Biomedical subjects

D C Shaw

Publications and source records attributed to D C Shaw.

At least 37 records · Page 2Linked to original sources

PubMed1991

The complete amino acid sequence of feline beta-lactoglobulin II and a partial revision of the equine beta-lactoglobulin II sequence.

The amino acid sequence of feline beta-lactoglobulin (designated II) has been determined. The protein chain is 163 amino acids long with a relative molecular mass of 18,558. The primary structure was determined by sequencing of native protein (residues 1-25), BPNS-skatole cleavage fragments and the peptides obtained by proteolytic cleavage with V8 proteinase and TPCK-trypsin. Feline beta-lactoglobulin II has 53 and 57% positional identities with bovine beta-lactoglobulin A and equine beta-lactoglobulin I, respectively, and approx. 68% with a revised sequence of equine beta-lactoglobulin II. The equine beta-lactoglobulin II sequence was re-examined between positions 78 and 122 resulting in a major revision in this area with only a single insertion to give a total of 163 residues.

Amino Acid Sequence↗

PubMed1991

Purification and characterization of acidic glutathione S-transferase 6 from human brain.

An acidic glutathione S-transferase (GST) isoenzyme termed GST6 has been isolated from human brain, characterized and compared with other isoenzymes. The N-terminal amino acid sequence of GST6 was found to be identical with that of GST4 previously purified from human muscle. GST6 cross-reacted with antibody raised against GST4, but not with antisera raised against GST1, GST2 or GST3. The subunit Mr and pI of GST6 were found to be different from those of GST4. The present results indicate that GST6 is another member of the Mu evolutionary class which in man also includes GST1, GST4 and GST5. A minor component that co-purified with GST6 was shown to have an N-terminal sequence similar to, but not identical with, that of GST3. This isoenzyme may be an additional member of the Pi evolutionary class.

Adult↗

PubMed1991

Isolation and nucleotide sequence of the hmp gene that encodes a haemoglobin-like protein in Escherichia coli K-12.

In the course of an attempt to identify genes that encode Escherichia coli dihydropteridine reductase (DHPR) activities, a chromosomal DNA fragment that directs synthesis of two soluble polypeptides of Mr 44000 and 46000 was isolated. These proteins were partially purified and were identified by determination of their N-terminal amino acid sequences. The larger was serine hydroxymethyltransferase, encoded by the glyA gene, while the smaller was the previously described product of an unnamed gene closely linked to glyA, and transcribed in the opposite direction. Soluble extracts of E. coli cells that overproduced the 44 kDa protein had elevated DHPR activity, and were yellow in colour. Their visible absorption spectra were indicative of a CO-binding b-type haemoprotein that is high-spin in the reduced state. The sequence of the N-terminal 139 residues of the protein, deduced from the complete nucleotide sequence of the gene, had extensive homology to almost all of Vitreoscilla haemoglobin. We conclude that E. coli produces a soluble haemoglobin-like protein, the product of the hmp gene (for haemoprotein). Although the protein has DHPR activity, it is distinct from the previously purified E. coli DHPR.

Amino Acid Sequence↗

PubMed1991

The equine major plasma serpin multigene family: partial characterization including sequence of the reactive-site regions.

The equine Pi system, which is highly polymorphic and was considered to be controlled by a single locus, has been shown to be controlled by four loci (named Spi 1-4). This system is the equine equivalent of the major human plasma serpin (serine protease inhibitor), human alpha 1 PI. Twenty-two haplotypes of the equine Pi system have been characterized by two-dimensional electrophoresis, resulting in the assignment of pI, Mr, and bovine trypsin and chymotrypsin inhibition characteristics to 109 proteins. These proteins have been analyzed further to determine their relatedness to each other as well as to human alpha 1 PI using immunochemical, structural, and functional criteria. The amino acid sequences of the N termini and reactive-site regions have been determined on proteins from each of the four equine Spi loci. This allowed the designation of the proteins from the Spi 1 locus as being METserpins and the functional equivalents of human alpha 1 PI. The Spi 4 proteins are ARGserpins, and by alignment the Spi 2 proteins are ILEserpins, the first so far described. The P1 residue for the Spi 3 proteins was unable to be determined. The limited peptide and immunopeptide mapping revealed that proteins from all four loci were closely related, but within the four there were two pairs (Spi 1 and 2 and Spi 3 and 4) which were more related. All were probably derived from the same gene that gave rise to human alpha 1 PI.

