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Biomedical subjects

M D Davison

Publications and source records attributed to M D Davison.

At least 19 recordsLinked to original sources
PubMed2001

Chromatographic separations as a prelude to two-dimensional electrophoresis in proteomics analysis.

Current methods of proteome analysis rely almost solely on two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) followed by the excision of individual spots and protein identification using mass spectrometry (MS) and database searching. 2-D PAGE is denaturing in both dimensions and, thus, cannot indicate functional associations between individual proteins. Moreover, less abundant proteins are difficult to identify. To simplify the proteome, and explore functional associations, nondenaturing anion exchange column chromatography was used to separate a soluble protein extract from Escherichia coli. Successive fractions were then analysed using 2-D PAGE and selected spots from both the gels for the start material and the fractionated material were quantified and identified by peptide mass fingerprinting using a MALDI-TOF mass spectrometer. Enrichments of up to 13-fold were attained for individual protein spots and peptide mass fingerprints were of significantly higher quality after chromatographic separation. The marked anomalies between predicted p/and column elution position contrasted with the almost perfect correlation with migration distance on isoelectric focusing (IEF) and were explored further for basic proteins.

Chromatography, Ion Exchange↗
PubMed2000

The regulation of AMP-activated protein kinase by phosphorylation.

The AMP-activated protein kinase (AMPK) cascade is activated by an increase in the AMP/ATP ratio within the cell. AMPK is regulated allosterically by AMP and by reversible phosphorylation. Threonine-172 within the catalytic subunit (alpha) of AMPK (Thr(172)) was identified as the major site phosphorylated by the AMP-activated protein kinase kinase (AMPKK) in vitro. We have used site-directed mutagenesis to study the role of phosphorylation of Thr(172) on AMPK activity. Mutation of Thr(172) to an aspartic acid residue (T172D) in either alpha1 or alpha2 resulted in a kinase complex with approx. 50% the activity of the corresponding wild-type complex. The activity of wild-type AMPK decreased by greater than 90% following treatment with protein phosphatases, whereas the activity of the T172D mutant complex fell by only 10-15%. Mutation of Thr(172) to an alanine residue (T172A) almost completely abolished kinase activity. These results indicate that phosphorylation of Thr(172) accounts for most of the activation by AMPKK, but that other sites are involved. In support of this we have shown that AMPKK phosphorylates at least two other sites on the alpha subunit and one site on the beta subunit. Furthermore, we provide evidence that phosphorylation of Thr(172) may be involved in the sensitivity of the AMPK complex to AMP.

AMP-Activated Protein Kinase Kinases↗
PubMed1998

Characterization of the protein encoded by gene UL49A of herpes simplex virus type 1.

Herpes simplex virus type 1 (HSV-1) gene UL49A potentially encodes a primary translation product of 91 residues with a signal sequence at the N terminus and a membrane anchor domain near the C terminus. Mutants were generated in this gene and utilized to characterize the encoded protein on SDS-PAGE as a 6.7 kDa species which fractionated with infected cell membranes, was a relatively abundant virion component, and was not detectably O-glycosylated. The protein was identified by microsequencing as a 68 residue polypeptide formed by removal of 23 residues from the N terminus of the primary translation product. Cleavage of the signal sequence was also demonstrated by in vitro transcription and translation in the presence of microsomal membranes. The UL49A protein was efficiently solubilized along with envelope proteins by treatment of virions with a non-ionic detergent but only in the presence of a reducing agent, suggesting that it may be an envelope protein that is disulphide-linked to the tegument. It is apparent from mutational analysis that the 10 amino acid residues at the C terminus are not essential for synthesis of the protein, signal sequence cleavage, targeting to membranes and virions, linkage to the tegument and growth of virus in cell culture.

Amino Acid Sequence↗
PubMed1996

Characterization of AMP-activated protein kinase beta and gamma subunits. Assembly of the heterotrimeric complex in vitro.

