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The mucin glycoproteins in tracheal mucus of patients with cystic fibrosis is more highly sulfated than the corresponding secretions from healthy individuals [16]. In order to further characterize these differences in sulfation and possibly also glycosylation patterns, we compared the structures of sulfated mucin oligosaccharides synthesized by continuously cultured human tracheal cells transformed by simian virus 40. The synthesis of highly sulfated oligosaccharide chains in mucins secreted by normal human epithelial and submucosal cell lines were compared with mucins formed by cystic fibrosis tracheal epithelial and submucosal cell lines. The epithelial cell lines from cystic fibrosis trachea showed a higher rate of sulfate uptake and a significantly higher rate of synthesis and sulfation of high molecular weight chains. Mucins synthesized by each cell line in the presence of 35SO4 were isolated and oligosaccharide chains were released by beta-elimination and separated by ion exchange chromatography and gel filtration. The sulfated high molecular weight chains synthesized by the cystic fibrosis cell lines were characterized by methylation analysis and sequential glycosidase digestion before and after desulfation. Carbohydrate analysis yielded Fuc, Gal and GlcNAc in a ratio of 1:2:2.2 and only one galactosaminitol residue for about every 150-200 sugar residues present. The average molecular size of oligosaccharide chains in these fractions was between 30,000-40,000 daltons. These studies show that increased sulfation of oligosaccharides in mucins synthesized by cells from cystic fibrosis trachea is accompanied by a significant increase in the extension of a basic branched structure present in many of the lower molecular weight oligosaccharides.

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UDP-GalNac: polypeptide N-acetylgalactosaminyltransferase from swine trachea epithelium was purified to homogeneity by procedures which included affinity chromatography on Sepharose 4B columns containing bound deglycosylated Cowper's gland mucin. The enzyme, purified 12,000-fold from microsomes with a yield of 40%, showed only a single band on dodecyl sulfate polyacrylamide gel electrophoresis. The homogenous enzyme has an apparent molecular mass of 70,000 Da, as determined by gel electrophoresis or gel filtration. The transferase has a broad pH optimum between 6.7-7.8 with maximal activity at pH 7.2, and required Mn2+ for activity with maximal activity at 5-7.5 mM. Higher concentrations of Mn2+, inhibited the enzyme. The purified transferase was specific for UDPGalNAc and glycosylated both threonine and serine residues in tryptic peptides prepared from deglycosylated Cowper's gland and swine and human trachea mucins. The apparent Km of the transferase for UDPGalNAc was 6.3 microM, and the Km values for deglycosylated Cowper's gland and human and swine trachea mucins were 0.83, 1.12 and 0.94 mg/ml, respectively. The Vmax of the purified enzyme was 2.1 micromol/min/mg with deglycosylated Cowper's gland mucin, as the glycosyl acceptor. However, the activities with peptides prepared from deglycosylated mucins by limited acid hydrolysis were 20-fold greater than the intact glycoprotein under identical conditions. The deglycosylated mucins and larger peptides aggregated with time of storage and precipitated from solution. Aggregation was accompanied by a corresponding loss of enzymatic activity even after dispersion of the aggregate by sonication. The deglycosylated mucins which were prepared by chemical treatment and periodate oxidation still contained about 20% of the N-acetylgalactosamine present in the intact mucin. When this residual amino sugar was removed by periodate oxidation the completely deglycosylated mucins became very poor substrates for the purified transferase. Data obtained in the current study indicate that the accessibility of serine and threonine in the polypeptide chains of mucin glycoproteins significantly influences the rate of glycosylation of these amino acids. The best substrates and affinity ligand for the enzyme were fragments of incompletely deglycosylated mucin polypeptide chains.

