The inhibitory effect of ATP on HMGCoA reductase

The inhibitory effect of ATP on HMGCoA reductase activity from rat liver microsomes in the system described by Beg $\text{et al.}$ was examined. The inhibition by magnesium ATP is confirmed but varies widely from zero to complete. A requirement for a cytosolic fraction to enhance the inhibition could not be established. ATP labeled uniformly with ¹⁴C in the adenine portion and ³²P in the terminal phosphate was incubated with the enzyme in a situation where strong inhibition was observed. The enzyme protein was precipitated with trichloroacetic acid, or subjected to column fractionation. No evidence of labeling was found in the protein. Finally, the enzyme protein was specifically isolated by immunoprecipitation with a specific antibody to the HMGCoA reductase. In no instance could labeling of the enzyme protein be detected. These results show that the mechanism of the inhibition does not involve phosphorylation or adenylation of the enzyme protein.     

HMGCoA reductase potentiates hedgehog signaling in Drosophila melanogaster

Drosophila HMGCoA reductase (hmgcr) catalyzes the biosynthesis of a mevalonate precursor for isoprenoids and has been implicated in the production of a signal by the somatic gonadal precursor cells (SGPs) that attracts migrating germ cells. Here, we show that hmgcr functions in the hedgehog (hh) signaling pathway. When hmgcr activity is reduced, high levels of Hh accumulate in hh-expressing cells in each parasegment, while the adjacent "Hh-receiving" cells cannot sustain wg expression and fail to relocalize the Smoothened (Smo) receptor. Conversely, ectopic Hmgcr upregulates Hh signaling when it is produced in hh-expressing cells, but has no effect when produced in the receiving cells. These findings suggest that Hmgcr might orchestrate germ cell migration by promoting the release and/or transport of Hh from the SGPs. Consistent with this model, there are substantial germ cell migration defects in trans combinations between hmgcr and mutations in different components of the hh pathway.     

Isoprenoids control germ cell migration downstream of HMGCoA reductase

3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoAr) provides attractive cues to Drosophila germ cells, guiding them toward the embryonic gonad. However, it remains unclear how HMGCoAr mediates this attraction. In a genomic analysis of the HMGCoAr pathway, we found that the fly genome lacks several enzymes required for cholesterol biosynthesis, ruling out cholesterol and cholesterol-derived proteins as mediators of PGC migration. Genetic analysis of the pathway revealed that two enzymes, farnesyl-diphosphate synthase and geranylgeranyl-diphosphate synthase, required for the production of isoprenoids, act downstream of HMGCoAr in germ cell migration. Consistent with a role in geranylgeranylation, embryos deficient in geranylgeranyl transferase type I show germ cell migration defects. Our data, together with similar findings in zebrafish, implicate an isoprenylated protein in germ cell attraction. The specificity and evolutionary conservation of the HMGCoAr pathway for germ cells suggest that an attractant common to invertebrates and vertebrates guides germ cells in early embryos.     

HMGCoA reductase and cholesterol 7 alpha-hydroxylase in human liver

The activities of HMGCoA reductase and cholesterol 7 alpha-hydroxylase were assayed in liver biopsies of patients with or without cholestyramine treatment. The active dephosphorylated form of HMGCoA reductase and the activity of cholesterol 7 alpha-hydroxylase were under the detection limits in untreated subjects. After cholestyramine treatment activities of both enzymes were stimulated and the active form of HMGCoA reductase became detectable in four out of the five tested patients. In two subjects who received cholestyramine, the effect of exogenously added [4-14C] cholesterol on cholesterol 7 alpha-hydroxylase was tested. In the presence of Tween 80, the detergent by which [14C]cholesterol was suspended, the enzyme activity was profoundly inhibited and synthesis of 7 alpha-hydroxycholesterol was extremely low.     

Seasonal commitment of HMGCoA reductase activity to vitellogenin production

In female frogs (Rana Esculenta) during gametogenesis the cholesterol synthesized in the liver by 3-hydroxy-3-methylglutaryl coenzyme A reductase is mostly exported into the blood and taken up by the oocytes.In order to understand the fate of the neosynthesized cholesterol, female and male frogs and estrogenized male controls were injected with the labelled precursor¹⁴C mevalonate.In females and in estrogenized controls, mevalonate-derived radioactivity is found in a plasmatic lipoprotein that has been identified as vitellogenin by immunological detection.The increased 3-hydroxy-3-methylglutaryl coenzyme A reductase activity present in females in Fall is likely to be committed to provide cholesterol for the lipidation of this cholesterol-rich protein.     

