Comparison of purified bovine heart and rat liver 6-phosphofructo-2-kinase. Evidence for distinct isoenzymes.
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ABSTRACT:
SUBMITTER: Rider MH
PROVIDER: S-EPMC1152722 | biostudies-other | 1985 Oct
REPOSITORIES: biostudies-other
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Project description:Purified bovine heart 6-phosphofructo-2-kinase can be phosphorylated in the presence of protein kinase C and dephosphorylated by alkaline phosphatase; changes in phosphorylation state have no effect on enzyme activity. By contrast, the rat liver enzyme is a poor substrate for protein kinase C. Unlike the liver enzyme, which is bifunctional and is phosphorylated by fructose 2,6-[2-32P]bisphosphate, the heart enzyme contains 10 times less fructose 2,6-bisphosphatase activity and is phosphorylated at a slower rate and to a lesser extent than the liver enzyme. Both rat liver and bovine heart enzymes catalyse a similar exchange reaction between [U-14C]ADP and ATP.
| S-EPMC1147375 | biostudies-other
Project description:In vertebrates, there are different adenylate kinases in the compartments cytosol, mitochondrial intermembrane space, and mitochondrial matrix. Here, we report the spatial structure of the intermembrane species established in two crystal forms by X-ray diffraction analyses at 1.92 and 2.1 A resolution. In both structures, the enzyme is unligated, and thus in an "open" conformation. The enzyme was prepared from bovine liver, containing at least five variants arisen from posttranscriptional and posttranslational modifications. It could only be crystallized after removing some of these variants. A comparison with the known structures of the adenylate kinases from cytosol and mitochondrial matrix reveals structural differences that should play a role in protein targeting because none of these enzymes contains a cleavable signal peptide. A further comparison with adenylate kinases from Gram-positive bacteria showed that the structural Zn2+ ion of these species is replaced by a strictly conserved assembly of hydrogen bonded residues.
| S-EPMC2143366 | biostudies-other
Project description:Purified bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) showed two bands with subunit M(r) of 58,000 and 54,000 when analysed by SDS/PAGE. Both the 58,000- and 54,000-M(r) forms were phosphorylated by cyclic AMP-dependent protein kinase (PKA) and by protein kinase C (PKC) in vitro. Phosphorylation by PKA decreased the apparent Km of PFK-2 for one of its substrates, fructose 6-phosphate, while phosphorylation by PKC did not correlate with any change in PFK-2 activity. The differences between the 58,000- and 54,000-M(r) forms were studied by electroblotting, peptide mapping and microsequencing. Residues 451-510, which correspond to exon 15 in the rat and contain phosphorylation sites for PKA (Ser-466) and PKC (Thr-475), were absent from the 54,000-M(r) form. Peptide mapping after phosphorylation by [gamma-32P]MgATP and PKC showed a phosphorylated peptide containing Thr-475, which was present in the 58,000-M(r) form but not in the 54,000-M(r) form. The fact that the latter form was phosphorylated by PKC and PKA suggests that other phosphorylation sites for PKA and PKC are located outside the region encoded by exon 15. Finally, analysis of RNA from bovine heart showed that the tissue contains two PFK-2/FBPase-2 mRNAs, only one of which was recognized by a probe specific to the region coding for Ser-466 and Thr-475. Taken together, these findings demonstrate that the 58,000- and 54,000-M(r) forms of bovine heart PFK-2/FBPase-2 result from alternative splicing of the same primary transcript.
| S-EPMC1132803 | biostudies-other
Project description:The nature of the PFK (6-phosphofructo-1-kinase) isoenzymes in many rat tissues was examined by immunological and chromatographic techniques and by measurement of their subunit compositions. It was revealed that, except for diaphragm and skeletal muscle, these complex isoenzymic populations contained different amounts of the three subunit types and were nearly tissue-specific. Apparently this tissue specificity is due to different concentrations of the tetramers, which in turn are controlled by the types and amounts of each subunit that are available to associate randomly.
| S-EPMC1147763 | biostudies-other
Project description:Isoelectric focusing of a cytosol fraction from human foetal liver revealed the existence of an acidic and a basic isoenzyme of GSH transferase. The acidic and basic forms of GSH transferase were purified in good yield by use of ion-exchange chromatography on DEAE-cellulose followed by affinity chromatography on S-hexyl-GSH coupled to epoxy-activated Sepharose 6B. The content of the acidic and the basic isoenzymes of GSH transferase together was calculated to constitute 1-2% of the soluble proteins in the hepatic cytoplasm. Physical, catalytic and immunological analyses of the acidic and the basic isoenzymes from foetal liver demonstrated unambiguously that the two forms are different structures with distinct properties. On the other hand, the results show clearly extensive similarities between the foetal acidic transferase and transferase pi from human placenta as well as between the foetal basic form and the basic isoenzymes isolated from adult liver. An exception is that both foetal enzymes seem to be considerably more efficient in catalysing the conjugation of GSH with styrene 7,8-epoxide than the corresponding adult forms of GSH transferase.