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The morphologic and functional properties of explant out-growth cells and epithelial cells isolated from swine trachea epithelium by proteolysis were examined. A mixed population of ciliated, serous, and basal cells, obtained from out-growths, from proteolysis of trachea epithelium, and from unattached explants in organ culture, all yielded cell cultures that werecomposed almost entirely of mucus-secreting cells. When the cells were grown in primary or secondary culture on a modified collagen matrix in supplemented HAM:DMEM (1:1) medium they expressed a mucus-secreting phenotype with numerous mucus granules at various stages of maturation and incorporated [3H]GlcN and 35SO4 into secreied mucin glycoproteins. Results obtained in these studies suggest that extensive transdifferentiation of ciliated and serous cells to mucus-secreting-cells occurs after the release and during subsequent attachment and culture. Ciliated cells containing mucus granules were seen in various stages of cilia resorption. Basal cells containing mucus granules were also frequently observed. The number of mucus-secreting cells and the synthesis of mucin glycoproteins increased dramatically with time of attachment and culture, whereas cell proliferation, population doubling time of 72 h, and incorporation of [3H]-thymidine into DNA increased much more slowly. The number of mucus-secreting cells correlated closely with the level of secretion of mucin glycoproteins. Taken collectively, these studies help to elucidate the transdifferentiation process, which dramatically increases the number of mucus-secreting cells after disruption and release of epithelial cells from swine tracheobronchial epithelium. A similar mechanism involving disruption of the extracellular matrix may be involved in the stimulation of hypersecretion of mucus and mucin glycoproteins by chemical and infections irritants.

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The structures of high molecular weight sulfated oligosaccharide chains in mucins purified from the sputum of a patient with cystic fibrosis and blood group H determinant were established. Reduced oligosaccharides released by treatment with alkaline borohydride were separated by ion exchange chromatography on DEAE-Agarose and a fraction containing multisulfated chains was further purified by lectin affinity chromatography to completely remove small amounts of sialylated chains. A major sulfated oligosaccharide fraction containing chains with an average of 160 to 200 sugar residues was isolated by gel filtration on BioGel P-10 columns and individual subfractions were characterized by methylation analysis, periodate oxidation and sequential glycosidase digestion before and after desulfation. Carbohydrate analysis yielded Fuc, Gal and GldNAc in a ratio of 1:2:2.1 and only one galactosaminitol residue for every 160- to 200 sugar residues. The average molecular weight of oligosaccharide chains in these fractions was between 27,000 and 40,000 daltons. Structural analysis showed that these high molecular weight chains contained varying amounts of the repeating unit shown in the following oligosaccharide. Only one in about every 10 repeating units contained sulfate esters. Several shorter chains which contain 2 to 3 sulfate esters were also isolated from this multisulfated oligosaccharide fraction. The structures proposed for these oligosaccharides indicate that they are lower molecular weight chains with the same general structure as those found in the high molecular weight sulfated oligosaccharides. Taken collectively, the results of these studies show that a major sulfated oligosaccharide fraction in respiratory mucin purified from the mucus of patients with cystic fibrosis contains high molecular weight branched chains that consist of a repeating oligosaccharide sequence with sulfate linked to the 6 positions of galactose and possibly GlcNAc residues in the side chains.

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Human respiratory mucin glycoproteins from patients with cystic fibrosis were purified and oligosaccharide chains were released by treatment with alkaline borohydride. A neutral oligosaccharide alditol fraction was isolated from mucin obtained from a patient with A blood group determinant by chromatography on DEAE-cellulose and individual oligosaccharide chains were then isolated by gel filtration on BioGel P-6 columns and high performance liquid chromatography with gradient and isocratic solvent systems. The structures of the purified oligosaccharides were determined by methylation analysis, sequential glycosidase digestion and 'H-NMR spectroscopy. The amount of each chain was determined by compositional analysis. A wide array of discrete branched oligosaccharide structures that contain from 3 to 22 sugar residues were found. Many of the oligosaccharides are related and appear to be precursors of larger chains. The predominant branched oligosaccharides which accumulate contain terminal blood group H (Fuc alpha 2Ga1 beta 4) or blood group A (Fuc alpha 2(Ga1NAc alpha 3) (Ga1 beta 4) determinants which stop further branching and chain elongation. The elongation of oligosaccharide chains in respiratory mucins occurs on the beta 3-linked G1cNAc at branch points, whereas the beta 6-linked G1cNAc residue ultimately forms short side chains with a Fuc alpha 2(Ga1NAc alpha 3) Ga1 beta 4 G1cNAc beta 6 structure in individuals with A blood group determinant. The results obtained in the current studies further suggest that even higher molecular weight oligosaccharide chains with analogous branched structures are present in some human respiratory mucin glycoproteins. Increasing numbers of the repeating sequence shown in the oligosaccharide below is present in the higher molecular weight chains. [formula: see text] This data in conjunction with our earlier observations on the extensive branching of these oligosaccharide chains helps to define and explain the enormous range of oligosaccharide structures found in human and swine respiratory mucin glycoproteins. Comparison of the relative concentrations of each oligosaccharide chain suggest that these oligosaccharides represent variations of a common branched core structure which may be terminated by the addition of alpha 2-linked fucose to the beta 3/4 linked galactose residue at each branch point. These chains accumulate and are found in the highest concentrations in these respiratory mucins.

