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D B McKay

Publications and source records attributed to D B McKay.

At least 73 records · Page 4Linked to original sources
PubMed1992

Cloning, expression, and crystallization of recoverin, a calcium sensor in vision.

Recoverin, a recently discovered 23-kDa calcium-binding protein, activates retinal rod guanylate cyclase when the calcium level is lowered in the submicromolar range. We report here the cloning and sequencing of a cDNA for recoverin from a bovine retinal expression library. The recoverin coding sequence was inserted into a pET-11a expression vector under control of the T7 phage promoter. A second expression system, in which the coding sequence was placed under control of the lambda phage PR promoter, gave 10-fold higher yields (10 mg of purified recoverin per liter of Escherichia coli culture). The finding that retinal recoverin is myristoylated at its amino terminus led us to coexpress the recombinant protein and N-myristoyltransferase (EC 2.3.1.97). Myristoylated recombinant recoverin formed in this way in E. coli is like retinal recoverin in exhibiting a large calcium-induced shift in its tryptophan fluorescence emission spectrum. The availability of abundant protein enabled us to crystallize unmyristoylated recombinant recoverin and initiate x-ray studies. The space group of tetragonal crystals obtained from 75% saturation ammonium sulfate is I4 with unit cell dimensions a = 85.1 A and c = 59.8 A. These crystals of the calcium-bound form of the protein diffracted to a resolution of 2.2 A. The expression systems described here open the door to high-resolution x-ray crystallographic and nuclear magnetic resonance studies of this new member of the EF-hand superfamily and to the elucidation of its precise mode of action as a calcium switch.

Amino Acid Sequence↗
PubMed1992

Molecular analysis of an esterase-encoding gene from a lipolytic psychrotrophic pseudomonad.

An esterase gene (estA) from a lipolytic psychotroph (Pseudomonas sp. LS107d2), was cloned in Escherichia coli and its nucleotide sequence was determined, revealing an ORF encoding a polypeptide of 389 amino acid residues, with a molecular mass of 42276 Da. Labelling of plasmid-encoded proteins with [35S]methionine, using the maxicell procedure, gave a single polypeptide of molecular mass 42 kDa, consistent with that calculated from the ORF. Colonies of E. coli cells containing estA produced a clear halo when grown on solid media containing tributyrin; no clearance was produced when cells were grown on media containing triolein. Extracts of cells containing estA also hydrolysed water-soluble nitrophenol esters, but were unable to cleave water-insoluble substrates. The preference for water-soluble substrates indicates that the gene product is an esterase.

Amino Acid Sequence↗
PubMed1992

Time-dependent inhibition of stimulated adrenal catecholamine release by staurosporine.

Staurosporine, a potent inhibitor of protein kinases, is used to study the involvement of protein kinases in cellular processes. In the present studies, the effect of prolonged staurosporine treatment on catecholamine secretion in cultured bovine adrenal chromaffin cells was examined. Staurosporine inhibits catecholamine release stimulated by 10 microM nicotine, depolarizing concentrations of potassium (56 mM KCl), and 2 mM BaCl2. The effects of staurosporine on KCl-stimulated release are time dependent, with a half-time of approximately 50 min and a maximal inhibition at 2 h. Our results indicate that activation of a staurosporine-sensitive protein kinase is not directly involved in the stimulus-secretion coupling process. This does not rule out the possibility that Ca2+/phospholipid-dependent protein kinase or other protein kinases may acutely modulate release. However, these results suggest that a protein(s), which is phosphorylated by a staurosporine-sensitive protein kinase(s), is required to maintain the integrity of the stimulus-secretion coupling process.

Adrenal Glands↗
PubMed1992

Macrophage functions are regulated by murine decidual and tumor extracellular matrices.

