Characterization of peptic inhibitory activity associated with sulfated glycoprotein isolated from gastric mucosa.

Sulfated glycoproteins were extracted and purified from porcine stomach mucous scraping. Four sulfated glycoprotein fractions were separated and subsequently purified. These compounds always accompanied the apparent peptic inhibitory activity and consisted of 15-18% (w/w) protein. The carbohydrate portions contained an equimolar ratio of galactose and hexosamine (mainly glucosamine), together with lesser amounts of fucose and sialic acid. The sulfate content of the above fractions was 2-9% (w/w) of the total sulfated glycoprotein. The mode of inhibition of the sulfated glycoproteins to peptic activity was investigated and suggested that there was binding of the sulfated glycoproteins to the substrate of pepsin, making the substrate resistant to peptic activity. The sulfated glycoproteins neither bound pepsin at pH 1.8 nor inhibited the hydrolysis of a synthetic dipeptide substrate of pepsin. Desulfation of the sulfated glycoproteins resulted in the loss of both the inhibitory activity and the precipitate formation. The precipitation curve for sulfated glycoprotein and porcine serum albumin showed that both bound in varying proportions and suggests that both components are multivalent in this precipitate formation.     

Effect of pepsin on partially purified pig gastric mucus and purified mucin

Partially purified native-pig gastric mucus and purified pig gastric mucin, prepared by column chromatography and caesium chloride (CsCl) density-gradient ultracentrifugation, were subjected to pepsin digestion. The products of peptic digestion were chromatographed on Sepharose CL-2B, and fractions were assayed for carbohydrate by the periodic acid-Schiff reaction. The polymeric gastric mucin in the purified mucin samples was readily degraded by pepsin. In sharp contrast, the polymeric mucin in the partially purified mucus was relatively resistant to pepsin digestion. In 45 min, pepsin degraded 40% of the polymeric mucin in the purified samples, whereas it produced no significant degradation (less than 10%) in the partially purified mucus samples. In partially purified gastric mucus, treated with CsCl but not fractionated by ultracentrifugation, digestion with pepsin was also slow and incomplete. This showed that differences in susceptibility between partially purified and purified preparations are not due to the chaotropic effects of CsCl. In addition, the recombination of low-density nonmucin fractions in CsCl ultracentrifugation with the mucin also resisted pepsin digestion. Finally, we have shown that the low-density fractions in mucus exhibited a strong inhibitory effect of peptic activity in vitro. We conclude that under our experimental conditions, pepsin has little effect on partially purified mucus, and our findings indicate an inhibitor of peptic digestion is present in native gastric mucus. It is likely, but unproven, that this inhibitor is a noncovalently bound lipid present in the low-density fraction.     

Purificaton and characterization of a pepsinogen and its pepsin from proventriculus of the Japanese quail

A crude extract of the proventriculus of the Japanese quail gave at least five bands of peptic activity at pH 2.2 on polyacrylamide gel electrophoresis. The main component, constituting about 40% of the total acid protease activity, was purified to homogeneity by hydroxyapatite and DEAE-Sepharose column chromatographies. At below pH 4.0, the pepsinogen was converted to a pepsin, which had the same electrophoretic mobility as one of the five bands of peptic activity present in the crude extract. The molecular weights of the pepsinogen and the pepsin were 40 000 and 36 000, respectively. Quail pepsin was stable in alkali up to pH 8.5. The optimal pH of the pepsin on hemoglobin was pH 3.0. The pepsin had about half the milk-clotting activity of purified porcine pepsin, but the pepsinogen itself had no activity. The hydrolytic activity of quail pepsin on N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine was about 1% of that of porcine pepsin. Among the various protease inhibitors tested, only pepstatin inhibited the proteolytic activity of the pepsin. The amino acid composition of quail pepsinogen was found to be rather similar to that of chick pepsinogen C, and these two pepsinogens possessed common antigenicity.     

