Terms Of Enzyme Activity Research Articles

Changes in Certain Antioxidative Enzymes and Growth Parameters as a Result of Complete Submergence and Subsequent Re‐aeration of Rice Cultivars Differing in Submergence Tolerance

An experiment was conducted on the three indica rice (Oryza sativa L.) cultivars IR 42, CR 383‐10 and FR 13A, which are susceptible to submergence, submergence‐avoiding and tolerant to submergence, respectively. A deleterious effect of submergence was noted as both above‐ground dry matter accumulation and chlorophyll content decreased during submergence and subsequent re‐aeration. However, the rate of reduction was lower in the tolerant cultivar. The tolerant cultivar FR 13A maintained higher dry weight during submergence and subsequent re‐aeration and accumulated lower proline and malondialdehyde contents than the other cultivars. The activities of the enzymes catalase, peroxidase and superoxide dismutase, expressed on a per g fresh weight of leaves basis, were higher in FR 13A than in the other cultivars, both under water and 24 h after the initiation of re‐aeration. However, ascorbic acid oxidase activity was lower in FR 13A. The cultivar CR 383‐10, which has the capacity to accumulate more above‐ground dry matter under normal conditions, showed a greater elongation ability under submergence and was similar to the susceptible cultivar IR 42 in terms of enzyme activities and the other parameters investigated during submergence and subsequent re‐aeration.

An experimental analysis of membrane bioreactor performances with immobilized chymosin

Aim of the present work is the experimental analysis of a continuos flow membrane bioreactor with immobilized chymosin for the hydrolysis of milk k-casein, one of the steps in the cheese production process. Chymosin has been immobilized on the bioreactor membrane by ultrafiltering under pressure its solutions, continuously fed to the system in a total recycle configuration. Bioreactor performances are evaluated in terms of two different parameters: chymosin activity and its release. The former, indirectly, measures the amount of k-casein converted into reaction products ( para-k-casein and GlycoMacroPeptide, GMP); the latter quantifies the amount of biocatalyst that is removed from the membrane by the milk flowing stream. A theoretical approach, based on the boundary layer theory, has also been attempted to estimate the dependence of chymosin release upon the wall shear stress and on the back-diffusion phenomena towards the milk bulk. The experimental analysis of bioreactor behavior has been carried out, searching for the operating conditions and system configurations that maximized chymosin activity and minimized its release. Particular attention has been devoted to the selection of the membrane material: PolySulphone membranes offered the best performance during experiments, as compared to those of PolyPropilene and PolyVinilDeneFluoride. The system configuration that gave best results, both in terms of enzyme activity and of its release, was based on a continuous milk recycle to the bioreactor. Enzyme losses were confined to 5–10 mg/l range, whereas its activity always attained values high enough (>2 g/l) to promote the coagulation in the next heating step of destabilized milk. During the experiments, an optimal value of the recycle ratio, was also found.

The multifunctional oxidase activity of ceruloplasmin as revealed by anion binding studies.

The effect of multiple binding of azide, N3-, on the structural and functional properties of ceruloplasmin (CP) has been reinvestigated by means of both spectroscopic and enzymatic techniques. High affinity binding of the anion to human CP resulted in a dramatic increase of the absorbance at 610 nm and in a concomitant decrease of the optical density at 330 nm. The oxidase activity toward Fe(II) was essentially unaffected, while turnover parameters versus nonferrous substrates dramatically changed, with an approximately 100-fold enhancement of the kcat/Km parameter. Chloride at physiological concentration proved to behave very similarly to N3- bound with high affinity, in that it not only induced the spectroscopic changes previously interpreted in terms of an intramolecular electron transfer from reduced type 1 to type 3 copper ions [Musci, G., Bonaccorsi di Patti, M.C. & Calabrese, L. (1995) J. Protein Chem. 14, 611-617], but it also enhanced some 60-fold the kcat/Km value. A different behavior was observed with chicken CP, where a decrease at 330 nm occurred without a concomitant modification at 603 nm. The chicken enzyme was less sensitive also in terms of enzymatic activity, which was nearly unchanged in the presence of either high affinity N3- or Cl-. At higher N3- concentrations, optical changes of both human and chicken CP were mainly focussed on the appearance of ligand-to-metal charge transfer bands below 500 nm, and the anion behaved as an inhibitor of the oxidase activity versus Fe(II) as well as noniron substrates. The well known bleaching of the blue chromophore could be observed, at neutral pH, only at very high N3-/CP ratios. The data presented in this paper are consistent with a mechanism of structural and functional modulation of CP by anions, that would be able to dictate the substrate specificity of the cuproprotein, and suggest the possibility that CP may act in vivo as a multifunctional oxidase.

