Phosphate Labeling Research Articles

Studies on the phosphorylation and synthesis of type I regulatory subunit of cyclic AMP-dependent protein kinase in intact S49 mouse lymphoma cells.

Phosphorylation and synthesis of type I regulatory subunit (RI) of cAMP-dependent protein kinase were studied using two-dimensional polyacrylamide gel electrophoresis of [35S]methionine-labeled proteins from intact S49 mouse lymphoma cells. [32P]Phosphate labeling, peptide mapping, and acid hydrolysis confirm that charge heterogeneity in RI results from phosphorylation of a single serine residue. In drug-free cells, phosphorylation proceeds to a steady state proportion of 90 to 95% of total RI with a half-time of about 25 min. The rate and steady state extent of RI phosphorylation are reduced by some, but not all, agents causing intracellular kinase activation. These results suggest that RI might assume different conformations in association with different amounts of cAMP or different analogs of cAMP. Endogenous kinase activation has no immediate effect on RI synthesis but leads to a moderate increase in RI synthesis after several hours; this induction occurs with all agents tested. Mutants of S49 cells lacking catalytic activity of cAMP-dependent protein kinase exhibit reduced phosphorylation and synthesis of RI. Comparative studies suggest that the phosphorylation of RI and its induction by kinase activation are fairly general phenomena; the extent of RI phosphorylation and the relative rate of RI synthesis are variable among cell types.

Phosphorylation of synaptic membranes.

Abstract: In vivo and in vitro phosphorylation of synaptic membrane proteins were compared. In vivo phosphorylation was carried out by injecting rats intraventricularly with 1 mCi of 32Pi‐orthophosphate. After a 40‐min isotope incorporation period, rats were decapitated and synaptic membranes isolated. In vitro phosphorylation was accomplished by incubating unlabeled synaptic membranes, isolated from noninjected rats, with 5 μM‐[γ‐32P]ATP. In vivo and in vitro32P‐labeled synaptic membranes were then fractionated electrophoretically on an SDS‐polyacrylamide (7.5%) slab gel. The Coomassie blue protein patterns for in vivo and in vitro32P‐labeled synaptic membranes were identical. In contrast, autoradiographs of these gels showed striking differences in the pattern of phosphate incorporation. Cyclic‐AMP (10 μM) maximally stimulated) the in vitro phosphate labeling of two protein bands (80,000 and 55,000 apparent molecular weight) which were not substantially labeled by the in vivo procedure.

Correction for non-uniform phosphate labeling of [formula omitted] and phage RNAs synthesized [formula omitted

E. coli cells grown to phosphate starvation incorporate 32PO 4 unequally into the α position of the four ribonucleotide triphosphates during a short period of labeling. A method for determining the relative specific activities of nucleotides in RNA molecules synthesized under these conditions and correcting sequence data is described.

The mechanistic significance of phosphate labeling of alkaline phosphatase.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMechanistic significance of phosphate labeling of alkaline phosphataseTed Warren Reid, Milos Pavlic, Daniel Joseph Sullivan, and Irwin B. WilsonCite this: Biochemistry 1969, 8, 8, 3184–3188Publication Date (Print):August 1, 1969Publication History Published online1 May 2002Published inissue 1 August 1969https://doi.org/10.1021/bi00836a008RIGHTS & PERMISSIONSArticle Views61Altmetric-Citations33LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (513 KB) Get e-Alerts Get e-Alerts

32P-Labeling of Mitochondrial Protein and Lipid Fractions and Their Relation to Oxidative Phosphorylation

Abstract Evidence is presented that phosphorylated proteins or lipids containing phosphohistidine, phosphoserine, or other phosphoryl groups stable to phenol extraction at acidic, near neutral, or alkaline pH do not serve as intermediates in the formation of adenosine triphosphate by oxidative phosphorylation in mitochondrial particles. Suggestions of phosphoproteins as intermediates are thus considered to be tenuous. It has been shown that an unidentified, apparently protein-bound, and very labile phosphate fraction rapidly formed from 32Pi in mitochondria and particles probably represents a specifically occluded Pi and not a labile phosphoryl derivative. This Pi does not appear to be at or near the site of oxidative phosphorylation. In intact mitochondria, bound phosphohistidine is labeled much more rapidly and initially to a greater extent than phosphoserine or lipid phosphate fractions, and independently from the labeling of these fractions. Phosphohistidine may be labeled directly from Pi, but labeling of the phosphoserine and lipid phosphate fractions by 32Pi appears to occur only via intermediate formation of AT32P. Transphosphorylation among the various fractions does not occur under conditions tested. Most of the lipid phosphate labeling from AT32P occurs near the mitochondrial surface in an atractyloside-insensitive locus.

Uridine phosphate labeling in relation to ribonucleic acid synthesis in early regenerating rat liver

1. (1) After intravenous injection of orotic acid-6- 14C, the labeling of uridine mono-, di-, and triphosphates was previously shown to be the same in livers of normal and 45-minute partially hepatectomized rats when the incorporation time was 2–4 minutes, but to differ by more than two-fold when the time was 16 minutes. 2. (2) The present study, aimed at explaining these findings, showed that (a) rats injected immediately after partial hepatectomy yielded values for 16-minute incorporation of orotic acid- 14C almost identical to those from 45-minute hepatectomized animals, and (b) blood levels of orotic acid- 14C were slightly higher at 2, 8, and 16 minutes after injection in the partially hepatectomized than in the normal animals. 3. (3) It was concluded that since the initial rates of incorporation of orotic acid- 14C were similar, 45-minute regenerating liver did not differ appreciably from normal liver in rate of synthesis of UTP (nor, on the basis of earlier experiments in synthesis of other uridine phosphate compounds and possibly RNA). The continuing increase in uridine phosphate labeling in the partially hepatectomized rats when incorporation times were prolonged was attributable to a longer lasting supply of labeled precursor due to the reduced complement of liver cells in these animals. 4. (4) Hence an increase in labeling does not necessarily connote an increase in biosynthesis. Similar considerations may apply to other situations where large changes in cell population occur.