Mechanism of action of rabbit liver phosphoglucomutase.
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ABSTRACT:
SUBMITTER: Jamil H
PROVIDER: S-EPMC1152685 | biostudies-other | 1985 Sep
REPOSITORIES: biostudies-other
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Project description:1. The kinetics of phosphoglucomutases from different sources are discussed and it is concluded that on the available evidence there are in all cases three possible mechanisms for the reaction. These are an indirect transfer of phosphate involving the phosphoenzyme (mechanism 1), a direct transfer of phosphate (mechanism 2), and an intermolecular transfer of phosphate from glucose 1,6-diphosphate to the substrate (mechanism 3). Conventional net flux measurements are shown not to differentiate between these mechanisms. 2. Flux equations are developed and it is shown that there are three flux ratios that characterize and distinguish between the mechanisms. 3. To examine these flux ratios induced-transport tests are described with (14)C- and (32)P-labelled substrates. The fluxes determined with (14)C- and (32)P-labelled substrates are also compared at chemical equilibrium. 4. With rabbit muscle phosphoglucomutase the results of these tests were completely consistent with mechanism 1 and unequivocally excluded any substantial part of the reaction proceeding by mechanism 2 or mechanism 3. Evidence was also obtained for an isomerization of the phosphoenzyme with an apparent rate constant about 4.5x10(7)sec.(-1). Taking into account the activity coefficients of the substrates the true rate constant appears to be about one-sixth of this value. 5. Isotope effects and non-ideal behaviour of the solutions are discussed and the activity coefficients of the substrates are shown to be equal by measurement of the depression of freezing point. It is concluded that these factors do not influence the tests significantly. 6. Alternative mechanisms are considered and it is concluded that the tests show that the glucose residue is transferred directly, that the phosphate is transferred indirectly with one intermediate phosphate, and that there is an isomerization of the free phosphoenzyme without reference to any other details of the reaction. Further, no assumptions are required about the constancy of rate constants. 7. The relative merits of induced transport and product inhibition for detecting isomerization of the enzyme are discussed. It is concluded that the induced-transport test is more sensitive and that its interpretation is less equivocal. 8. The application of the tests to other enzyme systems is briefly considered.
| S-EPMC1187194 | biostudies-other
Project description:Myocardial ischemia is associated with significant changes in action potential (AP) duration, which has a biphasic response to metabolic inhibition. Here, we investigated the mechanism of initial AP prolongation in whole Langendorff-perfused rabbit heart. We used glass microelectrodes to record APs transmurally. Simultaneously, optical AP, calcium transient (CaT), intracellular pH, and magnesium concentration changes were recorded using fluorescent dyes. The fluorescence signals were recorded using an EMCCD camera equipped with emission filters; excitation was induced by LEDs. We demonstrated that metabolic inhibition by carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) resulted in AP shortening preceded by an initial prolongation and that there were no important differences in the response throughout the wall of the heart and in the apical/basal direction. AP prolongation was reduced by blocking the ICaL and transient outward potassium current (Ito) with diltiazem (DTZ) and 4-aminopyridine (4-AP), respectively. FCCP, an uncoupler of oxidative phosphorylation, induced reductions in CaTs and intracellular pH and increased the intracellular Mg2+ concentration. In addition, resting potential depolarization was observed, clearly indicating a decrease in the inward rectifier K+ current (IK1) that can retard AP repolarization. Thus, we suggest that the main currents responsible for AP prolongation during metabolic inhibition are the ICaL, Ito, and IK1, the activities of which are modulated mainly by changes in intracellular ATP, calcium, magnesium, and pH.
| S-EPMC6095129 | biostudies-literature
Project description:The mechanism of the phosphoglucomutase from Micrococcus lysodeikticus was investigated. Induced-transport tests at low substrate concentrations (0.15mm) showed co-transport of the (32)P label but no induced transport of the (14)C label, which is in quantitative agreement with a phosphoenzyme mechanism with a rapid isomerization of the phosphoenzyme. The results excluded an intramolecular transfer of phosphate and could only have been compatible with a sequential mechanism if the K(m) for glucose 1-phosphate had been over 20 times smaller than the measured value. The results of induced-transport tests at intermediate concentrations (1mm) with both labels agreed quantitatively with a phosphoenzyme mechanism, and induced-transport tests with (14)C-labelled substrates at high concentrations (26mm) indicated that the rate constants for isomerization of the phosphoenzyme must be greater than about 3x10(6)s(-1). Consistent with these findings is the fact that (14)C label exchanged between the substrates twice as rapidly as the (32)P label at chemical equilibrium. Further, since the (14)C label exchanged between the substrates about ten times more rapidly than between the substrates and glucose 1,6-diphosphate, glucose 1,6-diphosphate is not an obligatory intermediate in the interconversion of the substrates. It is concluded that, contrary to previous evidence, the mechanism of the enzyme from M. lysodeikticus is essentially that of the rabbit muscle enzyme. To account for the rapid isomerization of the phosphoenzyme in both cases a mechanism is proposed in which there is no formal isomerization of the phosphoenzyme.
