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R Rodnight

Publications and source records attributed to R Rodnight.

At least 55 records · Page 3Linked to original sources
PubMed1990

Regional variations in protein phosphorylating activity in rat brain studied in micro-slices labeled with [32P]phosphate.

Regional variations in protein phosphorylating activity in the rat brain were studied. Micro-slices (1 mm diameter) were prepared from 19 brain areas, phosphoproteins labeled by incubation with [32P]phosphate, and the tissue analyzed by nonequilibrium two-dimensional electrophoresis and autoradiography. Attention was focused on three phosphorylating systems that showed consistent variation in activity. (1) A system that phosphorylates a substrate of 47 kDa (ppH-47) whose activity was highest in the hippocampus. The next highest activity of this system was observed in the globus pallidus, followed by the periventricular gray matter of the aqueduct, lateral septum, cerebellar cortex, entorhinal cortex, hypothalamus, mammillary nuclei, amygdala, and substantia nigra. Activity was low or undetectable in the cerebral cortex, neostriatum, and the colliculi. (2) A system that phosphorylates a substrate of 50 kDa (ppC-50) whose activity was highest in the caudate nucleus. The activity of this system was roughly inversely correlated with that of the ppH-47 system. (3) The protein kinase C system that phosphorylates an 82- to 87-kDa substrate known as MARCKS. The highest activity of this system was observed in the cerebellar cortex, followed by the hypothalamus, mammillary nuclei, periventricular gray matter of the aqueduct, and the superior colliculus. Activity of this system was relatively low in several regions of the cerebral cortex, the neostriatum, and the inferior colliculus.

Animals↗
PubMed1988

An investigation of experimental conditions for studying protein phosphorylation in micro-slices of rat brain by two-dimensional electrophoresis.

Procedures are described for studying protein phosphorylation in 1 mm diameter micro-slices of rat brain tissue using two-dimensional electrophoresis as analytical tool. The activity of several protein phosphorylating systems, including a major system phosphorylating a 40 kDa substrate complex, was highly dependent on the procedures used for micro-slice preparation and on the Ca2+-content of the preparation medium. Under optimal conditions the pattern of phosphorylation observed in micro-slices closely resembled that obtained by in vivo labelling.

Animals↗
PubMed1987

Demonstration by phase-partitioning in Triton X-114 solutions that phosphoprotein B-50 (F-1) from rat brain is an integral membrane protein.

The Triton X-114 phase separation technique was employed to fractionate phosphoproteins present in membrane fragments from rat brain. Membranes were labelled with [gamma-32P]ATP in media containing Ca2+, Ca2+ plus calmodulin or cyclic AMP, and then treated with Triton X-114. Phosphoproteins recovered in the detergent-insoluble fraction, aqueous and detergent phases were detected by SDS-polyacrylamide gel electrophoresis and autoradiography. Of the proteins solubilised by the detergent, a known substrate of protein kinase C, the B-50 phosphoprotein (45 kD; also known as F-1), partitioned quantitatively into the detergent-rich phase, making it very probable that this phosphoprotein is an integral membrane protein. The detergent-rich phase also contained an 80 kD phosphoprotein, which probably corresponds to the widespread acidic 87 kD substrate of protein kinase C.

Animals↗
PubMed1987

Effect of digestion with phospholipase A2 on endogenous protein phosphorylation in particulate fractions from rat brain synaptosomes.

The endogenous phosphorylation of synapsin 1 in cyclic AMP-containing media was greatly decreased by digestion of synaptic vesicles and synaptosomal membranes with phospholipase A2, suggesting that the system is functionally dependent on the membrane structure. Treatment of the synaptic vesicle fraction with phospholipase A2 also caused a small but significant inhibition of the Ca2+/calmodulin-dependent phosphorylation of the same protein. The Ca2+/calmodulin-dependent phosphorylation of other major acceptors, and the basal phosphorylation of a 52-kD acceptor enriched in the vesicle fraction, remained unchanged after cleavage of the membrane phospholipids with phospholipase A2. The significance of the selective effect of phospholipase A2 treatment on endogenous membrane phosphorylation is discussed.

Animals↗
PubMed1986

Acceptors for cyclic AMP-dependent and calcium ion-dependent protein kinases in rat brain cytosol fractions: a comparison of occluded (synaptosomal) cytosol with non-occluded cytosol.

