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Biomedical subjects

K J Wilson

Publications and source records attributed to K J Wilson.

At least 73 records · Page 4Linked to original sources
PubMed1981

Modification by simetryn sulphoxide of a specific thiol group in rat haemoglobin.

Native rat haemoglobins were found to bind simetryn sulphoxide to an extent 40-fold greater than human haemoglobin. This specific behaviour was studied by using only high-pressure ('performance') liquid chromatography for the preparative separation of globin chains and the isolation of peptides resulting from chemical and enzymic degradation. High recoveries (greater than 80%) of peptides throughout the procedures in combination with microsequence techniques, allow a definitive assignment of the residue undergoing modification. The haemoglobin beta-chain cystine-125 residue, with a stoichiometry of one per tetramer of rat haemoglobin, was found to be modified. Stereochemical implications of this finding are discussed. Simetryn sulphoxide would appear to be useful as a specific reagent for the mapping of exposed thiol residues in proteins.

Animals↗
PubMed1981

Muscle beta-actinin and serum albumin of the chicken are indistinguishable by physicochemical and immunological criteria.

Chicken muscle beta-actinin is considered to be one of the "true" myofibrillar components due to its specific binding to isolated myofibrils. Surprisingly, the direct comparison of this muscle protein with serum albumin, both isolated from chicken, showed that they behaved identically under several electrophoretic conditions. Furthermore, immunoreplica gels and double-immunodiffusion tests with antibodies prepared against beta-actinin established the serological identity of both proteins. No significant differences were found by circular dichroic spectroscopy or in amino acid composition. In addition, the amino-terminal sequences of both proteins were identical (H2N-Asp-Ala-Glu-His-Lys-Ser-Glu-Ile-Ala-His-Arg-Tyr-Asn-Asp-Leu-). Combined, these results strongly indicate that muscle beta-actinin and serum albumin are similar, if not identical.

Actinin↗
PubMed1980

Peptide fragmentation suitable for solid-phase microsequencing. Use of N-bromosuccinimide and BNPS-skatole (3-bromo-3-methyl-2-[(2-nitrophenyl)thio]-3H-indole).

BNPS-skatole (3-bromo-3-methyl-2-(2-nitrophenyl)thiol-3H-indole) and N-bromosuccinimide have been used to specifically cleave at peptide bonds after amino acids with available C-gamma=C-delta double bonds, i.e. tryptophan, tyrosine and histidine. The resulting C-terminal lactones conveniently attach to amino-glass supports for sequencing with DABITC (4-NN-dimethylaminoazobenzene 4'-isothiocyanate). Also, peptides having such C-termini, i.e. from a chymotryptic digest, can be readily made to react with these reagents and thus be easily attached and sequenced by solid-phase methods.

Amino Acid Sequence↗
PubMed1979

Fetal and adult albumins are indistinguishable by immunological and physicochemical criteria.

The existence of a functionally immature fetal albumin has been postulated to explain the reduced ability of newborn plasma to bind bilirubin and various drugs. In support of this, cord and adult albumin, isolated by a simple salting-out technique, were reported to differ in electrophoretic and chromatographic properties and in their resistance to alkali and proteolytic enzymes. However, the interpretation of these findings has since been questioned. To resolve this controversy, we have purified to homogeneity human serum albumins from pooled umbilical cord and adult donor plasma. The two albumins were compared and found to be indistinguishable by polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate, as well as by immunoelectrophoresis and double immunodiffusion using specific antibodies against both albumins. Furthermore, the amino acid compositions, the aminoterminal sequence (Asp-Ala-His-Lys-Ser-Glu-Val-Ala-), the carboxy terminus (Leu), and the peptide fingerprints were identical in the two albumins. No significant differences were found by circular dichroism in the ultraviolet (200-350 nm). Binding studies with bilirubin showed association constants of 3.7 +/- 0.7 x 10(7) M(-1) for cord and 2.9 +/- 0.3 x 10(7) M(-1) for adult albumin, respectively. The circular dichroic spectra of 1:1 bilirubin.albumin complexes showed considerable variation between the batches but were not significantly different. The only difference was found in the fluorescence spectra of the bilirubin.albumin complexes, in which complexes with adult albumin showed only 75% of the relative fluorescence exhibited with cord albumin. The combined results nevertheless strongly indicate that fetal and adult albumins are very similar, if not identical.

Amino Acid Sequence↗
PubMed1978

Isolation and characterization of the hemoglobin from the lanceolate fluke Dicrocoelium dendriticum.

