Don M. Carlson

Alkaline borohydride degradation of blood group H substance

Abstract Treatment of blood group H substance at a concentration of 1 mg/ml with O.05 n NaOH and 1 m NaBH 4 at 50 ° results in the release of oligosaccharide side chains with a minimum of degradation. A procedure for the partial fractionation as well as the chemical and immunochemical analyses of these reduced oligosaccharides is described.

Human respiratory tract secretions: Mucous glycoproteins of nonpurulent tracheobronchial secretions, and sputum of patients with bronchitis and cystic fibrosis

Abstract Mucous glycoproteins were isolated by agarose gel filtration from nonpurulent tracheobronchial secretions and purulent sputum which had been reduced, carboxymethylated and, in the case of purulent secretions, treated with deoxyribonuclease. The solubilized and purified glycoproteins were fractionated on diethylaminoethyl cellulose into two major (I, II) and two minor (Ia, III) blood group active components. Components I and II had similar carbohydrate and amino acid compositions which were typical for human blood group substances. These two components did differ in several respects. Component I contained 1.4–2.6% sulfate and did not inhibit influenza virus hemagglutination while component II contained 7.1–7.8% sulfate and was a potent inhibitor of virus hemagglutination. Component II also migrated more rapidly on sodium dodecyl sulfate-3.3% acrylamide gel electrophoresis. Components I and II in purulent secretions displayed only minor compositional differences from their counterparts in nonpurulent secretions. Component II was more abundant in two sputum samples from subjects with cystic fibrosis than in purulent bronchitic secretions or in nonpurulent secretions.

Isolation of a “proline-rich” protein from rat parotid glands following isoproterenol treatment

Abstract Prolonged treatment of rats with isoproterenol induces both hypertrophy and hyperplasia of the parotid glands. Daily injections of 5 mg dl-isoproterenol resulted in a 4–6 fold increase in gland wet weight within 7 days. The protein composition of parotid gland homogenates and saliva, as monitored by disc gel electrophoresis, was markedly altered. In particular, the concentration of a proline-rich protein increased dramatically. This protein was purified by Sephadex G-100 chromatography and preparative disc gel electrophoresis and was found to be comprised mainly of four amino acids (moles/100 moles): Pro, 29.5; Glx, 19.3; Gly, 17.1; Asx, 11.9. The molecular weight as determined by sedimentation equilibrium is about 25,000. When [ 14 C] glucosamine was injected intraperitoneally, a small amount of label was incorporated into the proline-rich protein. Upon stopping the isoproterenol treatment, the parotid glands and the polyacrylamide disc gel patterns of the soluble proteins both return to normal within 10 to 14 days.

Isolation and properties of four α-amylase isozymes from human submandibular saliva☆

Abstract Four isoamylases have been isolated from human submandibular secretions by gel filtration and isoelectric focusing. The isozymes (1A, 1B, 2A, 2B) were each purified about 8-fold and each yielded one major band on disc gel electrophoresis. In all cases the major protein band contained more than 95% of the protein and amylase activity recovered. The isoenzymes, in order of their relative positions on the polyacrylamide gels (from the anodal end), their isoelectric points, and percentage distribution in the submandibular secretion are as follows: isozyme 2A, pH 5.9, 9%; isozyme 1A, pH 5.9, 18%; isozyme 2B, pH 6.4, 63%; isozyme 1B, pH 6.4, 10%. Amino acid analyses showed that the protein compositions of the four isoamylases were essentially the same. Possible differences were noted in aspartic acid, serine, glutamic acid, and proline contents. Molecular weights, determined by SDS disc gel electrophoresis, were 57,000 for 1A and 1B, and 54,000 for 2A and 2B. This molecular weight difference is attributed mainly to the presence of bound carbohydrate on isozymes 1A and 1B. Gas Chromatographic analysis was used for determining the carbohydrate compositions. Molar ratios of sugars were similar for both glycoprotein amylases (moles sugar/mole enzyme): glucosamine, 3; mannose, 3; galactose, 2; fucose, 3. Isoamylase 1A, which had more carbohydrate than 1B, also contained about 2 moles of N -acetylneuraminic acid. Sialic acid was not detected in isozyme 1B.

