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J D Scott

Publications and source records attributed to J D Scott.

At least 163 records · Page 9Linked to original sources
PubMed1987

The molecular cloning of a type II regulatory subunit of the cAMP-dependent protein kinase from rat skeletal muscle and mouse brain.

A cDNA clone for a type II regulatory (R) subunit of the cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) was isolated from a rat skeletal muscle library using a specific 47-base oligonucleotide probe. The rat cDNA was 1.2 kilobases (kb) in length and contained an open reading frame of 1.113 kb representing 92% of the coding region of the molecule. Nick-translated rat cDNA was then used to isolate a mouse RII cDNA clone from a brain library that contained an open reading frame of 1.143 kb. Because both cDNAs lacked complete coding sequences, the remainder of the RII coding region was obtained from a 15-kb mouse genomic clone. The mouse RII coding region contains 1.2 kb corresponding to a 400-amino acid protein of 51.141 kDa. The mouse cDNA hybridizes to two mRNA species, a 2.4-kb form that was only observed in testis and a 6.0-kb form found in a wide range of tissues, including testis.

Amino Acid Sequence↗
PubMed1987

Cloning and characterization of BCY1, a locus encoding a regulatory subunit of the cyclic AMP-dependent protein kinase in Saccharomyces cerevisiae.

We have cloned a gene (BCY1) from the yeast Saccharomyces cerevisiae that encodes a regulatory subunit of the cyclic AMP-dependent protein kinase. The encoded protein has a structural organization similar to that of the RI and RII regulatory subunits of the mammalian cyclic AMP-dependent protein kinase. Strains of S. cerevisiae with disrupted BCY1 genes do not display a cyclic AMP-dependent protein kinase in vitro, fail to grow on many carbon sources, and are exquisitely sensitive to heat shock and starvation.

Amino Acid Sequence↗
PubMed1986

Association of calmodulin with peptide analogues of the inhibitory region of the heat-stable protein inhibitor of adenosine cyclic 3',5'-phosphate dependent protein kinase.

A 20-residue peptide analogue (IASGRTGRRNAIHDILVSSA) of the 8000-dalton heat-stable cAMP-dependent protein kinase inhibitor undergoes efficient calcium-dependent binding by calmodulin, with Kd approximately 70 nM when calcium is present. It is a potent inhibitor of smooth muscle myosin light chain kinase and of the calmodulin-dependent phosphatase activity of calcineurin. At concentrations above 3 microM, the peptide stimulates the basal activity of calcineurin. The native protein kinase inhibitor has no effect on the catalytic activity of myosin light chain kinase and is moderately inhibitory to both the calmodulin-dependent and -independent phosphatase activity of calcineurin. Competition experiments using excess concentrations of calcineurin and calmodulin suggest that the primary interaction of the native heat-stable inhibitor is with the catalytic subunit of protein kinase. Dansylcalmodulin exhibits only a weak interaction with the inhibitor. Observations on deletion peptides of the 20-residue analogue help to delineate the overlapping peptide binding specificities of the cAMP-dependent protein kinase [Scott, J. D., Glaccum, M. B., Fischer, E. H., & Krebs, E. G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 1613-1616] and calmodulin. In both cases, the most effectively bound peptides contain the RTGRR sequence.

Amino Acid Sequence↗
PubMed1986

Membrane fluidization increases low-affinity muscarinic receptor binding in brain: changes with aging.

