Loss Of Enzyme Protein Research Articles

Down regulation of kidney neutral endopeptidase mRNA, protein and activity during acute renal failure: possible mechanism for ischemia-induced acute renal failure in rats?

Neutral endopeptidase (NEP, 24.11) is an ectoenzyme involved in the degradation of peptide hormones such as endothelin (ET), atrial natriuretic factor and enkephalins. The current study was designed to assess the involvement of NEP in ischemia-induced acute renal failure (ARF). In unilaterally nephrectomized Sprague-Dawley rats, the left renal artery was occluded for 30 min under pentobarbital anesthesia (40 mg/kg, i.p.) at 37 degree C. In addition to plasma creatinine levels, NEP activity was determined in renal cortical membranes at 0, 2, 5, and 24 h following reperfusion. Plasma creatinine levels significantly increased at 2, 5 and 24 h. There was a significant decrease in NEP activity as early as 2 h following reperfusion that was maintained up to 24 h (57.9 +/- 4%) with a concomitant loss of enzyme protein shown by Western analysis. Northern analysis of kidney cortical RNA, probed with an NEP cDNA, showed a 45% decrease in NEP mRNA level by the end of the ischemic period and decreased further during reperfusion. Thus, decrease in NEP mRNA levels preceded the changes in protein level, enzyme activity and plasma creatinine levels. These data, along with the reported increase in the tissue level of ET in kidney cortex, and the beneficial effect of ET antibody as well as ET receptor antagonist in ARF, suggest that down regulation of NEP, one of the mechanisms leading to increased tissue level of ET, may be a contributing factor to ARF.

Modification of growth related enzymatic pathways and apparent loss of tumorigenicity of a ras-transformed bovine endothelial cell line by treatment with 5-iodo-6-amino-1,2-benzopyrone (INH2BP)

Bovine aortic endothelial cells were converted to a highly tumorigenic cell line by transfection with Ha-ras and stimulation with thrombin. Sustained pretreatment with a non-cytotoxic concentration (600 mu M) of 5-iodo-6-amino-1,2-benzopyrone (INH2BP), a lipophilic ligand of poly(ADP-ribose) polymerase, abrogated in vivo tumorigenicity, of 10(5) cells per inoculum an effect which developed progressively during 2 to 6 weeks of drug treatment. The initial action of the drug was cytostasis, consisting of an arrest in prophase, extreme cell enlargement consistent with cytoplasmic hypertrophy, as seen by EM, and dramatic morphologic changes. Although neither DNA, RNA or protein syntheses are directly affected by INH2BP, apparently newly synthesized cellular DNA is degraded by endonucleases, which are upregulated by the inhibition of their poly-ADP-ribosylation. The drug treated cells exhibited greatly increased respiration and aerobic glycolysis, due to an augmentation of,glycolytic and respiratory enzymes in enlarged cells. These responses to the drug were reversible in cell cultures following drug removal, within 5-10 days drug exposure but the progressive loss of tumorigenicity in nude mice that developed after 3-6 weeks of drug exposure of cells, prior to inoculation to nude mice, was not reversible in vivo. Drug treatment produced a sustained 70-80% inhibition of pADPRT in intact cells at 600 mu M extracellular concentration of INH2BP. The prerequisite for the abrogation of tumorigenicity was the maintenance of pADPRT inhibition. The arrest of cell multiplication and a large decrease of Topo I, especially of Topo II and MAP kinase activities occurred without loss of enzyme protein as assayed in cell extracts of drug-treated cells. However INH2BP had no direct effect on these enzymes. Drug treatment down-regulated DNA-methyltransferase, PKC, ODC proteins, diminished cyclin A protein, but the hypophosphorylated form of Rb protein was significantly augmented. None of the enzymatic components of signal pathways so far studied, were directly affected by INH2BP. The inhibition of pADPRT by INH2BP coincided with an induction or activation of alkaline phosphatase and leucyl and glutamyl peptidase. The pADPRT content or the expression of pADPRT gene were not influenced by drug treatment, but the expression of ras gene was completely absent in nontumorigenic drug-treated cells, without a loss of ras gene from genomic DNA. Telomerase activity was not directly influenced by INH2BP treatment when assayed in diluted cell extracts, but the addition of homogeneous pADPRT to cell extracts, to approach physiological concentration of this protein in the cell, inhibited telomerase activity by binding of the polymer-free pADPRT to telomer templates. We conclude that inhibition of pADPRT indirectly down-regulates growth stimulatory signal pathways and sustains growth-arrested cells in culture at a pre-apoptotic threshold which explains the absence of tumorigenicity in vivo.

