrate is described for the assay of cholesterol esterase in rabbit ovarian tissues. Activities of cholesterol esterase 100-200-fold higher than those previously reported in ovarian tissues were measured by using this method. In addition to that of cholesterol esterase, activities of cholesterol ester synthetase, cholesterol side-chain cleavage enzyme and 3beta-hydroxy steroid dehydrogenase were determined in rabbit ovarian interstitial tissue and corpora lutea. Activities of these enzymes are in general compatible with the flows through them measured under a variety of conditions both in vivo and in vitro. It is concluded that, in the rabbit ovarian tissues investigated, these enzymes are capable of catalysing the conversions usually attributed to them.

Acyltransferases↗
PubMed1996

Regulating expression of the gene for matrix metalloproteinase-1 (collagenase): mechanisms that control enzyme activity, transcription, and mRNA stability.

Matrix metalloproteinase-1 (MMP-1) is one of three collagenases that can degrade the interstitial collagens, types I, II, and III at neutral pH. As these collagens are the most abundant proteins in the body, collagenase plays a critical role in modeling and remodeling the extracellular matrix. Therefore, it is not surprising that MMP-1 gene expression can be regulated at multiple points. Procollagenase can be activated by mechanisms that generate an active enzyme with differing specific activities, and the active enzyme can be inhibited by complexing with either the tissue inhibitor of metalloproteinases (TIMPs) or alpha 2 macroglobulin. The activator protein-1 (AP-1) site in the collagenase promoter plays a prominent role in the transcriptional control of the collagenase gene. It is essential for basal transcription, and contributes to induction by phorbol esters, although other sites in the proximal promoter are essential. In contrast, transactivation by cytokines such as Interleukin-1 depends on sequences in more distal regions of the promoter. Posttranscriptional mechanisms also regulate gene expression, and several cytokines and growth factors increase the stability of the collagenase transcript. Finally, glucocorticoid hormones repress transcription of the collagenase gene by the interaction of glucocorticoid receptors with the AP-1 proteins, Fos and Jun. Retinoids also suppress transcription by mechanisms that involve down-regulation of fos and jun mRNA, sequestration of Fos and Jun proteins, and the formation of complexes of retinoic acid receptors (RAR/RXR heterodimers) and AP-1 proteins on the DNA. These multiple points of regulation assure precise control of collagenolytic activity in a variety of physiologic and pathologic conditions.

Amino Acid Sequence↗
PubMed1984

Species difference between rats and mice in activities of enzymes activating aromatic amines: effect of dietary 3-methoxy-4-aminoazobenzene.

Male Donryu rats and B6C3F1 mice were treated with dietary 3-methoxy-4-aminoazobenzene (3-MeO-AAB) (0.06% or 0.09%), and subcellular fractions of the liver were examined for ability to mutagenically activate 3-MeO-AAB and 2 amino acid pyrolysate components using Salmonella typhimurium TA 98 as a tester strain. The treatment resulted in striking induction of enzyme(s) for the mutagenic activation of these aromatic amines in rats, but the effect was smaller in mice. The enzyme(s) involved in the reaction was characterized as an isotype of microsomal cytochrome P-448.

Aminopyrine N-Demethylase↗
PubMed1991

Biosensor system for continuous flow determination of enzyme activities. II. Simultaneous determination of plural enzyme activities.

A biosensor system for continuous flow determination of plural enzyme activities was prepared from the combination of two pyruvate sensors, a prereactor and a flow cell. This system was applied to the simultaneous determination of lactic dehydrogenase (LDH) and glutamic-pyruvic transaminase (GPT) activities in the same sample. These enzyme activities can be determined by measuring pyruvate produced by the enzyme reactions as follows. The amount of pyruvic acid can also be determined from the amount of oxygen consumed upon oxidation of pyruvic acid by pyruvate oxidase. (Formula: see text). Therefore, both of the detectors for the determination of lactic dehydrogenase and glutamic-pyruvic transaminase activities were prepared from the combination of a pyruvate oxidase membrane and an oxygen electrode. Pyruvate oxidase was covalently immobilized on a membrane prepared from cellulose triacetate. A linear relation was obtained between the output current and LDH or GPT activities in the range of 50 to 3,600 IU l-1 or 6 to 1,000 IU l-1, respectively. Each assay of these enzyme activities was completed within 15 min. The results obtained had a precision of ca. 4%. The sensor was stable for more than 25 days at 5 degrees C.

Alanine Transaminase↗
PubMed1991

Chymotrypsinogen: an activating enzyme in the mitochondrial membrane of rat submaxillary gland.

A peripheral membrane protease was purified from mitochondria of rat submaxillary gland. On non-denaturing PAGE the purified enzyme showed a single protein band with the enzyme activity. It yielded two protein bands with molecular weights of 39 KDa and 20 KDa on SDS-PAGE, indicating that the enzyme is composed of two protein components. The enzyme activity was strongly inhibited by SBTI, aprotinin and benzamidine. PMSF, TLCK and EDTA did not produce inhibition. The enzyme could hydrolyze different synthetic substrates having arginine at the P1 position with highest affinity for the substrate Bz-Phe-Val-Arg-p-nitroanilide was noted. The enzyme showed significant activation of chymotrypsinogen A.

Animals↗
PubMed2002

Enzyme activation for nonaqueous media.

Highly active enzyme formulations can be prepared for use in nonaqueous media. Considerable progress has been made in the past two years on gaining an improved mechanistic understanding of enzyme function and activation in dehydrated environments. This increased fundamental understanding has led to the development of a broad array of techniques for generating active, stable, and enantioselective and regioselective tailored enzymes for synthetically relevant transformations. This, in turn, is resulting in an exponential increase in the opportunities for enzymatic processes to be developed on a commercial scale.