Because of their paternal antigens, the fetus and placenta may be considered an allograft in the maternal host. Understanding the mechanisms which prevent maternal immunological rejection of the fetus remains a fundamental unsolved problem in immunology. We have previously reported that macrophages are inhibited by maternal decidual stromal cells residing at the maternal-fetal interface. In view of the central role of macrophages in cell-mediated immunity, this inhibition may contribute to preventing maternal antifetal responses. We now report that it was the solid phase signals embedded in the extracellular matrix (ECM) made by decidual cells which are responsible for inhibiting macrophage-mediated lysis of TNF-alpha-resistant P815 mastocytoma cells. The latter macrophage function is acquired after stimulation by interferon gamma and endotoxin. All these macrophage functions were also inhibited by ECM isolated from the Engelberth-Holm-Swarme (EHS) tumor. This tumor ECM has a similar biochemical composition to decidual ECM. This ECM inhibited the effector, as opposed to the stimulator, phase of macrophage-mediated tumor lysis. Laminin, type IV collagen, and heparan sulfate proteoglycans, the major known components of decidual and EHS ECMs, did not inhibit the above macrophage functions. Altogether these data indicate that macrophages were inhibited by solid phase signals embedded in decidual and EHS ECMs. Whether the solid phase signals in these two ECMs are biochemically identical remains to be determined. To our knowledge, such signals are a novel pathway of inhibiting macrophage functions which may be important in understanding the maternal-fetal immunologic relationship, and the pathogenesis of perinatal infections. Furthermore, the ability of EHS tumor ECM to inhibit macrophage functions may indicate that some tumors may defend themselves against host macrophage responses using solid phase signals. This may be important in understanding some host-tumor relationships.

Animals↗
PubMed1992

Crystallographic structures of the elastase of Pseudomonas aeruginosa.

The elastase protein of Pseudomonas aeruginosa is a zinc metalloprotease which has been shown to be a member of the bacterial neutral protease family. Its overall tertiary structure is similar to that of thermolysin. The x-ray crystallographic structure of the elastase has been solved to high resolution in three different crystal forms. Substantial conformational differences are observed in the protein in different crystal forms. In the absence of ligand, and independently in the presence of a covalent noncompetitive inhibitor, the elastase is observed to have a relatively "open" substrate binding cleft, while in the presence of tight-binding competitive inhibitors, the active site cleft is "closed".

Amino Acid Sequence↗
PubMed1991

Similarity of the three-dimensional structures of actin and the ATPase fragment of a 70-kDa heat shock cognate protein.

Although there is very little sequence identity between the two proteins, the structures of rabbit skeletal muscle actin (375-amino acid residues) and the 44-kDa ATPase fragment of the bovine 70-kDa heat shock cognate protein (HSC70; 386 residues) are very similar. The alpha-carbon positions of 241 pairs of amino acid residues that are structurally equivalent within the two proteins can be superimposed with a root-mean-square difference in distance of 2.3 A; of these, 39 residues are identical, and 56 are conservative substitutions. In addition, the conformations of ADP are very similar in both proteins. A local sequence "fingerprint," which may be diagnostic of the adenine nucleotide beta-phosphate-binding pocket, has been derived. The fingerprint identifies members of the glycerol kinase family as candidates likely to have a similar structure in their nucleotide-binding domains. The structural differences between the two molecules mainly occur in loop regions of actin known to be involved in interactions with other monomers in the actin filament or in the binding of myosin; the corresponding regions in heat shock proteins may have functions that are as yet undetermined. Placing the Ca2+ ATP of actin on the ATPase fragment structure suggests Asp-206 (corresponding to His-161 of actin) as a candidate proton acceptor for the ATPase reaction.

Actins↗
PubMed1991

Three-dimensional structure of the elastase of Pseudomonas aeruginosa at 1.5-A resolution.

Pseudomonas aeruginosa elastase (PAE) is a zinc metalloprotease with 301 amino acids. We have crystallized and solved the three-dimensional structure of PAE, using data to 1.5-A resolution, and have refined the native molecular structure to R = 0.188. The overall tertiary structure of the PAE molecule is similar to that of thermolysin, with which it shares 28% amino acid sequence identity. Nearly all of the active site residues that might potentially interact with substrates are identical in the two proteins. However, the active site cleft is significantly more "open" in PAE than in thermolysin.

Amino Acid Sequence↗
PubMed1991

Characterization of muscarinic receptors of bovine adrenal chromaffin cells: binding, secretion and anti-microtubule drug effects.