Protein composition of human submandibular secretions

Abstract The protein composition of human submandibular saliva obtained from a single donor has been investigated, and 21 proteins have been resolved. On Sephadex G-100, submandibular secretions (unstimulated) separated into four fractions, I, II, III, and IV. Each fraction was analyzed further by isoelectric focusing and disc gel electrophoresis. The major components detected in each fraction along with their isoelectric point (p I ) are as follows: I, blood group specific substance (2.3), immunoglobulin A (5.0–6.0), and immunoglobulin G (4.5–6.5); II, albumin (4.9), two glycoproteins (5.0), and acid phosphatase (5.2); III, three phosphoproteins (4.3–4.4), isoamylase 1A (5.9), isoamylase 1B (6.4), unidentified protein (7.1), lysozyme (>10), and a basic protein (>10); and IV, isoamylase 2A (5.9) and isoamylase 2B (6.4). Isoamylase 1A and IB are glycoproteins. Stimulated submandibular secretions were also resolved into four protein fractions by gel filtration. Fraction III, compared with unstimulated secretions, showed the greatest percent increase in protein. Analysis of this fraction by disc gel electrophoresis demonstrated the presence of four protein bands which were not detected in the unstimulated secretion. One of these proteins is tentatively identified as a phosphoprotein and two as basic proteins (p I > 10). The protein composition of submandibular, parotid, and sublingual secretions is compared.

Composition and Molecular Weights of Butyrylcholinesterase from Horse Serum

Abstract The physical and chemical characterization of horse serum butyrylcholinesterase has been extended. The results show that the enzyme is a glycoprotein containing about 20% carbohydrate by weight. Mannose, glucosamine, galactose, and sialic acid are the sugar residues found. The extinction coefficient of butyrylcholinesterase, E1cm1% at 280 nm, was found to be 15.2 ± 0.3 by dry weight determination. The molecular weight of the protein in dilute phosphate buffer was determined to be (31.7 ± 1.2) × 104 by high speed equilibrium sedimentation with a redetermined partial specific volume of 0.723 ± 0.003 ml/g. Subunit molecular weights for the dissociated protein were found to be (7.9 ± 0.4) × 104 and (8.1 ± 0.1) × 104, respectively, in guanidine hydrochloride and in a solution at pH 11.8. The subunit molecular weight was also estimated to be (8.8 ± 0.2) × 104 by analytical sodium dodecyl sulfate-gel electrophoresis. This apparently higher subunit molecular weight from dodecyl sulfate gels is expected for glycoproteins containing significant amounts of carbohydrate. No free sulfhydryl group was detected, even though there are six half-cystines in each subunit. Therefore, it seems likely that there are three pairs of disulfide bonds per subunit. The available data indicate that native butyrylcholinesterase is a tetrameric glycoprotein consisting of subunits of equal molecular weight.

Studies of mucin-type glycoproteins: Olefinic amino acids, products of the β-elimination reaction

Abstract The carbohydrate moieties of mucin-type glycoproteins are attached to the polypeptide core by O -glycosyl linkages of N -acetylgalactosamine to the hydroxyl groups of both serine and threonine. Alkali catalyzes the release of the carbohydrate groups by a β-elimination reaction. Studies reported here on the relative rates of the β-elimination reaction have demonstrated that serine-linked glycosides are released more rapidly than threonine-linked glycosides. The ratio of glycosylated serine to glycosylated threonine can readily be determined after β-elimination. The olefinic amino acids, which are products of the β-elimination reaction, are converted into pyruvate and α-ketobutyrate by acid hydrolysis and then are assayed spectrophotometrically with lactate dehydrogenase. In addition, the olefinic amino acids formed upon alkali treatment of pig submaxillary mucin have been further identified by preparation of the dinitrophenylhydrazone derivatives of the α-keto acids and their conversion to α-amino acids by catalytic hydrogenolysis.

