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PubMed1978

Lipofuscin and melanin of human retinal pigment epithelium. Fluorescence, enzyme cytochemical, and ultrastructural studies.

The life history of melanin and lipofuscin granules of human retinal pigment epithelium (RPE) was studied in 30 human eyes spanning nine decades of life. Autofluorescent granules in the cytoplasm of eye over 30 years of age were shown, ultrastructurally and through lipid solvent extraction, to be lipofuscin granules. Sparse small fluorescent granules in infant eyes were secondary lysosomes containing small droplets of lipid. Flourescent substances in RPE granules of eyes less than 50 years old were more readily extracted with lipid solvents than those in very old eyes (greater than 70). Lipfuscin granules were positive for acid phosphatase and aryl sulfatase activity. Fusions between primary lysosomes and lipofuscin granules were common in older eyes, suggesting that the over-all degradative process involves repeated injection of lysosomal enzymes, i.e., the initial fusion of lysosomes with phagosomes (phagocytized outer segment disks) is only one of several attempts to hydrolyze the membranous material. Some melanin granules showed hydrolytic enzyme reactions. By use of enzyme cytochemistry, fluorescence microscopy, and lipid extraction two types of melanin-containing complex granules were identified: melanin with a cortex of lipofuscin (melanolipofuscin) and melanin with a cortex of nonlipid, enzyme reactive material (melanolysosomes). These findings indicate that melanin commonly becomes incorporated into the lysosomal system of the RPE cell and suggests that it undergoes modification or degradation there. These studies indicate that a dynamic, complex interrelationship exists between the various components of the phagolysosomal system and the melanin granules in the RPE cytoplasm. Also, the observed variation from one human eye to another in the content and lipid extractability of RPE lipofuscin granules suggests that there may be differences in lipid composition of phagocytized photoreceptor disks and/or differences in the degradation of these lipids in the phagolysosomal system of the RPE cell.

Acid Phosphatase↗
PubMed1984

Immunological assay of pancreatic ribonuclease in serum as an indicator of pancreatic cancer.

Serum levels of RNase activity, presumed to originate in the pancreas, have been suggested to be of use in the diagnosis of pancreatic cancer. We have used a radioimmunological assay of human pancreatic-like RNase to quantitate this protein in serum from normal blood donors and patients with a variety of diseases. Serum pancreatic-like RNase rises gradually with age, and its level is usually higher in males than females. Although many patients with pancreatic cancer show elevated serum levels of immunologically cross-reactive enzyme, others are apparently normal. In several other types of cancer, a similar pattern of elevated RNase is apparent. However, in kidney or bladder carcinoma and in patients with severe kidney disease, RNase levels are almost always greater than normal. Regardless of the nature of the disease, an elevated level of pancreatic-like enzyme is usually accompanied by above-normal levels of serum urea nitrogen. Hence, elevated circulating levels of pancreatic-like RNase are best related to kidney function and do not serve as a specific marker for cancers of the pancreas or other organs.

Adult↗
PubMed1977

Cholinesterase activity in the microfilaria of Wuchereria bancrofti Cobbold.

The distribution of cholinesterase activity in the microfilaria of the nocturnally periodic Wuchereria bancrofti has been described. Using histochemical methods, cholinesterase activity has been demonstrated in the amphids (Mundgebilde), phasmids (Schwanzgebilde), nerve-ring, excretory and anal vesicles of the microfilaria. It has been found that the enzyme activity disappeared completely after treatment with the specific cholinesterase inhibitor eserine. Evidence is presented that the greater amount of the cholinesterase activity detected is related to acetylcholinesterase and the functional significance of the enzyme in the enzyme reactive structures is discussed.

Acetylcholinesterase↗
PubMed1993

Enhanced oxidation of aniline derivatives by two mutants of cytochrome c peroxidase at tryptophan 51.

