Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This paper reports on the discovery of a protein kinase activity associated with the inner membrane of mammalian mitochondria. The enzyme does not respond to addition of cyclic AMP or cyclic GMP and has a preference for whole histone as phosphate acceptor. Some standard assay systems for the cyclic nucleotide-dependent cytosol protein kinases would be unable to pick up this activity of the orthophosphate concentration is higher than 25 mM and the pH or the assay lower than pH 6.5. The enzyme described here has an apparent pH optimum of 8.5. Activity in liver mitochondria is not evident unless the mitochondria are disrupted by either sonication or freezing and thawing. Distribution of kinase activity in centrifugal fractions of both liver and heart mitochondrial sonicates was parallel to that of the two inner membrane marker enzymes succinic dehydrogenase and cytochrome oxidase and quite different from that of the matrix enzyme malic dehydrogenase. Experiments with preparations enriched in outer or inner membranes confirmed the contention that this enzyme is located on the inner membrane. Since disruption of the inner membrane by a freeze-thaw treatment (after the outer membrane had been disrupted by swelling in phosphate) was necessary for full expression of activity by this enzyme, the tentative conclusion was reached that substrate is accepted only from the matrix side of the inner membrane.
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PMID:Protein kinase activity at the inner membrane of mammalian mitochondria. 1 32

In crude extracts of Candida maltosa, about 12 proteins are phosphorylated in the presence of cAMP or of a catalytic subunit of cAMP-dependent protein kinase. A strongly labelled protein spot occurred in the position of fructose-1,6-bisphosphatase both after electrophoresis of crude extracts incubated with cAMP and of a partially purified fructose-1,6-bisphosphatase incubated with a catalytic subunit of cAMP-dependent protein kinase. No phosphorylation of the cytoplasmic malate dehydrogenase could be detected. From these results it was concluded that cAMP-dependent phosphorylation plays an important role in the catabolite inactivation of fructose-1,6-bisphosphatase in Candida maltosa, as described for Saccharomyces cerevisiae.
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PMID:Cyclic AMP-dependent phosphorylation of fructose-1,6-bisphosphatase and other proteins in the yeast Candida maltosa. 196 37

Glucose-induced inactivation of the gluconeogenetic enzymes fructose-1,6-biphosphatase, cytoplasmic malate dehydrogenase and phosphoenolpyruvate carboxykinase was tested in yeast mutants defective in adenylate cyclase (cyr1 mutation) and in the cAMP-binding subunit of cAMP-dependent protein kinase (bcy 1 mutation). In the mutant AM7-11D (cyr1 mutation), glucose-induced cAMP overshoot was absent, and no significant inactivation of the gluconeogenetic enzymes was detected, thus supporting the role of cAMP in the process. Moreover, in the mutant AM9-8B (bcy1 mutation), no cAMP-dependent protein kinase activity was evidenced, and, in addition, a normal inactivation pattern was observed, thus indicating that other mechanisms evoked by glucose might be required in the process. In the double mutant AM7-11DR-4 (cyr1 bcy1 mutations), no inactivating effect was triggered by the sugar: this suggests that cAMP exerts some additional effect on the process, besides the activation of cAMP-dependent protein kinase. Furthermore, in AM7-11D, extracellular cAMP triggered about 50% of inactivation of fructose-1,6-bisphosphatase; this effect was largely reversed in acetate medium plus cycloheximide even after 150 min of incubation. However, an extensive and essentially irreversible inactivation was evidenced in the presence of glucose plus cAMP, whereas glucose alone was only slightly effective. Therefore, the reversible effect of cAMP, which probably corresponds to enzyme phosphorylation, seems to be required for the irreversible, probably proteolytic, glucose-stimulated inactivation of this enzyme. Cytoplasmic malate dehydrogenase and phosphoenolpyruvate carboxykinase in AM7-11D were also inactivated by cAMP, and much more by glucose plus cAMP, whereas glucose was practically ineffective. However, reversibility of the effect was not detected, and, in addition, no phosphorylation of phosphoenolpyruvate carboxykinase could be evidenced. Therefore, the sugar quite probably stimulates proteolysis of these enzymes, but the mechanism of cAMP in their degradation has still to be defined.
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PMID:Studies on glucose-induced inactivation of gluconeogenetic enzymes in adenylate cyclase and cAMP-dependent protein kinase yeast mutants. 609 42