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RNA was isolated from cultured swine trachea epithelial cells and mucus-secreting tumor cell lines from human pancreas, lung and colon by extraction with guanidine isothiocyanate. Poly(A)+mRNA rich fractions were purified by repeated chromatography on oligo (dT)-cellulose columns and they were translated in a cell-free rabbit reticulocyte system. Translation products labelled with 35S-methionine were isolated by immunoprecipitation with specific antibodies to the polypeptide chains of mucin glycoproteins and they were analyzed by SDS-PAGE and fluorography. A single principal polypeptide band of 67 kDa was found in all cases when the immunoprecipitates were washed with buffer containing bovine serum albumin and unlabeled deglycosylated mucin glycoprotein. The intensity of the 67 kDa band decreased when unlabeled deglycosylated mucin glycoprotein was added to the translation mixture before immunoprecipitation. Affinity purified monospecific antibodies elicited against chemically deglycosylated polypeptide chains of purified mucin glycoproteins from human and swine trachea and Cowper's gland were all equally effective in immunoprecipitating the 67 kDa translation product. Monospecific antibodies directed against the glycosylated and unglycosylated regions of the polypeptide chain yielded single bands with a molecular size of 67 kDa in each case. Peptide profiles obtained by digestion of the 67 kDa translation product with S. aureus V-8 protease were identical to those obtained with deglycosylated human and swine trachea mucin glycoproteins. These studies clearly demonstrate that the translation product of swine trachea and human lung, colon and pancreatic mucin glycoprotein gene is a single polypeptide chain of 67 kDa. The relative size and properties of the translation products synthesized with poly (A)+RNA isolated from mucus-secreting cells derived from three different tissues are similar to those of mucin glycoproteins purified directly from mucus secretions of human and swine trachea epithelium.

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Antibodies were raised in rabbits against purified swine and human trachea and Cowper's gland mucin glycoproteins and their deglycosylated polypeptide chains. Three monospecific antibody fractions that recognize the carbohydrate, the deglycosylated or unglycosylated regions of the polypeptide chains in these glycoproteins, were isolated by immunoaffinity chromatography. The human and swine trachea mucin glycoproteins showed extensive immunological homology in both their carbohydrate and polypeptide chains. The carbohydrate chains and unglycosylated region of the polypeptide chain in Cowper's gland mucin glycoprotein showed little or no cross-reaction with comparable regions in respiratory mucin glycoproteins. However, the polypeptide chains in the deglycosylated regions of all three mucin glycoproteins showed extensive homology. Five bands with molecular masses ranging from 40 to 46 kDa that differed by a constant molecular mass of approximately 1.5 kDa were detected in hydrolysates of all of the polypeptide chains after treatment with S. aureus V8 protease. Monospecific antibodies to the deglycosylated region of these chains reacted with the peptides, whereas those directed against the unglycosylated region did not. The results suggest that these chains contain tandem repeating sequences of amino acids.