3Fe-4S] to [4Fe-4S] cluster conversion in Desulfovibrio fructosovorans [NiFe] hydrogenase by site-directed mutagenesis.

The role of the high potential [3Fe-4S]1+,0 cluster of [NiFe] hydrogenase from Desulfovibrio species located halfway between the proximal and distal low potential [4Fe-4S]2+,1+ clusters has been investigated by using site-directed mutagenesis. Proline 238 of Desulfovibrio fructosovorans [NiFe] hydrogenase, which occupies the position of a potential ligand of the lacking fourth Fe-site of the [3Fe-4S] cluster, was replaced by a cysteine residue. The properties of the mutant enzyme were investigated in terms of enzymatic activity, EPR, and redox properties of the iron-sulfur centers and crystallographic structure. We have shown on the basis of both spectroscopic and x-ray crystallographic studies that the [3Fe-4S] cluster of D. fructosovorans hydrogenase was converted into a [4Fe-4S] center in the P238 mutant. The [3Fe-4S] to [4Fe-4S] cluster conversion resulted in a lowering of approximately 300 mV of the midpoint potential of the modified cluster, whereas no significant alteration of the spectroscopic and redox properties of the two native [4Fe-4S] clusters and the NiFe center occurred. The significant decrease of the midpoint potential of the intermediate Fe-S cluster had only a slight effect on the catalytic activity of the P238C mutant as compared with the wild-type enzyme. The implications of the results for the role of the high-potential [3Fe-4S] cluster in the intramolecular electron transfer pathway are discussed.

Structural and kinetic investigations on the 15-21 and 42-45 loops of muscle acylphosphatase: evidence for their involvement in enzyme catalysis and conformational stabilization.

The structural and catalytic importance of the 15-21 and 42-45 loop residues of the acylphosphatase muscular isoenzyme has been investigated by oligonucleotide-directed mutagenesis. Seven mutants involving conserved residues of the two loops have been prepared and characterized for structural, kinetic, and stability features by using different spectroscopic techniques and compared to the wild-type enzyme. The results are discussed in light of the crystal structure of the highly homologous common type acylphosphatase [Thunnissen et al. (1997) Structure 5, 69-79]. A differential role of the two loops has emerged: the 15-21 and the 42-45 loops appear mainly involved in active site formation and enzyme structural stabilization, respectively. These conclusions are supported by a strong impairment of the catalytic efficiency, in terms of enzymatic activity and substrate binding capability, for most of the 15-21 loop mutants. In particular, the Gly15Ala mutant is completely inactive and displays a native-like overall fold, indicating that the correct geometry of the 15-21 loop is an essential requisite for optimal enzymatic catalysis. Instead, the Gly45Ala mutant, though revealing unchanged catalytic properties, shows a considerably reduced conformational stability, as judged by circular dichroism and 1H NMR spectroscopy. This finding confirms previous results relative to Thr42 and Thr46 residues [Taddei et al. (1996) Biochemistry 35, 7077-7083] underlining the structural importance of the 42-45 loop as a linker for the two beta alpha beta units constituting the overall enzyme structure.

The Expression of an Intestinal Form of Glutathione Peroxidase (GSHPx-GI) in Rat Intestinal Epithelium