Slow accumulation of acetylcholinesterase in rat brain during enzyme inhibition by repeated dosing with chlorpyrifos

When given to rats, O, O′-diethyl- O-[3,5,6-trichloro-2-pyridyl]-phosphorothionate (chlorpyrifos), a common insecticide, causes an unusually lengthy dose-dependent fall in the activity of brain acetylcholinesterase (AChE; EC 3.1.1.7). To determine whether the slow recovery involves impaired AChE synthesis, experiments were designed to measure AChE activity, immunoreactive AChE protein (AChE-IR) and AChE mRNA. Male, Long-Evans rats, maintained at 350 ± 5 g, were dosed (s.c.) weekly for 4 weeks with 0, 15, 30, or 60 mg/kg chlorpyrifos in peanut oil. Brain tissue was harvested 1, 3, 5, 7 and 9 weeks after treatment began. AChE activity was measured by Ellman assay, and AChE-IR was estimated by two-site ELISA using monoclonal antibodies to rat brain AChE. While AChE activity fell significantly at all times and doses, AChE-IR increased at 3 and 5 weeks in the two higher dosage groups. Larger increases of AChE-IR were observed after chlorpyrifos was administered for 4 weeks by the oral route. Northern blots quantified with reference to cyclophilin were consistent with stable levels of AChE mRNA. Overall, it appears that chronically reduced brain AChE activity after chlorpyrifos reflects sustained enzyme inhibition, not loss of enzyme protein or suppression of AChE message.

Mechanism of superoxide dismutase loss from human sperm cells during cryopreservation.

Earlier studies on human sperm cryodamage have shown that plasma membrane stress is the primary process and that phospholipid peroxidation in cryopreserved samples is not inhibited by addition of antioxidants. One consistent effect of cryopreservation is loss of enzymatic activity of the peroxidation defense enzyme, superoxide dismutase (SOD). To clarify this aspect of the freeze-thaw process and to develop a more complete resolution of the reactions leading to cryodamage, we sought to identify which of the two most probable mechanisms, loss of enzyme protein from the cells of denaturation of the protein, operates. If the first operates, cellular enzymatic activity and enzyme protein as identified by immunocytochemistry should give a linear correlation. If the second operates, there should be no correlation. In this study, five individual samples were analyzed before and after cryopreservation for immunoreactive Cu/Zn SOD and cell intactness by flow cytometry, for SOD enzymatic activity by a highly sensitive fluorimetric method, and for motility characteristics by Hamilton-Thorn motility analyzer. Fresh samples were obtained by the "swim-up" method and had > 95% intact cells with > 78% motile cells. After freeze-thaw, about half the cells were intact. SOD enzymatic activity was determined on Triton X-100 cell extracts, a method that removes all enzymatic activity from the cell structure, and compared with immunoreactive SOD in the cells as determined by indirect immunofluorescence mean intensities. Residual immunofluorescence was observed in the cells after Triton X-100 treatment; if this was taken into account, a close linear correlation between SOD enzyme activity and SOD immunoreactivity was obtained (r = 0.90; P = 0.00014). There was no correlation between SOD enzyme activity ratios for cryopreserved and fresh cells and fraction of intact cells after freeze-thaw. We conclude that loss of SOD protein from the subset of cells undergoing acute membrane damage is the most probable primary mechanism of SOD enzymatic activity loss from the sample and that resistance to cryodamage and SOD activity in any given cell are quite independent of one another.