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Two specific beta-N-acetylglucosaminyltransferases involved in the branching and elongation of mucin oligosaccharide chains, namely, a beta 1,6 N-acetylglucosaminylsaminyltransferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal beta 3GalNAc-mucin to yield Gal beta 3(GlcNAc beta 6)GalNAc-Mucin and a beta 3-N-acetylglucosaminyl transferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal beta 3(GlcNAc beta 6)GalNAc-mucin to yield GlcNAc beta 3Gal beta 3 (GlcNAc beta 6)GalNAc-Mucin were purified from the microsomal fraction of swine trachea epithelium. The beta 1,6-N-acetylglucosaminyltransferase was purified about 21,800-fold by procedures which included affinity chromatography on DEAE columns containing bound asialo Cowper's gland mucin glycoprotein with Gal beta 1,3GalNAc side chains. The apparent molecular weight estimated by gel filtration was found to be about 60 Kd. The purified enzyme showed a high specificity for Gal beta 1,3GalNAc chains and the most active substrates were mucin glycoproteins containing these chains. The apparent Km of the beta 6-glucosaminyltrans-ferase for Cowper's gland mucin glycoprotein containing Gal beta 1,3GalNAc chains was 0.53 microM; for UDP-N-acetylglucosamine, 12 microM; and for Gal beta 1,3GalNAc alpha NO2 phi, 4 mM. The activity of the beta 6-glucosaminyltransferase was dependent on the extent of glycosylation of the Gal beta 3GalNAc chains in Cowper's gland mucin glycoprotein. The best substrate for the partially purified beta 3-Glucosaminyltransferase was Cowper's gland mucin glycoprotein containing Gal beta 1,3(GlcNAc beta 6)GalNAc side chains. This enzyme showed little or no activity with intact sialylated Cowper's gland mucin glycoprotein or derivatives of this glycoprotein containing GalNAc or Gal beta 1,3GalNAc side chains. The radioactive oligosaccharides formed by these enzymes in large scale reaction mixtures were released from the mucin glycoproteins by treatment with alkaline borohydride, isolated by gel filtration on Bio-Gel P-6 and characterized by methylation analysis and sequential digestion with exoglycosidases. The oligosaccharide products formed by the beta 6- and beta 3-glucosaminyltransferases were shown to be Gal beta 3(GlcNAc beta 6) GalNAc and GlcNAc beta 3 Gal beta 3(GlcNAc beta 6)GalNAc respectively. Taken collectively, these results demonstrate that swine trachea epithelium contains two specific N-acetylglucosaminyltransferases which catalyze the initial branching and elongation reactions involved in the synthesis of O-linked oligosaccharide chains in respiratory mucin glycoproteins. The first enzyme a beta 6-glucosaminyltransferase converts Gal beta 3GalNAc chains in mucin glycoproteins to Gal beta 3(GlcNAc beta 6)GalNAc chains.(ABSTRACT TRUNCATED AT 400 WORDS)

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The oligosaccharide chains in human and swine trachea and Cowper's gland mucin glycoproteins were completely removed in order to examine the subunit structure and properties of the polypeptide chains of these glycoproteins. The carbohydrate, which constitutes more than 70% of these glycoproteins, was removed by two treatments with trifluoromethanesulfonic acid for 3 h at 3 degrees and periodate oxidation by a modified Smith degradation. All of the sialic acid, fucose, galactose, N-acetylglucosamine and N-acetylgalactosamine present in these glycoproteins was removed by these procedures. The deglycosylated polypeptide chains were purified and characterized. The size of the monomeric forms of all three polypeptide chains were very similar. Data obtained by gel filtration, release of amino acids during hydrolysis with carboxypeptidase B and gel electrophoresis in the presence of 0.1% dodecyl sulfate showed that a major fraction from each of the three mucin glycoproteins had a molecular size of about 67 kDa. All of the deglycosylated chains had a tendency to aggregate. Digestion with carboxypeptidases showed that human and swine trachea mucin glycoproteins had identical carboxyl terminal sequences, -Val-Ala-Phe-Tyr-Leu-Lys-Arg-COOH. Cowper's gland mucin glycoprotein had a similar carboxyl terminal sequence, -Val-Ala-Tyr-Leu-Phe-Arg-Arg-COOH. The yield of amino acids after long periods of hydrolysis with carboxypeptidases showed that at least 85% of the polypeptide chains in each of the deglycosylated preparations have these sequences. These results suggested that the polypeptide chains in these deglycosylated mucin glycoprotein preparations were relatively homogeneous. The deglycosylated polypeptide chains as well as the intact mucin glycoproteins had blocked amino terminii. The purified polypeptide chains were digested with trypsin-TCPK, and S. aureus V8 protease and the resulting peptides were isolated by gel electrophoresis in the presence of 0.1% dodecyl sulfate and by HPLC. Two partial amino acid sequences from swine trachea mucin glycoprotein, two partial sequences from human trachea mucin glycoprotein and three partial sequences from Cowper's gland mucin glycoprotein were determined. The partial amino acid sequences of the peptides isolated from swine trachea mucin glycoprotein showed more than 70% sequence homology to a repeating sequence present in porcine submaxillary mucin glycoprotein. Five to eight immunoprecipitable bands with sizes ranging from about 40 kDa to 46 kDa were seen when the polypeptide chains were digested with S. aureus V8 protease. All of the bands had blocked amino terminii and differed by a constant molecular weight of about 1.5 kDa.(ABSTRACT TRUNCATED AT 400 WORDS)

