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PubMed1984

Enzyme histochemical differentiation of white adipose tissue in the rat.

Subcutaneous adipose tissues from fetal and young rats were studied with enzyme histochemical techniques. Lipid staining and histological evaluation were also utilized to compare the development of a wide variety of enzyme activities to cytoplasmic lipid deposition and morphological differentiation of adipocytes. Three distinct stages of adipose-tissue differentiation were postulated. In stage III, adipocytes were morphologically differentiated (rounded, basal-lamina positive) and enzyme reactive for many enzymes. In stage II, however, adipocytes were reactive for some enzymes but were not morphologically differentiated. Stage I adipose tissue was histologically distinct from connective tissue but did not contain lipid-laden cells or enzyme-reactive cells. Stages I and II (95%) were predominant in fetuses, whereas stage III (90%) was predominant in young animals. Histochemical analysis of adipocytes in newborn rats established the metabolic competence of these cells despite their small size. These studies indicate that enzymatic differentiation of adipocytes clearly precedes morphological differentiation.

Adipose Tissue↗
PubMed1993

AMP-deaminase from human uterine smooth muscle: the effect of DTNB treatment on kinetic and regulatory properties of the enzyme.

Reactivity of sulfhydryl groups of human uterine smooth muscle AMP-deaminase with DTNB, and the effect of their chemical modification on kinetic and regulatory properties of the enzyme were investigated. (1), Approx. 7 and 5 sulfhydryl groups per mol of the enzyme have been shown to be accessible for DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) titration in denaturated and native AMP-deaminase, respectively. (2), Titrated groups were not homogenous; some of them reacted with DTNB much faster than others. (3), The activity of the modified enzyme was very low, and the modified enzyme manifested unusual hyperbolic saturation kinetics with the substrate. (4), Exhaustive dialysis against a buffer containing 10 mM thioethanol reactivated the modified enzyme, and restored its original regulatory properties. Experimental results obtained indicate that modified sulfhydryl groups play a significant role in the maintenance of the proper, catalytically-efficient conformation of the enzyme.

AMP Deaminase↗
PubMed1991

Analysis of the metal requirement of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli.

The three isozymes of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli were overproduced, purified, and characterized with respect to their requirement for metal cofactor. The isolated isozymes contained 0.2-0.3 mol of iron/mol of enzyme monomer, variable amounts of zinc, and traces of copper. Enzymatic activity of the native enzymes was stimulated 3-4-fold by the addition of Fe2+ ions to the reaction mixture and was eliminated by treatment of the enzymes with EDTA. The chelated enzymes were reactivated by a variety of divalent metal ions, including Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Mn2+, Ni2+, and Zn2+. The specific activities of the reactivated enzymes varied widely with the different metals as follows: Mn2+ greater than Cd2+, Fe2+ greater than Co2+ greater than Ni2+, Cu2+, Zn2+ much greater than Ca2+. Steady state kinetic analysis of the Mn2+, Fe2+, Co2+, and Zn2+ forms of the phenylalanine-sensitive isozyme (DAHPS(Phe)) revealed that metal variation significantly affected the apparent affinity for the substrate, erythrose 4-phosphate, but not for the second substrate, phosphoenolpyruvate, or for the feedback inhibitor, L-phenylalanine. The tetrameric DAHPS(Phe) exhibited positive homotropic cooperativity with respect to erythrose 4-phosphate, phophoenolpyruvate, and phenylalanine in the presence of all metals tested.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed1988

[Effects of synthetic bioactive lipids on the activity of D-beta-hydroxybutyrate dehydrogenase, a membrane enzyme].

The structural requirements of lecithins analogs for purified D-beta-hydroxybutyrate dehydrogenase activation have been studied with chemically defined phospholipids. It appears that the trimethylamine group of choline can be changed by a pyridinium group. On the other hand, the decrease of density of the positive charges on the liposomes surface obtained by dilution of such bearing molecules with negative or non charged phospholipids increases the enzyme reactivation. Finally, the PAF acether, a lipid mediator, is able to reactivate the enzyme in similar conditions as these obtained with mitochondrial phosphatidylcholines.