Two hyperactive mutants of cytochrome c peroxidase (CCP), W51F and W51A, catalyze the enhanced oxidation of a number of substituted anilines. The reaction of CCP compound ES with mesidine is biphasic, while similar reactions using compound II give monophasic kinetics. These data, in addition to the ratio of the Fe4+ = O and free-radical species observed during steady-state turnover, indicate that reduction of the Trp-191 free radical of compound ES is more rapid than the reduction of the Fe4+ = O species. Transient kinetics were examined for the oxidation of eight mono-substituted anilines by CCP, W51F, and W51A. Each of the aniline derivatives were oxidized by the mutants at rates that exceeded that of the wild-type enzyme, and the rate constant for m-chloroaniline was 400-fold faster for W51F than for wild-type CCP. Variations in the rate constants for the different substrates follow a linear free-energy relationship using the Hammet substituent effect parameter sigma +, implicating electron transfer from the aniline ring in the transition state. For aniline oxidation, the free energy of activation is 3 kcal/mol lower for the mutants than for wild-type CCP, and this is due primarily to an increase in the activation entropy. These results indicate that the enhanced kinetics of W51F and W51A result from a generalized increase in enzyme reactivity characterized by an exo-entropic transition state such as dissociation of bound H2O from the Fe4+ = O center.

Amino Acid Sequence↗
PubMed1993

Hepatitis C virus infection among patients attending a clinic for sexually transmitted diseases.

OBJECTIVE: To evaluate the association between hepatitis C virus (HCV) infection and sexual behavior in a sexually active population. DESIGN: Cross-sectional study. SETTING: Inner-city clinic for sexually transmitted diseases. SUBJECTS: The study included 1292 patients attending the clinic for care during a 1-month period and having syphilis serologic tests performed. OUTCOME MEASURES: Antibody to HCV (anti-HCV) positivity as defined by a repeatedly-reactive enzyme immunoassay and a positive neutralization enzyme immunoassay (Abbott Laboratories, Chicago, Ill). RESULTS: Of 1292 patients screened for anti-HCV, 99 (7.7%) were positive. Logistic regression analysis found that patients who reported intravenous drug use, were positive for antibody to hepatitis B core antigen, reported a history of a blood transfusion, were black, or reported crack cocaine use were more likely to be anti-HCV-positive. Forty-five percent of patients who were anti-HCV-positive reported intravenous drug use. Sex with an intravenous drug user and a history of gonorrhea and syphilis were associated with anti-HCV positivity in a univariate analysis, but after controlling for confounding variables, no such associations remained. While having multiple sexual partners in the previous 3 months, being homosexual or bisexual, and engaging in receptive anal intercourse were associated with being positive for antibody to hepatitis B core antigen, those behaviors were not associated with anti-HCV positivity. CONCLUSIONS: While these results cannot exclude a role for the sexual transmission of HCV, they do suggest that, in this sexually active population, the sexual transmission of HCV occurs infrequently and that HCV is largely associated with intravenous drug use.

Adult↗
PubMed1995

Propagation of infectious salmon anaemia (ISA) virus in cell culture.

A long-term cell line supporting growth of the infectious salmon anaemia (ISA) virus has been established. The cell line (SHK-1) was developed from a culture of head kidney leucocytes from Atlantic salmon, and exhibited macrophage-like enzyme reactivities. By means of transmission experiments, ISA infectivity of cell culture medium could be demonstrated from day 5 after infection of SHK-1 cells with ISA-infective tissue homogenate. ISA infectivity of cell culture medium increased following repeated passages of virus. ISA-infected cell cultures develop cytopathic effects (CPE), making quantitation of virus possible. The development of CPE in ISA virus infected cells was inhibited by ammonium chloride, chloroquine and bafilomycin A, suggesting that infection of SHK-1 cells with ISA virus requires a low-pH step.

Anemia↗
PubMed1994

[Solidscreen II Enzyme: a complete test method using Solidscreen II].