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Proximal tubule cells were isolated from swine kidney and cultured for periods of more than 30 days. The cells formed confluent monolayers after plating on a collagen surface and they were passaged more than 5 times on this matrix. The cells maintain several metabolic functions of proximal tubule cells, including gluconeogenesis and the ability to respond to epinephrine and parathyroid hormone. Gluconeogenesis, a principal metabolic pathway in proximal tubule cells, was examined as a function of days in culture. The isolated cells showed a nearly constant rate of gluconeogenesis from 14C-lactate, 14C-alanine and 14C-glycerol with no significant loss of activity for at least 30 days in culture. Likewise, the activities of several cytosolic and membrane associated enzymes including, alkaline phosphatase, delta-glutamyltransferase, fructose-1,6-bisphosphatase and phosphofructokinase were nearly constant over the same time period. The cells responded to treatment with epinephrine and parathyroid hormone, and the rate of gluconeogenesis from 14C-lactate doubled in the presence of these hormones. The morphological and biochemical evidence obtained in these studies show that the proximal tubule cells isolated from swine kidney provide an excellent well defined system for studying the hormonal regulation of carbohydrate metabolism in this tissue.

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The structures of the major sialylated oligosaccharide chains in swine tracheal mucin glycoprotein were established. The oligosaccharide chains were released by treatment with alkaline borohydride and isolated by gel filtration on Bio-Gel P6 columns and chromatography on DEAE-cellulose. The neutral oligosaccharide chains in this glycoprotein have been characterized in previous studies (Rana, S.S., Chandrasekaran, E.V., Kennedy, J., and Mendicino, J. (1984) J. Biol. Chem. 259, 12899-12907; Chandrasekaran, E.V., Rana, S.S., Davila, M., and Mendicino, J. (1984) J. Biol. Chem. 259, 12908-12914). The present study reports the isolation of four monosialylated chains ranging in length from 6 to 14 sugar units, two disialylated chains containing 6 and 12 sugar units, and one trisialylated chain containing 9 sugar units. The structure of the sialylated oligosaccharides was determined by permethylation analysis and sequential hydrolysis with specific exoglycosidases. The following structures (where GalNAcol is N-acetylgalactosaminitol) were assigned to these oligosaccharides.

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The structures of large O-glycosidically linked oligosaccharides derived from swine trachea mucin glycoprotein were established. Reduced oligosaccharides released by treatment with alkaline borohydride were separated by gel filtration on Bio-Gel P-6 and the neutral oligosaccharides were isolated by chromatography on DEAE-cellulose. Eight oligosaccharides (DIa to BII), ranging in length from 8 to 15 sugar units, were isolated. On the basis of carbohydrate composition and analytical data from sequential treatment with exoglycosidases and permethylation analysis, the following structures were assigned to these oligosaccharides: (Formula: see text).

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Mucin glycoproteins were purified from extracts of swine trachea mucosa and Cowper's gland. The gelatinous extracts were solubilized by reduction and carboxymethylation and then purified by chromatography on Sepharose CL-6B and DEAE-Sepharose. The structure of some of the carbohydrate units in these glycoproteins were determined and compared. Alkaline borohydride treatment indicated that more than 85% of the carbohydrate chains in these glycoproteins were linked to serine or threonine residues in the polypeptide chain through O-glycosidic bonds with N-acetylgalactosamine. Reduced oligosaccharides released by treatment with alkaline borohydride were isolated by gel filtration on Bio-Gel P-6 and chromatography on DEAE-cellulose and paper. The structures of the oligosaccharides were established by methylation analysis, gas chromatography, and sequential hydrolysis with specific exoglycosidases. The major oligosaccharides in Cowper's gland mucin glycoproteins were sialylated short chains: NeuAc alpha 2,6GalNAcol and NeuAc alpha 2,3Gal beta 1,3(NeuAc alpha 2,6)GalNAcol. In marked contrast, branched chains containing a Gal beta 1,3(GlcNAc beta 1,6)GalNAc core unit were the major components of trachea mucin glycoprotein. Ten of these chains had the following structures: (Formula: see text).