Chemical Phenomena↗
PubMed1982

Cystamine-Sepharose. A probe for the active site of gamma-glutamylcysteine synthetase.

gamma-Glutamylcysteine synthetase, previously known to be potently inhibited by cystamine, has been found to bind covalently to cystamine-Sepharose. ATP facilitates, whereas glutamate plus magnesium ions inhibit, binding of the enzyme to cystamine-Sepharose. A large fraction of the enzyme applied to columns of cystamine-Sepharose binds by forming a disulfide bond between cysteamine-Sepharose and a sulfhydryl group at or near the active site of the enzyme. The enzyme may be released by treatment with dithiothreitol. Some of the enzyme applied to such columns is inactivated and not bound covalently to the column. That the enzyme does not bind to columns of S-(S-methyl)cysteamine-Sepharose, whereas free S-(S-methyl)cysteamine is a potent inhibitor, indicates that a cysteamine-S disulfide moiety derived from the external cysteamine residue of cystamine-Sepharose is the critical group recognized by the enzyme. The observed partitioning of the enzyme on columns of cystamine-Sepharose between covalently column-bound enzyme and nonbound inactivated enzyme suggests that the reactive enzyme sulfhydryl group forms a disulfide linkage with the sulfur atom at the immobilized end of cystamine to link the enzyme to the column and to liberate free cysteamine, and also that the enzyme interacts with the external cysteamine moiety of the bound cystamine. The latter may occur if the free cysteamine released is spontaneously oxidized to free cystamine followed by its inhibition of the enzyme, or if there is a direct reaction between the enzyme-reactive sulfhydryl group and the sulfur atom of the external cysteamine moiety of cystamine-Sepharose.

Animals↗
PubMedDate not supplied

Effect of vitamin E on glutathione-dependent enzymes.

Reactive oxygen species and various electrophiles are involved in the etiology of diseases varying from cancer to cardiovascular and pulmonary disorders. The human body is protected against damaging effects of these compounds by a wide variety of systems. An important line of defense is formed by antioxidants. Vitamin E (consisting of various forms of tocopherols and tocotrienols) is an important fat-soluble, chain-breaking antioxidant. Besides working as an antioxidant, this compound possesses other functions with possible physiological relevance. The glutathione-dependent enzymes form another line of defense. Two important enzymes in this class are the free radical reductase and glutathione S-transferases (GSTs). The GSTs are a family of phase II detoxification enzymes. They can catalyze glutathione conjugation with various electrophiles. In most cases the electrophiles are detoxified by this conjugation, but in some cases the electrophiles are activated. Antioxidants do not act in isolation but form an intricate network. It is, for instance, known that vitamin E, together with glutathione (GSH) and a membrane-bound heat labile GSH-dependent factor, presumably an enzyme, can prevent damaging effects of reactive oxygen species on polyunsaturated fatty acids in biomembranes (lipid peroxidation). This manuscript reviews the interaction between the two defense systems, vitamin E and glutathione-dependent enzymes. On the simplest level, antioxidants such as vitamin E have protective effects on glutathione-dependent enzymes; however, we will see that reality is somewhat more complicated.

Animals↗
PubMed1978

The role of ATP and divalent cations in the regulation of a cardiac phosphorylase phosphatase (phosphoprotein phosphatase) of Mr = 35,000.