1. Binding of [3H]QNB to adrenal membranes is saturable, specific and to a single class of receptors. 2. Tubulozole, and not other microtubule drugs, inhibits [3H]QNB binding. 3. Pretreating cultured chromaffin cells with oxotremorine, a muscarinic receptor agonist, has no effect on either basal, nicotine (10 microM) or K(+)-stimulated catecholamine release and failed to enhance secretion of submaximal concentrations of nicotine (3-5 microM). 4. These results confirm that binding of [3H]QNB is associated with muscarinic receptors on bovine adrenal medullary tissue. 5. These studies also demonstrate that although bovine adrenal chromaffin cells possess muscarinic receptors, these receptors do not appear to be coupled to secretory processes.

Adrenal Glands↗
PubMed1991

A randomized prospective trial of anti-Tac monoclonal antibody in human renal transplantation.

Patient entry is now complete in a prospective trial of anti-Tac, a murine IgG2a monoclonal antibody directed against the p55 chain of the human IL-2 receptor, for the prevention of renal allograft rejection. Recipients of primary cadaver allografts were randomized to receive either anti-Tac (20 mg q.d. x 10 days beginning POD 1) plus low-dose CsA (4 mg/kg/day), azathioprine (2 mg/kg/day), and prednisone (30 mg q.d.), or conventional triple therapy with CsA (8 mg/kg/day), azathioprine, and prednisone. Forty patients were entered in each group, with current followup from 6 to 26 months. The results show a significant reduction in early rejection episodes in the anti-Tac-treated patients. During the 10-day treatment, 5 of 40 anti-Tac patients had rejection episodes, compared with 21 of 40 control patients (P less than 0.001). Anti-Tac significantly delayed the time to the first rejection (12.5 +/- 6.3 vs. 7.6 +/- 6.7 days) (P less than 0.05). Despite these effects, there were no differences in either actual or actuarial graft or patient survival between the two groups. Pneumonia, primarily CMV, developed in 5 treated and 4 control patients. In patients with functioning grafts mean serum creatinine at 3 months was 1.8 +/- 0.7 in the anti-Tac group and 2.0 +/- 0.8 in the control group (P = NS); at 12 months the values were 2.3 +/- 1.5 and 1.8 +/- 0.5, respectively (P = NS). The peak expression of IL-2 receptors on circulating T-cells was significantly lower in anti-Tac patients (15.1 +/- 3.6%) than in controls (21.9 +/- 4.5%) (P less than 0.05). Seven of 10 patients tested to date developed antimouse immunoglobulin antibodies, with antiidiotype shown in 6. These antibodies do not preclude subsequent treatment with OKT3. Five patients in this and previous anti-Tac protocols have received OKT3 for acute rejection despite known pretreatment antimouse antibodies, with resolution of rejection in all cases.

Adolescent↗
PubMed1991

Listeriolysin as a virulence factor in Listeria monocytogenes infection of neonatal mice and murine decidual tissue.

Listeriolysin is a 60-kDa protein which allows the growth of Listeria monocytogenes in macrophages and other cells and has been shown to be a virulence factor in Listeria infections of adult mice. However, the neonate and fetoplacental unit are major populations susceptible to listeriosis. Recent data indicate that macrophage and T-cell functions are markedly inhibited in these young mice, and the virulence of listeriolysin-negative (HLY-) and listeriolysin-positive (HLY+) Listeria cells in the setting of such inhibited macrophage and T-cell functions has not previously been examined. We now compare CNL 85/162, a transposon-induced, HLY- Listeria strain, and CNL 85/163, a spontaneous HLY+ revertant. We found that all 18 neonates injected with CNL 85/163 (HLY+) died within 12 days after an injection of 10(4) Listeria cells per mouse. In contrast, all 16 neonates injected with 1,000 times more CNL 85/162 (HLY-) cells survived more than 14 days. Three days after injection, growth of CNL 85/163 (HLY+) in the internal organs was more than 5 log greater than that of CNL 85/162 (HLY-). We also found that CNL 85/162 (HLY-) did not proliferate well in decidual tissue, which is a major component of the placental region. Our studies indicate that HLY- bacteria are not virulent in the neonate and the fetoplacental unit despite the inhibited immune functions at these sites.