Inhibition of HMGCoA reductase by simvastatin protects mice from injurious mechanical ventilation

Background

Mortality from severe acute respiratory distress syndrome exceeds 40% and there is no available pharmacologic treatment. Mechanical ventilation contributes to lung dysfunction and mortality by causing ventilator-induced lung injury. We explored the utility of simvastatin in a mouse model of severe ventilator-induced lung injury.

Methods

Male C57BL6 mice (n = 7/group) were pretreated with simvastatin or saline and received protective (8 mL/kg) or injurious (25 mL/kg) ventilation for four hours. Three doses of simvastatin (20 mg/kg) or saline were injected intraperitoneally on days −2, −1 and 0 of the experiment. Lung mechanics, (respiratory system elastance, tissue damping and airway resistance), were evaluated by forced oscillation technique, while respiratory system compliance was measured with quasi-static pressure-volume curves. A pathologist blinded to treatment allocation scored hematoxylin-eosin-stained lung sections for the presence of lung injury. Pulmonary endothelial dysfunction was ascertained by bronchoalveolar lavage protein content and lung tissue expression of endothelial junctional protein Vascular Endothelial cadherin by immunoblotting. To assess the inflammatory response in the lung, we determined bronchoalveolar lavage fluid total cell content and neutrophil fraction by microscopy and staining in addition to Matrix-Metalloprotease-9 by ELISA. For the systemic response, we obtained plasma levels of Tumor Necrosis Factor-α, Interleukin-6 and Matrix-Metalloprotease-9 by ELISA. Statistical hypothesis testing was undertaken using one-way analysis of variance and Tukey’s post hoc tests.

Results

Ventilation with high tidal volume (HVt) resulted in significantly increased lung elastance by 3-fold and decreased lung compliance by 45% compared to low tidal volume (LVt) but simvastatin abrogated lung mechanical alterations of HVt. Histologic lung injury score increased four-fold by HVt but not in simvastatin-pretreated mice. Lavage pleocytosis and neutrophilia were induced by HVt but were significantly attenuated by simvastatin. Microvascular protein permeability increase 20-fold by injurious ventilation but only 4-fold with simvastatin. There was a 3-fold increase in plasma Tumor Necrosis Factor-α, a 7-fold increase in plasma Interleukin-6 and a 20-fold increase in lavage fluid Matrix-Metalloprotease-9 by HVt but simvastatin reduced these levels to control. Lung tissue vascular endothelial cadherin expression was significantly reduced by injurious ventilation but remained preserved by simvastatin.

Conclusion

High-dose simvastatin prevents experimental hyperinflation lung injury by angioprotective and anti-inflammatory effects.     

Germ cell migration in zebrafish is dependent on HMGCoA reductase activity and prenylation

Hydroxymethylglutaryl coenzyme A reductase (HMGCoAR) is required for isoprenoid and cholesterol biosynthesis. In Drosophila, reduced HMGCoAR activity results in germ cell migration defects. We show that pharmacological HMGCoAR inhibition alters zebrafish development and germ cell migration. Embryos treated with atorvastatin (Lipitor) exhibited germ cell migration defects and mild morphologic abnormalities. The effects induced by atorvastatin were completely rescued by prior injection of mevalonate, the product of HMGCoAR activity, or the prenylation precursors farnesol and geranylgeraniol. In contrast, squalene, a cholesterol intermediate further down the pathway, failed to rescue statin-induced defects. Moreover, pharmacologic inhibition of geranylgeranyl transferase 1 (GGT1) protein prenylation activity also resulted in abnormal germ cell migration. Thus, our pharmacological inhibition-and-rescue approach provided detailed information about the elements of isoprenoid biosynthesis that contribute to germ cell migration. Together with data from Drosophila (Santos and Lehmann, this issue), our results highlight a conserved role for protein geranylgeranylation in this context.     