The effects of ATP and divalent cations on a divalent cation-independent phosphorylase phosphatase of Mr = 35,000 (phosphatase S) purified from canine cardiac muscle have been studied. The enzyme can be rapidly inactivated by ATP or other nucleoside di- and triphosphates and PPi, but not by AMP, adenosine, adenine, Pi, EDTA, ethylene glycol bis(beta-aminoethyl ether)N,N' -tetraacetic acid, 1,10-phenanthroline, or 8-hydroxyquinoline. After removing the inactivating agent, such as ATP or PPi, by gel filtraiton followed by exhaustive dialysis, the inactivated enzyme (apophosphatase S) can be reactivated by preincubating with Mn2+ or Co2+, but not with Mg2+, Ca2+, Ni2+, Zn2+, Fe2+, Cu2+, Ba2+, Hg2+, Pb2+, or Cd2+. The Mn2+ -reactivated enzyme, which is less active than the Co2+ -reactivated enzyme, can be again inactivated by preincubating with ATP. The present findings indicate that phosphatase S contains a tightly bound divalent cation, probably Mn2+, in the active site. ATP and PPi, due to their structural similarity to the phosphoprotein substrate and their ability to chelate metal ions, can readily enter the active site to remove the divalent cation(s) essential for the catalytic function. The present findings also indicate that phosphatase S, a common catalytic subunit of several larger molecular forms of nospecific phosphoprotein phosphatase in cardiac muscle, can exist in two interconvertible forms, a metallized form (active) and a demetallized form (inactive). ATP and metal ions may regulate this class of isozymes by mediating the interconversions.

Adenosine Triphosphate↗
PubMed2001

DNA substrates containing defined oxidative base lesions and their application to study substrate specificities of base excision repair enzymes.

Reactive oxygen species generate structurally diverse base lesions in DNA. These lesions are primarily removed by base excision repair (BER) enzymes in prokaryotic and eukaryotic cells. Biochemical properties of BER enzymes such as substrate specificity, enzymatic parameters, and action mechanisms can be best studied by employing defined oligonucleotide and DNA substrates. Currently available methods are listed to prepare defined DNA substrates containing oxidative base damage and analogs. BER enzymes for oxidative base damage are classified into two subgroups that recognize pyrimidine lesions (Endo III homologs) and purine lesions (Fpg homologs), though E. coli Fpg exhibits weak repair activity for certain pyrimidine damage. Recently, several interesting findings have been reported in relation to the substrate specificity of BER enzymes. Saccharomyces cerevisiae Endo III homologs (NTG1 and NTG2) have been shown to recognize formamidopyrimidine (Fapy) lesions that are derived from purine. Endo III and Endo VIII have a very weak activity to dihydrothymine in comparison with thymine glycol. Excision of 7,8-dihydro-8-oxoguanine by Fpg and human OGG1 is paired-base-dependent, whereas that of Fapy is essentially paired-base-independent. The repair efficiency of BER enzymes is affected by surrounding sequence contexts. In general, the sequence context effect appears to be more pronounced for Fpg homologs than Endo III homologs.

Bacterial Proteins↗
PubMed2000

Superoxide regulation of endothelin-converting enzyme.

Reactive oxygen species (ROS) act as signaling molecules in the cardiovascular system, regulating cellular proliferation and migration. However, an excess of ROS can damage cells and alter endothelial cell function. We hypothesized that endogenous mechanisms protect the vasculature from excess levels of ROS. We now show that superoxide can inhibit endothelin-converting enzyme activity (ECE) and decrease endothelin-1 synthesis. Superoxide inhibits ECE but hydrogen peroxide and nitric oxide do not. Superoxide inhibits ECE by ejecting zinc from the enzyme, and the addition of exogenous zinc restores enzymatic activity. Superoxide may inhibit other zinc metalloproteinases by a similar mechanism and may thus play an important role in regulating the biology of blood vessels.

Animals↗
PubMed2001

V-Ha-Ras overexpression induces superoxide production and alters levels of primary antioxidant enzymes.

