Properties of acetate kinase activity inClostridium thermocellum cell extracts

1998 ◽  
Vol 69 (2) ◽  
pp. 137-145 ◽  
Author(s):  
Wenglong R. Lin ◽  
Claudia C. Lee ◽  
Janet J. Hsu ◽  
Jean-Francois Hamel ◽  
Arnold L. Demain
Keyword(s):  
Kinase Activity ◽  
Acetate Kinase ◽  
Cell Extracts

320 MPF ACTIVITY AND DEVELOPMENTAL COMPETENCE IN DIFFERENT SIZES OF PREPUBERTAL GOAT OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 267 ◽  
Author(s):  
B. Anguita ◽  
A. R. Jimenez-Macedo ◽  
D. Izquierdo ◽  
M. T. Paramio
Keyword(s):  
Embryonic Development ◽  
Kinase Activity ◽  
Developmental Competence ◽  
Kinase Assay ◽  
Oocyte Diameter ◽  
Cdc2 Kinase ◽  
Cell Extracts ◽  
Morphological Criteria ◽  
Blastocyst Rate ◽  
Cytoplasmic Maturation

Developmental competence of prepubertal goat oocytes recovered from a slaughterhouse is low, probably due to an incomplete cytoplasmic maturation. Regulation of cytoplasmic maturation is still unknown, although maturation-promoting factor (MPF) is suggested to play an important role in this process. To better understand the role of MPF in cytoplasmic maturation, we have studied MPF kinase activity in oocytes with different developmental competence. Ovaries were obtained from a slaughterhouse, and oocytes were recovered by slicing and were selected according to morphological criteria. Some oocytes were denuded and classified in diameter groups (<110 μm, 110–125 μm, 125–135 μm, and >135 μm), placed in lysis buffer (50 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 5 mM EDTA, 0.01% Brij35, 1 mM phenyl methyl sultonyl fluoride (PMSF), 0.05 mg/mL leupeptin, 50 mM 2-mercaptoethanol, 25 mM α-glycerophosphate, 1 mM Na-orthovanadate) and frozen in liquid N2. Cell extracts were stored at −80°C until use. The rest of oocytes were matured in vitro in medium TCM199 supplemented with hormones, 10% (DBS), and 400 μM cysteamine, for 27 h in 5% CO2 in air and 38.5°C. After IVM, a sample of oocytes were also denuded, classified by diameters, and frozen as described above. The rest of oocytes were used for IVF in mDM with spermatozoa capacitated with heparin and ionomicin. After 24 h, presumptive zygotes were cultured for 7 days in medium SOF in 5% CO2, 5% O2, and 90% N2 at 38.5°C. At 48 h post-insemination, we added 0.1 μL FBS per embryo. Embryonic development was evaluated with Hoechst staining after IVC. MPF kinase activity was detected using the MESACUP cdc2 kinase assay kit (MBL Woburn, MA, USA). Briefly, the oocyte extract corresponding to 10 oocytes was mixed with 10× reaction buffer (25 mM HEPES buffer (pH 7.5), 10 mM MgCl2) and 10% biotinylated MV peptide (SLYSSPGGAYC). We added 0.1 mM ATP to start the reaction. The mixture was incubated at 30°C for 30 min. The reaction was finished by adding 200 μL of PBS containing 50 mM EGTA. The phosphorylated MV peptide was detected by specific antibody using an ELISA procedure, and the OD was measured at 492 nm. Fisher's exact test was used to analyze IVC results, and ANOVA to analyze cdc2 kinase activity results. We considered differences statistically significant when P < 0.05. Results are shown in Table 1. We observed that embryonic cleavage and blastocyst rate increased with oocyte diameter. The MPF activity detected was higher in the largest oocytes after IVM. As a consequence, we could establish that oocytes with a higher MPF activity are more capable of maintaining embryonic development until the blastocyst stage, which may indicate the important role that MPF plays in cytoplasmic maturation. Table 1. Cleavage, blastocyst rate, and MPF kinase activity in different sizes of prepubertal goat oocytes

Ca2+/calmodulin-dependent protein kinase II activation and regulation of adrenal glomerulosa Ca2+ signaling

1995 ◽  
Vol 269 (6) ◽  
pp. F751-F760 ◽  
Author(s):  
R. J. Fern ◽  
M. S. Hahm ◽  
H. K. Lu ◽  
L. P. Liu ◽  
F. S. Gorelick ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Low Voltage ◽  
Extracellular Potassium ◽  
Dependent Protein Kinase ◽  
Calmodulin Antagonist ◽  
Cell Extracts ◽  
Protein Kinase Ii ◽  
Dependent Protein ◽  
A Cell