An extended application of the solid-phase antiglobulin test Solidscreen II consists in the use of enzyme-treated test cells. Beside the increased sensitivity in the detection of weak Rh antibodies, 'real enzyme-reactive antibodies' can be detected. A total of 818 serum samples and 404 EDTA plasma samples were tested for red cell antibodies. In addition to Solidscreen II, 12 antibodies (0.98%) were detected and identified.

Autoantibodies↗
PubMed1992

Effect of ozone on the activities of reactive oxygen scavenging enzymes in RBC of ozone exposed Japanese charr (Salvelinus leucomaenis).

The effect of ozone exposure on the activities of reactive oxygen scavenging enzymes (SOD, catalase, GSH-Px) in RBC of Japanese charr (Salvelinus leucomaenis) was examined. Ozone (0, 0.4 and 0.7 ppm as initial concentrations) was exposed to Japanese charr for 30 min, which definitely caused serious membrane damage to RBC of fish. Ozone exposure at 0.4 and 0.7 ppm decreased activities of both catalase and GSH-Px by 80 to 57% of the control. On the other hand, the activities of SOD remained unaffected even by 0.7 ppm ozone exposure. A hypothesis on the RBC membrane damage and participation of SOD and heme-iron was proposed.

Animals↗
PubMed2003

Effect of lanthanum ions (La3+) on the reactive oxygen species scavenging enzymes in wheat leaves.

Physiological effects of lanthanum ions on the activities of the enzymes in the reactive oxygen species (ROS) scavenging system in leaves of wheat (Triticum aestivum L.) seedlings were studied. Wheat leaves treated in Hogland solution with 0.1 mM LaCl(3) for 48 h showed increased levels of superoxide dismutase (SOD), catalase (CAT), ascorbate-specific peroxidase (AsA-POD), and dehydroascorbate reductase (DHAR). However, a minor effect was observed on the levels of monodehydroascorbate reductase (MDAR) and glutathione reductase (GR), which regulate the release of energy required by the ROS scavenging system. The whole system was linked up by H(+) transmission. Our results indicated that the activities of the enzymes that function directly to remove ROS were elevated by La(3+) treatment, which is consistent with the observations that La(3+)-treated plants had increased tolerance to environmental stresses. The remaining levels of MDAR and GR suggested that these two enzymes might be regulated differently from that of the other four enzymes studied.

Cations↗
PubMed1992

Rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase: half-of-the-sites reactivity of the enzyme modified at arginine residues.

Modification of a single arginine residue per subunit of rabbit muscle apo-D-Glyceraldehyde-3-phosphate dehydrogenase does not affect the rate of hydrolysis of p-nitrophenyl acetate catalyzed by the enzyme, but locks the tetramer in a conformation wherein only two active sites are functioning. The modified enzyme also exhibits half-of-the sites reactivity towards iodoacetate and iodoacetamide. On the other hand, its NAD(+)-binding characteristics remain unchanged. Evidence is presented supporting the view that mechanisms of half-of-the-sites reactivity and negative cooperativity in coenzyme binding are different. The results are consistent with a built-in asymmetry of the tetramer and suggest that the arginine residue (probably Arg-231) controls the conformational transition between the asymmetric and symmetric states of the tetramer.

Animals↗
PubMed1997

Reactivation of thermally inactivated enzymes by free and immobilized chaperonin GroEL/ES.

Thermally inactivated bovine deoxyribonuclease I (DNase I) and yeast enolase were reactivated by GroEL/ES from Escherichia coli. In both cases, GroEL/ ES was found to have the ability to reactivate inactivated enzymes in an ATP-dependent manner. GroEL/ ES can interact with the enzymes that were denatured at high temperature and convert them to the active conformations. To test the applicability of GroEL/ES to the reactivation processes of thermally inactivated enzymes, GroEL/ES was immobilized using formyl-Cellulofine (GroEL/ES-Cellulofine) and its performance was studied. GroEL/ES-Cellulofine retained a sufficiently high ability to reactivate enzymes. Moreover, GroEL/ES-Cellulofine could be used repeatedly, indicating high durability. These results indicate that immobilized chaperonin is effective for reactivation of enzymes that are thermally inactivated in various bioprocesses.

