Structural studies on dissociated porcine gastric mucus gel

Reduction of the insoluble porcine mucus gel with dithiothreitol, followed by molecular sieve chromatography, resulted in the recovery of the two fragments of molecular size 2.54 X 10(6) and 1.75 X 10(6). Both gastric mucus glycoproteins have been cleaved by pronase, papain or trypsin into fragments whose molecular sizes range from 5.60 X 10(5) to 1.87 X 10(6) daltons. On reduction, the enzyme-treated fragments were further dissociated into smaller fragments which have been characterized by SDS-PAGE. The evidence indicates that the pol: neric structure of gastric mucus differs from that of mucus of varying origins, in particular with respect to the size and number of component subunits per native molecule.     

Characterization of gastric mucoproteins isolated by equilibrium density-gradient centrifugation in caesium chloride

1. The mucoprotein from pig gastric mucus has been purified by equilibrium centrifugation in a CsCl gradient. 2. This procedure removes the non-covalently bound protein, which is closely associated with the mucoprotein and not easily removed from it by gel filtration. 3. The purified mucoprotein is separable by gel filtration into a high-molecular-weight mucoprotein A (mol.wt. 2.3×10⁶) and a low-molecular-weight mucoprotein B/C (mol.wt. 1.15×10⁶). 4. These two mucoproteins have the same chemical analysis namely fucose 11.3%, galactose 26%, glucosamine 19.5%, galactosamine 8.3% and protein 13.6%. 5. Mucoprotein A contains 3.1% ester sulphate. 6. These mucoproteins are isolated without enzymic digestion and have a higher protein content than the blood-group-substance mucoproteins from proteolytic digestion of gastric mucus. Detailed amino acid analysis shows that the extra protein in the non-enzymically digested material is composed of amino acids other than serine and threonine. 7. Mucoproteins A and B/C contain respectively 130 and 9 half-cystine residues per molecule of which about 78 and 6 residues are involved in disulphide linkages. 8. Cleavage of these disulphide linkages by mercaptoethanol splits both mucoproteins into four equally sized subunits of mol.wt. 5.2×10⁵ for mucoprotein A and 2.8×10⁴ for mucoprotein B/C. 9. The sole N-terminal amino acid of mucoprotein A is aspartic acid, whereas mucoprotein B/C has several different N-terminal amino acid residues.     

Polymeric structure of pig small-intestinal mucus glycoprotein. Dissociation by proteolysis or by reduction of disulphide bridges.

Pig small-intestinal mucus glycoprotein, of molecular weight 1.72 X 10(6), is cleaved by Pronase digestion into glycoprotein subunits of molecular weight 4.5 X 10(5). Of the protein component of the native glycoprotein 29% by weight was lost on Pronase digestion, with no loss of carbohydrate. The non-glycosylated region of the protein that was lost with proteolytic digestion had a broad spectrum of amino acid residues, in contrast with the glycosylated region of the protein, which was resistant to proteolysis and was rich in serine, threonine and proline residues. Reduction with 0.2M-mercaptoethanol dissociated the Pronase-digested glycoprotein subunits into smaller glycoprotein subunits of molecular weight 2.7 X 10(5). On reduction, the native glycoprotein was dissociated into subunits of molecular weight 2.4 X 10(5), a similar size to those obtained from reduction of the Pronase-digested glycoprotein. On reductive dissociation of the native glycoprotein, in addition to glycoprotein subunits, protein was also released principally as a component of 90000 molecular weight. This protein was separated quantitatively from the reduced glycoprotein in amounts compatible with one 90000-mol.wt. protein molecule per 1.72 X 10(6)-mol.wt. native glycoprotein molecule. No 90000-mol.wt. protein was released on reduction of the isolated Pronase-digested glycoprotein. Pig small-intestinal mucus glycoprotein is therefore a covalent polymer of glycoprotein subunits joined by disulphide bridges. This polymeric structure differs in important respects from that previously shown for gastric mucus, in particular with respect to the size and number of component subunits per native molecule.     