Specific cholinergic muscarinic receptor binding was determined with L-[3H]quinuclidinyl benzilate ([3H]QNB) in homogenates from crude synaptosomal pellets prepared from mouse whole-brain homogenates. Specific total (high- and low-affinity) binding was determined in the absence of the agonist carbachol and low-affinity binding in its presence. These membrane preparations were fluidized by adding in vitro aliphatic alcohols ranging from ethanol to hexanol and by increasing the incubation temperatures. At 23 degrees C hexanol (14.7 mM) nearly doubled the low-affinity binding in the presence of carbachol (0.32 mM) and decreased high-affinity binding by the same amount. This suggested a change of muscarinic receptors from high- to low-affinity conformation. Increase of incubation temperature from 24 degrees C to 37 degrees C nearly tripled low-affinity binding. Brain homogenates from female C57BL/6J mice, ages 6, 12, 18, and 30 months, showed a progressively lower stimulation by hexanol of low-affinity [3H]QNB binding in the presence of carbachol. We postulate that this diminished change with age of [3H]QNB-receptor binding in response to alcohols may be a result of increasing membrane rigidity with advancing age. Rigidity of membranes may link aging at the membrane level, synaptic receptors, and impaired learning behavior.

Aging↗
PubMed1986

Primary-structure requirements for inhibition by the heat-stable inhibitor of the cAMP-dependent protein kinase.

The amino acid sequence of the heat-stable inhibitor of the cAMP-dependent protein kinase (PKI) was determined recently [Scott, J. D., Fischer, E. H., Takio, K., Demaille, J. G. & Krebs, E. G. (1985) Proc. Natl. Acad. Sci. USA 82, 5732-5736]. An earlier report [Scott, J. D., Fischer, E.H., Demaille, J. G. & Krebs, E. G. (1985) Proc. Natl. Acad. Sci. USA 82, 4379-4383] showed that at least part of the inhibitory domain of PKI is located in a 20-residue segment extending from residue 11 to residue 30: Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile-His-Asp-Ile-Leu-Val-Ser- Ser-Ala . In the present study, we further mapped the inhibitory region of PKI by addition or deletion of residues at both ends of this peptide and by substitutions for specific amino acids. The results show that (i) deletion of residues 25-30 did not change inhibitory activity but addition of residues toward the amino terminus increased the inhibitory potency up to 150-fold (Ki 4.8 nM), to a level approaching that of PKI; (ii) replacement of alanine-21 by serine converted the inhibitor into a substrate having a relatively low affinity (Km 280 microM) for the enzyme; (iii) replacement of alanine-21 by phosphoserine or alpha-aminobutyric acid decreased inhibitory activity by a factor of 120 and 20, respectively; (iv) replacement of serine-13 had essentially no effect, whereas substitution of threonine-16 decreased inhibitory activity. The greatest decreases of inhibitory potency occurred with replacements of the arginines in positions 18 and 19.

Amino Acid Sequence↗
PubMed1985

Identification of an inhibitory region of the heat-stable protein inhibitor of the cAMP-dependent protein kinase.

The present study was undertaken in order to identify the inhibitory site of the heat-stable inhibitor of cAMP-dependent protein kinase (PKI) and to synthesize a peptide that could serve as a useful inhibitor of the enzyme. Digestion of purified PKI by mast cell proteinase II yielded a peptide fragment that retained inhibitory activity. A sequence of 20 amino acids of the peptide, (sequence in text) revealed the presence of a "pseudosubstrate site" (Arg-Arg-Asn-Ala-Ile) for the cAMP-dependent protein kinase in which alanine replaces the seryl or threonyl residue that is normally phosphorylated. Digestion of PKI with various other proteinases implicated the involvement of arginyl and hydrophobic residues as determinants for the inhibitory activity. The assumption that this region is part of the inhibitory site was confirmed by the synthesis of a corresponding duodecapeptide that displayed strong inhibitory activity. Inhibition by the peptide was competitive with a Ki of 0.8 microM as measured against a number of protein substrates. The sequence of this fragment bears a strong resemblance to the autophosphorylation site in the type II regulatory subunit of cAMP-dependent protein kinase, a region also postulated to interact with the catalytic subunit, and the analogous region of type I regulatory subunit. Neither intact PKI nor the synthetic peptide inhibit the cGMP-dependent protein kinase, phosphorylase kinase, myosin light-chain kinase, casein kinase II, or protein kinase C.