Amino Acid Sequence↗

PubMed1991

Donkey and horse alpha 1 B-glycoprotein: partial characterization and new alleles.

1. The donkey postalbumin protein has been shown to be the equivalent of human alpha 1 B-glycoprotein by protein immunoblotting and N-terminal amino acid sequence. 2. The horse A1B system (already identified as the homologue of human alpha 1 B-glycoprotein) and the donkey alpha 1 B-glycoprotein were characterized further for terminal sialic acid content, isoelectric point, amino acid composition and affinity for the dye-ligand, Cibacron Blue F3GA (known to bind human alpha 1 B-glycoprotein). 3. Two new alleles in the horse A1B system were found, bringing the total number of alleles to five. No polymorphism was found in the donkey alpha 1 B system. 4. As expected the first 20 N-terminal residues of the donkey and horse proteins are highly conserved with only two differences being found. 5. The polymorphism of the horse alpha 1 B-glycoproteins may be due in part to differing numbers of terminal sialic acid residues and the higher electrophoretic mobility of the donkey alpha 1 B-glycoprotein may be due in part to increased sialylation. 6. The horse and donkey alpha 1 B-glycoproteins exhibited differences in affinity for the dye-ligand, Cibacron Blue F3GA, with the donkey alpha 1 B-glycoprotein not being bound.

Alleles↗

PubMed1991

The tammar wallaby major plasma serpin: partial characterization including the sequence of the reactive site region.

1. The putative equivalent of the human major plasma serpin (alpha 1-proteinase inhibitor or alpha 1-antitrypsin) in the tammar wallaby (Macropus eugenii) has been further characterized by structural (peptide and immunopeptide mapping and sequence studies) and functional analyses revealing close homology of the wallaby proteins to human alpha 1-proteinase inhibitor. 2. A sixth allele, Pi J, was detected and its products characterized in terms of pI, Mr, inhibitory spectra and terminal sialic acid content. 3. A recently-developed electrophoretic in situ oxidation/binding method was adapted to provide protein suitable for sequence analysis of the N-terminus and reactive site region including assignment of the P1 and P'1 residues. 4. All sequence analyses were performed on proteins or peptides (approximately Mr 3500) blotted onto polybrene treated GF/C or polyvinylidene difluoride membrane respectively. 5. The P5 to P'4 residues of the reactive centre are identical with those of the human inhibitor thereby allowing the wallaby inhibitor also to be classified as a METserpin. 6. The P1 methionine is presumably responsible for the oxidation sensitivity observed in the electrophoretic in situ functional assay for the wallaby inhibitor. 7. The plasma concentration of the wallaby inhibitor is similar to that reported for human alpha 1-proteinase inhibitor.

Alleles↗

PubMed1991

The isolation and amino acid sequences of echidna (Tachyglossus aculeatus) milk lysozyme I and II.

Comparative studies of monotreme proteins are of particular value in gaining an understanding of the origin of mammals and their interrelationships. The presence of two lysozyme variants, echidna lysozyme I and II, has been confirmed in mature milk samples of Tachyglossus aculeatus multiaculeatus and Tachyglossus aculeatus aculeatus respectively. A simplified procedure is described for their isolation. Their amino acid sequences, the first determined for a monotreme secretory protein, are unusual. They are shown to be c-type lysozymes, each consisting of a single chain of 125 residues (terminating at Cys 125). The only other known c-type lysozyme with this termination is that of pigeon eggwhite. Echidna lysozyme is unique in having no Cys at position 6, but at position 9. It has precisely the residues relevant to the binding of Ca(II), and most of the residues implicated in the galactosyl transferase modifier action of alpha-lactalbumin. However, the weak modifier action previously observed for variant I, prepared by a different method, was not found for the present preparation. The evolutionary significance of the results is discussed.