Conformational changes in gastric mucoproteins induced by caesium chloride and guanidinium chloride

1. Caesium chloride and guanidinium chloride were shown to cause conformational changes in the high-molecular-weight mucoprotein A of water-soluble gastric mucus with no change in molecular weight. 2. Increasing concentrations of CsCl decrease the viscosity of the mucoprotein bringing about a transition which is essentially complete in 0.1m-CsCl. The shear-dependence of viscosity of the mucoprotein is abolished by low concentrations of CsCl. The normally highly expanded molecule becomes contracted in CsCl to a molecule having the same symmetry but a smaller volume and decreased solvation, in keeping with an increased sedimentation coefficient (18.7S-->33S). 3. This contracted form does not revert to the native conformation on removal of the CsCl. 4. A mechanism is discussed in terms of the effect of the Cs(+) and Cl(-)ions on water structure and the water-mucoprotein interaction. 5. Guanidinium chloride causes the CsCl-treated material to expand, in keeping with a decrease in s(0) (25,w) (33S-->26S). This is analogous to the known unfolding effect of guanidinium chloride on proteins and suggests that guanidinium chloride solubilizes groups involved in stabilizing the contracted structure. Removal of the guanidinium chloride results in a limited aggregation of four mucoprotein molecules. 6. These results show that caution must be exercised before interpreting the physical properties of mucoproteins which have been treated with CsCl and/or guanidinium chloride.     

The isolation and characterization of the high-molecular-weight glycoprotein from pig colonic mucus.

1. A high-molecular-weight glycoprotein constitutes over 80% by weight of the total glycoprotein from water-soluble pig colonic mucus. 2. It was isolated from from nucleic acid and non-covalently bound protein by nuclease digestion followed by equilibrium centrifugation in a CsCl gradient. 3. The glycoprotein has the following composition by weight: fucose 10.4%; glucosamine 23.9%; galactosamine 8.3%; sialic acid 9.9%; galactose 20.8%; sulphate 3.0%; protein 13.3%; moisture about 10%. 4. The native glycoprotein has the high mol.wt. of 15 X 10(6). 5. Reduction of the native glycoprotein with 2-mercaptoethanol results in a glycoprotein of mol.wt. 6 X 10(6). 6. Pronase digestion removes 29% of the protein (3% of the glycoprotein) but none of the carbohydrate. 7. The molecular weight of the Pronase-digested glycoprotein is 1.5 X 10(6), which is halved to 0.76 X 10(6) on reduction with 2-mercaptoethanol. 8. The contribution of non-covalent interactions, disulphide bridges and the non-glycosylated peptide core to the quaternary structure of the glycoprotein are discussed and compared with the known structure of pig gastric glycoportein.     

Preparation of 32P-labeled murine immune interferon and its binding to the mouse immune interferon receptor

Murine immune interferon (Mu-IFN-gamma) can be radiolabeled with [gamma-32P]ATP by the catalytic subunit of cAMP-dependent protein kinase. The resulting 32P-labeled Mu-IFN-gamma (32P-Mu-IFN-gamma) with high radiological specific activity (60-260 muCi/micrograms) retains biological activity. Acid hydrolysis of 32P-Mu-IFN-gamma or 32P-labeled human IFN-gamma leads to the release of [32P]phosphoserine but not phosphothreonine or phosphotyrosine. With 32P-Mu-IFN-gamma, we have demonstrated that there are 5 X 10(3) to 1.5 X 10(4) receptors per-cell on several murine cell lines of diverse origin and that the Kd at 24 degrees C for these cells is in the range of 1 X 10(-10) to 1 X 10(-9) M. Covalent binding of 32P-Mu-IFN-gamma to its receptor results in the formation of several specific high-molecular weight products, the major one of which has an apparent molecular weight of 90,000-100,000. If this represents a 1:1 complex of Mu-IFN-gamma and its receptor (or its binding subunit), the murine interferon gamma receptor has a molecular weight of 75,000-85,000.     