Amino Acid Sequence↗
PubMed1985

Amino acid sequence of the heat-stable inhibitor of the cAMP-dependent protein kinase from rabbit skeletal muscle.

The amino acid sequence of rabbit skeletal muscle heat-stable inhibitor of the cAMP-dependent protein kinase has been determined by microsequencing techniques. Proof of the structure involved a series of nonoverlapping tryptic fragments for primary identification of 86% of the amino acids. Complementary fragments generated by cleavage with chymotrypsin, Staphylococcus aureus V8 proteinase, and mast cell proteinase II contributed to proof of the structure. The inhibitor is a single polypeptide chain of 75 residues and has a molecular weight of 7829. It lacks tryptophan, proline, and sulfur-containing amino acids. The amino terminus of the inhibitor is blocked by an unidentified group. The amino-terminal region of the molecule contains the kinase inhibitory domain, and synthetic peptides based on the sequence of residues 11-30 are potent competitive inhibitors of the cAMP-dependent protein kinase [Scott, J. D., Fischer, E. H., Demaille, J. G. & Krebs, E. G. (1985) Proc. Natl. Acad. Sci. USA 82, 4379-4383]. Residues 14-22 show considerable homology to the "hinge-regions" of the regulatory subunits of the cAMP-dependent protein kinase. The remainder of the molecule shows no similarity to the known amino acid sequence of any protein.

Amino Acid Sequence↗
PubMed1982

A general method for affinity purification of complement component C3b using factor H-sepharose.

Complement component C3b has been purified from human, rabbit and bovine serum by affinity chromatography on human factor H-Sepharose after preliminary fractionation by poly(ethylene glycol) and DEAE-Sepharose. The yields are high (35--40%) and the whole process is rapid (3 days). Binding of C3b to factor H-Sepharose is equimolar, has a sharp optimum pH at 7.6 and is quite sensitive to ionic strength.

Animals↗
PubMed1982

Lens epithelial proliferation in retinal detachment.

That such profound epithelial changes occur might indicate the need to dwell less on protein changes in the formation of other kinds of cataract and more on epithelial alterations resulting in abnormal or absent lens fibre production. Complicated retinal detachment and particularly that due to giant retinal tear causes profound epithelial changes in the lens. These take the form of migration of equatorial and possible anterior cells posteriorly, metaplasia of these cells and of the anterior epithelium, cytoplasmic vaccuolation of anterior and migrated cells, swelling and destruction of migrated cells to form balloon cells and finally, cessation of normal lens fibre production. Many of these changes are related to those occurring in non-retinogenic cataract but are on a massive scale which in a predictable time leads to complete loss of metabolic control in the lens with the formation of acute cataract.

Cataract↗
PubMed1981

Arrest and regression of retinal neovascularization.

Retinal neovascularization is one of many forms of new vessel formation in the eye. The chief cause is diabetic retinopathy and the severe retinal and vitreous complications can now be significantly reduced by argon laser and xenon arc coagulation. Although the natural history of proliferative retinopathy is well described, clinical details of monitoring the progress and regression have not been described. This paper describes the signs of active proliferation of arrest and regression both spontaneously and as a result of laser photocoagulation. Vitreous detachment is considered in some detail together with the factors which control the visual outcome after this significant event in the natural history of the retinopathy. The purpose of the paper is to provide a more rational approach to photocoagulation of retinal neovascularization.

Diabetic Retinopathy↗
PubMed1980

Congenital myopia and retinal detachment.

Retinal detachment is unusual in children and is often associated with other serious ocular abnormalities. In more than 25 children varying in age from 3 to 16 years in whom the common ocular complication was congenital myopia, associated features included facial abnormalities and other changes often associated with familial and inherited conditions. A very high incidence of giant retinal breaks was found and these were associated with considerable difficulty in treatment.

Adolescent↗