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Biomedical subjects

B M Martin

Publications and source records attributed to B M Martin.

At least 145 records · Page 8Linked to original sources
PubMed1989

Peptide: N-glycosidase F: studies on the glycoprotein aminoglycan amidase from Flavobacterium meningosepticum.

Peptide: N-glycosidase from Flavobacterium meningosepticum was isolated in a homogeneous state and its physico-chemical characterization was accomplished. The reliability of the previously recorded assay procedures was assessed. Using an octaglycopeptide derived from ovomucoid a rapid and sensitive FPLC method was developed for the assay of enzymatic activity. Peptide: N-glycosidase was found to effect deglycosylation of glycoproteins bearing complex and/or multiantennary glycans even in their native state. In contrast, glycoproteins with high mannose and/or hybrid carbohydrates required denaturation to become susceptible to deglycosylation by the enzyme.

Amidohydrolases↗
PubMed1989

Human anti-endoplasmic reticulum antibodies in sera of patients with halothane-induced hepatitis are directed against a trifluoroacetylated carboxylesterase.

Previous studies have demonstrated that patients with halothane-induced hepatitis have serum antibodies that are directed against novel liver microsomal neoantigens and have suggested that these neoantigens may play an immunopathological role in development of the patients' liver damage. These investigations have further revealed that the antibodies are directed against distinct polypeptide fractions (100 kDa, 76 kDa, 59 kDa, 57 kDa, 54 kDa) that have been covalently modified by the reactive trifluoroacetyl halide metabolite of halothane. In this paper, the trifluoroacetylated (TFA) 59-kDa neoantigen (59-kDa-TFA) recognized by the patients' antibodies was isolated from liver microsomes of halothane-treated rats by chromatography on an immunoaffinity column of anti-TFA IgG. Antibodies were raised against the 59-kDa-TFA protein and were used to purify the native protein from liver microsomes of untreated rats. Based upon its apparent monomeric molecular mass, NH2-terminal amino acid sequence, catalytic activity, and other physical properties, the protein has been identified as a previously characterized microsomal carboxylesterase (EC 3.1.1.1). A similar strategy may be used to purify and characterized neoantigens associated with other drug toxicities that are believed to have an immunopathological basis.

Animals↗
PubMed1989

Isolation, partial amino acid sequence, and immunohistochemical localization of a brain-specific calcium-binding protein.

A calcium-binding protein (protein 10) having a molecular mass of 29 kDa and an isoelectric point of 5.3 was purified from guinea pig brain. The amino acid sequence of fragments from proteolytic digestion of protein 10 revealed an 86% sequence identity with a calcium-binding protein (calretinin) found in chicken retina. Polyclonal antibodies against protein 10 revealed a specific distribution of this protein within sensory neurons of auditory, visual, olfactory, nociceptive, and gustatory systems as well as other discrete neuronal circuits in rat and guinea pig brain, whereas no specific label was observed in any of several peripheral tissues examined.

Amino Acid Sequence↗
PubMed1989

Saposin A: second cerebrosidase activator protein.

Saposin A, a heat-stable 16-kDa glycoprotein, was isolated from Gaucher disease spleen and purified to homogeneity. Chemical sequencing from its amino terminus and of peptides obtained by digestion with protease from Staphylococcus aureus strain V-8 demonstrated that saposin A is derived from proteolytic processing of domain 1 of its precursor protein, prosaposin. Processing of prosaposin (70 kDa) also generates three other previously reported saposin proteins, B, C, and D, from its second, third, and fourth domains. Similar to saposin C, saposin A stimulates the hydrolysis of 4-methylumbelliferyl beta-glucoside and glucocerebroside by beta-glucosylceramidase and of galactocerebroside by beta-galactosylceramidase, mainly by increasing the maximal velocity of both reactions. Saposin A is as active as saposin C in these reactions. Saposin A has no significant effect on other sphingolipid and 4-methylumbelliferyl glycoside hydrolases tested. Saposin A has two potential glycosylation sites that appear to be glycosylated. After deglycosylation, saposin A had a subunit molecular mass of 10 kDa and was as active as native saposin A. However, reduction and alkylation abolished the activation. A three-dimensional model comparing saposins A and C reveals significant sequence homology between them, especially preservation of conserved acidic and basic residues in their middle regions. Each appears to possess a conformationally rigid hydrophobic pocket stabilized by three internal disulfide bridges, with amphipathic helical regions interrupted by helix breakers.