Reversible inactivation of maize leaf nitrate reductase

Preincubation of maize leaves crude extracts with NADH resulted in a progressive accumulation of nitrite which mimicked a rapid and lineal activation of nitrate reductase. Nevertheless, in partially purified preparations it was found that preincubation at pH 8.8 with NADH promoted a gradual inactivation of nitrate reductase. At pH 7.5, the enzyme was not inactivated by the presence of NADH alone, but, with the simultaneous presence of a low concentration of cyanide, a fast inactivation took place. The NADH-cyanide-inactivated nitrate reductase remained inactive after removing the excess of NADH and cyanide by filtration through Sephadex G-25. However, it could be readily reactivated by incubation with ferricyanide or by a short exposure to light in the presence of FAD. Prolonged irradiation caused a progressive inactivation of the photoreactivated enzyme.     

A plant oxysterol, 28-homobrassinolide binds HMGCoA reductase catalytic cleft: stereoselective avidity affects enzyme function

Understanding the influence of ubiquitously present plant steroids on mammalian cell biology is currently of interest. Feedback inhibition of HMGCoA reductase (HMGCR) catalytic activity in the transformation of HMG-CoA to mevalonate is a significant regulatory step in sterol biosynthetic pathway. To assess the role of dietary steroids in this biochemical transformation, the phytosteroid isoform 28-homobrassinolide (28-HB), 90 % pure, obtained from Godrej Agrovet (India) was used to determine its effect on mammalian HMG-CoA reductase. Photometric assay of pure human and select rat tissue HMGCR post 28-HB oral feed, PCR-HMGCR gene expression, and in silico docking of 28-HB and HMGCoA on HMGCR protein template were carried out. Using an oral feed regimen of pure 28-HB, we noted a decrease of 16 % in liver, 17.1 % in kidney and 9.3 % in testicular HMGCR enzyme activity, 25 % in HMGCR gene expression and 44 % in the activity of pure human HMGCR due to this plant oxysterol. In silico docking studies yielded binding metrics for 28-HB-HMGCR lower than for HMGCoA-HMGCR, indicating stronger binding of HMGCR by this ligand. 28-HB exerts differential effects on rat tissue HMGCR, down regulates liver HMGCR gene expression and significantly inhibits HMGCR activity.     

Structural determinant for cold inactivation of rodent L-xylulose reductase

L-Xylulose reductase (XR) is a homotetramer belonging to the short-chain dehydrogenase/reductase family. Human XR is stable at low temperature, whereas the enzymes of mouse, rat, guinea pig, and hamster are rapidly dissociated into their inactive dimeric forms. In order to identify amino acid residues that cause cold inactivation of the rodent XRs, we have here selected Asp238, Leu242, and Thr244 in the C-terminal regions of rodent XRs and performed site-directed mutagenesis of the residues of mouse XR to the corresponding residues (Glu, Trp, and Cys) of the human enzyme. Cold inactivation was prevented partially by the single mutation of L242W and the double mutation of L242W/T244C, and completely by the double mutation of D238E/L242W. The L242W and L242W/T244C mutants existed in both tetrameric and dimeric forms at low temperature and the D238E/L242W mutant retained its tetrameric structure. No preventive effect was exerted by the mutations of D238E and T244C, which were dissociated into their dimeric forms upon cooling. Crystallographic analysis of human XR revealed that Glu238 and Trp242 contribute to proper orientation of the guanidino group of Arg203 of the same subunit to the C-terminal carboxylate group of Cys244 of another subunit through the neighboring residues, Gln137 and Phe241. Thus, the determinants for cold inactivation of rodent XRs are Asp238 and Leu242 with small side chains, which weaken the salt bridges between Arg203 and the C-terminal carboxylate group, and lead to cold inactivation.     

