Isolation and characterization of latherin, a surface-active protein from horse sweat.

A protein, latherin, with unusual surface activity was isolated from horse sweat by gel filtration and ion-exchange chromatography. The protein has a Stokes radius, determined by gel filtration, of 2.47 nm, and in the ultracentrifuge sediments as a single species with S20,W 2.05 S, indicating an Mr of 24,400. On SDS/polyacrylamide-gel electrophoresis the molecule behaves as a single peptide chain of apparent Mr 20,000. Latherin contains a high proportion of hydrophobic amino acids (37.2%), and the leucine content (24.5%) is exceptionally high. The unusual composition of the protein may account for apparent anomalies in the Mr of latherin determined by empirical methods. Evidence indicating that latherin is responsible for much of the surface activity of horse sweat was obtained by a simple assay for surface tension and by contact-angle measurements. Latherin adsorbs very readily at hydrophobic surfaces, rendering them wettable. A possible role for latherin in thermoregulation is proposed.

Characterisation of glycoproteins in the sweat of the horse (Equus caballus).

The two major polypeptides H (Mr 49,000) and L (Mr 33,000) of equine sweat have been purified by gel filtration and characterised by gel electrophoresis and compositional analysis. Both H and L are glycoproteins containing sialic acid, neutral sugars, N-acetylglucosamine and N-acetylgalactosamine, but the two polypeptides differ considerably in the extent of glycosylation. H and L also differ in amino acid composition, but both contain only low levels of sulphur containing amino acids and histidine. These glycoproteins may behave as surfactants.

Polyamines in colorectal cancer--a clinical and experimental approach.

The urinary polyamines putrescine, spermidine and spermine were measured prior to operation in 10 patients with colorectal cancer and 10 control subjects. Carcinoembryonic antigen assays were also performed in an attempt to correlate these values with polyamine excretion. The total polyamine rates in patients with colorectal cancer were 3.2 +/- 1.5 (SD) mg/24 h and 2.6 +/- 1.2 (SD) mg/24 h in the controls. The difference between the group with colorectal cancer and the controls was not statistically significant. Urinary polyamines were also measured in an experimental animal model for colorectal cancer in which tumour cell mass could be assessed. Only marginal differences occurred in polyamine rates between animals with extensive tumours and controls. These findings suggest that urinary polyamine measurement is unlikely to be a useful procedure to assess tumour cell mass in patients with colorectal cancer.

Turtle-dove ovomucoid, a glycoprotein proteinase inhibitor with P1-blood-group antigen activity.

Ovomucoid from the egg white of turtle-dove (Streptopelia risoria) was purified and shown to be a glycoprotein of mol. wt. 29 400, with valine as N-terminal residue. It is an inhibitor of both trypsin and chymotrypsin, but has a lower affinity for trypsin than has hen ovomucoid. Turtle-dove ovomucoid contains antigenic activity cross-reacting with the blood-group-P1 antigen of human erythrocytes. Hen ovomucoid has no detectable blood group-P1 activity. The carbohydrate composition of turtle-dove ovomucoid differs from hen ovomucoid in having substantially higher galactose content. The possible relationship between carbohydrate composition and antigenic activity is discussed.

Location of the carbohydrate groups of ovomucoid.

Tryptic glycopeptides were purified from the sialic acid-free variant of ovomucoid, O1, and its CNBr fragments. The amino acid sequences adjacent to the four major sites of carbohydrate (Carb.) attachment were: (1), Phe-Pro-Asn(Carb.)-Ala-Thr-Asp-Lys-Glu-Gly-Lys; (2), Ala-Try-Ser-Ile-Glu-Phe-Gly-Thr-Asn (Carb.)-Ile-Ser-Lys; (3), Glu, Thr-Val-Pro-Met-Asn(Carb.)-cys-Ser; (4), Ser-Ser-Tyr-Ala-Asn (Carb.)-Thr-Thr-Ser-Glu-Asp-Gly-Lys, Glycosylated Asn residues were located at position 10, between residues 49 and 60, and at positions 69 and 75, in the primary sequence. All of these carbohydrate groups contained GlcNAc, Man and Gal in the approximate molar proprotions 5:3:0.5. A further glycopeptide containing His was isolated in low yield, suggesting that some carbohydrate is attached at a fifth site. Two of the carbohydrate-attachment sites (Asn-10 and Asn-75) occur in sequences that show internal homologies. These are presumed to have evolved as a consequence of partial gene duplication. Three of the carbohydrate-attachment sites occur in similar positions to the carbohydrate groups in quail ovomucoid [Laskowski (1976) Protides Biol. Fluids Proc. Colloq. 23, in the press]. Prediction of peptide conformation from the sequence data by the method of Chou & Fasman [(1974) Biochemistry 13, 222-225] indicated that four glycosylated Asn residues in hen ovomucoid are very close to groups of amino acids that occur with high frequency in beta-turns. The possible significance of peptide-chain conformation in the attachment of carbohydrate to glycoproteins is briefly discussed.

Active fragments obtained by cyanogen bromide cleavage of ovomucoid.

Cleavage of the two methionine residues in the glycoprotein trypsin inhibitor ovomucoid, variant O1, with CNBr resulted in two fragments whose mol.wts. were approx. 16 600 (fragment LS) and 11 000 (fragment M). Both fragments formed precipitates with antisera to ovomucoid. Fragment LS retained 56% of the trypsin-inhibitory activity of ovomucoid, but fragment M did not inhibit. After reduction and alkylation, the molecular weight of fragment M was unchanged, but fragment LS could be resolved into two segments of peptide chain with mol.wts. of approx. 12000 (fragment L) and 4700 (fragment S). Each of these peptides contained carbohydrate. Marked heterogeneity was observed in the hexose and hexosamine contents of fragment L. This may account for much of the heterogeneity in neutral carbohydrate occurring in ovomucoid preparations. It was found that fragment M was located at the N-terminal end, fragment S was in the centre and fragment L made up the C-terminal portion of the molecule.

The isolation of ovomucoid variants differing in carbohydrate composition.

Three major and two minor species of ovomucoid were separated by chromatography on sulphoethyl-Sephadex. The predominant sialic acid-free species was further resolved into three fractions by DEAE-cellulose chromatography. Although all species of ovomucoid had closely similar trypsin-inhibiting activity, immunochemical properties and amino acid composition, they differ in carbohydrate composition. Wide variation was observed in the content of galactose, N-acetylglucosamine and sialic acid. Charge heterogeneity was related, in part, to variation in sialic acid content. The implications of variable carbohydrate composition for the structure and function of ovomucoid are discussed.