Catalysis↗
PubMed2000

Pyruvate formate-lyase-activating enzyme: strictly anaerobic isolation yields active enzyme containing a [3Fe-4S](+) cluster.

Pyruvate formate-lyase-activating enzyme (PFL-AE) from Escherichia coli (E. coli) catalyzes the stereospecific abstraction of a hydrogen atom from Gly734 of pyruvate formate-lyase (PFL) in a reaction that is strictly dependent on the cosubstrate S-adenosyl-l-methionine (AdoMet). Although PFL-AE is an iron-dependent enzyme, isolation of the enzyme with its metal center intact has proven difficult due to the oxygen sensitivity and lability of the metal center. We report here the first isolation of PFL-AE under nondenaturing, strictly anaerobic conditions. Iron and sulfide analysis as well as UV-visible, EPR, and resonance Raman data support the presence of a [3Fe-4S](+) cluster in the purified enzyme. The isolated native enzyme, but not apo-enzyme, exhibits a high specific activity (31 U/mg) in the absence of added iron, indicating that the native cluster is necessary and sufficient for enzymatic activity.

Acetyltransferases↗
PubMed2001

Functional and phylogenetic analysis of the ubiquitylation system in Caenorhabditis elegans: ubiquitin-conjugating enzymes, ubiquitin-activating enzymes, and ubiquitin-like proteins.

BACKGROUND: The eukaryotic ubiquitin-conjugation system sets the turnover rate of many proteins and includes activating enzymes (E1s), conjugating enzymes (UBCs/E2s), and ubiquitin-protein ligases (E3s), which are responsible for activation, covalent attachment and substrate recognition, respectively. There are also ubiquitin-like proteins with distinct functions, which require their own E1s and E2s for attachment. We describe the results of RNA interference (RNAi) experiments on the E1s, UBC/E2s and ubiquitin-like proteins in Caenorhabditis elegans. We also present a phylogenetic analysis of UBCs. RESULTS: The C. elegans genome encodes 20 UBCs and three ubiquitin E2 variant proteins. RNAi shows that only four UBCs are essential for embryogenesis: LET-70 (UBC-2), a functional homolog of yeast Ubc4/5p, UBC-9, an ortholog of yeast Ubc9p, which transfers the ubiquitin-like modifier SUMO, UBC-12, an ortholog of yeast Ubc12p, which transfers the ubiquitin-like modifier Rub1/Nedd8, and UBC-14, an ortholog of Drosophila Courtless. RNAi of ubc-20, an ortholog of yeast UBC1, results in a low frequency of arrested larval development. A phylogenetic analysis of C. elegans, Drosophila and human UBCs shows that this protein family can be divided into 18 groups, 13 of which include members from all three species. The activating enzymes and the ubiquitin-like proteins NED-8 and SUMO are required for embryogenesis. CONCLUSIONS: The number of UBC genes appears to increase with developmental complexity, and our results suggest functional overlap in many of these enzymes. The ubiquitin-like proteins NED-8 and SUMO and their corresponding activating enzymes are required for embryogenesis.

Amino Acid Sequence↗
PubMed2004

Computational design of a biologically active enzyme.

Rational design of enzymes is a stringent test of our understanding of protein chemistry and has numerous potential applications. Here, we present and experimentally validate the computational design of enzyme activity in proteins of known structure. We have predicted mutations that introduce triose phosphate isomerase activity into ribose-binding protein, a receptor that normally lacks enzyme activity. The resulting designs contain 18 to 22 mutations, exhibit 10(5)- to 10(6)-fold rate enhancements over the uncatalyzed reaction, and are biologically active, in that they support the growth of Escherichia coli under gluconeogenic conditions. The inherent generality of the design method suggests that many enzymes can be designed by this approach.

Algorithms↗
PubMed1990

Effects of the tumor promoter 12-O-tetradecanoylphorbol 13-acetate on peroxisomal activities and enzyme activities involved in lipid metabolism in rat liver.

The effects of 12-O-tetradecanoylphorbol 13-acetate (TPA) on hepatic lipids and key enzymes involved in esterification, hydrolysis and oxidation of long-chain fatty acids at increasing doses were investigated in rats. TPA administration tended to decrease the mitochondrial activities of palmitoyl-CoA synthetase and carnitine palmitoyltransferase. The microsomal palmitoyl-CoA synthetase activity was increased. TPA administration was also associated with a dose-dependent increase of glycerophosphate acyltransferase activity both in the mitochondrial and microsomal fractions in particular. The data are consistent with a decreased catabolism of long-chain fatty acids at the mitochondrial level, and an increased capacity for esterification of fatty acids in the microsomal fraction. Peroxisomal beta-oxidation was increased about 2-fold in the peroxisome-enriched fraction of TPA-treated rats while the catalase and urate oxidase activities were only marginally affected. TPA administration revealed elevated capacity for hydrolysis of palmitoyl-CoA and palmitoyl-L-carnitine in the microsomal fraction. Neither increased cytosolic palmitoyl-CoA hydrolase activity nor increased hydroxylation of lauric acid nor changes of the hepatic content of cytochrome P-450 isoenzymic forms were observed in the TPA-treated animals. There was no induction of the protein content of the bifunctional enoyl-CoA hydratase. Thus, TPA behaves more like choline-deficient diet and ethionine treatment than well-known peroxisome proliferators. It seems possible that TPA selectively stimulated the peroxisomal activities, i.e., peroxisomal beta-oxidation rather than evoking a peroxisome proliferation capacity.

Animals↗