We have previously identified and characterized GSHPx-GI, which is a cellular selenium-dependent glutathione peroxidase (GSHPx) distinct from the classic GSHPx-1 and phospholipid hydroperoxide glutathione peroxidase (PHGPX). We have determined the level of GSHPx-GI mRNA expression in the rat gastrointestinal tract from esophagus to colon. Although GSHPx-GI mRNA is readily detectable throughout the GI tract, the highest level is detected in the ileum and cecum. We have also determined the levels of GSHPx-GI mRNA expression and several antioxidant enzyme activities along the villus-to-crypt axis in the rat small intestine by cell fractionation. GSHPx-GI mRNA is present at a similar level in all of the epithelial fractions, whereas GSHPx-1 mRNA is detectable only in the remnant. This suggests that GSHPx-GI is the major cellular tetrameric GSHPx expressed in intestinal epithelium, and the expression of GSHPx-GI in the GI tract is not likely regulated differentially through maturation of epithelial cells. In terms of enzymatic activity, although we detected lower glutathioneS-transferase activity in the crypt epithelium, there was a marginal increase of PHGPX activity, a twofold increase of GSHPx activity, and a three- to fivefold increase of catalase activity in the crypt relative to the distal villus. Thus, the crypt epithelial cells may be better protected from peroxidative damage.

Purification and characterization of a Dictyostelium protein kinase required for actin activation of the Mg2+ ATPase activity of Dictyostelium myosin ID.

We have isolated a protein from Dictyostelium with a molecular mass of 110 kDa as judged by SDS-gel electrophoresis that can stimulate the actin-activated MgATPase activity of Dictyostelium myosin ID (MyoD). In the presence of MgATP the 110-kDa protein incorporated phosphate into itself and into the heavy chain, but not the light chain, of MyoD. Phosphorylation to 0.5 mol of Pi/mol increased the MyoD actin-activated MgATPase rate from 0.2 to 3 mumol/min/mg. Renaturation following SDS-gel electrophoresis demonstrated that the 110-kDa protein contained intrinsic protein kinase and autophosphorylation activity. Autophosphorylation to 1 mol of Pi/mol enhanced the rate at which the 110-kDa protein kinase phosphorylated MyoD by 40-fold. The rate of autophosphorylation was strongly dependent on the 110-kDa protein kinase concentration, indicating an intermolecular reaction. Synthetic peptides of 9-11 residues corresponding to the heavy chain phosphorylation site of Acanthamoeba myosin IC and the homologous sites in Dictyostelium myosin IB (MyoB) and MyoD were poor substrates for the 110-kDa protein kinase. The 110-kDa protein kinase was unable to phosphorylate the MyoB isozyme suggesting that it may be specific for MyoD.

Hypometabolism with Fasting in the Yellow Perch (Perca flavescens): A Study of Enzymes, Hepatocyte Metabolism, and Tissue Size

This study analyzes the total potential of the liver in terms of enzyme activities and hepatocyteflux in fed and fasted yellow perch. A 7-wk fast decreased the hepatosomatic index from 1.99 to 0.85 with an equivalent increase in liver DNA concentration, indicating atrophy of the liver with fasting. Total contents of liver and muscle glycogen and protein declined. As a function of body weight, enzyme activities all decreased with the exception of the lipid-catabolizing enzymes; gluconeogenic enzymes declined less than those of glycolysis, the NADPH-producing enzymes, and citrate synthase. No enzyme activities increased with fasting as a function of body weight. Phosphorylation-related changes in regulatory enzymes support the protection or maintenance of gluconeogenesis. Lactate, alanine, and glycerolflux to glucose in isolated hepatocytes all decreased as a function of tissue DNA, as did alanine flux to CO₂. Again, no increases influx were noted with fasting. These data demonstrate that after a 7-wk fast yellow perch are hypometabolic. The importance of considering changes in tissue size in the analysis of environmental stressors is discussed.

Site-directed mutagenesis of conserved cysteine residues in Escherichia coli fumarate reductase: modification of the spectroscopic and electrochemical properties of the [2Fe-2S] cluster.

Site-directed mutants of Escherichia coli fumarate reductase in which each of the four N-terminal cysteine residues in the FrdB subunit, residues 57, 62, 65, and 77, was mutated individually to serine have been constructed, overexpressed, and investigated in terms of enzymatic activity as well as the EPR and redox properties of the iron-sulfur centers. In each case, the mutant contains a functional fumarate reductase in which all three of the constituent iron-sulfur clusters (i.e., center 1, [2Fe-2S]; center 2, [4Fe-4S]; center 3, [3Fe-4S]) have been assembled. The mutations affect the properties of center 1 only and demonstrate that the anomalously high redox potential of this [2Fe-2S] center is essential for optimal enzymatic activity. The results are consistent with cysteines 57, 62, 65, and 77 providing the ligands to center 1 but leave open the possibility of noncysteinyl coordination for the localized valence Fe(III) site of the reduced cluster. The implications of the results for the role of center 1 in the electron-transfer pathway and the valence localization of reduced center 1 are discussed.