| S-EPMC1166144 | biostudies-other
Project description:Background and purposeMarine sponges have evolved the capacity to produce a series of very efficient chemicals to combat viruses, bacteria, and eukaryotic organisms. It has been demonstrated that several of these compounds have anti-neoplastic activity. The highly toxic sponge Crambe crambe has been the source of several molecules named crambescidins. Of these, crambescidin-816 has been shown to be cytotoxic for colon carcinoma cells. To further investigate the potential anti-carcinogenic effect of crambescidin-816, we analysed its effect on the transcription of HepG2 cells by microarray analysis followed by experiments guided by the results obtained.Experimental approachAfter cytotoxicity determination, a transcriptomic analysis was performed to test the effect of crambescidin-816 on the liver-derived tumour cell HepG2. Based on the results obtained, we analysed the effect of crambescidin-816 on cell-cell adhesion, cell-matrix adhesion, and cell migration by Western blot, confocal microscopy, flow cytometry and transmission electron microscopy. Cytotoxicity and cell migration were also studied in a variety of other cell lines derived from human tumours.Key resultsCrambescidin-816 had a cytotoxic effect on all the cell lines studied. It inhibited cell-cell adhesion, interfered with the formation of tight junctions, and cell-matrix adhesion, negatively affecting focal adhesions. It also altered the cytoskeleton dynamics. As a consequence of all these effects on cells crambescidin-816 inhibited cell migration.Conclusions and implicationsThe results indicate that crambescidin-816 is active against tumour cells and implicate a new mechanism for the anti-tumour effect of this compound.
| S-EPMC3966746 | biostudies-literature
Project description:Liver disease, involving a wide range of liver pathologies from fatty liver, hepatitis, and fibrosis to cirrhosis and hepatocellular carcinoma, is a serious health problem worldwide. In recent years, many natural foods and herbs with abundant phytochemicals have been proposed as health supplementation for patients with hepatic disorders. As an important category of phytochemicals, natural polyphenols have attracted increasing attention as potential agents for the prevention and treatment of liver diseases. The striking capacities in remitting oxidative stress, lipid metabolism, insulin resistance, and inflammation put polyphenols in the spotlight for the therapies of liver diseases. It has been reported that many polyphenols from a wide range of foods and herbs exert therapeutic effects on liver injuries via complicated mechanisms. Therefore, it is necessary to have a systematical review to sort out current researches to help better understand the potentials of polyphenols in liver diseases. In this review, we aim to summarize and update the existing evidence of natural polyphenols in the treatment of various liver diseases by in vitro, in vivo, and clinical studies, while special attention is paid to the action mechanisms.
| S-EPMC5817364 | biostudies-literature
Project description:BackgroundAmoebic liver abscess (ALA) is the most frequent clinical presentation of extra-intestinal amoebiasis. The diagnosis of ALA is typically based on the developing clinical symptoms, characteristic changes on radiological imaging and serology. Numerous serological tests have been introduced for the diagnosis of ALA, either detecting circulating amoebic antigens or antibodies. However those tests show some pitfalls in their efficacy and/or the preparation of the tests are costly and tedious. The commercial IHA kit that used crude antigen was reported to be useful in diagnosis of ALA, however high antibody background in endemic areas may cause problems in its interpretation. Thus, discovery of well-defined antigen(s) is urgently needed to improve the weaknesses of current serodiagnostic tests.MethodsCrude antigen of E. histolytica was analysed by 2-DE and Western blot to identify a protein of diagnostic potential for ALA. The corresponding gene of the antigenic protein was then cloned, expressed and the purified recombinant protein was subsequently evaluated for serodiagnosis of ALA in an indirect ELISA format.ResultsAnalysis of crude antigen showed that phosphoglucomutase (PGM) has the diagnostic potential. Recombinant PGM (rPGM) showed 79.17% (19/24) sensitivity and 86.67% (195/225) specificity in diagnosis of ALA based on the COV of mean +1SD. There was no significant difference between rPGM-ELISA and IHA diagnostic kit in the diagnosis of ALA in terms of sensitivity and specificity at p-value < 0.05.ConclusionIn conclusion, rPGM-ELISA is found to be useful for serodiagnosis of ALA. Future studies will determine whether rPGM-ELISA also detects antibodies produced in amoebic dysentery and asymptomatic cases.