Adenosine Triphosphate↗
PubMed1989

Rapid determination of C-reactive protein by enzyme immunoassay using two monoclonal antibodies.

Several monoclonal antibodies for human C-reactive protein (CRP) were characterized, and two antibodies binding to separate domains were used to construct a rapid and simple immunoenzymometric assay for CRP. The assay consists of a single 15 min immunological reaction during which CRP forms a complex with a peroxidase-labelled antibody and with another antibody attached to the test-tube wall. The immobilized complex is detected by a 3 min colour reaction using peroxidase substrate. The quantitative measuring range of the assay is 0.04-5 mg/l, and no hook occurs at five-fold higher values. The sensitivity of the method allows reliable determination of low CRP levels, eg. in paediatric samples. The values obtained with the present assay correlated well with turbidimetric results.

Adult↗
PubMed1975

Further confirmation of carboxypeptidase Y as a metal-free enzyme having a reactive serine residue.

The metal content of carboxypeptidase Y was analyzed by the atomic absorption method. After exhaustive dialysis against an EDTA solution, the enzyme showed no loss of activity nor any significant content of metals (Zh,Mg,Ca,Cu,Mn,Ni,Fe, and Co). The activity was, however, rather sensitive to preincubation with various metals. The reactivity of a serine residue of the enzyme was also reevaluated. Diisopropyl fluorophosphate (DFP) and phenylmethanesulfonyl fluoride (PMSF) stoichiometrically and irreversively inhibited the enzyme. The rate of inactivation with DFP was much faster than that for typsin [EC 3.4.21.4] and chymotrypsin [EC 3.4.21.1.], while the rate with PMSF was one-fifteenth of that for chymotrypsin. The pH-dependence of the inactivation by DFP was similar to that of the enzymatic hydrolysis of acetylphenylalanine ethyl ester. The present results indicate that carboxypeptidase Y is free of metals and has a serine residue with a vital role in the catalytic process, though the functional role of this SH group remains to be clarified.

Apoenzymes↗
PubMed1983

Human methionine sulfoxide-peptide reductase, an enzyme capable of reactivating oxidized alpha-1-proteinase inhibitor in vitro.

The present study demonstrates the presence of methionine sulfoxide [Met(O)] peptide reductase activity in human lung homogenates and in lysates of polymorphonuclear leukocytes (PMN) and alveolar type II cells. Enzyme activity was not detected in human bronchoalveolar lavage fluid or in pulmonary alveolar macrophage lysates. The Met(O)-peptide reductase derived from PMN is capable of reactivating alpha-1-proteinase inhibitor (alpha 1Pl) oxidized by treatment with chloramine-T or a myeloperoxidase oxidizing system. However, the PMN-derived enzyme does not reactivate alpha 1Pl inactivated by treatment in vitro with aqueous solutions of cigarette smoke plus peroxide. In addition, after the instillation of oxidized human alpha 1Pl into lungs of normal or ozone-tolerant rats, no reactivated alpha 1Pl could be found in the pulmonary lavage obtained from these animals. Finally, patients with chronic obstructive pulmonary disease appear to have normal levels of PMN Met(O)-peptide reductase.

Animals↗
PubMed2004

Discovering disease-associated enzymes by proteome reactivity profiling.