Reactive oxygen species have been shown to play important roles in v-Ha-Ras mitogenic signaling. We hypothesized that v-Ha-Ras overexpression would induce superoxide production, and therefore modify expression of the primary antioxidant enzyme system. We have demonstrated that immortal rat kidney epithelial cells stably transduced with constitutively active v-Ha-ras produced significantly larger amounts of superoxide radical than wild-type or vector-transfected control cells. The levels of the primary antioxidant enzymes copper- and zinc-containing superoxide dismutase, manganese-containing superoxide dismutase, catalase, and glutathione peroxidase were increased in the superoxide-overproducing cells. DNA-binding activities of the transcription factors activator protein-1, activator protein-2, and nuclear factor-kappaB were all enhanced in the superoxide-overproducing cells. These v-Ha-ras transduced cells also had a shortened cell doubling time and higher plating efficiency, and displayed greater constitutive levels of phosphorylated mitogen-activated protein kinases. These data demonstrate that v-Ha-Ras overexpression increases superoxide production and this apparently affects a wide variety of cell signaling and redox systems.

Animals↗
PubMed2003

Developmental changes in murine brain antioxidant enzymes.

Reactive oxygen species produced in cells during normal aerobic metabolism have the ability to induce lipid peroxidation and protein oxidation; therefore, their detoxification and elimination are necessary for physiologic cellular activity and survival. The changes in neuronal antioxidant enzymes from fetal life to adulthood have not been fully described. We investigated protein expression, using Western blot analysis, and enzymatic activity of the antioxidant system-copper-zinc superoxide dismutase (SOD), manganese SOD, catalase, and glutathione peroxidase, as well as reduced glutathione level as an indicator of the nonenzymatic system-in CD1 murine brain at embryonic d 18 (E18), and postnatal d 1 (P1), d 4, d 7, d 14, and d 21. Copper-zinc SOD and glutathione peroxidase protein levels were low, whereas manganese SOD and catalase protein levels were high at E18 and P1. Total SOD activity was high at E18 and P1 and paralleled elevated manganese SOD activity; however, copper-zinc SOD activity was relatively unchanged throughout development. Catalase activity doubled and glutathione peroxidase activity tripled between E18 and P1. Reduced glutathione increased between E18 and P1. Except for catalase and manganese SOD, peak protein levels do not occur until later developmental ages. We suggest that as the fetus moves from an in utero hypoxic to a relatively hyperoxic environment with an approximate 4-fold elevation in oxygen concentration, these developmental changes in antioxidant enzymes are compensatory mechanisms aimed at protecting the newborn from oxidative stress. These data will be important in our future understanding of the mechanisms by which hypoxia mediates injury in the immature and the mature brain.

Animals↗
PubMed2000

Garlic ameliorates gentamicin nephrotoxicity: relation to antioxidant enzymes.

Reactive oxygen species are involved in gentamicin (GM) nephrotoxicity, and garlic is effective in preventing or ameliorating oxidative stress. Therefore, the effect of garlic on GM nephrotoxicity was investigated in this work. Four groups of rats were studied: (i) fed normal diet (CT), (ii) treated with GM (GM), (iii) fed 2% garlic diet (GA), and (iv) treated with GM and 2% garlic diet (GM + GA). Rats were placed in metabolic cages and GM nephrotoxicity was induced by injections of GM (75 mg/kg every 12 h) for 6 d. Lipoperoxidation and enzyme determinations were made in renal cortex on day 7. GM nephrotoxicity was made evident on day 7 by (i) tubular histological damage, (ii) enhanced BUN and urinary excretion of N-acetyl-beta-D-glucosaminidase, and (iii) decreased creatinine clearance. These alterations were prevented or ameliorated in GM + GA group. The rise in lipoperoxidation and the decrease in Mn-SOD and glutathione peroxidase (GPx) activities observed in the GM group, were prevented in the GM + GA group. Cu, Zn-SOD activity and Mn-SOD and Cu,Zn-SOD content did not change. CAT activity and content decreased in the GM, GA, and GM + GA groups. CAT mRNA levels decreased in the GM group. The protective effect of garlic is associated with the prevention of the decrease of Mn-SOD and GPx activities and with the rise of lipoperoxidation in renal cortex.