There is growing evidence that mammalian AMP-activated protein kinase (AMPK) plays a role in protecting cells from stresses that cause ATP depletion by switching off ATP-consuming biosynthetic pathways. The active form of AMPK from rat liver exists as a heterotrimeric complex and we have previously shown that the catalytic subunit is structurally and functionally related to the SNF1 protein kinase from Saccharomyces cerevisiae. Here we describe the isolation and characterization of the two other polypeptides, termed AMPKbeta and AMPKgamma, that together with the catalytic subunit (AMPKalpha) form the active kinase complex in mammalian liver. Sequence analysis of cDNA clones encoding these subunits reveals that they are related to yeast proteins that interact with SNF1, providing further evidence that the regulation and function of AMPK and SNF1 have been conserved throughout evolution. The amino acid sequence of the beta subunit is most closely related to SIP2 (35% identity), while the amino acid sequence of the gamma subunit is 35% identical with SNF4. We show that both AMPKbeta and AMPKgamma mRNA and protein are expressed widely in rat tissues. We show that AMPKbeta interacts with both AMPKalpha and AMPKgamma in vitro, whereas AMPKalpha does not interact with AMPKgamma under the same conditions. These results suggest that AMPKbeta mediates the association of the heterotrimeric AMPK complex in vitro, and will facilitate future studies aimed at investigating the regulation of AMPK in vivo.

AMP-Activated Protein Kinases↗
PubMed1995

Identification of structural proteins of channel catfish virus by mass spectrometry.

Emerging methods that couple mass spectrometry of fragments generated from isolated proteins with database searching offer a powerful means of identifying proteins and the genes that encode them. We have applied this technology to a herpesvirus, channel catfish virus, and have identified 12 genes, 11 viral and 1 cellular, that encode 16 principal structural proteins. These proteins include three components of the mature capsid and a potential scaffolding protein present in immature capsids, three protein kinases, a C3HC4 zinc-binding protein and cellular actin located in the tegument, and a multiply hydrophobic protein associated with the envelope.

Amino Acid Sequence↗
PubMed1995

The cell-coded polypeptide U90 increased by herpes simplex virus type 2 infection induces Fos and DNA synthesis.

Lytic infection with herpes simplex virus type 2 (HSV-2) induces synthesis of a cell-coded protein of molecular mass 90 kDa, termed U90. U90, whose expression is specific in tumour cells is, in addition, excreted from he cell. To determine if the function of U90 could be advantageous for the virus the protein was purified from the medium of HSV-2 infected cells, shown to be similar to the internalized form and its effect on cell growth studied. Addition of U90 to quiescent fibroblast cells stimulated teh expression of Fos, the product of the cellular transcription factor c-fos and increased the incorporation of [3H]thymidine into DNA, factors which may facilitate HSV-2 replication. However, U90 might also induce abnormal cells such as those in precancerous lesions to proliferate.

Animals↗
PubMed1994

Mammalian AMP-activated protein kinase is homologous to yeast and plant protein kinases involved in the regulation of carbon metabolism.

In mammals, an AMP-activated protein kinase (AMPK) phosphorylates both acetyl-CoA carboxylase and 3-hydroxy-3-methylglutaryl-CoA reductase in vitro and has been proposed to play a major role in the regulation of lipid metabolism in vivo. We report here the primary sequence of rat AMPK and show that antibodies raised against synthetic peptides based on the deduced sequence of AMPK immunoprecipitate AMPK activity from rat liver extracts. AMPK has a remarkable degree of sequence identity to the proteins encoded by the yeast SNF1 gene and the plant RKIN1 gene. SNF1 protein kinase activity is essential for release of genes from glucose repression in Saccharomyces cerevisiae. Expression of cRKIN1 in yeast snf1 mutants restores SNF1 function. These results indicate that AMPK, SNF1, and RKIN1 form part of a family of protein kinases that have been highly conserved throughout evolution. Our results suggest that AMPK may be involved in the regulation of a wide range of metabolic pathways.

AMP-Activated Protein Kinases↗
PubMed1994

Isolation of a cDNA encoding 5T4 oncofetal trophoblast glycoprotein. An antigen associated with metastasis contains leucine-rich repeats.