Endogenous protein phosphorylation patterns were compared in occluded and non-occluded cytosol fractions prepared from rat forebrain. The occluded fraction was taken as representative of synaptosomal cytosol. One- and two-dimensional autoradiographs revealed the presence in non-occluded cytosol of a substrate for cAMP- and Ca2+/calmodulin-dependent protein kinase activities of Mr 300kD, corresponding to phosphorylated microtubule-associated protein-2 (MAP-2); this protein was absent in occluded cytosol. In contrast, a major substrate for protein kinase C was observed exclusively in occluded cytosol after phosphorylation under basal conditions. However, after phosphorylation in the presence of exogenous lipids, approximately equal amounts of the 82kD substrate were detected in both fractions, suggesting that protein kinase C in the occluded fraction was present in a partially activated state. Other minor differences in phosphorylation patterns between the two fractions were observed.

Animals↗
PubMed1986

Ca2+ sensitivity of Ca2+-dependent protein kinase activities toward intrinsic proteins in synaptosomal membrane fragments from rat cerebral tissue.

The Ca2+ and calmodulin sensitivity of endogenous protein kinase activity in synaptosomal membrane fragments from rat brain was studied in medium containing Ca2+ plus EGTA using a modified computer programme to calculate free Ca2+ concentrations that took into account the effect of all competing cations and chelators. The Ca2+-dependent phosphorylation of 10 major polypeptide acceptors with Mr values ranging from 50 to 360 kilodaltons required calmodulin in reactions that were all equally sensitive to Ca2+; half-maximal phosphorylation required a free Ca2+ concentration of 45 nM and maximal phosphorylation approximately 110 nM. The significance of these values in relation to published data on the intracellular concentration of free Ca2+ in the nervous system is discussed. One acceptor of 45 kilodaltons was phosphorylated in a Ca2+-dependent reaction that did not require calmodulin. This polypeptide appeared to correspond to the B-50 protein, an established substrate of the lipid-dependent protein kinase C. Further study of this phosphorylating system showed that the reaction was only independent of calmodulin at saturating concentrations of Ca2+; at subsaturating concentrations (in the range 50-130 nM), a small but significant stimulation of the enzyme by calmodulin was demonstrated. The possible significance of this finding is discussed.

Animals↗
PubMed1986

Protein phosphorylation and synaptic transmission: receptor mediated modulation of protein kinase C in a rat brain fraction enriched in synaptosomes.

Aspects of protein phosphorylation related to events occurring during synaptic transmission were briefly reviewed. High resolution two-dimensional electrophoresis was used to study protein phosphorylation catalysed by protein kinase C in a fraction from rat brain enriched in synaptosomes. Incubation of 32P-labelled synaptosomes with 4 beta-phorbol 12 beta-myristate 13 alpha-acetate resulted in an increase in the phosphorylation of a 45 K polypeptide (generally known as B-50) and an 82 K polypeptide; other major phosphoproteins in the preparation were unaffected by this treatment. It appears therefore that the 45 K and 82 K polypeptides are the only significant substrates for protein kinase C in synaptosomes. Depolarisation of labelled synaptosomes by high K+ increased the phosphorylation of the 82 K polypeptide, synapsin I and several unknown phosphoproteins. Incubation of labelled synaptosomes with the cholinergic agonist carbachol resulted in a modest, but statistically significant, increase in the phosphorylation of the 45 K (B-50) and 82 K polypeptides. This effect was blocked by atropine. The results are discussed in relation to a possible role for the B-50 phosphoprotein in regulating the resynthesis of polyphosphoinositides following cholinergic stimulation.

Animals↗
PubMed1985

A simple and economical method for studying protein phosphorylation in vivo in the rat brain.

A simple and economical procedure, capable of routine application, is described for the labelling of cerebral phosphoproteins in vivo. [32P]Orthophosphate, in high concentration, was infused into selected brain areas of anaesthetised rats under stereotaxic control. The animals were frozen with liquid N2 and the labelled tissue punched out of frozen thick sections. [32P]Polypeptides were analysed by high-resolution two-dimensional gel electrophoresis. Several phosphoproteins on the gels were provisionally identified, including synapsin I, MAP-2 and an 82-87 kdalton substrate of protein kinase 'C'.

Animals↗
PubMed1984

On the disposition of a phosphorylated protein ("synapsin I") and its associated kinases in synaptosomes from rat brain.