Acetylglucosaminidase↗
PubMed2006

Tertiary-butyl hydroperoxide induced oxidative stress and male reproductive activity in mice: role of transcription factor NF-kappaB and testicular antioxidant enzymes.

Reactive oxygen species (ROS) have been proposed as a major factor affecting male reproductive capacity. The present study has evaluated the possible role of oxidative stress during testicular pathogenesis in male mice exposed to tertiary-butyl hydroperoxide (TBHP). TBHP was administered by daily intraperitoneal injection for 2 weeks. Treatment greatly increased lipid peroxidation in the testis and led to a significant decrease in sperm concentration and motility and a reduction in litter size relative to controls. An increase in testicular mRNA abundance of redox-regulated p50 and p65 subunits of NF-kappaB was observed after TBHP treatment. Evaluation of NF-kappaB regulated antioxidant enzymes in the testis revealed an increase in superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and glutathione-S-transferase (GST) activities and corresponding mRNA abundance. These results suggest a potential role of NF-kappaB in oxidative stress mediated changes in the physiology of male reproductive system.

Animals↗
PubMed1993

Proteinase 3: substrate specificity and possible pathogenetic effect of Wegener's granulomatosis autoantibodies (c-ANCA) by dysregulation of the enzyme.

Reactivity of proteinase 3 (PR3) was tested against various amino acid and thioester substrates. The best substrate is Boc-Ala-Ala-Nva-SBzl with a kcat/Km value of 1.0 x 10(6) M-1.s-1. We also studied the effect of C-ANCA on PR3 proteolytic activity towards elastin and inactivation by alpha 1-antitrypsin (alpha 1AT). C-ANCA IgG from 8 patients with active Wegener's granulomatosis were tested and found to inhibit elastin degradation by PR3 and to prevent the inactivation of PR3 by alpha 1AT.

Amino Acid Sequence↗
PubMed1951

A lipolytic enzyme in reactive histiocytes of guinea pigs with experimental encephalomyelitis.

A lipolytic enzyme has been demonstrated by means of a cytochemical technique and by direct chemical assay in granulomatous tissues of guinea pigs with encephalomyelitis and demyelinization resulting from the injection of an emulsion comprised of brain tissue and adjuvants, including Mycobacterium butyricum. Combined histologic and cytochemical studies showed that the lipolytic enzyme was present in the cytoplasm of a large proportion of the reactive histiocytes in the granulomatous tissue around the site of injection in the diseased animals, and that the enzyme-containing histiocytes were even more numerous in the inflamed regional lymph nodes. In control experiments, when emulsions lacking either brain tissue or M. butyricum were injected in previously normal guinea pigs, the experimental condition did not develop; under these circumstances the lipolytic enzyme was found in only a small proportion of the cells of the granulomatous tissue around the injection sites, and it was almost negligible in the regional lymph nodes of these animals. It was absent from the cells of the lymph nodes of normal animals, and from the cells of talcum and aluminum hydroxide granulomas produced experimentally in guinea pigs. The lipolytic enzyme may be a factor in the pathogenesis of the experimental encephalomyelitis and demyelinization.

Animals↗
PubMed1993

Enzyme immunoassay of beta-hexosaminidase A and B in serum: carrier detection of GM2-gangliosidoses, and equivalence of enzyme activity and enzyme protein reactivity.