The monoclonal antibody 5T4 defines a human oncotrophoblast antigen expressed by a variety of carcinomas but with a restricted pattern of expression in normal adult tissues. The 5T4 antigen has been isolated from term placenta as a 72-kDa glycoprotein consisting of a 42-kDa core protein with extensive N-linked glycosylation. A cDNA has been isolated from a human placental library using pools of oligonucleotides based on amino acid sequence obtained from purified 5T4 molecules. The predicted open reading frame encodes a protein of 420 amino acids with a molecular mass of 46 kDa and 8 potential N-glycosylation sites. There are N- and C-terminal hydrophobic segments corresponding to putative signal and membrane anchorage sequences, respectively. Northern analysis has demonstrated a major 2.5-kilobase mRNA present in cell lines serologically reactive with the monoclonal antibody 5T4. Comparison of the 5T4 protein sequence with current sequence data bases has identified the presence of leucine-rich repeats, which are found in a variety of proteins from yeast, insects, and mammals. The 5T4 antigen expression is strongly associated with metastasis in colorectal and gastric cancer, and, hence, the possible functions of the gene product and its relationship to tumor growth and progression are discussed.

Amino Acid Sequence↗
PubMed1993

Phosphorylation of recombinant tau by cAMP-dependent protein kinase. Identification of phosphorylation sites and effect on microtubule assembly.

Tau protein is an integral component of paired helical filaments, a pathological feature of Alzheimer's disease. tau extracted from these filaments displays decreased electrophoretic mobility due to aberrant phosphorylation. Here we show that recombinant human tau can be phosphorylated by cAMP-dependent protein kinase resulting in decreased electrophoretic mobility. Phosphorylation of tau by cAMP-dependent protein kinase caused a 92% decrease in the maximum rate of tau-induced microtubule assembly. The sites of phosphorylation were identified by digesting phosphorylated tau with proteases, separating the peptides by reversed-phase HPLC, and analyzing the isolated peptides by liquid-secondary ion mass spectrometry and solid-phase N-terminal sequencing. Five phosphorylation sites were identified, two of which were located within microtubule binding domains. One site was previously shown to be the sole phosphorylation site for CaM kinase II; phosphorylation at this site by CaM kinase II was sufficient to cause decreased electrophoretic mobility (Steiner, B., Mandelkow, E. M., Biernat, J., Gustke, N., Meyer, H. E., Schmidt, B., Mieskes, G., Soling, H. D., Drechsel, D., Kirschner, M. W., Goedert, M., and Mandelkow, E. (1990) EMBO J. 9, 3539-3544). Thus two different second messenger-dependent protein kinases can phosphorylate tau at the same site and induce a shift in tau mobility like that seen in Alzheimer's disease.

Adenosine Triphosphate↗
PubMed1993

Antileukoprotease in psoriatic scales.

Antileukoprotease is known to be an antiproteolytic compound of mucous secretions in humans. While searching for peptide-like inhibitors of neutrophil-derived serine proteases in horny layers of human skin, we isolated a potent inhibitor of human leukocyte elastase (EC 3.4.21.37) and cathepsin G (EC 3.4.21.20) from psoriatic scales. This inhibitor showed inhibitory constants for human leukocyte elastase of approximately 0.5-2 x 10(-10) M and for cathepsin G of 2-4 x 10(-9) N. The N-terminal amino acid sequence of the purified peptide matched the sequence of antileukoprotease and both peptides showed the same M(r) on SDS-PAGE. Therefore, antileukoprotease may not only regulate serine protease activities in mucous secretions, but also in skin.