HMGCoA reductase inhibition partially mediates tumor necrosis factor alpha-induced apoptosis in human U-937 and HL-60 cells

We have examined the effects of tumor necrosis factor alpha (TNF alpha) and its second messenger, ceramide, on HMGCoA reductase, the rate-limiting enzyme in the mevalonate pathway. Treatment of human U-937 and HL-60 cells with TNF alpha or C2-ceramide inhibited both expression and activity of HMGCoA reductase in a time-dependent manner. Maturation of p21(ras) was also inhibited in a mevalonate-dependent fashion. The addition of mevalonate to both U-937 and HL-60 cells could also partially prevent TNF alpha and ceramide-induced apoptosis. These results support the hypothesis that the inhibition of HMGCoA reductase expression and the subsequent decrease in prenylation of proteins such as p21(ras) are part of the mechanism by which TNF alpha induces apoptosis in these cells.     

Mechanism of enteroviral inactivation by ozone

The mechanism of enteroviral inactivation by ozone was investigated with poliovirus 1 (Mahoney) as the model virus. Ozone was observed to alter two of the four polypeptide chains present in the viral protein coat of poliovirus 1. However, the alteration of the protein coat did not significantly impair virus adsorption or alter the integrity of the virus particle. Damage to the viral RNA after exposure to ozone was demonstrated by velocity sedimentation analysis. It was concluded that the damage to the viral nucleic acid is the major cause of poliovirus 1 inactivation by ozone.     

Reversible inactivation of Saccharomyces cerevisiae glutathione reductase under reducing conditions

Glutathione reductase from Saccharomyces cerevisiae was rapidly inactivated following aerobic incubation with NADPH, NADH, and several other reductants, in a time- and temperature-dependent process. The inactivation had already reached 50% when the NADPH concentration reached that of the glutathione reductase subunit. The inactivation was very marked at pH values below 5.5 and over 7, while only a slight activity decrease was noticed at pH values between these two values. After elimination of excess NADPH the enzyme remained inactive for at least 4 h. The enzyme was protected against redox inactivation by low concentrations of GSSG, ferricyanide, GSH, or dithiothreitol, and high concentrations of NAD(P)+; oxidized glutathione effectively protected the enzyme at concentrations even lower than GSH. The inactive enzyme was efficiently reactivated after incubation with GSSG, ferricyanide, GSH, or dithiothreitol, whether NADPH was present or not. The reactivation with GSH was rapid even at 0 degree C, whereas the optimum temperature for reactivation with GSSG was 30 degrees C. A tentative model for the redox interconversion, involving an erroneous intramolecular disulfide bridge, is put forward.     

Reversible inactivation of nitrate reductase in Chlorella vulgaris in vivo

Summary The NADH-nitrate oxidoreductase of Chlorella vulgaris has an inactive form which has previously been shown to be a cyanide complex of the reduced enzyme. This inactive enzyme can be reactivated by treatment with ferricyanide in vitro. In the present study, the activation state of the enzyme was determined after different prior in vivo programs involving environmental variations. Oxygen, nitrate, light and CO₂ all affect the in vivo inactivation of the enzyme in an interdependent manner. In general, the inactivation is stimulated by O₂ and inhibited by nitrate and CO₂. Light may stimulate or inhibit, depending on conditions. Thus, the effects of CO₂ and nitrate (inhibition of reversible inactivation) are clearly manifested only in the light. In contrast, light stimulates the inactivation in the presence of oxygen and the absence of CO₂ and nitrate. Since the inactivation of the enzyme requires HCN and NADH, and it is improbable that O₂ stimulates NADH formation, it is reasonable to conclude that HCN is formed as the result of an oxidation reaction (which is stimulated by light). The formation of HCN is probably stimulated by Mn²⁺, since the formation of reversibly-inactivated enzyme is impaired in Mn²⁺-deficient cells. The prevention of enzyme inactivation by nitrate in vivo is in keeping with previous in vitro results showing that nitrate prevents inactivation by maintaining the enzyme in the oxidized form. A stimulation of nitrate uptake by CO₂ and light could account for the effect of CO₂ (prevention of inactivation) which is seen mainly in the presence of nitrate and light. Ammonia added in the presence of nitrate has the same effect on the enzyme as removing nitrate (promotion of reversible inactivation). Ammonia added in the absence of nitrate has little extra effect. It is therefore likely that ammonia acts by preventing nitrate uptake. The uncoupler, carbonylcyanide-m-chloro-phenylhydrazone, causes enzyme inactivation because it acts as a good HCN precursor, particularly in the light. Nitrite, arsenate and dinitrophenol cause an enzyme inactivation which can not be reversed by ferricyanide in crude extracts. This suggests that there are at least two different ways in which the enzyme can be inactivated rather rapidly in vivo.     