beta-Hexosaminidase (Hex; EC 3.2.1.52) isoenzymes A and B were analyzed in sera from a control group of 22 apparently healthy subjects, 13 obligate carriers of Tay-Sachs disease (TSD), 10 obligate carriers of Sandhoff disease (SHD), and 4 affected TSD patients by enzyme immunoassay methods based on enzyme activity. No Hex A activity was detected in the sera of patients with TSD. The activities of Hex A in the obligate carriers of TSD and SHD tended to be lower (nonsignificantly) than in the control group. Hex B activities tended to be higher in TSD patients as well as in carriers of TSD, although the mean activities did not significantly differ from the corresponding mean for the control group. However, Hex B activities were decreased in the carriers of SHD in comparison with the other groups. Sera from 900 postmenopausal women, all of age 55 years, were also analyzed for Hex isoenzymes; the results indicated a carrier frequency of about 1 in 200 for both TSD and SHD. We also compared the enzyme immunoassay method based on enzyme activity with one based on the antigenic (enzyme protein) reactivity alone. Because both methods yielded similar information, we conclude that no significant amounts of inactive enzyme protein are present in the circulation.

Adolescent↗
PubMed1985

Mammalian chymotrypsin-like enzymes. Comparative reactivities of rat mast cell proteases, human and dog skin chymases, and human cathepsin G with peptide 4-nitroanilide substrates and with peptide chloromethyl ketone and sulfonyl fluoride inhibitors.

The extended substrate binding sites of several chymotrypsin-like serine proteases, including rat mast cell proteases I and II (RMCP I and II, respectively) and human and dog skin chymases, have been investigated by using peptide 4-nitroanilide substrates. In general, these enzymes preferred a P1 Phe residue and hydrophobic amino acid residues in P2 and P3. A P2 Pro residue was also found to be quite acceptable. The S4 subsites of these enzymes are less restrictive than the other subsites investigated. The substrate specificity of these enzymes was also investigated by using substrates which contain model desmosine residues and peptides with amino acid sequences of the physiologically important substrates angiotensin I and angiotensinogen and alpha 1-antichymotrypsin, the major plasma inhibitor for chymotrypsin-like enzymes. These substrates were less reactive than the most reactive tripeptide reported here, Suc-Val-Pro-Phe-NA. The thiobenzyl ester Suc-Val-Pro-Phe-SBzl was found to be an extremely reactive substrate for the enzymes tested and was 6-171-fold more reactive than the 4-nitroanilide substrate. The four chymotrypsin-like enzymes were inhibited by chymostatin and N-substituted saccharin derivatives which had KI values in the micromolar range. In addition, several potent peptide chloromethyl ketone and substituted benzenesulfonyl fluoride irreversible inhibitors for these enzymes were discovered. The most potent sulfonyl fluoride inhibitor for RMCP I, RMCP II, and human skin chymase, 2-(Z-NHCH2CONH)C6H4SO2F, had kobsd/[I] values of 2500, 270, and 1800 M-1 s-1, respectively. The substrates and inhibitors reported here should be extremely useful in elucidating the physiological roles of these proteases.

Amino Acid Chloromethyl Ketones↗
PubMed1994

Utility of quantitative enzyme immunoassay reactivity for predicting human immunodeficiency virus seropositivity in low- and high-prevalence populations.

To assess the utility of quantitative enzyme immunoassay (EIA) reactivity for predicting human immunodeficiency virus seropositivity, we evaluated 22,823 serum samples from homo- and bisexual men, heterosexual intravenous drug users, and other heterosexuals with initial screening by EIA, retesting of reactive samples in duplicate, and confirmatory Western blot (immunoblot) testing. Quantitative EIA reactivity was determined by a mean of the optical density ratio of the three assays performed for each reactive specimen. A total of 1,773 samples (7.8%) were repeatedly reactive, and 1,747 (7.7%) were confirmed Western blot positive. All 26 EIA-reactive-Western blot-negative samples had low-level EIA reactivity (ratio < 2.2), while most (86%) of the Western blot-positive samples had high-level reactivity (ratio, > 3.0). The positive predictive value for samples with moderate-to-high-level EIA reactivity (ratio, > 2.2) was 100% for all risk groups. These results support the value of quantitative EIA reactivity in predicting human immunodeficiency virus seropositivity and suggest that confirmatory testing of specimens with high-level reactivity is not necessary in all situations.

Algorithms↗