Amino Acid Sequence↗
PubMed1993

Isolation and pharmacological characterization of omega-grammotoxin SIA, a novel peptide inhibitor of neuronal voltage-sensitive calcium channel responses.

omega-Grammotoxin SIA, a peptidergic blocker of voltage-sensitive calcium channel (VSCC) responses, was purified from Grammostola spatulata (tarantula spider) venom by reverse phase high performance liquid chromatography. Protease-sensitive biological activity was monitored by determining the inhibition of K(+)-stimulated influx of 45Ca2+ into rat brain synaptosomes. Electrospray mass spectrometry indicated an average molecular mass of 4109.2 Da for the native peptide. Chemical reduction of omega-grammotoxin SIA indicated the presence of three disulfide bridges. Primary sequence data confirmed the existence of six cysteine residues and 36 residues in total, with an average theoretical molecular mass of 4109.7 Da for the amidated carboxyl-terminal species. The biological profile of omega-grammotoxin SIA indicated virtually complete blockade of presynaptic vertebrate N-type as well as P-type VSCC responses. Specifically, omega-grammotoxin SIA caused a concentration-dependent and virtually complete inhibition of K(+)-evoked influx of 45Ca2+ into either rat or chick brain synaptosomes. Similar inhibition profiles were generated for the inhibition of release of either D-[3H]aspartate or [3H]norepinephrine from rat hippocampal or [3H]norepinephrine from chick cortical brain slice preparations evoked by K+ depolarization. As reported earlier, omega-grammotoxin SIA did not inhibit 125I-omega-conotoxin GVIA, [3H]PN 200-110, or [3H]desmethoxyverapamil binding to neuronal membrane fragments. To our knowledge, omega-grammotoxin SIA is the first ligand identified to block putative N-channel function without displacement of 125I-omega-conotoxin GVIA. omega-Grammotoxin SIA thus represents a novel vertebrate VSCC antagonist that inhibits neuronal N- and P-type VSCC responses.

Amino Acid Sequence↗
PubMed1992

Identification of genes encoding two capsid proteins (VP24 and VP26) of herpes simplex virus type 1.

The capsid of herpes simplex virus type 1 is composed of seven proteins. VP5, VP19C, VP22a and VP23 have been shown previously to be the products of genes UL19, UL38, UL26.5 and UL18, respectively. The genes encoding VP21, VP24 and VP26 have not been identified to date. We have determined amino acid sequences of fragments of isolated capsid proteins generated by partial cleavage with CNBr. The results confirm the gene assignments for VP5, VP19C and VP23. They also show that VP26 is the product of gene UL35 and that VP24 contains the protease domain present in the N-terminal portion of the UL26-encoded protein. VP21 was not investigated, but we suggest that it is the C-terminal portion of the UL26-encoded protein remaining after release of VP24 and that it thus corresponds to a form of VP22a extended at the N terminus.

Amino Acid Sequence↗
PubMed1990

N-terminal sequence similarities between components of the multicatalytic proteinase complex.

The multicatalytic proteinase complex is a high molecular weight nonlysosomal proteinase which is composed of many different types of subunit. As part of a study of the possible relationships between subunits, polypeptides derived from the multicatalytic proteinase from rat liver have been subjected to N-terminal amino acid sequence analysis. Although several of the subunits are blocked at their N-termini, sequences have been obtained for 7 of the polypeptides. Each of the 7 sequences is unique but they show considerable sequence similarity, suggesting that the proteins are encoded by members of the same gene family.

Amino Acid Sequence↗
PubMed1990

Identification of the genes encoding two capsid proteins of herpes simplex virus type 1 by direct amino acid sequencing.

Amino-terminal amino acid sequencing was carried out on proteins purified from herpes simplex virus type 1 capsids. The sequences of two capsid proteins (VP19C and VP23) showed them to be encoded by genes UL38 and UL18, respectively. The product of UL38 has been shown to be essential for capsid assembly, but no role has previously been assigned to the product of UL18.

Amino Acid Sequence↗
PubMed1989

Primary sequence and domain structure of chicken vinculin.