Amino Acid Sequence↗
PubMed1988

Rat liver carboxylesterase: cDNA cloning, sequencing, and evidence for a multigene family.

A cDNA clone was isolated from a rat liver lambda gt11 expression library by screening with polyclonal antibodies raised against a rat liver microsomal carboxylesterase. This clone of 1.8 kb contained an open reading frame encoding a mature protein of 531 amino acids with a predicted molecular weight of 58,084. The 5' portion of the clone coded for 9 amino acids of a putative signal peptide. The 3' end of the clone included an untranslated region and a poly (A) tail. Carboxylesterase active site regions, five potential N-linked glycosylation sites, and 2 postulated cystine disulfide bridges were found in the cDNA-deduced amino acid sequence. Sequences obtained from tryptic peptides and the NH2-terminus of the purified native carboxylesterase were aligned with the deduced amino acid sequence, and the overall identity was 84%. Southern blot analysis suggested the presence of multiple genes. Thus it is concluded that we have cloned a rat liver carboxylesterase, and that this enzyme is a member of a multigene family.

Amino Acid Sequence↗
PubMed1988

Saposin D: a sphingomyelinase activator.

Saposin D, a newly discovered heat-stable, 10 kDa glycoprotein, was isolated from Gaucher spleen and purified to homogeneity. Chemical sequencing from its amino terminus demonstrated colinearity between its amino acid sequence and the deduced amino acid sequence of the fourth domain of prosaposin, the precursor of saposin proteins. Saposin D specifically stimulates acid sphingomyelinase but has no significant effect on the other hydrolases tested.

Enzyme Activation↗
PubMed1988

The isolation of angiotensin-converting enzyme cDNA.

Angiotensin-converting enzyme (ACE) is an Zn(II)-containing dipeptidyl carboxypeptidase that converts angiotensin I to the potent vasoconstrictor, angiotensin II. Using oligonucleotide probes based on the amino acid sequence of mouse kidney ACE, cDNA encoding this protein has been isolated. One cDNA, ACE.31, encodes the N-terminal 332 amino acids of mouse ACE, a portion of the protein containing a putative 34-amino acid leader sequence and the N terminus of the mature protein. Northern analyses with cloned ACE cDNA revealed that both mouse kidney and lung express two ACE mRNAs, one of 4900 and another of 4150 bases. Southern analysis suggests that cDNA ACE.31 is the product of a single gene, and thus these data add evidence to the hypothesis that the converting enzymes produced by epithelial and endothelial cells are identical.

Amino Acid Sequence↗
PubMed1988

Expression of human tyrosine hydroxylase cDNA in invertebrate cells using a baculovirus vector.

A human cDNA containing the complete coding sequence for a human tyrosine hydroxylase (EC 1.14.16.2, form 2) was introduced into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) downstream to the polyhedrin promoter. Infection of Spodoptera frugiperda cells (SF9) with recombinant virus resulted in the expression of human tyrosine hydroxylase in these invertebrate cells. Characterization of tyrosine hydroxylase activity in infected SF9 cells demonstrated both substrate and cofactor kinetics that were characteristic of those previously reported for the native human enzyme. Both 3-iodotyrosine and alpha-methyl-p-tyrosine competitively inhibited the recombinantly produced tyrosine hydroxylase with Ki values of 1.2 and 16 microM, respectively, similar to those previously reported for the rat and human enzymes. Western blot analysis of extracts of SF9 cells infected with the recombinant baculovirus containing human tyrosine hydroxylase cDNA revealed a major immunoreactive band with an apparent Mr of 60 kDa, identical to the size of the immunoreactive protein from rat adrenal and caudate nucleus. The use of the baculovirus expression system to produce abundant quantities of each of the multiple forms of active human tyrosine hydroxylase in eukaryotic cells should facilitate structural analysis and help clarify the physiological significance of each of the isoenzymes.