Studies on gastric mucoproteins. The isolation and characterization of the mucoprotein of the water-soluble mucus from pig cardiac gastric mucosa

1. Gel filtration of the water-soluble radioactive mucus produced three radioactive fractions, fraction A excluded on Sepharose 4B, fraction B included on Sepharose 4B but excluded on Sephadex G-200, and fraction C included on Sephadex G-200. 2. The specific radioactivities of fractions A and B were the same, with fraction C a little lower, whether the material was labelled with (14)C-labelled carbohydrate or with (3)H-labelled protein prepared by incubation of mucosal scrapings in vitro with [U-(14)C]glucose or [G-(3)H]threonine respectively. 3. Fractions A and B had an analysis of protein 22%, hexose 28%, hexosamine 28%, fucose 10% and sialic acid 1%; fraction C had an analysis closely similar to this, except that it contained about 10% of a protein contaminant. 4. All three fractions had closely similar A and H blood-group activities. 5. Ultracentrifuge studies showed fractions A, B and C were polydisperse with s(0) (25,w) values of 18.7S, 4.9S and 3.9S respectively. 6. The unfractionated water-soluble mucus contained only two peaks, fraction A 18.7S and a peak of 4.4S, which was a combination of fractions B and C. 7. The radioactive mucoprotein accounted for 85% by weight of the soluble mucus and the results show that it consisted of two distinct fractions A and B-C, which were chemically, biosynthetically and immunologically very similar.     

L-Glutamate-glyoxylate aminotransferase in Lactobacillus plantarum.

Glutamate-glyoxylate aminotransferase which mediates the reaction of glyoxylic acid with glutamic acid to yield glycine and alpha-oxoglutaric acid has been isolated and purified 84-fold from extracts of Lactobacillus plantarum. Purified enzyme requires the addition of pyridoxal phosphate and magnesium ions for its activity. The molecular weight of the enzyme estimated by Sepharose 4B gel filtration amounts to 37.000. Micaelis constants for glyoxylate and glutamate are corresponding to 6.25 X 10(-3) M and 2.75 X 10(-3) M, respectively. Optimal pH in phosphate and veronal buffers is 8.0 and optimal temperature 35--37 degrees C.     

Studies on gastric mucoproteins. The production of radioactive mucoproteins by pig gastric mucosal scrapings in vitro

1. Optimum conditions, including the effect of media of different pH values, were determined for the incorporation of radioactive precursors into mucoproteins by pig gastric mucosa in vitro. 2. Mucosal scrapings incorporated radioactivity from [U-¹⁴C]-glucose and from [G-³H]threonine or [G-³H]serine solely into the carbohydrate and protein portions respectively of the mucoprotein molecules. 3. Of the radioactive mucoprotein 22% was water-soluble and up to 80% of the remainder was soluble in other solvents. 4. Pronase was the most successful proteolytic enzyme tested for making the mucoprotein water-soluble, up to 94% dissolving after digestion. 5. The Pronase digestion products of the mucoproteins were separated from protein by equilibrium-density-gradient centrifugation in a CsCl gradient. 6. These Pronase-digested mucoproteins were further fractionated on Sepharose 4B and the isolated fractions analysed by chemical and sedimentation-velocity methods. 7. Pronase digestion and solvent extraction of mucosal scrapings labelled with ¹⁴C in the carbohydrate and ³H in the protein showed that one type of mucoprotein was the only non-diffusible biosynthetic product of the scrapings in vitro, and that this mucoprotein was the only mucoprotein constituent of the water-soluble and water-insoluble mucus.     