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Two specific N-acetylglucosaminyltransferases, alpha-1,3-mannoside:beta-2-N-acetylglucosaminyltransferase (transferase I) and alpha-1,6-mannoside:beta-2-N-acetylglucosaminyltransferase (transferase II), which catalyze the transfer of N-acetylglucosamine (GlcNAc) from uridine diphospho-GlcNAc to terminal branched alpha-mannosyl (Man) residues, were purified from liver metastases of human colon adenocarcinoma. Transferase I was assayed with Man alpha 1,6(Man alpha 1,3)Man alpha 1,6(Man alpha 1,3)Man beta 1,4GlcNAc beta 1, 4GlcNAc-Asn (Km 0.35 mM), and transferase II was assayed with Man alpha 1,6(GlcNAc beta 1,2Man alpha 1,3)Man beta 1,4GlcNAc beta 1,4-Glc-NAc-Asn (Km 1.0 mM), in which Asn is asparagine. The Km of transferase I for Man alpha 1,6(Man alpha 1,3)Man beta 1,4GlcNAc-beta 1,4)-(Fuc alpha 1,6)GlcNAc-Asn was 1 mM. The specificity of the interaction of transferase I with ovalbumin, ovomucoid, the modified heavy chain of porcine immunoglobulin G and glycopeptides prepared from these glycoproteins was examined by kinetic and structural analysis. The best macromolecular substrates for transferase I were ovalbumin devoid of terminal GlcNAc and some mannose, a solubilized preparation of the heavy chain of porcine immunoglobulin G, devoid of sialic acid, galactose, and terminal GlcNAc, and untreated ovomucoid. The apparent KmS were 45, 19, and 390 microM for ovalbumin, the modified heavy chain of immunoglobulin G, and untreated ovomucoid, respectively. The apparent Km of the enzyme for uridine diphospho-GlcNAc was not significantly influenced by the nature of the glycoprotein acceptor, and it varied between 14 and 20 microM for the different glycoproteins. The structures of the oligosaccharide chains in these glycoproteins which acted as acceptors for the purified enzyme were determined. A major glycopeptide product with the structure Man alpha 1,3(Man alpha 1,6)Man alpha 1,6(14C-GlcNAc beta 1,2Man-alpha 1,3)Man beta 1,4GlcNAc-beta 1,4-GlcNAc-Asn was isolated from both ovalbumin and ovomucoid following incubation with transferase I. The specificity of the enzyme for terminal branched mannosyl residues attached to a beta-linked mannose unit greatly restricts the action of this transferase to this juncture in the synthesis of complex-type oligosaccharide chains of N-asparagine-linked glycoproteins.(ABSTRACT TRUNCATED AT 400 WORDS)

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The influence of fructose 2,6-bisphosphate on the activation of purified swine kidney phosphofructokinase as a function of the concentration of fructose 6P, ATP and citrate was investigated. The purified enzyme was nearly completely inhibited in the presence of 2 mM ATP. The addition of 20 nM fructose 2,6-P2 reversed the inhibition and restored more than 80% of the activity. In the absence of fructose 2,6-P2 the reaction showed a sigmoidal dependence on fructose-6-phosphate. The addition of 10 nM fructose 2,6-bisphosphate decreased the K0.5 for fructose 6-phosphate from 3 mM to 0.4 mM in the presence of 1.5 mM ATP. These results clearly show that fructose 2,6-bisphosphate increases the affinity of the enzyme for fructose 6-phosphate and decreases the inhibitory effect of ATP. The extent of inhibition by citrate was also significantly decreased in the presence of fructose 2,6-phosphate. The influence of various effectors of phosphofructokinase on the binding of ATP and fructose 6-P to the enzyme was examined in gel filtration studies. It was found that kidney phosphofructokinase binds 5.6 moles of fructose 6-P per mole of enzyme, which corresponds to about one site per subunit of tetrameric enzyme. The KD for fructose 6-P was 13 microM and in the presence of 0.5 mM ATP it increased to 27 microM. The addition of 0.3 mM citrate also increased the KD for fructose 6-P to about 40 microM. AMP, 10 microM, decreased the KD to 5 microM and the addition of fructose 2,6-phosphate decreased the KD for fructose 6-P to 0.9 microM. The addition of these compounds did not effect the maximal amount of fructose 6-P bound to the enzyme, which indicated that the binding site for these compounds might be near, but was not identical to the fructose 6-P binding site. The enzyme bound a maximum of about 12.5 moles of ATP per mole, which corresponds to 3 moles per subunit. The KD of the site with the highest affinity for ATP was 4 microM, and it increased to 15 microM in the presence of fructose 2,6-bisphosphate. The addition of 50 microM fructose 1,6-bisphosphate increased the KD for ATP to 5.9 microM. AMP increased the KD to 5.9 microM whereas 0.3 mM citrate decreased the KD for ATP to about 2 microM.(ABSTRACT TRUNCATED AT 400 WORDS)