Base Sequence↗
PubMed1988

Charybdotoxin and noxiustoxin, two homologous peptide inhibitors of the K+ (Ca2+) channel.

We show that noxiustoxin (NTX), like charybdotoxin (CTX) described by others, affects Ca2+-activated K+ channels of skeletal muscle (K+(Ca2+) channels). Chemical characterization of CTX shows that it is similar to NTX. Although the amino-terminal amino acid of CTX is not readily available, the molecule was partially sequenced after CNBr cleavage. A decapeptide corresponding to the C-terminal region of NTX shows 60% homology to that of CTX, maintaining the cysteine residues at the same positions. While CTX blocks the K+ (Ca2+) channels with a Kd of 1-3 nM, for NTX it is approx. 450 nM. Both peptides can interact simultaneously with the same channel. NTX and CTX promise to be good tools for channel isolation.

Animals↗
PubMed1988

beta-VLDL-induced alterations in growth potentiating activity produced by mononuclear phagocytes.

This report describes the enhancement of growth potentiating activity produced by mononuclear phagocytes that were incubated with beta-migrating very low density lipoproteins (beta-VLDL). Conditioned media harvested from cultured human peripheral blood monocytes incubated in the presence or absence of the lipoprotein were evaluated for their ability to stimulate DNA synthesis ([3H]thymidine incorporation) of sparsely seeded quiescent BHK-21 (BHK) cells as well as neonatal rat aortic smooth muscle cells (NRSMC). Conditioned media from monocytes incubated in the presence of beta-VLDL enhanced [3H]thymidine incorporation into DNA of both BHK and NRSMC, when compared to conditioned media harvested from monocytes incubated in the absence of beta-VLDL. Studying NRSMC, this effect was evident using media collected from monocytes incubated with lipoprotein for 2 days; however, a longer incubation of monocytes plus lipoprotein was necessary to induce changes in growth potentiating activity for BHK cells. Likewise, the effect of beta-VLDL treatment of thioglycollate broth elicited BALB/c mouse peritoneal macrophages was evaluated. Conditioned media from lipoprotein-treated macrophages exhibited greater growth-stimulating activity for both BHK cells and NRSMC when compared to conditioned media from macrophages incubated in the absence of the lipoprotein. beta-VLDL did not affect viability of the mononuclear cells. These findings further implicate the involvement of the monocyte-derived foam cell in the development of atherosclerosis.

Animals↗
PubMed1988

Isolation of several toxins from the venom of the scorpion Centruroides limpidus tecomanus Hoffmann.

The soluble venom of the scorpion of Colima, Mexico, was fractionated by Sephadex G-50 gel filtration followed by separation of the toxic fraction (number II) with carboxymethyl-cellulose columns in 20 mM ammonium acetate buffer, pH 4.7. Of 24 fractions five were toxic to mice and were further separated in 50 mM phosphate buffer, pH 6.0 using the same ion exchange resin. Final separation included chromatography of the toxic subfractions in the same resin, but in 75 mM ammonium acetate buffer, pH 5.0. By this procedure at least five distinct toxins were obtained in pure form according to gel electrophoresis analysis. Amino acid composition of toxin 1 (component II.20.3.4) and toxic component II.22.5 is included. The N-terminal amino acid sequence of component II.22.5 was shown to be: Lys-Glu-Gly-Tyr-Ile-Val-Asn-Tyr-His-Thr-Gly-Cys-Lys-Tyr-Thr-Cys-Ala-Lys- Leu-Gly - Asp-Asn-Asp-Tyr-Cys-Leu-Arg-Glu-Cys-Lys-.