Characterization of mucus glycoprotein fatty acyltransferase from gastric mucosa

A fatty acyltransferase activity which catalyzes the transfer of palmitic acid from palmitoyl coenzyme A to gastric mucus glycoprotein has been demonstrated in the rat gastric mucosa. Subcellular fractionation studies revealed that the enzyme activity was present in a Golgi-rich membrane fraction. Optimum enzymatic activity for acylation of mucus glycoprotein was obtained with 0.5% Triton X-100, 25 mM NaF, and 2 mM dithiothreitol at a pH of 7.4. The enzymatic activity increased proportionally, over a given range, with increased concentrations of both substrates and of enzyme. The apparent Km of the enzymes for the undegraded mucus glycoprotein was 4.5 X 10(-7) M and for palmitoyl-CoA, 3.8 X 10(-5) M. The 14C-labeled product of the reaction cochromatographed on Bio-Gel A-50 column and migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with gastric mucus glycoprotein. Treatment of this 14C-labeled glycoprotein with mild alkali released hexane-extractable product which was identified as [14C]palmitate. The enzyme was also capable of fatty acylation of the deglycosylated glycoprotein, but did not catalyze the transfer of palmitic acid to the proteolytically degraded mucus glycoprotein. This indicates that the acceptor site for fatty acyltransferase is situated in the protease-susceptible nonglycosylated region of the mucus glycoprotein polymer.     

Identification of virus-coded nonstructural polypeptides in bunyavirus-infected cells.

Analyses of bunyavirus-infected cell extracts identified at least two virus-induced nonstructural polypeptides. With snowshoe hare (SSH), La Crosse (LAC), and six SSH-LAC reassortant viruses, it was shown that one of these nonstructural polypeptides (NSs, approximate molecular weight, 7.4 X 10(3)) is coded by the SSH small (S)-size viral RNA species. This nonstructural polypeptide was not detected (at least in the same relative abundancies) in LAC virus-infected cells or in cells infected with reassortants having LAC S RNA. For SSH virus, tryptic peptide analyses of either [3H]leucine- or [3H]arginine-labeled NSs indicated that it contains unique sequences not present in the SSH nucleocapsid (N) polypeptide (also coded by the S RNA; J. R. Gentsch and D. H. L. Bishop, J. Virol. 28:417-419, 1978). Analyses of SSH virus-infected cell extracts and extracts of cells infected with SSH-LAC reassortants having SSH medium (M)-size RNA species indicated that a nonstructural polypeptide (NSM; approximate molecular weight, 12 X 10(3)) is coded by the SSH M RNA species. In extracts of LAC virus-infected cells (or cells infected with SSH-LAC reassortants having LAC M RNA), a polypeptide with an electrophoretic mobility slightly faster than that of the SSH NSM polypeptide was observed (approximate molecular weight, 11 X 10(3)); it has been designated LAC NSM. The relationships of the NSM polypeptides to the other M RNA-coded polypeptides (G1 and G2; J. R. Gentsch and D. H. L. Bishop, J. Virol. 30;767-770, 1979) have not been determined. Two additional polypeptides present in both LAC- and SSH-infected cell extracts also appear to be virus induced (one with an approximate molecular weight of 10 X 10(3), p10; the other with an approximate molecular weight of 18 X 10(3), p18). Whether these polypeptides are virus coded has not been determined.     

Viscosity and molecular weight of hyaluronic acids.

The intrinsic viscosity ([eta]) and the molecular weight (M) by sedimentation equilibrium were determined for hyaluronic acids of low (M=104--7.2X10(4)) and high (M=3.1X10(5)--1.5X10(6)) molecular weights. Double logarithmic plot of [eta] against M gave different lines for the two groups. The relationship between [eta] and M was [eta]=3.0X10(6)XM1,20 for the former and [eta]=5.7X10(-4)XM0.46 for the latter group. The molecular weight at the point of intersection of the two lines was about 1.5X10(5). The rheological behavior of the hyaluronic acids below M=2.1X10(4), for which the value of reduced viscosity was independent of concentration, was different from that of the hyaluronic acids above M=5.1X10(4), for which the value of reduced viscosity increased with concentration.     

Purification and properties of L-glutaminase-L-asparaginase from Pseudomonas acidovorans.