Catalytic properties and chemical composition of pepsins from Atlantic cod (Gadus morhua)

1. Three pepsins were purified from the gastric mucosa of Atlantic cod (Gadus morhua). 2. The enzymes, called Pepsin I and Pepsin IIa and b, had isoelectric points 6.9, 4.0 and 4.1, respectively, and digested hemoglobin at a maximal rate at a pH of approximately 3. 3. They resembled bovine cathepsin D in being unable to digest the mammalian pepsin substrate N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine. 4. Specificity constants (kcat/Km) for the cod pepsins were lower than for porcine pepsin, and they expressed higher substrate affinity and physiological efficiency at pH 3.5 than at pH 2. 5. The cod pepsins are glycoproteins, and their amino acid composition resembles that of porcine cathepsin D more than that of porcine pepsin. 6. The N-terminal sequence of Atlantic cod pepsins is substantially different from that of porcine pepsin. This indicates a significant evolutionary gap between fish and mammalian pepsins.     

Mucus glycoprotein of human saliva: differences in the associated and covalently bound lipids with caries

A high molecular weigh mucus glycoprotein has been isolated from submandibular saliva of caries-resistant and caries-susceptible individual by a procedure involving fractionation on Bio-Gel P-100 and A-50 columns followed by equilibrium density-gradient centrifugation in CsCl. The purified caries-resistant mucus glycoprotein displayed a buoyant density of 1.50 and accounted for 9.5% of the dry weight of caries-resistant saliva. The caries-susceptible mucus glycoprotein representd 14.1% of the dry weight of caries-susceptible saliva and gave a buoyant density of 1.43. Both glycoproteins exhibited similar protein and carbohydrate content, but the caries-resistant mucus glycoprotein contained 28.7% less associated lipids and 3-times less covalently bound fatty acids than the caries-susceptible mucus glycoprotein. The associated lipids were represented by neutral lipids, glycolipids and phospholipids, whereas the covalently bound fatty acids consisted mainly of hexadecanoate, octadecanoate and docosanoate. Extraction of associated lipids caused the caries-resistant glycoprotein to band in CsCl gradient at the density of 1.54 and caused the caries-susceptible glycoprotein to band at the density of 1.52. A further shift in the buoyant densities occurred following removal of the covalently bound fatty acids, and both glycoproteins banded at the density of 1.57. While the intact caries-resistant and caries-susceptibel glycoproteins were susceptible to proteolysis by pronase, the lipid-rich caries-susceptible glycoprotein was degraded to a lesser extent. Extraction of associated lipids increased the degradation of both glycoproteins, but the caries-susceptible glycoprotein still remained 25% less susceptible. However, the susceptibility to pronase of the delipidated and deacylated caries-resistant and caries-susceptible glycoproteins was essentially identical. The caries-resistant and caries-susceptible mucus glycoproteins also differed in susceptibility to peptic degradation. The apparent K_m values for intact caries-resistant and caries-susceptible glycoproteins were 10.5 · 10⁻⁷ M and 8.1 · 10⁻⁷ M, while the values for the delipidated and deacylated caries-resistant and caries-susceptible glycoproteins were 13.0 · 10⁻⁷ M and 12.4 · 10⁻⁷ M. The results suggest that the differences in the content of associated lipids and covalently bound fatty acids are responsible for the different physicochemical characteristics of caries-resistant and caries-susceptible salivary mucus glycoproteins, which may be determining falctors in the resistance to caries.     

Age-related changes in chemical composition and physical properties of mucus glycoproteins from rat small intestine.

