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D B McKay

Publications and source records attributed to D B McKay.

At least 55 records · Page 3Linked to original sources
PubMed1995

Degradation of triglycerides by a pseudomonad isolated from milk: the roles of lipase and esterase studied using recombinant strains over-producing, or specifically deficient in these enzymes.

The roles of lipase and esterase in causing hydrolytic spoilage of milk by a highly lipolytic psychrotrophic strain of Pseudomonas fluorescens, LS107d2, has been studied. Strains of LS107d2 have been constructed that over-produce, or are specifically deficient in, a lipase (encoded by lipA) and an esterase (encoded by estA). Southern blot analysis reveals that LS107d2 contains only one esterase and one lipase (encoded by estA and lipA) and this was confirmed by the phenotypes of mutants on triolein and tributyrin-containing agar. Analysis of broth cultures showed that the lipase is secreted into the culture medium; in contrast, the esterase is not secreted. Free fatty acid (FFA) levels in whole milk cultures of wild-type, over-producing and the mutant strains of LS107d2 have been examined. From these studies it is concluded that esterase is not involved in the accumulation of FFA by hydrolysing short chain fatty acid esters; that the highly lipolytic phenotype of LS107d2 is due solely to a single secreted lipase; and that the main FFA accumulated in milk cultures of LS107d2 are C4, C16, C18 and C18: 1. Evidence is also presented demonstrating that FFA degradation, as well as production, determines the level of FFA in milk contaminated with lipolytic organisms.

Animals↗
PubMed1994

ATPase kinetics of recombinant bovine 70 kDa heat shock cognate protein and its amino-terminal ATPase domain.

Steady-state kinetic, pre-steady-state kinetic, and equilibrium binding measurements have been applied to determine the rate constants of individual steps of the ATPase cycle for the recombinant bovine 70 kDa heat shock cognate protein and its amino-terminal 44 kDa ATPase fragment. At 25 degrees C, pH 7.0, in the presence of 75 mM KCl and 4.5 mM Mg2+, the measured association rate constants for MgATP approximately hsc70 and MgADP approximately hsc70 are (2.7 +/- 0.5) x 10(5) and (4.1 +/- 0.5) x 10(5) M-1 s-1, respectively, while the dissociation rate constants are 0.0114 (+/- 0.0002) and 0.0288 (+/- 0.0018) s-1, respectively. MgATP (Kd = 0.042 microM) therefore binds to hsc70 more tightly than MgADP (Kd = 0.11 microM). ADP release is inhibited by inorganic phosphate (Pi), suggesting that product dissociation is ordered with Pi released first and ADP second. The rate of chemical hydrolysis of ATP is 0.0030 (+/- 0.0003) s-1 for hsc70 and 0.0135 (+/- 0.0033) s-1 for the 44 kDa fragment. The rate of Pi release is 0.0038 (+/- 0.0010) s-1 for hsc70 and 0.0051 (+/- 0.0006) s-1 for the 44 kDa fragment. For the 44 kDa fragment, Pi release is the slowest step in the ATPase cycle, while for hsc70, Pi release and chemical hydrolysis of MgATP have similar rates; in both cases, ADP release is a relatively rapid step in the ATPase cycle.

Adenosine Diphosphate↗
PubMed1994

Three-dimensional structure of a hammerhead ribozyme.

The hammerhead ribozyme is a small catalytic RNA motif made up of three base-paired stems and a core of highly conserved, non-complementary nucleotides essential for catalysis. The X-ray crystallographic structure of a hammerhead RNA-DNA ribozyme-inhibitor complex at 2.6 A resolution reveals that the base-paired stems are A-form helices and that the core has two structural domains. The first domain is formed by the sequence 5'-CUGA following stem I and is a sharp turn identical to the uridine turn of transfer RNA, whereas the second is a non-Watson-Crick three-base-pair duplex with a divalent-ion binding site. The phosphodiester backbone of the DNA inhibitor strand is splayed out at the phosphate 5' to the cleavage site. The structure indicates that the ribozyme may destabilize a substrate strand in order to facilitate twisting of the substrate to allow cleavage of the scissile bond.

Base Sequence↗
PubMed1994

Model for an RNA tertiary interaction from the structure of an intermolecular complex between a GAAA tetraloop and an RNA helix.