| S-EPMC3606622 | biostudies-literature
Project description:Human phosphoglucomutase 1 (PGM1) plays a central role in cellular glucose homeostasis, mediating the switch between glycolysis and gluconeogenesis through the conversion of glucose 1-phosphate and glucose 6-phosphate. Recent clinical studies have identified mutations in this enzyme as the cause of PGM1 deficiency, an inborn error of metabolism classified as both a glycogen storage disease and a congenital disorder of glycosylation. Reported here are the first crystal structures of two disease-related missense variants of PGM1, along with the structure of the wild-type enzyme. Two independent glycine-to-arginine substitutions (G121R and G291R), both affecting key active site loops of PGM1, are found to induce regions of structural disorder, as evidenced by a nearly complete loss of electron density for as many as 23 aa. The disordered regions are not contiguous in sequence to the site of mutation, and even cross domain boundaries. Other structural rearrangements include changes in the conformations of loops and side chains, some of which occur nearly 20 Å away from the site of mutation. The induced structural disorder is correlated with increased sensitivity to proteolysis and lower-resolution diffraction, particularly for the G291R variant. Examination of the multi-domain effects of these G➔R mutations establishes a correlation between interdomain interfaces of the enzyme and missense variants of PGM1 associated with disease. These crystal structures provide the first insights into the structural basis of enzyme dysfunction in PGM1 deficiency and highlight a growing role for biophysical characterization of proteins in the field of precision medicine.
| S-EPMC5802404 | biostudies-literature
Project description:The purpose of this study was to assess the cytotoxic potential of a novel piperazine derivative (PCC) against human liver cancer cells. SNU-475 and 423 human liver cancer cell lines were used to determine the IC50 of PCC using the standard MTT assay. PCC displayed a strong suppressive effect on liver cancer cells with an IC50 value of 6.98 ± 0.11 µM and 7.76 ± 0.45 µM against SNU-475 and SNU-423 respectively after 24 h of treatment. Significant dipping in the mitochondrial membrane potential and elevation in the released of cytochrome c from the mitochondria indicated the induction of the intrinsic apoptosis pathway by PCC. Activation of this pathway was further evidenced by significant activation of caspase 3/7 and 9. PCC was also shown to activate the extrinsic pathways of apoptosis via activation of caspase-8 which is linked to the suppression of NF-κB translocation to the nucleus. Cell cycle arrest in the G1 phase was confirmed by flow cytometry and up-regulation of glutathione reductase expression was quantified by qPCR. Results of this study suggest that PCC is a potent anti-cancer agent inducing both intrinsic and extrinsic pathways of apoptosis in liver cancer cell lines.
| S-EPMC4806608 | biostudies-literature
Project description:(32)P-labelled phosphoglucomutase was digested with trypsin after denaturation and two peptides were isolated that contained the bulk of the radioactivity bound to peptides. Both peptides appeared to derive from an identical section of the molecule. Peptic and subtilisin digests of the tryptic peptides were prepared. The resulting radioactive peptides were purified and their sequences studied. The presence of a single serine [(32)P]phosphate residue was clearly established. Difficulties in purification and low yields, especially of the tryptic peptide, prevented exhaustive sequence studies, but a tentative sequence is proposed as:Ala-Ile-Gly-Gly-Ile-Ile-Leu-Thr-Ala-SerP-His-Asx-Pro-Gly-Gly-Pro-(Asx(2),Gly)-Phe-Gly-Ile-Lys(where SerP represents serine phosphate and Asx represents aspartic acid or asparagine). The results do not support the presence of two serine phosphate residues in the denatured enzyme, but confirm previous results of a unique sequence around a single serine phosphate residue.
| S-EPMC1186757 | biostudies-other
Project description:Rabbit haemorrhagic disease virus (RHDV) belongs to the family Caliciviridae, genus Lagovirus and is used in Australia as a biocontrol tool to keep the population of european rabbits low. This virus has a positive-sense single-stranded RNA genome that encodes structural (capsid) and non-structural proteins. Due to the lack of an established cell culture system for this virus, some of the non-structural proteins are yet awaiting characterisation and their function is unknown. This work attempted to characterise the process of RHDV infection and identify pathways that alterate in RHDV-infected rabbit liver at the proteome level. Young rabbits were infected with RHDV2 (genotype GI.1bP-GI.2) and humanely killed 24 hours post-infection. 25% liver homogenates were prepared in RNAlater buffer and stored at -20C. Uninfected rabbit liver samples served as a control. Samples from three RHDV2-infected animals (K375, K376, K378) and three uninfected animals (K3, K14, K12) were used in this study.
2022-10-14 | PXD031027 | Pride