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P Sheterline

Publications and source records attributed to P Sheterline.

28 records · Page 2Linked to original sources
PubMed1977

Phosphorylation of pig brain microtubule proteins. General properties and partial characterization of endogenous substrate and cyclic AMP-dependent protein kinase.

1. A simple purification procedure for microtubule proteins is described, which involves a single assembly step in vitro in the absence of glycerol, followed by centrifugation through sucrose. 2. The preparation contains 80% tubulin (mol.wt. 54000), 15-20% of a 280000-mol.wt. protein and several other minor components of intermediate molecular weight after polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate and 2-mercaptoethanol. 3. In the presence of [gamma-32P]ATP, [32P]phosphate was incorporated into the 280000-mol.wt. component reaching half-maximal incorporation at 1-2 min, but no phosphorylation of tubulin was detected. Cyclic AMP (Km 0.8 micrometer) increased both the initial rate and the extent of incorporation of [32P]phosphate into this component. 4. About half of the endogenous protein kinase activity did not require cyclic AMP and was not inhibited by a heat-stable inhibitor protein from muscle. The remainder of the activity was cyclic AMP-dependent and sensitive to the inhibitor protein. A regulatory subunit was not dissociable from microtubules assembled in vitro in the presence of saturating concentrations of cyclic AMP. 5. The endogenous substrate and the endogenous protein kinase activity could be partially resolved chromatography on phosphocellulose. 6. The data show that cyclic AMP can moduate the activity of an endogenous protein kinase(s) with unusual properties and which phosphorylates a prominent microtubule-associated protein.

Animals↗
PubMed1975

Colchicine binding to bovine anterior pituitary slices and inhibition of growth-hormone release.

The uptake of [ring C-methoxyl-3H]colchicine into bovine anterior pituitary slices was studied. The data suggest that more than one site exists for the binding of colchicine. At low concentrations colchicine binds to saturable trypsin-sensitive site(s), with a dissociation constant of 3.1 +/- 0.69 mug. The binding capacity of these sites is 8.58 +/- 0.60 pmol of colchicine/mg of wet pituitary. At higher colchicine concentrations binding occurs predominantly to sites which exhibit non-saturation kinetics. Subcellular fractionation of colchicine-labelled slices shows that 90% of the saturable sites are present in the fraction containing cytosol, where the binding protein has a molecular weight of about 11.9 x 10(4) and constitutes 0.7% of the protein present. The nuclear fraction contains 10% of the saturable sites, and the mitochondria and granule fraction contain only non-saturable sites. The rate of colchicine uptake was studied at 0.84 mm- and 2mum-colchicine. At both concentrations the colchicine space exceeded the total tissue water within 10 min. Equilibration with the saturable binding sites was complete in 120 min at 2mum-colchicine. A concentration of colchicine (13.4 mum) which would give 81% maximum binding was found to decrease the length of observable microtubules in tissue fixed at 37 degrees C in glutaraldehyde by 83 +/- 4%. The colchicine-binding protein could be partially purified by using a standard procedure for isolation of brain tubulin. Colchicine inhibits the release of growth hormone in the presence of 3-isobutyl-1-methylxanthine (0.1 mm), but does not alter basal release. The concentration-dependence of colchicine inhibition is similar to that of colchicine binding, but maximum inhibition is only 35%.

Animals↗