Amino Acid Sequence↗

PubMed1990

Feline whey proteins: identification, isolation and initial characterization of alpha-lactalbumin, beta-lactoglobulin and lysozyme.

1. Both alpha-lactalbumin and beta-lactoglobulin-like proteins were detected in the whey fraction of feline milk by immunoblotting with rabbit antisera to alpha-lactalbumin and beta-lactoglobulin, respectively. 2. alpha-Lactalbumin was found to occur in both glycosylated and unglycosylated forms in approximately equal concentrations. No polymorphism of feline alpha-lactalbumin was found. 3. Feline beta-lactoglobulin-like proteins produced complex electrophoretic patterns that appear to be determined by three distinct loci. Between two and five genetic variants are expressed by each locus. 4. Lysozyme was detected at levels of approximately 1 mg/ml in skim milk. 5. The identifications of the proteins as alpha-lactalbumin, beta-lactoglobulin and lysozyme were confirmed by determination of N-terminal amino acid sequences.

Amino Acid Sequence↗

PubMed1990

Haemoglobin, serum albumin and transferrin variants of Bali (Banteng) cattle, Bos (Bibos) javanicus.

1. Individual blood samples from 144 Bali (Banteng) cattle [Bos (Bibos) javanicus] in the Northern Territory of Australia and from 61 Bali cross cattle, were examined by zone electrophoresis to determine the variants of haemoglobin, serum albumin and transferrin that are present. 2. Of the common cattle haemoglobin variants (A and B) only variant B occurs in the Bali cattle samples. A second variant, designated CBali, occurs in Bali cattle either as the heterozygote (B CBali) or as the homozygote, the frequencies of occurrence indicating a two-allele system of inheritance without dominance. The CBali cross samples may exhibit the homozygous or heterozygous A variant. 3. The CBali variant has an electrophoretic mobility intermediate between those of the A and B variants at pH 8.6 and 9.1 but closer to B than to A (B greater than C greater than A). It appears to be similar in mobility to the C variants found in Indian Khillan (CKhillan) by Naik, Sukumaran and Sanghvi (Anim. Prodn, 1965 I, 275-277), and in Asian cattle by Oishi, Abe and Namikama (Immunogenet. Lett., 1968 5, 170-173) and Abe, Mogi, Oishi, Tanaka and Suzuki (Proc. XIIth Europ. Conf. Anim. Blood Groups Biochem. Polymorphisms 1972, pp. 225-228), but appreciably different from those in Kenyan and Rhodesian cattle (CRhodesia) found by Braend (Anim. Blood Grps Biochem. Genet., 1971 2, 15-21) and Carr (Rhod. J. agric. Res., 1964 3, 62-62A), respectively. It is also different in mobility from the C variant found by Winter, Mayr, Schleger, Dworak, Krutzler and Burger (Res. vet. Sci., 1984 36, 276-283) in the mithun.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1990

Sequence studies of rabbit serum transferrin.

Large fragments of rabbit serum transferrin have been prepared by enzymatic digestion with subtilisin and by chemical cleavage with BNPS-skatole. Sequence determinations on fragments from the N-terminal lobe lead to the assignment of 273 residues and those from the C-terminal lobe 267 residues. Together with previous determinations, a total of 614 of the ca 679 residues in rabbit transferrin have been assigned. A number of corrections are made to the preliminary sequence assignments of O.U. Beg, H.A. McKenzie and D.C. Shaw (1988) Biochemistry International 17, 1135-1142.

