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D Ogreid

Publications and source records attributed to D Ogreid.

48 records · Page 3Linked to original sources
PubMed1984

Characterization of the interchain and intrachain interactions between the binding sites of the free regulatory moiety of protein kinase I.

The interaction between the four binding sites (two A sites and two B sites) of the regulatory subunit dimer of protein kinase I (RI2) was studied. The rate of association of c[3H]AMP to site B was slower when site A had already been occupied. Occupation of site A also retarded the rate of dissociation of c[3H]AMP from site B. This site A-B interaction was intrachain since it was observed also for a monomeric fragment of RI2. Thus, each monomer of RI2 must have one A site and one B site. Quantitative analysis of the rate constants for cAMP binding to variously liganded RI2 suggested little or no thermodynamic coupling between site A and B. This conclusion was supported by equilibrium binding data. Occupation of one A site retarded the dissociation of c[3H]AMP from the A site of the other subunit (interchain interaction). The rate kinetic constants as well as equilibrium binding data indicated a positively cooperative site A-A interaction. The interaction between cAMP and either site was enthalphy-driven (25 degrees C), the process being accompanied by a loss of entropy. The thermodynamic parameters did not support the occurrence of an abrupt conformational change at a certain level of ligandation of RI2. Half-maximal saturation of either site occurred at 1-2 nM cAMP (37 degrees C, pH 7.0, 0.15 M KCl). The concentration of RI2 did not detectably influence any binding parameters. Aging of RI2 produced a form with minimally, if at all, altered Mr, but which showed a more rapid release of c[3H]AMP bound to site B.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1983

Cyclic nucleotides modulate the release of [3H] adenosine cyclic 3',5'-phosphate bound to the regulatory moiety of protein kinase I by the catalytic subunit of the kinase.

The rate of release of bound c[3H]AMP from the two types (A and B) of cAMP binding sites on the regulatory subunit dimer (R2I) of rabbit muscle protein kinase I was studied in the presence of the catalytic (C) subunit of protein kinase. Rebinding of released c[3H]AMP was avoided by using highly diluted reactants or adding unlabeled cAMP or its analogues. No significant C-induced dissociation of R2I-(c[3H]AMP)4 occurred in the absence of Mg2+-ATP. Of the two options that one or two molecules of C are required to induce the release of c[3H]AMP bound to R2I, only the first one was compatible with the first-order dependence on [C] of the rate of release of c[3H]AMP observed over a wide range of C concentrations. In the absence of added unlabeled cyclic nucleotide, the rate of the C-induced release of c[3H]AMP was the same from site A and site B. The apparent second-order rate constant for the association of C to R2I(c[3H]AMP)4 was 6 X 10(6) M-1 s-1 (37 degrees C, 0.15 M KCl). Raising the concentration of unlabeled cAMP in the medium up to 1 microM decreased by up to 50% the rate of the C-induced release of bound c[3H]AMP from both sites. This is explained by assuming that the association of one molecule of C to R2I(c-[3H]AMP)4 leads to the release of c[3H]AMP first from one R subunit and subsequently, by a process that can be blocked by about 1 microM cAMP, from the other R subunit. A further rise of the cAMP concentration decreased the rate of release from site B only, so that the C-induced release of c[3H]AMP occurred almost exclusively from site A at very high concentrations of cAMP. This suggests that c[3H]AMP is released first from site A and that this vacant site by interacting with cAMP inhibits the release of c[3H]AMP from site B of the same R subunit. The role of site A in controlling the C-induced release was further supported by the finding that several cAMP analogues inhibited the release with potencies correlating with their affinities for site A. The C-induced release of c[3H]AMP from aged R2I was about 10 times slower than that from fresh R2I. No significant C-induced release of c[3H]AMP was observed from the monomeric fragment obtained by limited trypsin treatment of R2(1).

Animals↗
PubMed1983

Evidence that cyclic nucleotides activating rabbit muscle protein kinase I interact with both types of cAMP binding sites associated with the enzyme.

Eighty different adenine-modified cAMP analogs were tested as activators of rabbit muscle protein kinase I (cAKI) in an in vitro phosphotransferase assay. All the analogs tested were able to activate completely the kinase. The affinities of the cAMP derivatives for the two types (A and B) of binding sites associated with the regulatory moiety of cAKI were determined under conditions similar to those used in the phosphotransferase assay. The potency of the cAMP analogs as cAKI activators was found to correlate with the mean affinity for sites A and B, rather than to the affinity for only one of the sites. This was true whether cAKI was assayed at low or near physiological ionic strength, whether the concentration of cAKI binding sites was 0.2 or 400 nM, and whether the kinase substrate was mixed histones or homogeneous phenylalanine-4-monooxygenase. Furthermore, site A-selective and site B-selective cAMP analogs activated cAKI synergistically. Finally, it was shown that the degree of synergism between cAMP analogs in activating cAKI correlated with their degree of site selectivity. It is concluded that cyclic nucleotides interact with both types of binding sites in the process of cAKI activation.

Animals↗
PubMed1982

Activation of protein kinase isoenzymes under near physiological conditions. Evidence that both types (A and B) of cAMP binding sites are involved in the activation of protein kinase by cAMP and 8-N3-cAMP.

cAMP-dependent protein kinase I and II (cAKI and cAKII) were incubated under near physiological conditions in the presence of various concentrations of 8-N3-c[3H]AMP or c[3H]AMP. Both types (A and B) of cyclic nucleotide binding sites of cAKI or cAKII were occupied to a similar extent and the degree of their occupation correlated with the degree of kinase activation. cAKI and cAKII bound cAMP in an apparent positively cooperative manner in the presence of Mg2+, ATP. 8-N3-c[3H]AMP dissociated several orders of magnitude faster from site A than site B of the regulatory moiety of cAKII, and was photo-incorporated only when bound to site B.

Azides↗
PubMedDate not supplied

Myocardial cAMP and calcium levels in the calcium paradox.

In a graded model (minimal, subtotal, total) of the calcium paradox phenomenon induced by a progressive increase in the flow rate and volume (5 ml, 10 ml, 45 ml) of calcium-free perfusion (5 min) the severity of tissue injury on calcium repletion was related to a potential elevation of cAMP during calcium depletion. In the subtotal and total models of the calcium paradox a 50% increase was found for tissue cAMP after calcium-free perfusion, but no such rise could be associated with the minimal calcium paradox. In the study tissue injury, as assessed by whole tissue and mitochondrial accumulation of calcium and by myocardial leakage of creatine kinase, could only partly be related to cAMP changes. It is concluded that calcium-free coronary perfusion induces a complex series of events favouring excessive calcium entry, only one of which may be related to a change in cAMP.

Animals↗
PubMedDate not supplied

Low-frequency mutation of Ha-ras and Ki-ras oncogenes in transitional cell carcinoma of the bladder.

UNLABELLED: BACKGROUND, PATIENTS AND METHODS: The objective was to study the frequency of mutations of Ha-ras and Ki-ras oncogenes in bladder tumours. Transitional cell tumours of the bladder from 55 patients were subjected to analyses of Ha-ras and Ki-ras oncogenes using a variety of techniques including sequencing to detect mutations. RESULTS: Two tumours (4%) exhibited mutation of the Ki-ras oncogene, both tumours were fast growing and invasive. We did not detect Ha-ras mutation in any of the samples. Nineteen tumours (35%) with established invasion of the detrusor muscle, did not reveal any mutation. CONCLUSION: Analyses of ras oncogenes seem to be of limited value for the biological assessment of transitional cell carcinomas.

Aged↗