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Mucus-producing cells were isolated from swine trachea mucosa by a method that included enzymatic digestion of the epithelial surface with Dispase, a neutral protease from Bacillus polymyxa, and differential attachment of the washed cells to culture flasks coated with collagen. Epithelial cells were the major cell type isolated by these procedures. Ciliated cells that did not attach to the flasks were removed by decantation , and fibroblasts were destroyed by the bacterial protease. The isolated cells synthesized respiratory mucins and the rate of secretion was increased about threefold when tracheas were exposed to sulfur dioxide. The cultured cells incorporated both [35S]O4 and [I-14C]N-acetylglucosamine into secreted mucin glycoproteins. The secretion of glycoprotein increased for about 3 d until the cells became confluent, and then a constant rate was observed for a period of at least 7 d. This increase in the output of mucin glycoprotein during the initial 3 d of culture was accompanied by a corresponding increase in the number of mucus-producing cells in the flasks. The results obtained in these and subsequent studies suggest that the rate of formation of mucus-producing cells may be a rate limiting step in the regulation of mucin glycoprotein synthesis in tracheal epithelium. The chemical, physical, and immunological properties of the glycoprotein secreted by isolated tracheal epithelial cells were very similar to the mucin glycoprotein purified from washes of swine trachea epithelium. The purified mucin glycoproteins showed complete cross-reaction with antibodies to trachea mucin glycoprotein. They were eluted near the void volume during gel filtration of Sepharose CL-6B columns. The glycoprotein isolated from culture media under the standard assay conditions had nearly the same carbohydrate composition as samples purified from washes of trachea epithelium. Reduced oligosaccharides released by beta-elimination with dilute alkaline borohydride showed similar elution profiles during chromatography on Bio Gel P-6 columns. Taken collectively, these results suggest that the isolated epithelial cells secreted mucin glycoproteins that were very similar to those synthesized by the intact trachea epithelium under standard incubation conditions.

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Swine tracheal epithelium has been cultured as explants in a chemically defined medium for periods of up to 2 wk. The viability of the explants was shown by the preservation of the ultrastructural features of cells in the epithelial layer and by the active incorporation of radioactive glucosamine and sulfate into secreted mucin glycoproteins. The rate of secretion of mucin glycoprotein was about 0.035 mg per cm2 per d. After initial 24 h lag period was shown to be due to the equilibration of intracellular mucin glycoprotein pools with radioactive precursors. The rate of secretion of glycoprotein showed a linear dependence on the area of the explant, and maximal incorporation was observed at 200 microM glucosamine. A higher concentration of 35SO4, 1000 microM, was required for maximal incorporation of the precursor. Insulin at 0.1 to 1 microgram/ml increased the rate of secretion twofold, whereas 0.1 to 100 micrograms/ml of hydrocortisone and 0.1 to 100 micrograms/ml of epinephrine significantly decreased the rate of secretion. Vitamin A had little or no effect of normal trachea explants at low concentrations, and, at higher concentrations, 10(-5) M, it decreased the secretion of mucin glycoproteins. Vitamin A, at a concentration of 10(-9) M, increased the rate of synthesis of glycoprotein at least fourfold in trachea explants from vitamin A-deficient rats. Mucus secretions collected from the surface of swine trachea and from the culture medium of trachea explants were purified. The mucus was solubilized by reduction and carboxymethylation, and the high molecular weight mucin glycoproteins were purified by chromatography on Sepharose CL-6B columns under dissociating conditions in 2 M guanidine HCl. The mucin glycoproteins purified from swine trachea and from the culture medium of trachea explants were virtually indistinguishable. They showed the same properties when examined by gel electrophoresis and immunoprecipitation. The purified glycoproteins contained about 25% protein, and serine, threonine, and proline were the principal amino acids present. More than 80% of the carbohydride chains in both samples were released by treatment with alkaline borohydride. Nearly the same molar ratio of N-acetylgalactosamine, N-acetylglucosamine, galactose, fucose, sulfate, and sialic acid was found in both preparations.