Amino Acid Sequence↗
PubMed1988

Amino acid sequence and physiological characterization of toxins from the venom of the scorpion Centruroides limpidus tecomanus Hoffmann.

The complete amino acid sequence of the major toxic component (II.20.3.4), named toxin 1, from the venom of the Mexican scorpion C. l. tecomanus is reported. The sequence (66 amino acids) was obtained by direct Edman degradation of reduced and alkylated toxin, followed by sequence determination of selected peptides separated after enzymatic cleavage with S. aureus V8 protease. In cultured chick dorsal root ganglion cells, 0.5 microM toxin 1 slowed down specifically the time course of Na+ current inactivation, while Ca2+ currents from the same preparation were little affected. In neonatal rat ventricular heart cells, toxin 1, at concentrations between 0.1 and 0.5 microM, reduced Na+ currents without changing the kinetics and Ca2+ currents were unaffected. Comparative analysis of the primary structure of this toxin with other scorpion toxins shows a high degree of similarity with the north American scorpion toxins. This analysis suggests that the 'fine tuning' of the molecular mechanism of action of these toxins is related to variations in the primary structure as well as to the type of membrane under study (tissue specificity).

Action Potentials↗
PubMed1988

Isolation and characterization of two toxins from the Mexican scorpion Centruroides limpidus limpidus Karsch.

1. Several toxic polypeptides were found in the venom of the scorpion Centruroides limpidus limpidus. Comparative studies of the potency of the venom in different strains of mice were conducted. 2. A new type of toxin (component II.9), specific for crustaceans (crayfish and isopods), was isolated from this scorpion and was shown to have the following N-terminal amino acid sequence: Lys-Lys-Asp-Gly-Tyr-Leu-Val-Asn-Lys-Tyr-Thr-Gly-Cys-Lys-Val-Asn-Cys- Tyr-Lys-Leu-Gly-Glu-Asn-Lys-Phe-Cys-Asn-Arg-Glu-. 3. A polypeptide toxic to mice (component II.6) from this venom was shown to have the following N-terminal sequence: Lys-Glu-Gly-Tyr-Leu-Val-Asn-His-Ser-Thr-Gly-Cys-Lys-Tyr- Glu-Cys-Tyr-Lys-Leu-Gly-Asp-Asn-Asp-Tyr-Cys-Leu-Arg-Glu-Cys-Lys-. 4. In cultured chick dorsal root ganglion cells, 1 microM of toxin II.6 was shown to reduce the size of sodium currents and to slow-down their activation-inactivation kinetics. The toxin had also a depressive action on the classical Ca2+ current activated at high membrane potentials (greater than 0 mV).

Amino Acid Sequence↗
PubMed1988

Partial protein sequence of mouse and bovine kidney angiotensin converting enzyme.

Angiotensin converting enzyme (ACE) plays an important role in the regulation of renal blood pressure by the hydrolysis of the inactive precursor peptide angiotensin I to the potent vasopressor angiotensin II. Renal ACE is a surface membrane protein of both endothelium and tubular epithelium. Enzymatically active ACE was isolated from renal homogenates by chromatography using an affinity column constructed by linking an ACE inhibitor, lisinopril, to Affi-Gel 15. Analysis of eluates from this column showed that ACE activity was increased greater than 500-fold. SDS-polyacrylamide gel electrophoresis demonstrated a single band of molecular weight 144 kD (mouse) and 149 kD (bovine). N-terminal amino acid sequence analysis revealed: (formula; see text) Though bovine ACE has one additional N-terminal amino acid, these two partial sequences are highly homologous (16 of 20 positions are identical). Mouse ACE was digested with trypsin and the peptides were isolated by reverse phase HPLC. Analysis of the amino acid sequences showed that these tryptic peptides were unique to ACE. Thus, we were able to isolate ACE from bovine and mouse kidneys and show that they had substantial structural homology. They were also quite similar to that from rabbit lung.