| S-EPMC1146750 | biostudies-other
Project description:Two different enzymes exhibiting 6-phosphofructo-1-kinase (PFK1) activity were isolated from the mycelium of Aspergillus niger: the native enzyme with a molecular mass of 85 kDa, which corresponded to the calculated molecular mass of the deduced amino acid sequence of the A. niger pfkA gene, and a shorter protein of approximately 49 kDa. A fragment of identical size also was obtained in vitro by the proteolytic digestion of the partially purified native PFK1 with proteinase K. When PFK1 activity was measured during the proteolytic degradation of the native protein, it was found to be lost after 1 h of incubation, but it was reestablished after induction of phosphorylation by adding the catalytic subunit of cyclic AMP-dependent protein kinase to the system. By determining kinetic parameters, different ratios of activities measured at ATP concentrations of 0.1 and 1 mM were detected with fragmented PFK1, as with the native enzyme. Fructose-2,6-biphosphate significantly increased the Vmax of the fragmented protein, while it had virtually no effect on the native protein. The native enzyme could be purified only from the early stages of growth on a minimal medium, while the 49-kDa fragment appeared later and was activated at the time of a sudden change in the growth rate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of sequential purifications of PFK1 enzymes by affinity chromatography during the early stages of the fungal development suggested spontaneous posttranslational modification of the native PFK1 in A. niger cells, while from the kinetic parameters determined for both isolated forms it could be concluded that the fragmented enzyme might be more efficient under physiological conditions.
| S-EPMC1065176 | biostudies-literature
Project description:Foetal and adult liver 6-phosphofructo-2-kinase (PFK-2) were purified by identical protocols. The native molecular masses of both enzymes were determined by gel filtration and were 89.1 and 100.0 kDa respectively. No differences were found in SDS/PAGE in 10%-acrylamide gel (55 kDa per subunit). The kinetic properties displayed by both enzymes were similar, except for the sensitivity to inhibition by sn-glycerol 3-phosphate. Foetal PFK-2 was a good substrate for phosphorylation by cyclic AMP-dependent protein kinase and protein kinase C, whereas the adult enzyme was phosphorylated only by cyclic AMP-dependent protein kinase. However, the phosphorylation affected only the kinetic properties of the adult enzyme, suggesting the presence in both enzymes of different sites of phosphorylation by cyclic AMP-dependent protein kinase. These differences in primary structure were consistent with the distinct chromatographic profiles of the phosphopeptides after digestion of the protein with CNBr. Western-blot analysis with antibodies specific for the N-terminal region of the liver-type PFK-2 poorly recognized the foetal enzyme, suggesting that both enzymes differ at least in the N-terminal sequence.
| S-EPMC1130707 | biostudies-other
Project description:Total adenylate kinase activity was determined in developing rat liver. The activity was 18 units/g wet weight of tissue in foetal liver; this increased to 41 units/g immediately after birth and continued increasing until adult activities of 150 units/g were reached after two weeks. The adenylate kinase activity was separated into four isoenzymes. Only isoenzymes II and III were observed in foetal rat liver. Isoenzyme II activity was 2 units/g in the foetal liver and increased to 25 units/g in adult liver. Adenylate kinase III activity was 20 units/g in the foetal liver and increased to 118 units/g in adult liver. The possible role that adenylate kinase might have in regulating the energy flow in the developing liver cell is discussed.
| S-EPMC1176813 | biostudies-other
Project description:1. The mitochondrial malate dehydrogenase from rat liver has been purified to a state of homogeneity as judged by starch-gel electrophoresis and the cytoplasmic isoenzyme has been obtained in a partically purified state. 2. Inhibition of the isoenzymes by sulphite has been studied. 3. In mitochondria loaded with sulphite, the catalytic activity of the (partially inhibited) internal malate dehydrogenase has been measured by addition of oxaloacetate to the suspension medium and observation of the consequent decrease in fluorescence of NADH. 4. Addition of mitochondrial malate dehydrogenase to suspensions of mitochondria loaded with sulphite resulted in an increase in the level of intramitochondrial enzymic activity as measured by the above technique. Addition of the cytoplasmic isoenzyme did not result in such an increase. 5. These results show that mitochondria in suspension are permeable to the mitochondrial malate dehydrogenase but not to the cytoplasmic isoenzyme. 6. This conclusion has been confirmed by direct measurement of a decrease of enzyme activity in solution and an increase inside the mitochondria after incubation of organelles in solutions containing mitochondrial malate dehydrogenase. No such effect was observed with the cytoplasmic isoenzyme. 7. Some features of the permeation process have been studied.
| S-EPMC1162381 | biostudies-other
Project description:The heart-specific isoform of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB2) is an important regulator of glycolytic flux in cardiac cells. Here, we present the crystal structures of two PFKFB2 orthologues, human and bovine, at resolutions of 2.0 and 1.8 Å, respectively. Citrate, a TCA cycle intermediate and well-known inhibitor of PFKFB2, co-crystallized in the 2-kinase domains of both orthologues, occupying the fructose-6-phosphate binding-site and extending into the γ-phosphate binding pocket of ATP. This steric and electrostatic occlusion of the γ-phosphate site by citrate proved highly consequential to the binding of co-complexed ATP analogues. The bovine structure, which co-crystallized with ADP, closely resembled the overall structure of other PFKFB isoforms, with ADP mimicking the catalytic binding mode of ATP. The human structure, on the other hand, co-complexed with AMPPNP, which, unlike ADP, contains a γ-phosphate. The presence of this γ-phosphate made adoption of the catalytic ATP binding mode impossible for AMPPNP, forcing the analogue to bind atypically with concomitant conformational changes to the ATP binding-pocket. Inhibition kinetics were used to validate the structural observations, confirming citrate's inhibition mechanism as competitive for F6P and noncompetitive for ATP. Together, these structural and kinetic data establish a molecular basis for citrate's negative feed-back loop of the glycolytic pathway via PFKFB2. Proteins 2016; 85:117-124. © 2016 Wiley Periodicals, Inc.
| S-EPMC5193105 | biostudies-literature