[45] Isolation of protein kinases from reticulocytes and phosphorylation of initiation factors

Hathaway, Gary M.; Lundak, Tina S.; Tahara, Stanley M.; Traugh, Jolinda A.

doi:10.1016/s0076-6879(79)60047-2

Cited by 129 publications

(36 citation statements)

References 17 publications

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“…Clostridium pasteurianum strain W5 was a gift from Professor J. S. Chen (Virginia Polytechnic Institute and State University) and was grown as described (18). Nitrogenase Fe and MoFe proteins were purified from each bacterial source as described (19) to apparent homogeneity as judged by migration on SDS-polyacrylamide gel with Coomassie Blue staining (20). Protein concentrations were determined by a modified biuret method using bovine serum albumin as the standard (21).…”

Section: Methodsmentioning

confidence: 99%

Hydrolysis of Nucleoside Triphosphates Other than ATP by Nitrogenase

Ryle¹,

Seefeldt

2000

Journal of Biological Chemistry

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The hydrolysis of ATP to ADP and P i is an integral part of all substrate reduction reactions catalyzed by nitrogenase. In this work, evidence is presented that nitrogenases isolated from Azotobacter vinelandii and Clostridium pasteurianum can hydrolyze MgGTP, MgITP, and MgUTP to their respective nucleoside diphosphates at rates comparable to those measured for MgATP hydrolysis. The reactions were dependent on the presence of both the iron ( The hydrolysis of ATP to ADP and P i has long been known to be an integral part of all nitrogenase substrate reduction reactions (1-4). The stoichiometry for coupling ATP hydrolysis to substrate reduction varies depending on reactions conditions, with a minimum of 2 ATP molecules hydrolyzed for each electron transferred to substrate (5, 6). Thus, the reduction of N 2 to 2NH 3 by nitrogenase requires the hydrolysis of at least 16 ATP molecules (Equation 1). There have been isolated examinations of the interactions of nucleotides other than adenine nucleotides with nitrogenase. To summarize, no nucleotide other than ATP has been shown to support substrate reduction, although there is a recent report that protons can be reduced at extremely low rates without ATP within an Fe protein-MoFe protein complex created with AlF 4 Ϫ ⅐ADP (10). The hydrolysis of ATP to ADP and P i is the predominant reaction observed. Apparently, the MoFe protein alone or the Fe protein-MoFe protein complex can catalyze the further hydrolysis of ADP at extremely low rates (11,12), although these reactions do not appear to be part of the substrate reduction mechanism. A divalent metal-bound form of ATP is required for hydrolysis, with MgATP showing the highest rates of substrate reduction (13). There is, however, evidence suggesting that nucleotides other than adenosine di-or triphosphates interact with nitrogenase. The Fe protein is known to bind 2 molecules of either MgATP or MgADP, with each resulting in distinct protein conformational changes (1). These protein conformational changes have been detected by a variety of methods. For example, the line shape of the EPR signal of the S ϭ 1/2 state of the reduced 1ϩ cluster of the Fe protein changes upon MgATP or MgADP binding (14). MgGTP was observed to induce small changes in the EPR line shape of the Fe protein, suggesting that MgGTP binds to the Fe protein and induces at least partial protein conformational changes (14). Another technique that reports protein conformational changes in the Fe protein is the sensitivity of the [4Fe-4S] cluster to chelation by iron-specific chelators like bathophenanthroline or ␣,␣Ј-dipyridyl. In the absence of nucleotides, the [4Fe-4S] cluster of the Fe protein is resistant to chelation by chelators (15, 16). Upon binding MgATP, however, a timedependent release of all of the iron atoms from the cluster to

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Section: Methodsmentioning

confidence: 99%

Hydrolysis of Nucleoside Triphosphates Other than ATP by Nitrogenase

Ryle¹,

Seefeldt

2000

Journal of Biological Chemistry

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“…[y-"PI-ATP (2500-3500 dis. min-' pmol-l) was prepared as described elsewhere [22]. The heat-stable inhibitor protein was a gift of Dr D. A.…”

Section: Methodsmentioning

confidence: 99%

“…The type I and type 11 CAMP-dependent protein kinases were isolated from reticulocytes by chromatography on DEAE-cellulose and phosphocellulose as previously described [22]. Highly purified cGMP-dependent protein kinase from bovine lung was generously provided by D r G. N. Gill, University of California, San Diego, and the catalytic subunit of the type I1 CAMP-dependent protein kinase from bovine cardiac muscle by Drs E. H. Fischer, E. G. Krebs and S. B. Smith, University of Washington, Seattle.…”