In large structured RNAs, RNA hairpins in which the strands of the duplex stem are connected by a tetraloop of the consensus sequence 5'-GNRA (where N is any nucleotide, and R is either G or A) are unusually frequent. In group I introns there is a covariation in sequence between nucleotides in the third and fourth positions of the loop with specific distant base pairs in putative RNA duplex stems: GNAA loops correlate with successive 5'-C-C.G-C base pairs in stems, whereas GNGA loops correlate with 5'-C-U.G-A. This has led to the suggestion that GNRA tetraloops may be involved in specific long-range tertiary interactions, with each A in position 3 or 4 of the loop interacting with a C-G base pair in the duplex, and G in position 3 interacting with a U-A base pair. This idea is supported experimentally for the GAAA loop of the P5b extension of the group I intron of Tetrahymena thermophila and the L9 GUGA terminal loop of the td intron of bacteriophage T4 (ref. 4). NMR has revealed the overall structure of the tetraloop for 12-nucleotide hairpins with GCAA and GAAA loops and models have been proposed for the interaction of GNRA tetraloops with base pairs in the minor groove of A-form RNA. Here we describe the crystal structure of an intermolecular complex between a GAAA tetraloop and an RNA helix. The interactions we observe correlate with the specificity of GNRA tetraloops inferred from phylogenetic studies, suggesting that this complex is a legitimate model for intramolecular tertiary interactions mediated by GNRA tetraloops in large structured RNAs.

Crystallography↗
PubMed1994

Structural basis of the 70-kilodalton heat shock cognate protein ATP hydrolytic activity. I. Kinetic analyses of active site mutants.

The 70-kDa heat shock cognate protein is a member of a highly conserved family of molecular chaperones in which the binding and release of target polypeptides are coupled to the chaperones' ATPase activity. The ATPase activity resides in the amino-terminal 44-kDa fragment of the protein. Four acidic residues of the ATPase fragment which might participate in catalysis (Asp-10 and Asp-199, which are Mg2+ ion ligands; Glu-175 and Asp-206, which are candidates for a role as catalytic base) have been individually mutated to both the cognate amide residue (aspartate to asparagine, glutamate to glutamine) and to serine, and the effects of the mutations on the kinetics (this manuscript) and structure (Flaherty, K.M., Wilbanks, S. M., DeLuca-Flaherty, C., and McKay, D. B. (1994) J. Biol. Chem. 269, 12899-12907) have been determined. Changes at Asp-10 and Asp-199 reduced kcat to approximately 1% of wild type; changes at Glu-175 and Asp-206 reduced kcat to approximately 10% of wild type. Changes to Asp-199 and Asp-206 had little effect on Km; changes to Asp-10 and Glu-175 increased Km 10-100-fold. These data suggest that the bound magnesium ion and its local environment are crucial to catalysis; they argue against a single residue acting as the sole essential general base catalyst in the hydrolytic reaction.

Adenosine Triphosphate↗
PubMed1994

Structural basis of the 70-kilodalton heat shock cognate protein ATP hydrolytic activity. II. Structure of the active site with ADP or ATP bound to wild type and mutant ATPase fragment.

The ATPase fragment of the bovine 70-kDa heat shock cognate protein is an attractive construct in which to study its mechanism of ATP hydrolysis. The three-dimensional structure suggests several residues that might participate in the ATPase reaction. Four acidic residues (Asp-10, Glu-175, Asp-199, and Asp-206) have been individually mutated to both the cognate amine (asparagine/glutamine) and to serine, and the effects of the mutations on the kinetics of the ATPase activity (Wilbanks, S. M., DeLuca-Flaherty, C., and McKay, D. B. (1994) J. Biol. Chem. 269, 12893-12898) and the structure of the mutant ATPase fragments have been determined, typically to approximately 2.4 A resolution. Additionally, the structures of the wild type protein complexed with MgADP and Pi, MgAMPPNP (5'-adenylyl-beta, gamma-imidodiphosphate) and CaAMPPNP have been refined to 2.1, 2.4, and 2.4 A, respectively. Combined, these structures provide models for the prehydrolysis, MgATP-bound state and the post-hydrolysis, MgADP-bound state of the ATPase fragment. These models suggest a pathway for the hydrolytic reaction in which 1) the gamma phosphate of bound ATP reorients to form a beta, gamma-bidentate phosphate complex with the Mg2+ ion, allowing 2) in-line nucleophilic attack on the gamma phosphate by a H2O molecule or OH- ion, with 3) subsequent release of inorganic phosphate.

Adenosine Diphosphate↗
PubMed1994

Staurosporine-induced reduction of secretory function in cultured bovine adrenal chromaffin cells.

Staurosporine, a potent inhibitor of protein kinases, has been used to investigate the involvement of protein kinases in cellular processes such as secretory function and differentiation. We have been examining the effects of staurosporine on secretory function under the same conditions it induces dramatic changes in cell morphology in cultured bovine adrenal chromaffin cells. Our results show that treatment with 100 nM staurosporine reduces catecholamine release stimulated by 56 mM KCl, 10 microM nicotine, and 2 mM BaCl2 in a time-dependent manner (t1/2s, 42, 32, and 31 min, respectively). However, we demonstrate that the time-dependent effects on secretory function are not the direct result of staurosporine-induced changes in cell morphology. The effects of staurosporine on secretion stimulated by KCl, nicotine, and BaCl2 are concentration-dependent (IC50s, 6.3, 29.3, and 34.9 nM, respectively). Staurosporine pretreatment does not inhibit activated 45Ca2+ influx, but does reduce catecholamine release stimulated directly by Ca2+ from permeabilized cells. Furthermore, staurosporine also inhibits basal release with time- and concentration-dependencies (IC50, 9.3 nM and t1/2, 21 min) similar to those found for stimulated release. These results suggest that prolonged staurosporine pretreatment may result in the depletion/alteration of a component essential for the more terminal steps of the secretory process.