Amino Acid Sequence↗
PubMed1988

Characterization of a cDNA clone encoding the calmodulin-binding domain of mouse brain calcineurin.

A cDNA clone corresponding to a portion of the catalytic subunit of calmodulin (CaM)-dependent phosphoprotein phosphatase (calcineurin) was isolated from a murine brain library by expression vector immunoscreening. A beta-galactosidase fusion protein that reacted on Western blots with anti-calcineurin antibodies and biotinylated CaM was purified in preparative amounts using CaM-Sepharose affinity chromatography. Partial digestion of the hybrid protein with Staphylococcus aureus V-8 protease produced several immunoreactive peptides that appeared identical to fragments generated from authentic brain calcineurin. The 1111-base-pair (bp) EcoRI insert contained an open reading frame encoding a protein of 35 kDa followed by a 190-bp 3' noncoding region; seven peptides obtained by partial amino acid sequencing of the bovine brain enzyme were found in the deduced sequence. A domain approximately 12 kDa from the carboxyl terminus was deduced to be the CaM-binding site based on consensus structural features and a sequence of seven amino acids highly related to smooth muscle myosin light-chain kinase. Two regions with identity to protein phosphatases 1 and 2A were found in the amino half of the cloned sequence; however, the intervening sequence contained apparent insertions, suggesting splicing of subdomains. Thus, the structure of calcineurin is chimeric, consisting of conserved catalytic elements and a regulatory CaM-binding domain.

Amino Acid Sequence↗
PubMed1988

Genetic heterogeneity in type 1 Gaucher disease: multiple genotypes in Ashkenazic and non-Ashkenazic individuals.

Nucleotide sequence analysis of a genomic clone from an Ashkenazic Jewish patient with type 1 Gaucher disease revealed a single-base mutation (adenosine to guanosine transition) in exon 9 of the glucocerebrosidase gene. This change results in the amino acid substitution of serine for asparagine. Transient expression studies following oligonucleotide-directed mutagenesis of the normal cDNA confirmed that the mutation results in loss of glucocerebrosidase activity. Allele-specific hybridization with oligonucleotide probes demonstrated that this mutation was found exclusively in the type 1 phenotype. None of the 6 type 2 patients, 11 type 3 patients, or 12 normal controls had this allele. In contrast, 15 of 24 type 1 patients had one allele with this mutation, and 3 others were homozygous for the mutation. Furthermore, some of the Ashkenazic Jewish type 1 patients had only one allele with this mutation, suggesting that even in this population there is allelic heterozygosity. These findings indicate that there are multiple allelic mutations responsible for type 1 Gaucher disease in both the Jewish and non-Jewish populations. Allelic-specific hybridization demonstrating this mutation in exon 9, used in conjunction with the Nci I restriction fragment length polymorphism described as a marker for neuronopathic Gaucher disease, provides a tool for diagnosis and genetic counseling that is approximately equal to 80% informative in all Gaucher patients studied.

Adolescent↗
PubMed1988

Glycosylation and processing of high levels of active human glucocerebrosidase in invertebrate cells using a baculovirus expression vector.

A human cDNA containing the complete coding region for the lysosomal glycoprotein glucocerebrosidase (EC 3.2.1.45) was introduced into the genome of Autographa californica nuclear polyhedrosis virus downstream from the polyhedrin promoter. Infection of Spodoptera frugiperda cells (SF9) with recombinant virus produced high levels of glucocerebrosidase, 40% of which was in the culture medium. The amino-terminal amino acid sequence of the recombinantly produced enzyme was identical to that of mature, human placental glucocerebrosidase, demonstrating that the signal sequence of the human preenzyme was recognized and appropriately removed in the SF9 invertebrate cells. The glucocerebrosidase in both the culture supernatant and SF9 cell pellet was glycosylated and contained, in part, high mannose oligosaccharide. These results demonstrate that insect cells can be used to produce abundant quantities of active mature human glucocerebrosidase that contains high mannose oligosaccharide as a consequence of post-translational processing.

Animals↗