We recently reported that elevations in the intracellular Ca2+ concentration ([Ca2+]i) enhance low-voltage-activated, T-type, Ca2+ channel activity via Ca2+/calmodulin-dependent protein kinase II (CaMKII). Here, we document CaMKII activity in bovine adrenal glomerulosa (AG) cells and assess the importance of CaMKII in depolarization-induced Ca2+ signaling. AG cell extracts exhibited kinase activity toward a CaMKII-selective peptide substrate that was dependent on both Ca2+ [half-maximal concentration for Ca2+ activation (K0.5) = 1.5 microM] and calmodulin (K0.5 = 46 nM) and was sensitive to a calmodulin antagonist and a CaMKII peptide inhibitor. On cell treatment with elevated extracellular potassium (10-60 mM) or angiotensin II, Ca(2+)-independent CaMKII activity increased to 133-205% of basal activity. Ca(2+)-independent kinase activity in agonist-stimulated extracts was inhibited by the CaMKII inhibitor peptide, 1(-)[N,O-bis(1,5- isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), a cell-permeable inhibitor of CaMKII, reduced the agonist-induced stimulation of Ca(2+)-independent CaMKII activity. KN-62 also diminished depolarization-induced increases in [Ca2+]i without affecting the membrane potential. These observations suggest that CaMKII is activated in situ by aldosterone secretagogues and augments Ca2+ signaling through voltage-gated Ca2+ channels.

scholarly journals Identification of RamA, a Novel LuxR-Type Transcriptional Regulator of Genes Involved in Acetate Metabolism of Corynebacterium glutamicum

2006 ◽  
Vol 188 (7) ◽  
pp. 2554-2567 ◽  
Author(s):  
Annette Cramer ◽  
Robert Gerstmeir ◽  
Steffen Schaffer ◽  
Michael Bott ◽  
Bernhard J. Eikmanns
Keyword(s):  
Corynebacterium Glutamicum ◽  
Isocitrate Lyase ◽  
Glyoxylate Cycle ◽  
Transcriptional Regulator ◽  
Malate Synthase ◽  
Acetate Kinase ◽  
Acetate Metabolism ◽  
Promoter Regions ◽  
Cell Extracts ◽  
Peptide Mass

ABSTRACT In Corynebacterium glutamicum, the acetate-activating enzymes phosphotransacetylase and acetate kinase and the glyoxylate cycle enzymes isocitrate lyase and malate synthase are coordinately up-regulated in the presence of acetate in the growth medium. This regulation is due to transcriptional control of the respective pta-ack operon and the aceA and aceB genes, brought about at least partly by the action of the negative transcriptional regulator RamB. Using cell extracts of C. glutamicum and employing DNA affinity chromatography, mass spectrometry, and peptide mass fingerprinting, we identified a LuxR-type transcriptional regulator, designated RamA, which binds to the pta-ack and aceA/aceB promoter regions. Inactivation of the ramA gene in the genome of C. glutamicum resulted in mutant RG2. This mutant was unable to grow on acetate as the sole carbon and energy source and, in comparison to the wild type of C. glutamicum, showed very low specific activities of phosphotransacetylase, acetate kinase, isocitrate lyase, and malate synthase, irrespective of the presence of acetate in the medium. Comparative transcriptional cat fusion experiments revealed that this deregulation takes place at the level of transcription. By electrophoretic mobility shift analysis, purified His-tagged RamA protein was shown to bind specifically to the pta-ack and the aceA/aceB promoter regions, and deletion and mutation studies revealed in both regions two binding motifs each consisting of tandem A/C/TG4-6T/C or AC4-5A/G/T stretches separated by four or five arbitrary nucleotides. Our data indicate that RamA represents a novel LuxR-type transcriptional activator of genes involved in acetate metabolism of C. glutamicum.

Properties and Regulation of Adenylate Kinase From Zea mays Leaf Operating in C4 Pathway Photosynthesis

1982 ◽  
Vol 9 (3) ◽  
pp. 287 ◽  
Author(s):  
MD Hatch
Keyword(s):  
Zea Mays ◽  
Adenylate Kinase ◽  
Kinase Activity ◽  
Mesophyll Cell ◽  
Reverse Reaction ◽  
Mesophyll Cells ◽  
Ph Optimum ◽  
Strong Inhibitor ◽  
Cell Extracts ◽  
Regulatory Properties