We have determined the complete sequence of chick vinculin from two overlapping cDNA clones. The vinculin mRNA consists of 262 bp of 5' untranslated sequence, an open reading frame of 3195 bp (excluding the initiation codon) and a long 3' untranslated sequence (greater than 2 kb). Chick vinculin contains 1066 amino acid residues, and has a deduced molecular mass of 116,933 Da. Analysis of the domain structure of vinculin shows that the molecule can be cleaved by V8 proteinase into a 90 kDa globular head and a 32 kDa tail region, the latter of which could further be cleaved into a 27 kDa polypeptide. The 90 kDa globular head contains the N-terminus of vinculin, three 112-residue repeats (residues 259-589), and extends to approximately residue 850. Gel overlay experiments show that it also contains a binding site for the cytoskeletal protein talin. The talin-binding domain was further localized to the N-terminal 398 amino acid residues of the protein by expression in vitro of this region from a vinculin cDNA cloned into the Bluescript SK+ vector. The head and tail domains are apparently separated by a proline-rich region that contains V8-proteinase-cleavage sites and a candidate tyrosine (822)-phosphorylation site. Secondary-structure prediction suggests that the head and tail domains contain alpha-helical regions separated by short stretches of turn/coil. Comparison of the chick with a partial human sequence reveals that vinculin is a highly conserved protein. In chickens Southern-blot analysis is consistent with a single vinculin gene, and it is therefore likely that vinculin, and its higher-molecular-mass isoform termed metavinculin, arise through alternative splicing.

Amino Acid Sequence↗
PubMed1989

Comparison of the binding characteristics of two different preparations of tetanus toxin to rat brain membranes.

Two different preparations of tetanus toxin (HTT and WTT) were iodinated, and their binding to rat brain membranes characterized. Under optimal binding conditions (25 mM Tris-acetate, pH 6.0), both preparations bound to a large number of high affinity sites, thought to be gangliosides. Binding constants were identical. However, in a physiological buffer (Krebs-Ringer, pH 7.4) binding of the two toxin preparations showed a number of differences. Under these conditions we have previously shown that HTT binding is markedly reduced, and that there are two classes of sites, a small number of heat-, sialidase- and protease-sensitive high affinity sites, and a larger number of sialidase-sensitive, heat- and protease-resistant lower affinity sites, probably gangliosides (PIERCE et al. (1986) Biochem. J. 236, 845-852). Although WTT bound to these same two sites, it displayed a higher affinity for the protease-resistant site than did HTT. WTT also bound to free or immobilized trisialoganglioside with higher affinity than HTT, consistent with the view that the protease-resistant site represents binding to ganglioside. In contrast, both toxin preparations bound to the protease-sensitive site with similar affinities. These observations may explain the four to five-fold higher levels of WTT binding to brain membranes, and the fact that a smaller percentage of total WTT binding is protease sensitive. Despite their different ganglioside-binding properties, both toxin preparations showed comparable neurotoxic activities, and appeared identical on SDS gels.

Animals↗
PubMed1989

Structural analysis of homologous repeated domains in alpha-actinin and spectrin.

The amino acid sequences of chick and slime mould alpha-actinin each contain four repeats of approximately 122 residues. These repeats are homologous to the 18-22 repeats, each of approximately 106 residues, found in the alpha and beta subunits of spectrin and fodrin, and to the multiple repeats of approximately 110 residues found in the Duchenne muscular dystrophy protein (dystrophin). The repeats correspond to the elongated rod-like portion of these molecules. We present a multiple sequence alignment of 21 repeats from this superfamily (8 alpha-actinin and 13 spectrin/fodrin), based on optimal pairwise alignments, from which a characteristic consensus pattern of amino acid types is deduced. Trp 46 is invariant in all but one repeat, and physicochemical classes of amino acids are conserved at 25 other positions. Secondary structure prediction on both the alpha-actinin and spectrin repeats taken together with the distribution of proline residues in the sequences, strongly suggest that each repeated domain consists of a four-helix structure. Our predictions differ significantly from previous three-helix models based on analyses of fewer sequences. To determine possible interdomain regions, sites of limited proteolysis of the native chick alpha-actinin dimer were determined and located in the amino acid sequence. The majority of these sites were in corresponding positions in different repeats within a segment predicted as a long helix. We propose a model, consistent with the overall dimensions of the rod-like portions of the molecules, in which these long, probably interrupted helices, link adjacent domains.

Actinin↗