The hemoglobin of the flatworm Dicrocoelium dendriticum, a lanceolate fluke which infests the hepatic ducts of certain mammals, has been isolated by gel filtration and ion-exchange chromatography. The molecular weight of the denatured protein was found to be 15500, a value in the same range as hemoglobin subunits. The fact that the native hemoglobin has an apparent molecular weight of 22000 in 0.01 M phosphate buffer, pH 7.4, suggests limited aggregation. The protein contains, as all other myoglobins and hemoglobins, one molecule of non-covalently associated ferroprotoporphyrin IX per polypeptide chain. It forms the same ligand derivatives with very similar spectral properties as vertebrate hemoglobins. The high oxygen affinity (p50 is 0.07--0.1 mmHg or 9.3--13.3 Pa at 20 degrees C and pH 7.0) and the absence of heme-heme interaction of (Hill coefficient nH=1.0) are properties which this heme protein shares with other monomeric hemoglobins from invertebrate and lower vertebrate organisms. The native hemoglobin exists in two forms, having isoelectric points of 4.51 and 4.53, which do not differ in their amino-acid compositions. Dansylation indicated that the amino-terminal amino-acid residue is alanine. The carboxy-terminal sequence, determined by carboxypeptidase A digestion of the globin, is -His-Ala-Leu.

Amino Acid Sequence↗
PubMed1977

Revised amino acid sequence of Qbeta coat protein between positions 1 and 60.

A reinvestigation of the primary structure of Qbeta coat protein between positions 1 and 60 was undertaken to resolve two discrepancies between the published amino acid seuqence (Maita, T., and Konigsberg, W. (1971) J. Biol. Chem. 246, 5003-5024) and the cognate nucleotide sequence recently determined in our laboratory (C. Escarmis and M. A. Billeter, unpublished results). The 22nd amino acid was asparagine rather than aspartic acid, and an additional amino acid, serine, was present between proline in position 55 and arginine in position 56. The revised structure agrees with the nucleotide sequence determined so far.

Amino Acid Sequence↗
PubMed1976

Affinity labeling of the primary bilirubin binding site of human serum albumin.

A label for the bilirubin binding sites of human serum albumin was synthesized by reacting 2 mol of Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate) with 1 mol of bilirubin. This yielded a water-soluble derivative in which both carboxyl groups of bilirubin were converted to reactive enol esters. Covalent labeling was achieved by reacting the label with human serum albumin under nitrogen at pH 9.4 and 20 degrees. Under the same conditions, no covalent binding to the monomers of several proteins could be demonstrated. The number of binding sites for bilirubin and the label were found to be the same, and competition experiments with bilirubin showed inhibition of covalent labeling. The absorption, fluorescence and CD spectra of the label in a complex with human serum albumin were similar to those of the bilirubin human serum albumin complex. However, following covalent attachment to the spectral properties were changed, indicating loss of conformational freedom of the chromophore. Labeling ratios were selected to result in the incorporation of less than 1 mol of label/mol of human serum albumin. Under these conditions, labeling is thought to occur primarily at the high affinity binding site.

Azo Compounds↗
PubMed1976

Heat loss from feet of herring gulls at rest and during flight.

The role of the feet of herring gulls (Larus argentatus) in heat dissipation was estimated during rest and wind-tunnel flight. We determined the blood flow to the feet and the arteriovenous temperature difference and thus estimated heat loss from the feet. Determinations of oxygen consumption and respiratory water loss at rest gave a heat production of about 8 W; 37-56% of this heat was lost from the feet (air temp = 10-35 degrees C). During flight heat production was estimated to be about 57 W and heat loss from the feet was 46 W, about 80% of the heat production in flight. Thus the webbed feet are an important avenue of heat loss in the herring gull.

Animals↗
PubMed1975

Dihydrofolate reductase from bovine liver. Enzymatic and structural properties.

Dihydrofolate reductase from bovine liver has been purified 5000-fold employing conventional techniques and methotrexate/aminohexyl/Sepharose affinity chromatography. Electrophoresis of the isolated enzyme on polyacrylamide gels resulted in the separation of two enzymatically active protein components which were not interconvertible by treatment with dihydrofolate and/or the coenzyme. The two forms, present in a ratio of 20:1, were found by isoelectric focusing to have isoelectric points of 7.15 and 5.94. They had identical specific activities toward dihydrofolate (26.1-27.0 U/mg) and folate (1.3-2.2 U/mg), and had identical molecular weights (23500) and amino acid compositions. Due to the small quantity of the acidic form and the similarity of the two forms, the amino-terminal sequence (19 residues) was determined on a mixture of carboxymethylated reductase. The single sulfhydryl group of the enzyme can be modified by several sulfhydryl reagents in the native enzyme without loss of activity. Modification of the same residue occurs in the denaturated state and partially inhibits renaturation to the fully acitve enzyme. One disulfide bridge was detected by reduction and alkylation. The cleavage of this bond did not effect the enzymatic activity.

Amino Acid Sequence↗