An enzyme that catalyzes the hydrolysis of both glutamine and asparagine has been purified to homogeneity from extracts of Pseudomonas acidovorans. The enzyme having a ratio of glutaminase to asparaginase of 1.45:1.0 can be purified by a relatively simple procedure and is stable upon storage. The glutaminase-asparaginase has a relatively high affinity for L-asparagine (Km=1.5 X 10(-5) M) and L-glutamine (Km=2.2 X 10(-5) M) and has a molecular weight of approximately 156,000 the subunit molecular weight being approximately 39,000. Injections of the enzyme produced only slight increases in the survival time of C3H/HE mice carrying the asparagine-requiring 6C2HED Gardner lymphoma and of white Swiss mice carrying the glutamine-requiring Ehrlich lymphoma.     

The thermal depolymerization of porcine submaxillary mucin

The time dependence of the molecular weight, radius of gyration, and hydrodynamic size distribution for porcine submaxillary mucin (PSM) in solution have been studied using static and dynamic light scattering. The weight average molecular weight (Mw) of PSM in 6 M guanidine HCl, pH 7, is initially 3 X 10(6) and decreases with time in three phases: rapidly from 3-2 X 10(6), less rapidly from 2-0.9 X 10(6), and slowly below 0.9 X 10(6). The rates of decrease are much greater at pH 2. The energy of activation associated with each phase is 20 kcal/mol, which is similar to that reported for peptide bond cleavage at an aspartic acid residue. Addition of mercaptoethanol to PSM in 6 M guanidine HCl leads to a rapid decrease in Mw to 0.9 X 10(6), followed by a very slow further decrease. These results suggest that native PSM consists of subunits (Mw = 0.9 X 10(6] that are linked by disulfide bonds to form dimers (Mw = 2 X 10(6] and then higher aggregates. This cross-linking appears to occur at unglycosylated regions of the protein core, which are believed to be richer in aspartic acid than the rest of the molecule.     

Structure, subunit composition, and molecular weight of RD-114 RNA.

The properties and subunit composition of the RNA extracted from RD-114 virions have been studied. The RNA extracted from the virion has a sedimentation coefficient of 52S in a nondenaturing aqueous electrolyte. The estimated molecular weight by sedimentation in nondenaturing and weakly denaturing media is in the range 5.7 X 10(6) to 7.0 X 10(6). By electron microscopy, under moderately denaturing conditions, the 52S molecule is seen to be an extended single strand with a contour length of about 4.0 mum corresponding to a molecular weight of 5.74 X 10(6). It contains two characteristic secondary structure features: (i) a central Y- or T-shaped structure (the rabbit ears) with a molecular weight of 0.3 X 10(6), (ii) two symmetreically disposed loops on each side of and at equal distance from the center. The 52S molecule consists of two half-size molecules, with molecular weight 2.8 X 10(6), joined together within the central rabbit ears feature. Melting of the rabbit ears with concomitant dissociation of the 52S molecule into subunits, has been caused by either one of two strongly denaturing treatments: incubation in a mixture of CH3HgOH and glyoxal at room temperature, or thermal dissociation in a urea-formamide solvent. When half-size molecules are quenched from denaturing temperatures, a new off-center secondary structure feature termed the branch-like structure is seen. The dissociation behavior of the 52S complex and the molecular weight of the subunits have been confirmed by gel electrophoresis studies. The loop structures melt at fairly low temperatures; the dissociation of the 52S molecule into its two subunits occurs at a higher temperature corresponding to a base composition of about 63% guanosine plus cytosine. Polyadenylic acid mapping by electron microscopy shows that the 52S molecule contains two polyadenylic acid segments, one at each end. It thus appears that 52S RD-114 RNA consists of two 2.8 X 10(6) dalton subunits, each with a characteristic secondary structure loop, and joined at the 5' ends to form the rabbit ears secondary structure feature. The observations are consistent with but do not require the conclusion that the two 2.8 X 10(6) dalton subunits of 52S RD-114 RNA are identical.     

Remarkable substrate-inhibitor properties of nicotine enantiomers towards a guinea pig lung aromatic azaheterocycle N-methyltransferase

The kinetics of nicotine methylation by guinea pig lung homogenates has been investigated. An interesting stereospecificity has been observed for nicotine enantiomers. R-(+)-Nicotine is a substrate Km = 1.42 X 10(-5)M for an SAM-dependent guinea pig lung aromatic azaheterocycle N-methyltransferase, whereas S-(-)-nicotine acts as a competitive inhibitor (Ki = 6.25 X 10(-5)M) of the N-methylation of its antipode.     