Rapid decrease in the expression of 3-hydroxy-3-methylglutaryl-CoA reductase protein owing to inhibition of its rate of synthesis after Ca2+ mobilization in rat hepatocytes. Inability of taurolithocholate to mimic the effect

The mechanisms through which Ca2+ mobilization in rat hepatocytes results in the loss of total activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase [Zammit & Caldwell (1990) Biochem. J. 269, 373-379] were investigated. The loss of total activity was shown to be paralleled by an equal loss of immunoreactive HMG-CoA reductase protein after exposure of hepatocytes to optimal concentrations of vasopressin plus glucagon for 40 min. This loss of enzyme protein was due to an inhibition of enzyme synthesis; the rate of degradation was unaffected. Other Ca(2+)-mobilizing conditions (phenylephrine, glucagon, vasopressin added singly and A23187) also resulted in graded inhibition of synthesis of HMG-CoA reductase. These effects were accentuated by omission of Ca2+ from the cell incubation medium, suggesting that it is the depletion of an intracellular InsP3-sensitive pool of Ca2+ to which synthesis of HMG-CoA reductase is sensitive. In agreement with this we found that t-butylhydroxybenzoquinone, which inhibits the activity of the Ca(2+)-ATPase of the endoplasmic-reticular membrane, mimicked the action of Ca(2+)-mobilizing hormones. However, taurolithocholate, which transiently mobilizes Ca2+ from the same pool, was ineffective. All these effects on HMG-CoA reductase were accompanied by parallel inhibition of 35S incorporation from [35S]methionine into total protein, suggesting that inhibition of reductase synthesis formed part of a generalized response of the hepatocyte to Ca2+ mobilization. Inhibition of the rate of synthesis of HMG-CoA reductase was, however, more responsive to Ca2+ mobilization in the absence of added Ca2+ from the extracellular medium. The concentrations of vasopressin required to elicit the inhibition of synthesis of HMG-CoA reductase were of the same order as those that elicited activation of glycogen phosphorylase in hepatocytes.

Glucagon and ammonia influence the long-term regulation of phosphate-dependent glutaminase activity in primary cultures of rat hepatocytes

1. Glutaminase activity was measured in primary cultures of hepatocytes. 2. Enzyme activity decreased markedly after 24-40 h in culture, and this loss of activity was accompanied by loss of enzyme protein. 3. The loss of activity was delayed by high concentrations of glutamine, and was abolished by the continuous presence of NH4Cl in the culture medium. 4. In cells from rats fed on high-carbohydrate protein-free diet, glutaminase activity was increased by glucagon, but not by dexamethasone. This induction was observed only in the continuous presence of NH3 or high concentrations of glutamine. 5. It is concluded that NH3 and glutamine are essential for the stabilization and induction of glutaminase activity in hepatocytes. The inactivation of glutaminase in hepatocytes and in vivo under certain conditions may be due to lack of NH3 in the extracellular medium.

Translocation of 5-lipoxygenase to the membrane in human leukocytes challenged with ionophore A23187.

Challenge of human peripheral blood leukocytes with ionophore A23187 resulted in leukotriene (LT) synthesis, a decrease in total cellular 5-lipoxygenase activity, and a change in the subcellular localization of the enzyme. In homogenates from control cells, greater than 90% of the 5-lipoxygenase activity and protein was localized in the cytosol (100,000 X g supernatant). Ionophore challenge (2 microM) resulted in a loss of approximately 55% of the enzymatic activity and 35% of the enzyme protein from the cytosol. Concomitantly, there was an accumulation of inactive 5-lipoxygenase in the membrane (100,000 X g pellets) which accounted for at least 45% of the lost cytosolic protein. There was a good correlation between the quantities of LT synthesized and 5-lipoxygenase recovered in the membrane over an ionophore concentration range of 0.1-6 microM. The time course of the membrane association was similar to that of LT synthesis. Furthermore, although the pellet-associated enzyme recovered from ionophore-treated leukocytes was inactive, an irreversible, Ca2+-dependent membrane association of active 5-lipoxygenase could be demonstrated in cell-free systems. To determine whether ionophore treatment induced proteolytic degradation of 5-lipoxygenase, the total activity and protein content of 10,000 X g supernatants from control and ionophore-treated cells were examined. These supernatants, which included both cytosolic and membrane-associated enzyme, showed a 35% loss of 5-lipoxygenase activity but only an 8% loss of enzyme protein as a result of ionophore challenge (2 microM). Therefore, the majority of the loss of 5-lipoxygenase activity was most likely due to suicide inactivation during the LT synthesis, rather than to proteolytic degradation. Together these results are consistent with the hypothesis that ionophore treatment results in a Ca2+-dependent translocation of 5-lipoxygenase from the cytosol to a membrane-bound site, that the membrane-associated enzyme is preferentially utilized for LT synthesis, and that it is consequently inactivated. Thus, membrane translocation of 5-lipoxygenase may be an important initial step in the chain of events leading to full activation of this enzyme in the intact leukocyte.