Proteomics aims to identify new markers and targets for the diagnosis and treatment of human disease. To realize this goal, methods and reagents are needed to profile proteins based on their functional properties, rather than mere abundance. Here, we describe a general strategy for synthesizing and evaluating structurally diverse libraries of activity-based proteomic probes. Quantitative screening of probe-proteome reactions coupled with bioinformatic analysis enabled the selection of a suite of probes that exhibit complementary protein reactivity profiles. This optimal probe set was applied to discover several enzyme activities differentially expressed in lean and obese (ob/ob) mice. Interestingly, one of these enzymes, hydroxypyruvate reductase, which was 6-fold upregulated in ob/ob livers, participates in the conversion of serine to glucose, suggesting that this unusual metabolic pathway may contribute to gluconeogenesis selectively in states of obesity.

Acetamides↗
PubMed1992

Changes in lipid peroxide levels and activity of reactive oxygen scavenging enzymes in skin, serum and liver following UVB irradiation in mice.

The effect of acute UVB on the generation of reactive oxygen species (ROS) in the skin and the induction of ROS scavenging enzymes in situ was examined. Lipid peroxide levels and the activities of superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px) and D-glucose-6-phosphate dehydrogenase (G-6-P-D) were determined in the skin, serum, and liver of ICR mice subjected to 1400 mJ/cm2 of acute UVB irradiation. In irradiated skin, lipid peroxides were increased at 3 and 24 hr after irradiation, whereas the four ROS scavenging enzymes were generally decreased during the first 48 hr after irradiation. In the serum, lipid peroxides showed an increase at 3 hr, but enzyme activities remained negligible. In the liver, lipid peroxides showed similar behaviour to that in skin. GSH-Px activity in the liver was decreased during the first 24 hr, whereas G-6-P-D showed substantial fluctuation and SOD and catalase activities showed no change. These data are consistent with a model in which lipid peroxides generated in the UVB-irradiated lesions are transported to the liver and there metabolized by the scavenging enzymes induced in situ.

Animals↗
PubMed1983

Kinetics of dissociation of reduced nicotinamide adenine dinucleotide phosphate from its complexes with malic enzyme in relation to substrate inhibition and half-of-the-sites reactivity.

Malic enzyme of pigeon liver binds NADPH at four equivalent enzyme sites and binds Mn2+ and malate each at two sets of "tight" and "weak" sites with negative cooperativity [Pry, T. A., & Hsu, R. Y. (1980) Biochemistry 19, 951-962]. Stopped-flow studies on the displacement of NADPH from the malate-enzyme complexes E4-NADPH4, E4-Mn2(2+)-NADPH4, E4-Mn2(2+)-NADPH4-dimalate, and E4-Mn2(2+)-NADPH4-tetramalate by large excess NADP+ or its analogue phosphoadenosine(2')diphospho(5')ribose show that NADPH dissociates from the binary complex rapidly with a first-order rate constant of 427 s-1. Dissociation from the ternary E4-Mn2(2+)-NADPH4 complex containing two tightly bound Mn2+ ions can be described by a single first-order process with a rate constant of 135 s-1, or more satisfactorily by two simultaneous first-order processes attributable to the reactions of Mn2+-deficient (k congruent to 427 s-1) and Mn2+-liganded (k = 96 s-1) subunits. The latter equals twice the maximum steady-state turnover rate of 53.2 + 3.0 s-1 assigned to dissociation of the reduced nucleotide from transient E-Mn2+-NADPH, and this 2:1 ratio strongly supports our proposed "half-of-the-sites" model [Hsu, R. Y., & Pry, T. A. (1980) Biochemistry 19, 962-968]. Dissociation from the E4-Mn2(2+)-NADPH4-dimalate complex (k = 100 s-1) follows only the slower process, suggesting that occupancy of malate at two sites tightens enzyme-bound NADPH on the adjacent sites. Binding of malate at two additional weak sites yields E4-Mn2(2+)-NADPH4-tetramalate and a NADPH dissociation rate constant of 2.69 s-1. The 97% decrease in NADPH dissociation parallels the observed 93% maximal inhibition by malate and is the cause of substrate inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