Section: Preparation Of Protein Kinusesmentioning

confidence: 99%

“…The concentration of 40-S ribosomal subunits was determined using 11.4 Az60/mg and a molecular weight of 1.3 x 10'. Reactions to be analyzed by slab gel electrophoresis in sodium dodecyl sulfate were terminated by the addition of 0.030 ml of the reaction mixture to 0.015 ml of sample buffer [22]. For analysis by one-dimensional and two-dimensional polyacrylamide gel electrophoresis in urea, the reactions were terminated by the addition of one volume of 8 M urea: 6 M LiCI.…”

Section: Phosphorylation O J Ribosomal Subunitsmentioning

confidence: 99%

“…Gel electrophoresis in a slab gel apparatus similar to that described by Studier [26] was performed using a modification of the Laemmli system [27] as previously described [22], except the acrylamide concentration of the resolving gel was 10% or 12.5 % as indicated.…”

Section: Slub Gel Electrophoresismentioning

confidence: 99%

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Phosphorylation of 40‐S Ribosomal Subunits by cAMP‐Dependent, cGMP‐Dependent and Protease‐Activated Protein Kinases

Grande

Traugh

1982

European Journal of Biochemistry

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The phosphorylation of 40-S ribosomal subunits by cyclic-nucleotide-dependent and protease-activated protein kinases from rabbit reticulocytes was studied in vitro. Under optimal conditions the CAMP-dependent protein kinases incorporated up to 2 mol phosphate/mol S6. The electrophoretic mobility of S6 following phosphorylation indicated that this value was not an average for a population of maximally phosphorylated and non-phosphorylated S6 but represented a uniform population of diphosphorylated 40-S ribosomal subunits. Tryptic digests of S6 were analyzed by two-dimensional fingerprinting following phosphorylation with the CAMP-dependent protein kinase; two phosphopeptides, A and B, were observed. When 40-S ribosomal subunits were examined with the cGMP-dependent protein kinase, 1 mol phosphate was incorporated/mol S6. Upon analysis of the phosphopeptides obtained with the cGMP-dependent protein kinase, only peptide A was observed. S6 was also modified by a cyclic-nucleotide-independent protein kinase, protease-activated kinase 11, following activation of the enzyme by limited proteolytic digestion. These findings suggest that a multiple protein kinase system may regulate the phosphorylation-state of S6. A second ribosomal protein, S10, was phosphorylated by a different cyclic-nucleotide-independent protein kinase, protease-activated kinase I, and up to 1 mol phosphate was incorporated.

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Phosphorylation of the insulin receptor by casein kinase I

Tuazon

Pang

Shafer

et al. 1985

J of Cellular Biochemistry

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Insulin receptor was examined as a substrate for the multipotential protein kinase casein kinase I. Casein kinase I phosphorylated partially purified insulin receptor from human placenta as shown by immunoprecipitation of the complex with antiserum to the insulin receptor. Analysis of the phosphorylated complex by polyacrylamide gel electrophoresis under nonreducing conditions showed a major phosphorylated band at the position of the alpha 2 beta 2 complex. When the phosphorylated receptor was analyzed on polyacrylamide gels under reducing conditions, two phosphorylated bands, Mr 95,000 and Mr 135,000, were observed which corresponded to the alpha and beta subunits. The majority of the phosphate was associated with the beta subunit with minor phosphorylation of the alpha subunit. Phosphoamino acid analysis revealed that casein kinase I phosphorylated only seryl residues. The autophosphorylated alpha 2 beta 2 receptor purified by affinity chromatography on immobilized O-phosphotyrosyl binding antibody was also a substrate for casein kinase I. Reduction of the phosphorylated alpha 2 beta 2 receptor indicated that casein kinase I incorporated phosphate into seryl residues only in the beta subunit.

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[45] Isolation of protein kinases from reticulocytes and phosphorylation of initiation factors

Cited by 129 publications

References 17 publications

Hydrolysis of Nucleoside Triphosphates Other than ATP by Nitrogenase

Hydrolysis of Nucleoside Triphosphates Other than ATP by Nitrogenase

Phosphorylation of 40‐S Ribosomal Subunits by cAMP‐Dependent, cGMP‐Dependent and Protease‐Activated Protein Kinases

Phosphorylation of the insulin receptor by casein kinase I

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