Synthesis and secretion of lipoprotein lipase in 3T3-L1 adipocytes. Demonstration of inactive forms of lipase in cells.

3T3-L1 adipocytes in culture incorporated [35S]methionine into a protein which could be immunoprecipitated with chicken antiserum to bovine lipoprotein lipase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed this protein had an Mr of 55,000, similar to that of bovine lipoprotein lipase, and accounted for 0.1-0.5% of total protein synthesis in the adipocytes. Lipoprotein lipase protein was present in small amounts in confluent 3T3-L1 fibroblasts, and the amount increased many-fold as the cells differentiated into adipocytes. This increase was accompanied by parallel increases in cellular lipase activity and secretion. When cells were grown with [35S]methionine, the amount of label incorporated into lipoprotein lipase increased for 2 h and then leveled off. Pulse-chase experiments showed that half-life of newly synthesized lipase was about 1 h. Turnover of lipoprotein lipase in control cells involved both release to the medium and intracellular degradation. When N-linked glycosylation was blocked by tunicamycin, the cells synthesized a form of lipase that had a smaller Mr (48,000), was catalytically inactive, and was not released to the medium. Radioimmunoassay demonstrated that 3T3-L1 adipocytes contained an unexpectedly large amount of lipoprotein lipase protein. 55% of the enzyme protein in acetone/ether powder of the cells was insoluble in 50 mM NH3/NH4Cl at pH 8.1, a solution commonly used to extract lipoprotein lipase; 27% of the lipase protein was soluble but did not bind to heparin-Sepharose and had very low lipase activity; and the remaining 13% was soluble, bound to heparin-Sepharose, and had high lipolytic activity. About one-half of the lipase released spontaneously to the medium was inactive, and lipase inactivation proceeded in the medium with little loss of enzyme protein. Lipoprotein lipase released heparin, in contrast, was fully active and more stable. When protein synthesis was blocked by cycloheximide, the level of lipoprotein lipase activity in adipocytes decreased more rapidly than the amount of lipase protein in the cells. Most of the inactive lipoprotein lipase in adipocytes probably results from dissociation of active dimeric lipase, but some could be a precursor of active enzyme.

The disappearance of isocitrate lyase from the green alga Chlorella fusca studied by immunoprecipitation

When acetate-adapted cultures of Chlorella fusca were transferred to nitrogen-free medium containing glucose, isocitrate lyase activity was lost over a period of about 25 h. Using a combination of in vivo isotope labelling and immunoprecipitation with anti-isocitrate lyase IgG it was shown that: 1. The onset of loss of enzyme activity preceeded the complete cessation of enzyme synthesis. 2. Disappearance of isocitrate lyase activity was accompanied by loss of enzyme protein, without accumulation of antigenic protein distinguishable from the normal subunit polypeptide of the enzyme, as judged by SDS gel electrophoresis of immunoprecipitated samples from supernatant cell-free extracts. 3. SDS gel electrophoresis of immunoprecipitated isocitrate lyase revealed the presence of antigenic protein bands of Mr about twice that of the normal subunit polypeptide, but the appearance of these apparent dimer forms did not obviously correlate with enzyme degradation. 4. Isoelectric focusing of immunoprecipitated isocitrate lyase showed that the enzyme became progressively more oxidised during the period of its degradation in vivo. 5. By titrating crude broken cell suspensions with anti-isocitrate lyase antibody, preliminary evidence was obtained for transfer of the enzyme from the soluble fraction to an insoluble form as part of the process of disappearance.

Alteration of tyrosine hydroxylase activity in PC12 cells infected with herpes simplex virus type 1.