Adenylate kinase from the mesophyll cells of Zea mays was partially purified and its kinetic and regulatory properties determined. Fractionation of mesophyll cell extracts by (NH4)2SO4 precipitation, filtration on Sephacryl S-200 and diethylaminoethylcellulose chromatography gave a fraction purified about 90-fold. For the reaction in the direction of ADP synthesis (forward reaction), activity was influenced by a complex interaction between Mg2+, ATP and pH. Optimal activity was observed with a Mg2+ to ATP ratio of about unity and the pH optimum under these conditions was about 8.0. With an excess of Mg2+, activity was reduced and the pH optimum was shifted to lower values. Varying ATP or AMP in a lower range of concentrations gave simple Michaelis- Menten responses. However, at higher concentrations there was a complex response to increasing ATP, and AMP became inhibitory. For the reverse reaction the Km (ADP) was about 70 �M and the pH optimum was between 8.0 and 8.5. AMP was a strong inhibitor of the reverse reaction (competitive with respect to ADP), the KI being about 40 �M. Several other metabolites tested had no effect on adenylate kinase activity. Adenylate kinase extracted from washed mesophyll chloroplasts had essentially identical properties.

scholarly journals Glycogen synthase kinase-3 is rapidly inactivated in response to insulin and phosphorylates eukaryotic initiation factor eIF-2B

1993 ◽  
Vol 294 (3) ◽  
pp. 625-629 ◽  
Author(s):  
G I Welsh ◽  
C G Proud
Keyword(s):  
Kinase Activity ◽  
Glycogen Synthase ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Glycogen Synthase Kinase ◽  
Initiation Factor ◽  
Glycogen Synthase Kinase 3 ◽  
Eukaryotic Initiation Factor ◽  
Nucleotide Exchange ◽  
Cell Extracts

We have studied the control of insulin-regulated protein kinases in Chinese hamster ovary cells transfected with the human insulin receptor (CHO.T cells). Among these enzymes is one that is obtained after chromatography of cell extracts on Mono-S, whose activity is decreased (7.3 +/- 1.9-fold) within 10 min of insulin treatment. This enzyme phosphorylates glycogen synthase and the largest subunit of protein synthesis eukaryotic initiation factor (eIF)-2B (the guanine nucleotide exchange factor). The kinase appears to be glycogen synthase kinase-3 (GSK-3), on the basis of: (1) its ability to phosphorylate a peptide based on the phosphorylation sites for GSK-3 in glycogen synthase, and (2) the finding that the fractions possessing this activity contain immunoreactive GSK-3, whose peak is coincident with that of kinase activity, as judged by immunoblotting using antibodies specific for the alpha- and beta-isoforms of GSK-3. The decrease in kinase activity induced by insulin was reversed by treatment of the column fractions with protein phosphatase-2A. These data indicate that insulin rapidly causes inactivation of GSK-3 and that this is due to phosphorylation of GSK-3. The implications of these findings for the control of glycogen and protein metabolism are discussed.

scholarly journals Leishmania mexicana p12cks1, a homologue of fission yeast p13suc1, associates with a stage-regulated histone H1 kinase

1996 ◽  
Vol 316 (3) ◽  
pp. 833-839 ◽  
Author(s):  
Jeremy C. MOTTRAM ◽  
Karen M. GRANT
Keyword(s):  
Fission Yeast ◽  
Kinase Activity ◽  
Histone H1 ◽  
Leishmania Mexicana ◽  
Cdc2 Kinase ◽  
Agarose Beads ◽  
Functional Homology ◽  
Large Gene ◽  
Cell Extracts ◽  
High Level

We have isolated a Leishmania mexicana homologue of the fission yeast suc1 gene using PCR with oligonucleotides designed to conserved regions of cdc2 kinase subunits (cks). The product of cks1 is a 12 kDa polypeptide, which has 70% identity with human p9cks1 and 44% identity with fission yeast p13suc1. p12cks1 was detected in the three life-cycle stages of L. mexicana by immunoblotting. Recombinant p12cks1 (p12cks1his) bound to agarose beads was used as a matrix to affinity-select histone H1 kinase complexes from Leishmania, yeast and bovine extracts. Immunoblotting showed that yeast and bovine cdc2 kinase bound to p12cks1his, thus demonstrating functional homology between L. mexicana p12cks1 and yeast p13suc1. Histone H1 kinase activity was found at a high level in the proliferative promastigote and amastigote forms of L. mexicana, but at a low level in the non-dividing metacyclic form. These activities are likely to be the same as the leishmanial p13suc1 binding kinase (SBCRK) described previously [Mottram, Kinnaird, Shiels, Tait and Barry (1993) J. Biol. Chem. 268, 21044–21051]. A distinct cdc2-related kinase, L. mexicana CRK1, was also found to associate with p12cks1his but affinity-depletion experiments showed that CRK1 was not responsible for the histone H1 kinase activity associating with p12cks1his in promastigote cell extracts. The finding that p12cks1 associates with at least two cdc2-related kinases, SBCRK and CRK1, is consistent with the presence of a large gene family of cdc2-related kinases in trypanosomatids, a situation thought to be more similar to higher eukaryotes than yeast.