Adrenal Medulla↗
PubMed1994

Comparison of the effects of antimitotic drugs on alpha-tubulin mRNA, microtubules and nicotinic receptor-mediated catecholamine secretion in adrenal chromaffin cells in culture.

1. Four hour treatments of adrenal chromaffin cells with colchicine (10, 100 microM and 1 mM), tubulozole (10 microM) or podophyllotoxin (100 microM) decreases alpha-tubulin mRNA content. Vinblastine (10 microM) and taxol (10 microM), however, do not decrease alpha-tubulin mRNA content. 2. Immunocytochemical techniques demonstrate that 4 hr treatments with all of the antimitotic drugs (colchicine, podophyllotoxin, taxol, tubulozole and vinblastine) produce abnormal microtubule arrays. 3. The effects of 4 hr treatments with the antimitotic drugs on adrenal catecholamine secretion are all qualitatively the same; each drug selectively inhibits adrenal nicotinic acetylcholine receptor-stimulated catecholamine release, while having no inhibitory actions on release stimulated through noncholinergic mechanisms. 4. These studies demonstrate that autoregulation of tubulin synthesis occurs in cultured adrenal chromaffin cells. 5. All of the antimitotic drugs selectively inhibit nicotinic receptor-mediated adrenal catecholamine release under treatment conditions that affect adrenal microtubules. These results support the possibility that the actions of the antimitotic drugs on adrenal nicotinic receptors may involve microtubules.

Adrenal Medulla↗
PubMed1993

Definition and spatial location of mouse interleukin-2 residues that interact with its heterotrimeric receptor.

The high affinity receptor for interleukin-2 (IL-2) contains three subunits called IL-2R alpha, beta and gamma. A biological and receptor binding analysis based on 1393 different mutant mouse IL-2 (mIL-2) proteins was used to define the function of each of the 149 residues. By this genetic analysis, 44 residues were assigned important functions, 21 of which were structural. The remaining 23 residues consisted of 19 residues, from three separate regions, that were important for IL-2R alpha interaction; three residues, from two separate regions, that were important for IL-2R beta interaction; and a single residue important for IL-2R gamma interaction. We built a model mIL-2 structure based on the homologous human IL-2 (hIL-2) crystal structure. The roles of the 21 residues presumed to be important for structure were consistent with the model. Despite discontinuity in the primary sequence, the residues specific for each IL-2R subunit interaction were clustered and located to three disparate regions of the tertiary mIL-2 structure. The relative spatial locations of these three surfaces are different from the two receptor binding sites known for the structurally related human growth hormone and the significance of this observation is discussed.

Amino Acid Sequence↗
PubMed1993

Three-dimensional structure of recoverin, a calcium sensor in vision.

Recoverin, a recently discovered member of the EF hand superfamily, serves as a calcium sensor in vision. We report here the crystal structure of recombinant unmyristoylated recoverin at 1.9 A resolution. The four EF hands of the protein are arranged in a compact array that contrasts with the dumbbell shape of calmodulin and troponin C. A calcium ion is bound to EF hand 3, while EF hand 2 can bind samarium but not calcium in this crystal form. The other two EF hands have novel structural features that prevent or impair calcium binding. A concave hydrophobic surface formed by EF hands 1 and 2 may participate in the read out of calcium signals by recoverin and its homologs.

Amino Acid Sequence↗
PubMed1993

Threonine 204 of the chaperone protein Hsc70 influences the structure of the active site, but is not essential for ATP hydrolysis.

The chaperone protein Hsc70 is an ATPase of unknown mechanism, although the crystal structure of the 44-kDa ATPase domain has been solved. This structure shows that the hydroxyl of threonine 204 is located close to the gamma-phosphate of ATP, in a position where it might be an intermediate phosphate acceptor in the hydrolysis reaction. We made two point mutations at residue 204 of Hsc70, threonine to valine (T204V) and threonine to glutamic acid (T204E). The wild-type ATPase domain had a Km for ATP of approximately 1 microM; the mutants had Km values of approximately 90 microM. The kcat values for the mutant proteins were also increased. After crystallization, the structures of the T204V and T204E proteins were solved and refined with data to 2.3- and 2.4-A resolution, respectively. The overall tertiary structure of the mutants showed little change from the wild type; however, significant changes were observed in the active site. Analysis of the structures suggested possible reasons for the changes in kinetic constants. Threonine 204 does not seem to be an obligatory intermediate phosphate acceptor in the hydrolysis reaction since the mutants retained appreciable ATPase activity.