During infection with herpes simplex virus type 1 (HSV-1) the activity of tyrosine hydroxylase (TH) in PC12 pheochromocytoma cells was initially depressed reaching a nadir at 6 hours post-inoculation, but recovered rapidly with a return to baseline activity by 8 to 9 hours post-inoculation. Subsequently, TH activity again fell with a second more variable rise in activity occurring at 24 hours post-inoculation. Studies with metabolic inhibitors and 2 temperature-sensitive viral mutants indicated that these alterations of TH activity were dissociated from morphological cytopathology and likely required expression of "late" viral gene products. Immunotitration using anti-TH antibody suggested that early depression of TH activity resulted principally from loss of enzyme protein rather than simple enzyme inactivation, and that reconstitution of activity at 9 hours was related to augmented enzyme synthesis. These observations illustrate the complexity of perturbed cellular metabolism during HSV-1 infection and suggest involvement of two unexpected processes: alteration of a specialized cell function as a result of viral genes expressed late in the replicative cycle, and augmented synthesis of a cell-coded gene product during the course of infection.

Effect of 1,3-diaminopropane on ornithine decarboxylase enzyme protein in thioacetamide-treated rat liver.

A radioimmunoassay for ornithine decarboxylase was used to study the regulation of this enzyme in rat liver. The antiserum used reacts with ornithine decarboxylase from mouse, human or rat cells. Rat liver ornithine decarboxylase enzyme activity and enzyme protein (as determined by radioimmunoassay) were measured in thioacetamide-treated rats at various times after administration of 1,3-diaminopropane. Enzyme activity declined rapidly after 1,3-diaminopropane treatment as did the amount of enzyme protein, although the disappearance of enzyme activity slightly preceded the loss of immunoreactive protein. The loss of enzyme protein after cycloheximide treatment also occurred rapidly, but was significantly slower than that seen with 1,3-diaminopropane. When 1,3-diaminopropane and cycloheximide were injected simultaneously, the rate of disappearance of enzyme activity and enzyme protein was the same as that seen with cycloheximide alone. These results show that the rapid loss in enzyme activity after 1,3-diaminopropane treatment is primarily due to a loss in enzyme protein and that protein synthesis is needed in order for 1,3-diaminopropane to exert its full effect. A macromolecular inhibitor of ornithine decarboxylase that has been termed antizyme is induced in response to 1,3-diaminopropane, but our results indicate that the loss of enzyme activity is not due to the accumulation of inactive ornithine decarboxylase-antizyme complexes. It is possible that the antizyme enhances the degradation of the enzyme protein. Control experiments demonstrated that the antiserum used would have detected any inactive antizyme-ornithine decarboxylase complexes present in liver since addition of antizyme to ornithine decarboxylase in vitro did not affect the amount of ornithine decarboxylase detected in our radioimmunoassay. Anti-(ornithine decarboxylase) antibodies may be useful in the purification of antizyme since the antizyme-ornithine decarboxylase complex can be immunoprecipitated, and antizyme released from the precipitate with 0.3 M-NaCl.

Inactivation of yeast ornithine decarboxylase by polyamines [formula omitted] does not result from the incorporation of polyamines into enzyme protein

We have previously reported that addition of spermidine and spermine to a culture of Saccharomyces cerevisiae results in loss of ornithine decarboxylase activity even though there is no loss of enzyme protein. These findings indicate that the loss of activity is due to a post-translational modification of the enzyme. Since transglutaminase has been shown to catalyze the incorporation of putrescine into ornithine decarboxylase in vitro with resultant loss of enzymatic activity [ Russell, D. H. (1981) Biochem. Biophys. Res. Commun. 99 , 1167–1172], we decided to see if the inactivation we observed in vivo was caused by such incorporation. We found that this is not the case; i.e., after growth of yeast with added amines, the inactivated ornithine decarboxylase contains no covalently-bound spermidine or spermine. Thus, we conclude that transamidation by transglutaminase does not play a role in vivo in the polyamine-mediated post-translational modification of yeast ornithine decarboxylase.

Inactivation of Extracellular Laccase During Fruiting of Agaricus bisporus

SUMMARY: The decline in activity of extracellular laccase of Agaricus bisporus which occurs during fruit body development was shown to be due to both enzyme inactivation and proteolysis. Extracts of high activity, from cultures during mycelial growth, and of low activity, from cultures during fruiting, remained unchanged on prolonged incubation and did not cross-activate or inhibit on mixing. No activity changes occurred after dialysis, gel filtration or ultrafiltration of both forms. Molecular weights, pH optima, substrate specificities and immunological properties of the two forms were identical. Differences were found in the electrophoretic profiles of the two forms of enzyme. Loss of enzyme activity was shown to be greater than loss of enzyme protein by separate immunological and electrophoretic methods.