Quantitation of submandibular proteins resolved from normal individuals and children with cystic fibrosis.

Submandibular secretions collected from children with cystic fibrosis (CF) showed increased protein concentration (milligrams/milliliter) and increased amylase specific activity (units/milligram of protein) relative to normal secretions. These differences between normal (N) and CF secretions were as follows: protein, 1.25 ± 0.51 (N), 1.75 ± 0.35 (CF) (P < 0.02); and amylase, 58 ± 18 (N), 80 ± 19 (CF) (P < 0.001). To determine the basis for elevated protein in CF saliva, several major proteins resolved by polyacrylamide disc gel electrophoresis were quantitated by densitometry. These included four phosphoproteins (PP), serum albumin, an acid phosphatase-containing fraction, amylase, and an unidentified protein referred to as PI-7.1. Together, these proteins comprise greater than 75% of the total protein in the secretion. Differences in individual protein concentrations (milligrams/milliliter) resolved from normal and CF secretions, respectively, were as follows: PP2, 0.02 ± 0.01, 0.03 ± 0.02 (NS, not significant); PP3, 0.06 ± 0.04, 0.05 ± 0.03 (NS); acid phosphatase fraction, 0.06 ± 0.04, 0.12 ± 0.07 (P < 0.05); amylase, 0.09 ± 0.04, 0.27 ± 0.16 (P < 0.01); and pI-7.1, 0.04 ± 0.02, 0.13 ± 0.08 (P < 0.02). Amylase, the most significant contributor to the elevated protein, comprised 26% of the total protein of normal secretions and 38% of the total protein of CF secretions. Thus, our results do not support the concept of a generalized increase in all organic components in CF submandibular secretions but, rather, increases in specific proteins, namely amylase, component pI-7.1, and an acid phosphatase-containing fraction.

Quantitative enzymatic synthesis of 2-acetamido-2-deoxy-α-d-glucopyranosyl-1,6-diphosphate☆

Abstract Phosphoacetylglucosamine mutase (EC 2.7.5.2) catalyzes the interconversion of N-acetylglucosamine-1-phosphate and of N-acetylglucosamine-6-phosphate. Glucose-1,6-diphosphate activates this mutase through the formation of N-acetylglucosamine-1,6-diphosphate and glucose-6-phosphate. By adding glucose-6-phosphate dehydrogenase and an excess of N-acetylglucosamine-6-phosphate the reaction is pulled to completion with stoichiometric formation of N-acetylglucosamine-1,6-diphosphate with respect to the amount of glucose-1,6-diphosphate present. The isolation, chemical analysis, molar rotation, and cofactor activity of N-acetylglucosamine-1,6-diphosphate are described.

CARBOHYDRATE COMPOSITIONS OF EPITHELIAL MUCINS11This work was supported by Grants AM-08305 and AM-10335, National Institutes of Health, and by research support from the Greater Cleveland Health Fund.

The structures and immunochemical properties of oligosaccharides isolated from pig submaxillary mucins (PSM) are described. Oligosaccharides from A+-PSM, i.e., mucin which exhibits blood group A activity, contain a terminal N-acetylgalactosamine (GalNAc) residue which is absent in A−-PSM. The enzymic transfer of 14C-GalNAc from UDP-GalNAc onto A−-PSM yields immunochemically-active A+-PSM Studies on the incorporation of 14C-GalNAc into A−-PSM and 14C-galactose into a possible precursor material of blood group substance are given as support for the hypothesis that the addition of sialic acid regulates oligosaccharide chain growth. Investigations on the alkaline borohydride cleavage of mucins and a model disaccharide, N-acetylchondrosine, are discussed.