The role of proteinase on the metastasis of melanoma—Interaction of tumor and surrounding tissue in vivo and effect of protease inhibitor in vitro in terms of enzyme activity of B16 melanoma variants

The role of proteinase on the metastasis of melanoma—Interaction of tumor and surrounding tissue in vivo and effect of protease inhibitor in vitro in terms of enzyme activity of B16 melanoma variants

Biochemical basis of prolidase deficiency. Polypeptide and RNA phenotypes and the relation to clinical phenotypes.

Cultured skin fibroblasts or lymphoblastoid cells from eight patients with clinical symptoms of prolidase deficiency were analyzed in terms of enzyme activity, presence of material crossreacting with specific antibodies, biosynthesis of the polypeptide, and mRNA corresponding to the enzyme. There are at least two enzymes that hydrolyze imidodipeptides in these cells and these two enzymes could be separated by an immunochemical procedure. The specific assay for prolidase showed that the enzyme activity was virtually absent in six cell strains and was markedly reduced in two (less than 3% of controls). The activities of the labile enzyme that did not immunoprecipitate with the anti-prolidase antibody were decreased in the cells (30-60% of controls). Cell strains with residual activities of prolidase had immunological polypeptides crossreacting with a Mr 56,000, similar to findings in the normal enzyme. The polypeptide biosynthesis in these cells and the controls was similar. Northern blot analyses revealed the presence of mRNA in the polypeptide-positive cells, yet it was absent in the polypeptide-negative cells. The substrate specificities analyzed in the partially purified enzymes from the polypeptide-positive cell strains differed, presumably due to different mutations. Thus, there seems to be a molecular heterogeneity in prolidase deficiency. There was no apparent relation between the clinical symptoms and the biochemical phenotypes, except that mental retardation was present in the polypeptide-negative patients. The activities of the labile enzyme may not be a major factor in modifying the clinical symptoms.

Interaction of tumor and surrounding tissue of mice inoculated B16 melanoma variants in terms of enzyme activity

1. 1. The interactions of B16-F1 and B16-F10 tumors with their surrounding tissues in terms of enzyme activities such as cathepsin B, hemoglobin(Hb)-hydrolase, acid phosphatase, β-glucuronidase and plasminogen activator were investigated when said tumors proliferated locally and at secondary sites throughout the host's circulatory system. 2. 2. In the case of B16-F1 and B16-F10 tumor cells proliferating under the skin, statistical differences were not detected between the enzyme activities of the skin surrounding the tumors and control skin, nor between B16-F1 and B16-F10 tumors, except for β-glucuronidase. 3. 3. In the case of B16-F1 and B16-F10 tumor cells metastasizing to lung, statistical differences were detected between numerous enzyme activities of the lung tissues surrounding the tumors and control lung tissue, and also between B16-F1 and B16-F10 tumors. 4. 4. The activities of cathepsin B and acid phosphatase of lung tissue surrounding B16-F1 tumor were lower than those of the control lung. 5. 5. β-Glucuronidase activity of lung tissue surrounding B16-F10 tumor was higher than that of the control lung. 6. 6. The activities of cathepsin B, Hb-hydrolase and β-glucuronidase of the B16-F10 tumor were higher than those of the B16-F1 tumor. 7. 7. Results indicate that metastasized B16 melanoma tumor cells interact with surrounding lung tissues, and that cathepsin B, Hb-hydrolase and β-glucuronidase might play important roles in the metastasis of the malignant tumor.

Preventive effect of pretreatment with zinc on cadmium nephrotoxicity in rats--evaluation by changes of urinary enzyme activities.