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Two forms of alpha 1-acid glycoprotein with common immunological determinants and almost identical amino acid compositions but different amounts of carbohydrate were isolated from liver metastases of primary colon, lung, and breast tumors by extraction with perchloric acid, gel filtration on Sepharose CL-6B and Sephadex G-200, and affinity chromatography on concanavalin A:agarose and Ricinus communis agglutinin l:agarose. Both forms of the antigen yielded single bands which stained for protein and carbohydrate when examined by disc gel electrophoresis and immunodiffusion. The molecular weights of the two forms were 45,000 and 37,000 respectively. The larger form contained about five to six oligosaccharide chains, whereas the smaller form had only three to four chains. The composition and structures of the oligosaccharide chains in the two forms of this glycoprotein were very similar. Each contained di-, tri-, and tetraantennary complex-type oligosaccharide chains. The diantennary oligosaccharide chains caused both forms of alpha 1-acid glycoprotein to be retained by concanavalin A-agarose columns. The lower-molecular-weight form contained fewer chains and correspondingly fewer terminal galactosyl residues. This resulted in the separation of this species from the higher-molecular-weight form on columns containing R. communis agglutinin I. Three types of reduced oligosaccharides were released from the light and heavy forms of alpha 1-acid glycoprotein by treatment with alkaline borohydride or by hydrazinolysis. These chains were isolated by chromatography on concanavalin A:agarose and Bio-Gel P-6 columns. The arrangement and linkage of sugars in the purified oligosaccharides were determined by periodate oxidation, sequential hydrolysis with glycosidases, and methylation analysis. The major oligosaccharide chain, comprising 50 to 55% of the carbohydrate, had a triantennary structure as shown in the structure: (formula; see text) in which NeuNAc is N-acetylneuraminic acid, Gal is galactose, GlcNAc is N-acetylglucosamine, Man is mannose, GlcNAcol is N-acetylglucosaminitol, and Fuc is fucose. Tetraantennary chains comprised about 25 to 30% of the carbohydrate, and the additional outer chain was attached to the alpha 1,6-mannosyl residue through a beta 1,6-linked GlcNAc unit. The remaining 15 to 20% of the oligosaccharide chains had a diantennary structure. The extent of sialylation of these chains varied in samples isolated from tumors of the same histological type from different individuals. However, a relatively constant proportion of the three types of chains was present in different forms of the glycoprotein isolated from liver metastases.

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Phosphofructokinase (PFK) from swine kidney was purified by a procedure which included affinity chromatography on Cibacron blue F3GA-Sepharose 4B and ATP-Sepharose 4B columns in order to examine its binding properties. The homogeneous enzyme was purified more than 3000-fold with a yield of 30% and it had a specific activity of 39.8 mumol/min/mg of protein at 25 degrees C. The molecular weight of the native enzyme was 360 000 and it contained 4 identical subunits of molecular weight 88 000. The principal catalytically reacting form of the enzyme had a S20,w of 13.7 S which corresponds to a molecular weight of 360 000 +/- 6 000. The initial velocity patterns in the forward and reverse directions suggested a sequential mechanism for the reaction. The Km values for fructose 6-phosphate, ATP, fructose, 1,6-bisP and ADP were 33 microM, 8.3 microM, 460 microM and 110 microM, respectively. The homogeneous native enzyme binds specifically to phosphoryl groups immobilized in cellulose phosphate columns. ATP and fructose 6-phosphate interacted with the enzyme and decreased its affinity for phosphoryl binding sites. Other metabolites including fructose 1,6-bisP, glucose 6-phosphate and various nucleotides, alone or in various combinations, were ineffective in promoting the dissociation of the enzyme. Allosteric effectors of the enzyme, such as citrate and AMP were also inactive. However, the cooperatively altered the concentration of ATP required to dissociate the enzyme from phosphoryl groups. The bound enzyme was enzymatically inactive. The enzyme was also inactivated when it was treated with pyridoxal 5'-phosphate and reduced with sodium borohydride and the inactive enzyme no longer bound to cellulose phosphate. These effects were not observed when treatment with pyridoxal 5'-phosphate was carried out in the presence of fructose 6-phosphate. These observations and the results of similar studies with swine kidney fructose 1,6-bisphosphatase (FBPase) show that both enzymes share the unique property of binding specifically to phosphoryl groups. FBPase interacts through its allosteric AMP binding site and PFK binds through its fructose 6-P binding site. This specific binding of both enzymes through these sites result in the inactivation of PFK and the desensitization of FBPase to allosteric inhibition by AMP. In the unbound state PFK may be active and FBPase can be inhibited by AMP. Taken collectively, these binding effects could play a role in the reciprocal regulation of these enzymes during gluconeogenesis in kidney.

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