Amino Acid Sequence↗

PubMed1989

Purification of a marsupial insulin: amino-acid sequence of insulin from the eastern grey kangaroo Macropus giganteus.

Insulin has been purified from kangaroo pancreas by acidic ethanol extraction, diethyl ether precipitation and gel filtration. The amino-acid sequence of this, the first marsupial insulin to be studied, is reported. It differs from human insulin by only four amino-acid substitutions, all in regions of the molecule previously known to be variable. However, it should be noted that one of these, asparagine for threonine at A8, has not been reported before. Computer comparisons of all 43 insulin sequences reported to date with kangaroo insulin show it to be most closely related to a group of mammalian insulins (dog, pig, cow, human) known to be of high biological potency. The measurement of blood glucose lowering in the rabbit by kangaroo insulin is consistent with this conclusion. Comparisons of amino-acid sequences of other proteins with their kangaroo counterparts show a greater difference, in line with the time of divergence of marsupials. The limited differences observed in insulin and cytochrome c suggest that their structures need to be closely conserved in order to maintain function.

Amino Acid Sequence↗

PubMed1989

Heat-stable proteins and abscisic Acid action in barley aleurone cells.

[(35)S]Methionine labeling experiments showed that abscisic acid (ABA) induced the synthesis of at least 25 polypeptides in mature barley (Hordeum vulgare) aleurone cells. The polypeptides were not secreted. Whereas most of the proteins extracted from aleurone cells were coagulated by heating to 100 degrees C for 10 minutes, most of the ABA-induced polypeptides remained in solution (heat-stable). ABA had little effect on the spectrum of polypeptides that were synthesized and secreted by aleurone cells, and most of these secreted polypeptides were also heatstable. Coomassie blue staining of sodium dodecyl sulfate polyacrylamide gels indicated that ABA-induced polypeptides already occurred in high amounts in mature aleurone layers having accumulated during grain development. About 60% of the total protein extracted from mature aleurone was heat stable. Amino acid analyses of total preparations of heat-stable and heat-labile proteins showed that, compared to heat-labile proteins, heat-stable intracellular proteins were characterized by higher glutamic acid/glutamine (Glx) and glycine levels and lower levels of neutral amino acids. Secreted heat-stable proteins were rich in Glx and proline. The possibilities that the accumulation of the heat-stable polypeptides during grain development is controlled by ABA and that the function of these polypeptides is related to their abundance and extraordinary heat stability are considered.

Journal Article↗

PubMed1988

Dihydropteridine reductase from Escherichia coli.

A dihydropteridine reductase from Escherichia coli was purified to apparent homogeneity. It is a dimeric enzyme with identical subunits (Mr 27000) and a free N-terminal group. It can use NADH (Vmax./Km 3.36 s-1) and NADPH (Vmax./Km 1.07 s-1) when 6-methyldihydro-(6H)-pterin is the second substrate, as well as quinonoid dihydro-(6H)-biopterin (Vmax./Km 0.69 s-1), dihydro-(6H)-neopterin (Vmax./Km 0.58 s-1), dihydro-(6H)-monapterin 0.66 s-1), 6-methyldihydro-(6H)-pterin and cis-6,7-dimethyldihydro-(6H)-pterin (Vmax./Km 0.66 s-1) when NADH is the second substrate. The pure reductase has a yellow colour and contains bound FAD. The enzyme also has pterin-independent NADH and NADPH oxidoreductase activities when potassium ferricyanide is the electron acceptor.

Amino Acid Sequence↗

PubMed1988

Human muscle glutathione S-transferase (GST-4) shows close homology to human liver GST-1.

Human muscle specific glutathione S-transferase (RX: glutathione R-transferase, EC 2.5.1.18) (GST-4) and liver GST-1 have been purified and subjected to N-terminal sequence analysis. These two isozymes show close homology and only differ in 3 residues within the first 24. The N-terminal sequences of GST-1 and GST-4 differ significantly from those of GST-2 and GST-3. Although antiserum raised against native GST-1 did not cross-react with GST-4, cross-reactivity was obtained with antiserum raised against denatured GST-1. The homology between GST-1 and GST-4 indicates that they are both members of the mu evolutionary class.