The preventive effect of pretreatment with zinc (Zn) on cadmium (Cd) nephrotoxicity in rats was evaluated by the measurement of enzyme activity in urine. The enzymes examined were γ-glutamyltranspeptidase (γ-GTP, EC 2.3.2.2), glycylproline-diaminopeptidase (GP-DAP, EC 3.4.14.4), alkaline phosphatase (Al-P, EC 3.1.3.1) and leucine aminopeptidase (LAP, EC 3.4.11.1).Three groups of male Sprague-Dawley rats were used. The first group received a single subcutaneous injection of 5.0 mg/kg body weight (5 rats, Cd Group), the second received a dosage of 250 μmol Zn/kg 24 hours prior to the same dosage of Cd (5 rats, Zn-Cd Group) and the third received the same dosage of Zn (5 rats, Zn Group).On the 1st day after Cd injection, significant elevation of both γ-GTP and GP-DAP activities in urine were observed in Cd Group, while in Zn-Cd Group the two enzyme activities were not elevated significantly compared to the reference values. On the 2nd day, γ-GTP and GP-DAP activities of Cd Group were reduced comparing with the first increase and those of Zn-Cd Group showed first a significant increase. On the 3rd day and after, both enzyme activities were almost the same as the reference value. The increase of enzyme activities in urine was explained to be caused by the leakage from renal cells. In Zn Group, both enzyme activities were observed little change in the course of the experiment.The excretion pattern of Al-P and LAP into urine differed from those of γ-GTP and GP-DAP in all groups.No histopathological changes under optical microscopy were observed in the livers and the kidneys of all groups.From these results, it appears that Zn Pretreatment showed a preventive effect on Cd nephrotoxicity in terms of enzyme activities in urine before any histophthological changes were seen.

Further studies of the β-lactamases ofBacteroides species

The β-lactamases of individual strains ofBacteroides fragilis, B. thetaiotaomicron, andB. melaninogenicus were examined to characterize their enzymatic activity and the relation between the periplasmic and cytoplasmic forms of the enzymes. Km and Vmax values indicate that all strains examined were very similar in terms of enzymatic activity with the antibiotics tested. Electrophoretic analysis and treatment with phospholipase D suggest the presence of a cytoplasmic form of the enzyme that is modified upon entry into the periplasmic space.

THE ADENINE NUCLEOTIDE (AdN) TRANSLOCASE (T): A ROLE IN DEVELOPMENTAL CHANGES

Although developmental changes occurring during the first days of extrauterine life have been well-characterized in terms of enzymatic activities, little attention has been given to overall cellular energy metabolism. Investigations of energy metabolism in guinea pig liver mitochondria (m) have shown that m from the newborn (N) have membrane pH gradients and membrane electrical potentials similar to those found in adults. During the first 24 hours after birth, State 3 rates of oxygen consumption (OC) increase 3 fold whereas the uncoupled rates of (OC) increase only 30%. The m AdN increases 3 fold with no change in the ATP/ADP ratio. Titrations with carboxyatractyloside (CAT), a stoichiometric inhibitor of the m AdNT show that the N has only 50% of the AdNT activity of the adult. Furthermore, it is possible to evaluate the role of the AdNT on the regulation of respiration using flux control theory (Eur. J. Biochem. 42:97-105). Briefly, the degree of control (control strength) exerted by a particular step in a pathway is defined as the fractional change in pathway flux produced by a fractional change in the activity of the enzyme comprising that step; the sum of all control strengths in a pathway being equal to 1.0. By applying this theory to inhibitor titrations of the AdNT, it has been shown that at any rate of OC the AdNT has greater control strength over respiration in the N than in the adult. At maximal rates of OC the AdNT has a control strength of 0.98 in the N and 0.56 in the adult. Since the AdNT plays a central role in cellular metabolism and the N period is a time of high energy demand, it seems probable that the AdNT plays a critical role in development.

Postmortal Histomorphologic and Histoenzymatic Alterations in Rats Brain

Summary Alterations in tissue specimens taken from rat's brain during certain postmortal intervals are described. The length of the intervals were 1, 2, 8, 16, 24, 32, 40, and 48 hours. Hematoxylin and eosin, cresyl-violet, and Kliiver-Barrera myelin stains, as well as the PAS and peroxidase reactions were used. The following enzymes were demonstrated: alkaline and acid phosphatase, alphanaphthyl acetate esterase, naphthol AS-D chloroacetate esterase, adenosine triphosphatase, uridine diphosphatase, tri- and diphosphopyridine nucleotide tetrazolium reductase, succinic dehydrogenase, acetylcholine esterase. The primary histomorphologic alterations 'observed were dehydration and swelling and/or autolysis of the nerve cells. The autolysis rate for nerve cells varied, depending on their localization. This variation tends to indicate a biocline process. Alterations in enzymatic activity were characterized both by diffusion of enzymes into the surrounding tissue and only a slight decrease in enzymatic activity which is apparently related to the autolytic deterioration. Since progressively fewer cells with a positive enzyme activity were identified, the authors suggested that the known distinct biochemical decline in activity is due primarily to a decrease in the number of positive cells. Histochemical studies 48 hours postmortem showed the enzyme activity in a group of cells to be virtually unchanged; the activity in other cells however was negative. The authors therefore assumed that, in terms of enzyme activity, the autolytic process may be defined as an all-ornothing law.