Hemin-mediated oxidative inactivation of 3-hydroxy-3-methylglutaryl CoA reductase

Summary Addition of hemoglobin, methemoglobin, hemin or hematin in the assay mixture of rat liver 3-hydroxy-3-methylglutaryl CoA (HMGCoA) reductase inhibited the activity of the enzyme. The inhibition by hemin was rapid, without any apparent dependence on time of preincubation. At 20 M hemin, a maximum of about 50% inhibition was obtained in the case of the microsomal enzyme while the solubilized enzyme showed almost 80%6 inhibition. Dithiothreitol at high concentrations or either of the two substrates of the enzyme (HMGCoA and NADPH) could afford partial protection when added before hemin. The K_m for both the substrates increased in the presence of hemin. The inhibition by hemin appeared to be irreversible, the presence of KCN or NaN₃ being the only means of preventing the inhibition. Molecular oxygen was required for the inhibition. Oxygen radicals and H₂O₂, however, did not seem to be involved. This offered a clue that an oxidation reaction of the reductase protein may be the likely mechanism of its inactivation. The enzyme protein did not, however, get degraded under the conditions of inhibition.Abbreviations HMGCoA 3-Hydroxy-3-methylglutaryl coenzyme - DTT Dithiothreitol - DTNB 5,5-Dithiobis-(2-nitrobenzoic acid) - SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Mechanism of inactivation of trypsin by antithrombin

General aspects of the mechanism of antithrombin action were elucidated by a comparison of the inactivation of trypsin by antithrombin with the inactivation of coagulation proteinases by the inhibitor. Bovine antithrombin and bovine trypsin were shown to form an inactive equimolar complex. A non-complexed, proteolytically modified form of antithrombin, electrophoretically identical with that formed in the reaction with coagulation proteinases, was also produced in the reaction with trypsin. In the absence of heparin, the inactivation of trypsin by antithrombin was 20 times faster than the inactivation of thrombin; the second-order rate constant was 1.5 x 10(5)m(-1).s(-1) at 25 degrees C and pH 7.4. However, the inhibition of thrombin was accelerated about 30 times more efficiently by small amounts of heparin than was trypsin inhibition. Dissociation of the antithrombin-trypsin complex at pH 7.4 followed first-order kinetics with a half-life for the complex of about 80h at 25 degrees C. The complex was rapidly and quantitatively dissociated at pH 11, resulting in the liberation of a modified two-chain form of the inhibitor, cleaved at the same Arg-Ser bond as in modified antithrombin released from complexes with thrombin, Factor Xa and Factor IXa. This supports the previous proposal that this bond is the active-site bond of antithrombin. Antisera specific for thrombin-modified antithrombin reacted with purified antithrombin-trypsin complex, indicating that the inhibitor was present in the complex in a form immunologically identical with thrombin-modified antithrombin. The results thus suggest a common mechanism, but different kinetics, for the inhibition of trypsin and coagulation proteinases by antithrombin.     

Mechanism of dehydration inactivation of Lactobacillus plantarum

The mechanism of dehydration inactivation of Lactobacillus plantarum cells after vacuum-drying above saturated salt solutions was studied. The method used is based on the hypothesis that DNase diffuses into cells with damaged cell membranes/walls and hydrolyses the intracellular DNA. Intact, undamaged cells and cells inactivated by either dehydration or heat treatent were incubated in the presence of DNase. The release of DNA hydrolysis products into the incubation medium was measured. It was shown that dehydration inactivation of L. plantarum, but not thermal inactivation, was associated with clear evidence of membrane damage. The residual glucose-fermenting activity of the dehydrated cells related to the release of hydrolysed DNA in the medium, but there was no such relationship with heat-treated cells. Addition of sorbitol to cells before dehydration increased the residual glucose-fermenting activity after drying and this was associated with a reduced rate of DNA hydrolysis. It is concluded that cell wall and/or cell membrane damage is an important mechanism of dehydration inactivation, but that thermal inactivation (up to 60°C) occurs by a different mechanism.Correspondence to: K. van't Riet