Amino Acid Sequence↗

PubMed1988

Long-term survival after resection of ampullary carcinoma is associated independently with tumor grade and a new staging classification that assesses local invasiveness.

Long-term survival characteristics after resection for ampullary carcinoma are documented poorly. We have reviewed the clinical and histopathologic features of 23 long-term survivors who underwent resections between 1972 and 1984 (5-year survival rate, 52.1%). Twenty patients (87%) had intestinal type tumors and only two (9%) had papillary tumors. Associated adenomata were present in eight cases (35%) and distant ductular dysplasia was present in nine cases (39%). Long-term survival was correlated independently with tumor grade (P = 0.0031) and a new staging system that assesses local invasiveness (P = 0.0055). No correlation was found between survival and sex, tumor size, or presence of adenoma. Age was significant in univariate analysis (P = 0.0322) but not in multivariate analysis. A simple scoring system based on the grade and stage increased the predictability of survival (P = 0.0004). Application of this scoring system may allow an objective comparison of long-term survival results after resection from different series.

Adenocarcinoma↗

PubMed1988

Studies of rabbit serum transferrin.

Rabbit serum transferrin is isolated by a procedure designed to preserve its conformation and disulfide linkages. A progress report is presented on the determination of its amino acid sequence, as part of studies on its primary, secondary and tertiary structure. The sequence of 378 residues, of the approximately 680 residues in the molecule, are determined. Observations are made on the site of carbohydrate attachment, iron binding sites and half-cystine residue location. The results are discussed in relation to the X-ray crystallographic studies of human lactoferrin (lactotransferrin) and of rabbit serum transferrin being made in other laboratories.

Amino Acid Sequence↗

PubMed1988

Studies on a partially purified bovine milk lysozyme.

Bovine milk lysozyme has been partially purified by a method developed in this laboratory. We have shown, by preliminary sequential analysis, and by gel filtration on HPLC, that the product is a mixture of two components. One of these, the enzymically active one, differs in its N-terminal sequence from that of "lysozyme 2", a bovine stomach mucosal enzyme, by 7 residues within the first 39 residues. However, some of its properties differ markedly from those of lysozyme 2. The other component, comprising 70% by weight of the total mixture, bears no sequential resemblance to any protein known to us. Our two component system appears to be the same as the preparation of Chandan et al. (Biochim. Biophys. Acta 110, 289 (1965], which they concluded was an homogeneous preparation of lysozyme.

Amino Acid Sequence↗

PubMed1987

A novel whey protein synthesized only in late lactation by the mammary gland from the tammar (Macropus eugenii).

A major whey protein which appears in milk from the tammar wallaby (Macropus eugenii) only during the second half of lactation (late lactation protein-A, LLP-A) was purified to apparent homogeneity by ion-exchange chromatography and gel filtration. An Mr of 21,600 +/- 2000 was calculated from its amino acid composition. A computer-based comparison of the sequence of the first 69 amino acid residues with the Atlas of Protein Sequence data base showed no significant homology with known proteins. Antiserum to LLP-A was prepared in rabbits, and single radial immunodiffusion was used to measure the amounts of LLP-A in milk during the first 40 weeks of lactation. LLP-A was first detected at 26 weeks; thereafter its concentration increased abruptly, to reach a maximum of 26 g/l at approx. 36 weeks of lactation. Explants prepared from mammary gland biopsies at 20 and 35 weeks of lactation were exposed to [3H]amino acids for 8 h; immunoprecipitation of tissue extracts showed that, whereas the rate of casein synthesis was the same at both stages of lactation, LLP-A was synthesized only by the 35-week mammary gland.

Amino Acid Sequence↗