scholarly journals Cks1 Is Required for G1Cyclin–Cyclin-Dependent Kinase Activity in Budding Yeast

2000 ◽  
Vol 20 (16) ◽  
pp. 5858-5864 ◽  
Author(s):  
Gregory J. Reynard ◽  
William Reynolds ◽  
Rati Verma ◽  
Raymond J. Deshaies
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Budding Yeast ◽  
Protein Kinase Activity ◽  
Cyclin Dependent Kinase ◽  
Multiple Substrates ◽  
Cell Extracts

ABSTRACT p13suc1 (Cks) proteins have been implicated in the regulation of cyclin-dependent kinase (CDK) activity. However, the mechanism by which Cks influences the function of cyclin-CDK complexes has remained elusive. We show here that Cks1 is required for the protein kinase activity of budding yeast G1 cyclin-CDK complexes. Cln2 and Cdc28 subunits coexpressed in baculovirus-infected insect cells fail to exhibit protein kinase activity towards multiple substrates in the absence of Cks1. Cks1 can both stabilize Cln2-Cdc28 complexes and activate intact complexes in vitro, suggesting that it plays multiple roles in the biogenesis of active G1cyclin-CDK complexes. In contrast, Cdc28 forms stable, active complexes with the B-type cyclins Clb4 and Clb5 regardless of whether Cks1 is present. The levels of Cln2-Cdc28 and Cln3-Cdc28 protein kinase activity are severely reduced in cks1-38 cell extracts. Moreover, phosphorylation of G1 cyclins, which depends on Cdc28 activity, is reduced in cks1-38 cells. The role of Cks1 in promoting G1 cyclin-CDK protein kinase activity both in vitro and in vivo provides a simple molecular rationale for the essential role of CKS1 in progression through G1 phase in budding yeast.

scholarly journals Novel Pathway for Alcoholic Fermentation of δ-Gluconolactone in the Yeast Saccharomyces bulderi

2002 ◽  
Vol 184 (3) ◽  
pp. 672-678 ◽  
Author(s):  
Johannes P. van Dijken ◽  
Arjen van Tuijl ◽  
Marijke A. H. Luttik ◽  
Wouter J. Middelhoven ◽  
Jack T. Pronk
Keyword(s):  
Pentose Phosphate Pathway ◽  
Alcoholic Fermentation ◽  
Kinase Activity ◽  
Glucose Dehydrogenase ◽  
Sole Source ◽  
Pentose Phosphate ◽  
Anaerobic Growth ◽  
Ph Values ◽  
Cell Extracts ◽  
Dehydrogenase Reaction

ABSTRACT Under anaerobic conditions, the yeast Saccharomyces bulderi rapidly ferments δ-gluconolactone to ethanol and carbon dioxide. We propose that a novel pathway for δ-gluconolactone fermentation operates in this yeast. In this pathway, δ-gluconolactone is first reduced to glucose via an NADPH-dependent glucose dehydrogenase (EC 1.1.1.47). After phosphorylation, half of the glucose is metabolized via the pentose phosphate pathway, yielding the NADPH required for the glucose-dehydrogenase reaction. The remaining half of the glucose is dissimilated via glycolysis. Involvement of this novel pathway in δ-gluconolactone fermentation in S. bulderi is supported by several experimental observations. (i) Fermentation of δ-gluconolactone and gluconate occurred only at low pH values, at which a substantial fraction of the substrate is present as δ-gluconolactone. Unlike gluconate, the latter compound is a substrate for glucose dehydrogenase. (ii) High activities of an NADP+-dependent glucose dehydrogenase were detected in cell extracts of anaerobic, δ-gluconolactone-grown cultures, but activity of this enzyme was not detected in glucose-grown cells. Gluconate kinase activity in cell extracts was negligible. (iii) During anaerobic growth on δ-gluconolactone, CO2 production exceeded ethanol production by 35%, indicating that pyruvate decarboxylation was not the sole source of CO2. (iv) Levels of the pentose phosphate pathway enzymes were 10-fold higher in δ-gluconolactone-grown anaerobic cultures than in glucose-grown cultures, consistent with the proposed involvement of this pathway as a primary dissimilatory route in δ-gluconolactone metabolism.

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