A cellular protein of apparent Mr 34,000--36,000 was suggested as a possible physiological substrate for the protein kinase (EC 2.7.1.37) activity associated with the transforming gene product of Rous sarcoma virus. We find this protein to migrate with an apparent Mr of 38,000 in NaDodSO4/polyacrylamide gels. It was not separable from cytosolic malic dehydrogenase activity when purified by chromatography on DEAE-Sephacel, hydroxylapatite, poly(A)-Sepharose, and blue Sepharose, by gel filtration, and by isoelectric focusing. The Mr 38,000 protein as well as cytosolic malic dehydrogenase activity focused with a pI of 7.5. In gel filtration experiments, both displayed an apparent native Mr of 68,000. The male dehydrogenase activity contained in homogeneous preparations of the Mr 38,000 protein had a specific activity of up to 130 units/mg of protein. The recovery of the enzyme was 5--10% of the activity in the extract. Antiserum against the Mr 38,000 protein inactivated the malic dehydrogenase activity associated with the Mr 38,000 protein.
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PMID:Cytosolic malic dehydrogenase activity is associated with a putative substrate for the transforming gene product of Rous sarcoma virus. 628 57

Transformation of fibroblasts by several retroviruses that produce transforming gene products associated with protein kinase activity results in the phosphorylation of a normal cellular protein with an Mr of 34,000 (the 34K protein). Evidence is presented here that, as extracted from chicken embryo fibroblasts, this protein exists in two forms that differ both in their elution from hydroxylapatite and in their native molecular weight. The form that eluted from hydroxylapatite at 210 to 295 mM potassium phosphate displayed a native molecular weight of 30,000 to 40,000, whereas the form that eluted at 320 to 440 mM displayed a native molecular weight of 60,000 to 70,000. The latter form copurified with a low-molecular-weight protein with an approximate Mr of 6,000 (6K). Both forms of 34K were completely separable from malate dehydrogenase activity. Phosphorylated 34K, isolated from Rous sarcoma virus-transformed cells, was also present in two forms; hence, in the cell neither form serves as a preferential substrate for pp60v-src. We found that the expression of 34K differed greatly in various avian tissues. In particular, it was present in the highest concentration in cultured fibroblasts and in very low concentration in brain tissue. Its expression in this tissue seems to be controlled at the level of transcription, since 34K mRNA in brain tissue was barely detectable. The expression of 6K was similar to that of 34K.
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PMID:Biochemical characterization of a 34-kilodalton normal cellular substrate of pp60v-src and an associated 6-kilodalton protein. 632 54

ATP-citrate lyase (CL) catalyzes the conversion of citrate and CoA to oxaloacetate (OA) and acetyl-CoA. As the coupled malic dehydrogenase (MDH) assay is not able either to study the effect of oxaloacetate (OA) on CL activity or to measure accurately CL activity in biological samples, a new assay was developed. The CL-citrate coupled CAT assay measures the amount of acetyl-CoA formed by transferring radiolabeled acetyl-CoA synthesized from [14C]citrate to chloramphenicol with chloramphenicol acetyltransferase (CAT). Employing this assay, the rate of increase in acetyl-CoA synthesis from citrate is linear with respect to added CL. Kinetic values for ATP, CoA and citrate are similar to those obtained using the MDH assay. The effect of CL phosphorylation on enzyme activity was determined. CL phosphorylated by cAMP-dependent protein kinase or by this kinase and glycogen synthase kinase-3 (GSK-3) decreases the apparent Vmax without changing the apparent Km. The effect of OA, a product of the enzyme reaction, on CL activity was also determined. Computational analysis of the data obtained without added OA and at three concentrations of OA indicate that the apparent Km for the substrate is not altered even though the apparent Vmax is decreased. The effect of OA on the activity of phosphorylated enzyme was also determined. OA decreases the apparent Vmax of the phosphorylated enzyme to the same extent as in control CL. This assay is able to measure CL activity in cytosol from 3T3-L1 adipocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effect of oxaloacetate and phosphorylation on ATP-citrate lyase activity. 766 53

A 13421 bp fragment located near the left telomere of chromosome XV (cosmid pEOA461) has been sequenced. Seven non-overlapping open reading frames (ORFs) encoding polypeptides longer than 100 residues have been found (AOB859, AOC184, AOE375, AOX142i, AOE423, AOA476 and AOE433). An additional ORF (AOE131) is found within AOA476. Three of them (AOC184, AOA476 and AOE433) show no remarkable identity with proteins deposited in the data banks. ORF AOB859 is quite similar to a hypothetical yeast protein of similar size located in chromosome VI, particularly within the C-terminal half. AOE375 encodes a new member of the glycogen synthase kinase-3 subfamily of Ser/Thr protein kinases. AOX142i is the gene encoding the previously described ribosomal protein L25. AOE423 codes for a protein virtually identical to the MDH2 malate dehydrogenase isozyme. However, our DNA sequence shows a single one-base insertion upstream of the reported initiating codon. This would produce a larger ORF by extending 46 residues the N-terminus of the protein. The existence of this insertion has been confirmed in three different yeast strains, including FY1679.
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PMID:Sequence analysis of a 13.4 kbp fragment from the left arm of chromosome XV reveals a malate dehydrogenase gene, a putative Ser/Thr protein kinase, the ribosomal L25 gene and four new open reading frames. 889 65