Endogenous phosphorylation of synapsin I (protein I), a phosphoprotein located on the surface of synaptic vesicles, was studied in vesicles prepared from synaptosomes lysed in the absence (control) or presence of 50 microM-cyclic AMP ("cAMP-treated"). Compared to synaptic plasma membrane (SPM) fractions prepared in parallel, and confirming previous work, the vesicle fractions were highly enriched on a unit protein basis in Ca2+-calmodulin-dependent kinase activity towards synapsin I. In contrast, with control vesicles the magnitude of the total phosphorylation of synapsin I in the presence of cyclic AMP was similar to that observed in SPM, but regulation by cyclic AMP was only partial. In "cAMP-treated" vesicles, however, synapsin I phosphorylation was highly enriched compared to SPM and the activity was virtually independent of cyclic AMP. The results show that while the free catalytic subunit of the cyclic AMP-dependent kinase remains associated with synapsin I during vesicle isolation the holoenzyme remains bound to membrane fragments, probably through its regulatory subunit.

Animals↗
PubMed1983

Stimulation and inhibition by magnesium ions of intrinsic protein phosphorylating systems in synaptosomal membrane fragments from rat brain.

Synaptosomal membrane fragments from rat brain were incubated with [gamma-32P]ATP in the presence of cyclic AMP or Ca2+ plus calmodulin and a range of Mg2+ concentrations. Incorporation of 32P into membrane polypeptides was examined by electrophoresis and radioautography. Cyclic AMP-stimulated reactions were stimulated by low concentrations and inhibited to varying degrees by high concentrations of Mg2+ in the range 1-50 mM. In general the Ca2+ plus calmodulin-stimulated reactions were maximally active in the range 30-50 mM Mg2+, but the Ca2+ plus calmodulin dependent phosphorylation of Protein I was progressively inhibited by concentrations of Mg2+ above 5 mM. These results emphasize the importance of establishing optimum Mg2+ concentrations in the study of specific membrane protein phosphorylating systems.

Animals↗
PubMed1982

Intrinsic protein phosphorylation in synaptic plasma membrane fragments from the rat. General characteristics and migration behaviour on polyacrylamide gels of the main phosphate acceptors.

Preparations enriched in synaptic membrane fragments from rat cerebral cortex contain protein kinases which phosphorylate membrane proteins in reactions dependent on cAMP, Ca2+ (in the absence of presence of calmodulin) or independent of these factors. In the present work characteristics of the main phosphorylated acceptors were studied and compared with the results of other investigations. Apparent molecular weights were estimated by determining electrophoretic mobility on gels of different acrylamide concentration. Irregular migration behaviour was detected by measuring free mobilities from Ferguson plots. Certain phosphate acceptors were found to exhibit anomalously low free mobilities and it was concluded that estimates of molecular weight for these acceptors were unreliable.

Animals↗
PubMed1982

Intrinsic protein phosphorylation in synaptosomal plasma membrane fragments: a comparison of cerebral cortex tissue from several species, including human biopsy specimens.

Intrinsic protein phosphorylation was studied in synaptosomal membrane fragments made from cerebral cortex tissue taken from the following species: human (biopsy specimens), ox, rat, rabbit, guinea pig and mouse. Membrane fragments from all species exhibited a qualitatively similar range of protein acceptors phosphorylated by cyclic AMP-dependent protein kinase activity; contrary to a previous report, no evidence for cyclic GMP-dependent protein kinase activity was found in the human material. With the exception of membrane fragments prepared from ox brain, all the preparations exhibited the same range of Ca2+-dependent protein kinase activity. Ox brain obtained from a slaughterhouse yielded membranes containing no Ca2+-dependent protein kinase activity, but this may have been due to unavoidable postmortem losses.

Animals↗
PubMed1981

Cyclic nucleotide-dependent phosphorylation of endogenous proteins in bovine adrenocortical cell membranes.

Activation of one or more cyclic AMP-dependent protein kinases has been suggested as an intermediate step in ACTH-stimulated adrenal cell steroidogenesis. Phosphorylation of a number of proteins from different subcellular fractions has been reported but those phosphorylation events which are relevant to the steroidogenic process have not yet been identified. In this paper we report that plasma membrane enriched fractions from bovine adrenal cortex retain the ability to phosphorylate endogenous membrane proteins and that phosphorylation of these acceptors is markedly enhanced by cyclic AMP or, to a lesser extent, by cyclic GMP. Cyclic nucleotide-dependent phosphorylation was most marked in protein acceptors of 191 000, 148 000, 138 000, 107 000, 65 000, 60 000 and 27 000 daltons. Cyclic nucleotide stimulation of phosphorylation was rapid (within 10 s), and is consistent with the rapid onset of ACTH-stimulated steroidogenesis.

Adrenal Cortex↗