Animals↗
PubMed1990

Uncoating protein (hsc70) binds a conformationally labile domain of clathrin light chain LCa to stimulate ATP hydrolysis.

Uncoating of clathrin-coated vesicles is mediated by the heat shock cognate protein, hsc70, and requires clathrin light chains (LCa and LCb) and ATP hydrolysis. We demonstrate that purified light chains and synthetic peptides derived from their sequences bind hsc70 to stimulate ATP hydrolysis. LCa is more effective than LCb in stimulating hsc70 ATPase and in inhibiting clathrin uncoating by hsc70. These differences correlate with high sequence divergence in the proline- and glycine-rich region (residues 47-71) that forms the hsc70 binding site. For LCa, but not LCb, this region undergoes reversible conformational changes upon perturbation of the ionic strength or the calcium ion concentration. Our results show that LCa is more important for interactions with hsc70 than is LCb and suggest a model in which the LCa conformation regulates coated vesicle uncoating.

Adenosine Triphosphatases↗
PubMed1990

Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein.

The three-dimensional structure of the amino-terminal 44K ATPase fragment of the 70K bovine heat-shock cognate protein has been solved to a resolution of 2.2 A. The ATPase fragment has two structural lobes with a deep cleft between them; ATP binds at the base of the cleft. Surprisingly, the nucleotide-binding 'core' of the ATPase fragment has a tertiary structure similar to that of hexokinase, although the remainder of the structures of the two proteins are completely dissimilar, suggesting that both the phosphotransferase mechanism and the substrate-induced conformational change intrinsic to the hexokinases may be used by the 70K heat shock-related proteins.

Adenosine Triphosphatases↗
PubMed1990

Effect of noncompetitive nicotinic receptor blockers on catecholamine release from cultured adrenal chromaffin cells.

Nicotinic acetylcholine receptors are found on both muscle tissue and neuronal tissue, including adrenal chromaffin cells. Certain drugs, such as the noncompetitive ion-channel blockers and the vinca alkaloids, have been demonstrated to interact with muscle-type nicotinic receptor-associated ion channels. The objectives of these studies were: (1) to determine the effects of the noncompetitive blockers of the nicotinic receptor-associated ion-channel drugs on adrenal chromaffin cells, and (2) to establish whether the vinca alkaloids and the ion-channel drugs have similar actions on adrenal nicotinic receptors. The ion-channel drugs, adiphenine, tetracaine, quinacrine, amantadine, lidocaine and procaine, inhibited receptor-stimulated catecholamine release from cultured adrenal chromaffin cells in a concentration-dependent manner (IC50s: 2, 4, 6, 41, 55 and 87 microM, respectively). The inhibitory effects of these drugs appeared to be noncompetitive. These drugs had little or no effects on catecholamine release stimulated by depolarizing concentrations of K+ or on basal release. Our results demonstrate that the ion-channel blockers interact with adrenal nicotinic receptors in a manner similar to their interaction with muscle-type nicotinic receptors and they interfere with adrenal receptor function in a manner similar to the vinca alkaloids.

Animals↗
PubMed1990

Macrophage functions are regulated by the substratum of murine decidual stromal cells.

Because of their paternal antigens, the fetus and placenta may be considered an allograft in the maternal host. Local properties of the maternal-fetal interface, the placenta and decidua basalis, are important in preventing maternal immunologic rejection of the fetoplacental allograft. However, the exact nature of these local properties remains a fundamental unsolved problem in immunology. We now report that three macrophage functions were inhibited by the substratum formed by monolayers of decidual stromal cells via a novel pathway. Solid-phase inhibitors blocked macrophage adhesion, spreading, and lysis of tumor necrosis factor-alpha-resistant P815 mastocytoma tumor cells. Inhibition was not solely attributable to an inability of macrophages to adhere to decidual substratum because there were differences in macrophage functions on this surface versus polyhema where no adherence occurred. Because macrophages play a central role in cell-mediated immunity, including allograft rejection, inhibiting their function in the decidua basalis may help prevent maternal antifetal responses.

Animals↗