Conversion of glucose to sorbitol and fructose by liver-derived cells in culture.

Conversion of glucose to fructose and sorbitol is documented in rat hepatoma-derived cultured cells (HTC cells). After addition of 5.5 mM [U-14C]glucose to incubation medium, labeled sorbitol and fructose accumulated intracellularly at a linear rate over a period of 60 min. The sugars were isolated, identified, and quantitated by paper chromatography, gas-liquid chromatography, and enzymatic phosphorylation of fructose. Primary culture of adult rat hepatocytes was analyzed similarly and demonstrated no significant accumulation of labeled fructose or sorbitol. The basis for this difference between HTC cells and primary hepatocyte culture was examined both in terms of enzyme activities that mediate the formation of sorbitol and fructose and in terms of the catabolism of these sugars. Both types of culture (as well as extracts of intact rat liver) exhibited enzymatic activities catalyzing the conversion of glucose to sorbitol (aldose reductase) and sorbitol to fructose (sorbitol dehydrogenase). However, the cultures differed strikingly with regard to the catabolism of sorbitol and fructose. The conversion of labeled sorbitol to metabolites in HTC cells was negligible; by contrast, hepatocytes in primary culture utilized the sugars at rates comparable to that of glucose, which may account for the lack of their accumulation in primary culture. The findings suggest that the conversion of glucose to sorbitol and fructose by HTC cells may represent a retained normal liver function, one which is amplified by the inability of HTC cells to dispose of these sugars.

Frog lysozyme. I. Its identification, occurrence as isozymes, and quantitative distribution in tissues of the leopard frog, Rana pipiens.

In the course of examining the etiology of the Lucké renal adenocarcinoma of the frog, Rana pipiens, it was found that organs of the normal adult contain bacteriolytic enzymes. These enzymes all satisfied the six criteria for the identification of lysozymes and at least eight forms were separable by polyacrylamide gel electrophoresis. Their qualitative and quantitative distribution was organ-specific. All eight isozymes were found in normal kidney, while liver and spleen contained seven forms; skin, six; ovarian egg, five; and serum, two. In quantitative assays using a radial diffusion test, spleen had the greatest lysozyme concentration, followed in descending order by kidney, liver, skin, and ovary. Serum contained very low amounts. In terms of enzyme activity per animal, ovary was the highest ranking organ. As such a large number of lysozyme isozymes has not been reported in any other organism, their origins and functions are considered in the context of their presence in an ectotherm.

Peritoneal dialysis in mercurial diuretic intoxication

Similar concerns have been noted previously for a different disease and distinct enzyme deficiency. In 1973 Vidgoff and associates 1~ reported a family which included a woman with a marked deficiency of hexosominidase A in all tissues studied. She actually represents the carrier state for Tay-Sachs disease, though her hexosominidase A level taken alone would have suggested that she was an affected homozygote. There are thus at least two heritable disorders for which there is overlap between the carrier and the affected person in terms of enzyme activity. Based on these findings, and considering the greater availability and less strict indications for prenatal diagnostic amniocentesis, it is suggested that great care be exercised in the diagnosis of these diseases, particularly in the interpretation of data for intrauterine diagnosis. The data and conclusions discussed here were presented in preliminary form to the Society for Pediatric Research, Denver, April 18, 1975.11

A Urea Electrode Based on the Ammonia Probe

Abstract An enzyme electrode specific for the substrate urea is described. The electrode consists of a layer of urease polymerized directly on to the surface of the gas diffusion membrane of an ammonia probe by means of glutaraldehyde. The calibration curves obtained when the electrode is allowed to measure in buffered solutions are discussed in terms of enzyme activity and pH-gradients in the membrane.