Adenosine Triphosphate↗
PubMed1993

Crystals of a hammerhead ribozyme.

Hammerhead ribozyme-inhibitor complexes consisting of an RNA "enzyme" strand and a DNA "inhibitor" strand have been crystallized in four different crystal forms. The crystal form that is most attractive for structure determination diffracts to approximately 3.2 A resolution; it is trigonal, space group P3(1)21 or enantiomorph, a = 92.5 A, c = 185.0 A.

Base Composition↗
PubMed1993

Three-dimensional structure of the alkaline protease of Pseudomonas aeruginosa: a two-domain protein with a calcium binding parallel beta roll motif.

The three-dimensional structure of the alkaline protease of Pseudomonas aeruginosa, a zinc metalloprotease, has been solved to a resolution of 1.64 A by multiple isomorphous replacement and non-crystallographic symmetry averaging between different crystal forms. The molecule is elongated with overall dimensions of 90 x 35 x 25 A; it has two distinct structural domains. The N-terminal domain is the proteolytic domain; it has an overall tertiary fold and active site zinc ligation similar to that of astacin, a metalloprotease isolated from a European freshwater crayfish. The C-terminal domain consists of a 21-strand beta sandwich. Within this domain is a novel 'parallel beta roll' structure in which successive beta strands are wound in a right-handed spiral, and in which Ca2+ ions are bound within the turns between strands by a repeated GGXGXD sequence motif, a motif that is found in a diverse group of proteins secreted by Gram-negative bacteria.

Amino Acid Sequence↗
PubMed1993

Nicotinic and nonnicotinic receptor-mediated actions of vinblastine.

Vinblastine has been demonstrated to inhibit nicotinic acetylcholine receptor (nAChR) activity in adrenal chromaffin cells and superior cervical ganglia and to alter agonist binding affinity to nAChR of the electric organ of Torpedo californica. In cultured chromaffin cells, vinblastine (IC50, 8.9 microM) is significantly more potent than hexamethonium (IC50, 16 microM) and decamethonium (IC50, 18 microM) and significantly less potent than d-tubocurarine (IC50, 2 microM), pentolinium (IC50, 0.6 microM), and mecamylamine (IC50, 0.1 microM) in inhibiting nAChR-stimulated catecholamine release. These results demonstrate that vinblastine has moderately potent anti-nAChR activity on adrenal nAChR. On the other hand, vinblastine does not interfere with phrenic nerve stimulation of rat diaphragm musculature in concentrations up to 200 microM. However, in relatively high doses, vinblastine (10-200 microM) produces an increase in baseline tension of diaphragm muscle. This effect is concentration related (EC50, approximately 88 microM), reversible, and independent of phrenic nerve stimulation. The elevation in baseline tension is unaffected by nAChR blockade via d-tubocurarine, but is dependent upon the presence of extracellular calcium. The results suggest that vinblastine's antinicotinic actions are selective for neuronal-type nAChR and do not extend to nAChR of mammalian skeletal muscle. High concentrations of vinblastine appear to elicit contractures of skeletal muscle that are unrelated to nAChR.

Adrenal Glands↗
PubMed1992

Structural comparison suggests that thermolysin and related neutral proteases undergo hinge-bending motion during catalysis.

Crystal structures are known for three members of the bacterial neutral protease family: thermolysin from Bacillus thermoproteolyticus (TLN), the neutral protease from Bacillus cereus (NEU), and the elastase of Pseudomonas aeruginosa (PAE), both in free and ligand-bound forms. Each enzyme consists of an N-terminal and C-terminal domain with the active site formed at the junction of the two domains. Comparison of the different molecules reveals that the structure within each domain is well conserved, but there are substantial hinge-bending displacements (up to 16 degrees) of one domain relative to the other. These domain motions can be correlated with the presence or absence of bound inhibitor, as was previously observed in the specific example of PAE [Thayer, M.M., Flaherty, K.M., & McKay, D.B. (1991) J. Biol. Chem. 266, 2864-2871]. The binding of inhibitor appears to be associated with a reduction of the domain hinge-bending angle by 6-14 degrees and a closure of the "jaws" of the active site cleft by about 2 A. Crystallographic refinement of the structure of thermolysin suggests that electron density seen in the active site of the enzyme in the original structure determination probably corresponds to a bound dipeptide. Thus, the crystal structure appears to correspond to an enzyme-inhibitor or enzyme-product complex, rather than the free enzyme, as has previously been assumed.

Amino Acid Sequence↗