Isolation and Composition of Oligosaccharide Cores from Endotoxins of Two Serratia marcescens Strains

Hydrophobic binding properties of purified human respiratory mucins were studied by the fluorescence probe technique using mansylphenylalanine (Mns-Phe) as the fluorescent probe. Mucins were purified from tracheobronchial secretions of cystic fibrosis (CF) and asthmatic patients, as well as from individuals with normal lungs, according to a protocol earlier established in our laboratory. Purified mucins were subjected to reduction-alkylation and Pronase digestion to study the effects of these treatments on the hydrophobic properties of the mucins. In addition, the effects of increased NaCl concentration on the hydrophobic properties of native and reduced-alkylated mucins were also investigated. Native mucins showed evidence of a large number of low-affinity (KD approximately 10(-5) M) binding sites for the hydrophobic ligand Mns-Phe and had between 40 and 50 binding sites/mg of mucin. Reduction of mucin using dithiothreitol in the presence of 6 M guanidine hydrochloride and subsequent alkylation with iodoacetamide apparently caused marked conformational changes in the mucin molecules as revealed by the presence of both high-affinity (KD approximately 10(-6) M) and low-affinity (KD approximately 10(-5) M) binding sites for the probe and an increase in the number of probe binding sites. Pronase digestion of the native and reduced-alkylated mucins almost completely eliminated binding of the fluorescent probe to the mucins, showing that the binding sites are on the nonglycosylated, Pronase-sensitive portion of the mucin molecules. Increasing NaCl concentrations (0.03-1.0 M) did not appreciably alter the native mucin-induced Mns-Phe fluorescence, while that of the reduced-alkylated mucin-induced Mns-Phe fluorescence was progressively increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Section: Methodsmentioning

confidence: 99%

Evidence of hydrophobic domains in human respiratory mucins. Effect of sodium chloride on hydrophobic binding properties

Shankar¹,

Naziruddin²,

Rocha³

et al. 1990

“…Sialic acid was determined by the resorcinol method (Svennerholm, 1957) using -acetylneuraminic acid as the standard. Fucose, galactose, deoxyribose, iV-acetylgalactosamine, and iV-acetylglucosamine were determined by gas-liquid chromatography as described previously (Sachdev et al, 1978;Wang et al, 1974). DNA was estimated by a fluorometric method using calf thymus DNA as the standard (Thomas & Farquhar, 1978).…”

Section: Methodsmentioning

confidence: 99%

Comparison of physicochemical properties of purified mucus glycoproteins isolated from respiratory secretions of cystic fibrosis and asthmatic patients

Chace¹,

Flux²,

Sachdev³

1985

The major nonreduced mucus glycoproteins (mucins) from sputa of cystic fibrosis (CF) and asthmatic patients have been purified to electrophoretic homogeneity and subjected to physical and chemical characterization. The sputum specimens were solubilized in buffer containing 0.22 M KSCN and fractionated on Bio-Gel A-5m, followed by digestion with DNase, rechromatography on the same column, and chromatography on hydroxylapatite. Sodium dodecyl sulfate gel electrophoresis of purified mucins gave a single band. Carbohydrate analyses of the purified mucins showed no significant differences in the sugar components from the two mucins. However, the CF mucin contained substantially higher (11%) sulfate content than that observed for the asthmatic mucin (5.9%). Amino acid analyses indicated that the CF mucin had higher levels of serine plus threonine (35%) as compared to the asthmatic mucin (29%). In contrast, CF mucin contained a lower content of aspartate, glutamate, and glycine than that observed for the asthmatic mucin. Molecular weights of 3.8 X 10(6) and 3.5 X 10(6) were obtained for CF and asthmatic mucins, respectively, from light-scattering studies of mucins in the presence of 6 M guanidine hydrochloride. Reduction of the disulfide bonds of the two mucins did not alter their molecular weights. Liquid chromatographic studies on Sepharose CL2B showed that CF mucin forms aggregates sufficiently large to be excluded from the gel. As compared to the CF mucin, the asthmatic mucin formed fewer of these large aggregates under identical experimental conditions. Reduction and alkylation of the mucins resulted in their inability to form aggregates. The higher state of aggregation of CF mucin may influence the viscoelastic properties of the CF lung's mucus secretions.

“…Carbohydrate Analyses. Neutral and amino sugars were determined by a modification (Wang et al, 1974) of the gas-liquid chromatographic procedure of Griggs et al (1971). Sialic acid was determined by the same procedure after mild acid hydrolysis (0.01 N HC1 for 90 min at 30 °C) of ApoD.…”

Section: Methodsmentioning

confidence: 99%

Studies on the isolation and partial characterization of apolipoprotein D and lipoprotein D of human plasma

McConathy¹,

Alaupovic²

1976

160

This report describes further studies on the characterization of apolipoprotein D (ApoD), a recently recognized human plasma apolipoprotein, and presents results on the isolation and distribution of its lipoprotein form, lipoprotein D (LP-D). ApoD, isolated by a procedure combining hydroxylapatite and Sephadex G-100 column chromatography, migrated on 7% polyacrylamide gel as a single band with a mobility intermediate between those of A-II and C-II polypeptides. On double diffusion and immunoelectrophoresis, ApoD reacted only with antiserum to ApoD. It was characterized by the presence of all common amino acids including half-cystine. The amino terminal acid was blocked. Carbohydrate analysis demonstrated that ApoD is a glycoprotein with glucose, mannose, galactose, glucosamine, and sialic acid accounting for 18% of the dry weight of ApoD. The estimated molecular weight of ApoD IS 22 100. ApoD occurs in the serum as a lipoprotein which was isolated from high density lipoproteins3 by two different chromatographic procedures. In the first procedure, high density lipoproteins3 were treated with neuraminidase and chromatographed on concanavlin A. The retained fraction containing LP-D was purified by hydroxylapatite column chromatography. Alternatively, LP-D was isolated by a procedure combining chromatography of high density lipoproteins3 or whole serum on an immunosorber containing antibodies to ApoD, and hydroxylapatite column chromatography. LP-D displayed a single, symmetrical boundary in the analytical ultracentrifuge and a single band on 7% polyacrylamide gel electrophoresis. When injected into rabbits it produced antisera that reacted only with ApoD. On immunoelectrophoresis LP-D had a mobility different from that of lipoprotein A (LP-A). A direct immunological comparison of LP-D and LP-A showed a reaction of nonidentity. LP-D consists of 65-75% protein and 25-35% lipid. The lipid moiety contains cholesterol, cholesterol ester, triglyceride, and phospholipid. The phospholipid. composition is characterized by a relative high content of lysolecithin and sphingomyelin and a relatively low content of lecithin. We have concluded from these studies that ApoD is a unique apolipoprotein that exists in the form of a distinct lipoprotein family with a macromolecular distribution extending from very low density lipoproteins into very high density lipoproteins, but with a maximum concentration in high density lipoproteins3 and a minimum concentration in high density lipoproteins.