A Salmonella typhimurium endonuclease that converts native DNA to fragments of about 8 X 10(5) daltons.

Crude extracts of Salmonella typhimurium were found to contain an endonuclease that degraded double-stranded linear DNA from bacteria and phages to fragments with a molecular weight of about 8 X 10(5). The nuclease did not have an absolute requirement for Mg2+. One discrete intermediate product had a molecular weight of 6-6 X 10(6). Extracts from two different mutants were tested: one completely lacked the endonuclease activity (strain DB5575), and the other showed an absolute requirement for Mg2+ (strain 4543). No biological role has yet been found for this endonuclease of S. typhimurium.     

Two types of rat gastric mucus glycoprotein subunits

Gastric mucus glycoproteins were extracted with 2% Triton X-100 from rat gastric corpus and antrum and purified by CsCl equilibrium centrifugation. Corpus mucus glycoproteins were degraded into what appeared to be two "subunits" (Mw 4.4 x 10(5) and 6 x 10(6)) by the reduction of disulfide bonds. Papain digestion of the latter produced glycopeptides with a molecular weight of approximately 4.4 x 10(5). This type of subunit had carbohydrate chains with about 9 sugars attached to every 2 amino acid residues. Papain digestion of the former type of subunit revealed no change in the elution profile on Bio-Gel A-15m. This type of subunit had carbohydrate chains with 17-19 sugars attached to every 3 amino acid residues. The subunit of antral mucus glycoproteins was essentially the same as the former type of corpus subunits in molecular weight (Mw 4.4 x 10(5)) and average oligosaccharide chain length. These results suggest that there are two distinct types of mucus glycoprotein subunits in rat stomach.     

Purification and properties of pig liver kynureninase.

Kynureninase [L-kynurenine hydrolase, EC 3.7.1.3] was purified from pig liver by a procedure including DEAE-cellulose chromatography, hydroxyapatite chromatography, ammonium sulfate fractionation, DEAE-Bio Gel chromatography, Sephacryl S-200 gel filtration, kynurenine-Sepharose affinity chromatography, and Sephadex G-200 gel filtration. The enzyme was found to be homogeneous by the criterion of disc-gel electrophoresis. The enzyme has a molecular weight of about 100,000 and exhibits absorption maxima at 280 and 420 nm. The optimum pH and the isoelectric point of the enzyme are 8.5 and 5.0, respectively. The Michaelis constants were determined to be as follows: L-kynurenine, 7.7 X 10(-4) M; L-3-hydroxykynurenine, 1.3 X 10(-5) M; and pyridoxal 5'-phosphate, 1.8 X 10(-6) M. L-3-Hydroxykynurenine is hydrolyzed more rapidly than L-kynurenine; the liver enzyme can be regarded as a 3-hydroxy-kynureninase.     

Radiolysis of chromatin extracted from cultured mammalian cells: production of alkali-labile strand damage in DNA.

Chromatin has been isolated from cultured Chinese-hamster lung fibroblasts as an expanded aqueous gel. The DNA in isolated chromatin has been examined by sedimentation on alkaline sucrose gradients. The average molecular weight of the DNA has been determined to be 50 million. gamma-irradiation of isolated chromatin degrades the DNA to lower molecular weight. The yield of single-strand breaks in the DNA is 0.02 single-strand breaks per krad-10(6) dalton, calculated from a dose-range of &--400 krad and covering a DNA molecular weight range of 2 X 10(7)-1.4 X 10(5). There is a considerable difference in the efficiency of the formation of single-strand breaks in DNA irradiated as isolated chromatin compared with chromatin irradiated in whole cells before isolation. For isolated chromatin, values of 6 dV per break have been calculated compared with about 80 eV per break for chromatin irradiated in whole cells, which suggest a large contribution from indirect action by aqueous radicals in isolated chromatin.