Maize leaf phosphoenolpyruvate carboxylase [PEPC; orthophosphate:oxaloacetate carboxy-lyase (phosphorylating), EC 4.1.1.31] protein-serine kinase (PEPC-PK) phosphorylates serine-15 of its target enzyme, thus leading to an increase in catalytic activity and a concomitant decrease in malate sensitivity of this cytoplasmic C4 photosynthesis enzyme in the light. We have recently demonstrated that the PEPC-PK activity in maize leaves is slowly, but strikingly, increased in the light and decreased in darkness. In this report, we provide evidence that cycloheximide, an inhibitor of cytoplasmic protein synthesis, when fed to detached leaves of C4 monocots (maize, sorghum) and dicots (Portulaca oleracea) in the dark or light, completely prevents the in vivo light activation of PEPC-PK activity regardless of whether the protein kinase activity is assessed in vivo or in vitro. In contrast, chloramphenicol, an inhibitor of protein synthesis in chloroplasts, has no effect on the light activation of maize PEPC-PK. Similarly, treatment with cycloheximide did not influence the light activation of other photosynthesis-related enzymes in maize, including cytoplasmic sucrose-phosphate synthase and chloroplast stromal NADPH-malate dehydrogenase and pyruvate, Pi dikinase. These and related results, in which detached maize leaves were treated simultaneously with cycloheximide and microcystin-LR, a potent in vivo and in vitro inhibitor of the PEPC type 2A protein phosphatase, indicate that short-term protein turnover of the PEPC-PK itself or some other essential component(s) (e.g., a putative protein that modifies this kinase activity) is one of the primary levels in the complex and unique regulatory cascade effecting the reversible light activation/seryl phosphorylation of PEPC in the mesophyll cytoplasm of C4 plants.
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PMID:Protein turnover as a component in the light/dark regulation of phosphoenolpyruvate carboxylase protein-serine kinase activity in C4 plants. 1160 71

Numerous inhibitors of cyclin-dependent kinases and glycogen synthase kinase-3 (GSK-3) are being developed in view of their potential applications against cancers and neurodegenerative disorders. Among these, paullones constitute a family of potent and apparently selective cyclin-dependent kinase and GSK-3 inhibitors. However, their actual intracellular targets remain to be identified. To address this issue we have immobilized a paullone, gwennpaullone, on an agarose matrix. Extracts from various cell types and tissues were screened for proteins interacting with this matrix. This approach validated GSK-3alpha and GSK-3beta as major intracellular paullone targets and also mitochondrial, but not cytoplasmic, malate dehydrogenase (MDH). Mitochondrial MDH was indeed inhibited by micromolar concentrations of paullones. Mitochondrial MDH was the major paullone-binding protein in the parasitic protozoon Leishmania mexicana, and paullones inhibited growth of the parasite. This simple batchwise affinity chromatography approach constitutes a straightforward method for the identification of intracellular targets of this particular class of novel anti-mitotic compounds. It has revealed an unexpected target, mitochondrial MDH, the inhibition of which may participate in the pharmacological effects of paullones.
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PMID:Intracellular Targets of Paullones. Identification following affinity purification on immobilized inhibitor. 1196 10

The MADS box transcription factor SrfA is required for spore differentiation in Dictyostelium discoideum. srfA null strains form rounded spores that do not resist adverse environmental conditions. Five genes whose expression is dependent on SrfA have been isolated by differential hybridization. One of these genes, sigC, is identical to phg1b, previously characterized in mutants with altered adhesive properties and found to encode a nine-transmembrane-domain protein. This gene is transcribed into two mRNAs as the result of alternative splicing of two internal exons. The slower-migrating mRNA codes for a shorter protein that lacks the first transmembrane fragment and is not expressed in srfA null strains. The other four genes (sigA, sigB, sigD, and 45D) are expressed only during late developmental stages. In situ hybridization experiments showed that expression of sigA, sigB, and sigD is restricted to the sorus of developing structures. sigA codes for a homologue of malate dehydrogenase that converts pyruvate to malate to replenish the tricarboxylic acid cycle. sigB encodes a protein with significant similarity to the GP63 metalloproteinase of Leishmania, leishmanolysin. The sequence of SigD is highly similar to that of several spore coat proteins of D. discoideum, and it may play a role in that structure. The gene 45D codes for an RNA-binding protein homologue whose expression is also dependent on the GATA transcription factor stalky (StkA). The expression of sigB is also dependent on both SrfA and StkA. The expression of 45D, but not of sigA, sigB, sigC, and sigD, can be induced in srfA null cells by constitutive protein kinase A activation. Strains in which either sigA, sigB, or sigD is disrupted were isolated and found to form spores that are not detectably different from those of wild-type strains.
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PMID:Dictyostelium discoideum developmentally regulated genes whose expression is dependent on MADS box transcription factor SrfA. 1466 66


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