Mucus glycoproteins from newborn and adult rat small intestine were radiolabelled in vivo with Na2 35SO4 and isolated from mucosal homogenates by using Sepharose 4B column chromatography followed by CsCl-density-gradient centrifugation. Non-covalently bound proteins, lipids and nucleic acids were not detected in the purified glycoproteins. Amino acid, carbohydrate and sulphate compositions were similar to chemical compositions reported for other intestinal mucus glycoproteins, as were sedimentation properties. There were, however, important differences in the chemical and physical characteristics of the mucus glycoproteins from newborn and adult animals. The buoyant density in CsCl was higher for the glycoproteins from newborn rats (1.55 g/ml versus 1.47 g/ml). On sodium dodecyl sulphate/polyacrylamide/agarose-gel electrophoresis, the glycoprotein from newborn rats had a greater mobility than the adult-rat sample. Although both preparations had similar general amino acid compositions, variations were observed for individual amino acids. The total protein content was greater in the glycoprotein from newborn animals (27%, w/w, versus 18%, w/w). The molar ratio of carbohydrate to protein was less in the newborn, primarily owing to a decreased fucose and N-acetylgalactosamine content. Comparison of the molar ratio of fucose and sialic acid to galactose for both glycoproteins demonstrated a reciprocal relationship similar to that described by Dische [(1963) Ann. N.Y. Acad. Sci. 106, 259-270]. The sulphate content was greater in the glycoprotein from newborn rats (5.5%, w/w, versus 0.9%, w/w). Both had similar sedimentation coefficients in a dissociative solvent. These results suggest an age-related difference in the types of mucus glycoproteins synthesized by small intestine.     

Purification and identification of novel angiotensin-I converting enzyme (ACE) inhibitory peptides from cultured marine microalgae (Nannochloropsis oculata) protein hydrolysate

Isolation of bioactive compounds and commercialization of marine microalgae sources are interesting targets in future marine biotechnology. Cultured biomass of the marine microalga, Nannochloropsis oculata, was used to purify angiotensin-I converting enzyme (ACE) inhibitory peptides using proteases including pepsin, trypsin, α-chymotrypsin, papain, alcalase, and neutrase. The pepsin hydrolysate exhibited the highest ACE inhibitory activity, compared to the other hydrolysates and then was separated into three fractions (F1, F2, and F3) using Sephadex G-25 gel filtration column chromatography. First fraction (F1) showed the highest ACE inhibitory activity and it was further purified into two fractions (F1-1 and F1-2) using reverse-phase high-performance liquid chromatography. The IC₅₀ value of purified ACE inhibitory peptides were 123 and 173 μM and identified as novel peptides, Gly-Met-Asn-Asn-Leu-Thr-Pro (GMNNLTP; MW, 728 Da) and Leu-Glu-Gln (LEQ; MW, 369 Da), respectively. In addition, nitric oxide production level (%) was significantly increased by the purified peptide (Gly-Met-Asn-Asn-Leu-Thr-Pro) compared to the purified peptide (Leu-Glu-Gln) and other treated pepsin hydrolysate fractions on human umbilical vein endothelial cells (HUVECs). Cell viability assay showed no cytotoxicity on HUVECs with the treated purified peptides and fractions. These results suggest that the isolated peptides from cultured marine microalga, N. oculata protein sources may have potentiality to use commercially as ACE inhibitory agents in functional food industry.     

Gastric proteases of the Greenland cod Gadus ogac. I. Isolation and kinetic properties

The zymogens of three gastric proteases of the Greenland cod (Gadus ogac) were isolated by exclusion chromatography and chromatofocusing. The cod zymogens were activated more rapidly at lower temperatures than porcine pepsinogen and, after activation, were further purified by exclusion chromatography. The cod proteases had more alkaline pH optima and were active over a wider range of pH than porcine pepsin. The specific activity of porcine pepsin on protein substrates was greater than that of the individual cod proteases. However, the cod proteases had cumulative activity on protein substrates that was greater than the sum of their individual activities. Cod protease 1 was active on pepsin-specific substrates, and cod proteases 2 and 3 were active as gastricsin-specific substrates. All three cod proteases had greater milk-clotting activity and hydrolysed hemoglobin to a greater extent than porcine pepsin. The Vmax and Km,app of the cod proteases were dependent upon the substrate, and Vmax/Km,app values of the cod proteases were generally lower than porcine pepsin. It is suggested that the cod proteases together exhibit broad substrate specificity and maintain activity over a wide range of conditions to enhance protein digestion in the cod stomach.     

Changes in mucus glycoprotein synthesized in rat gastric mucosa exposed to ethanol

The resistance to proteolysis by pepsin of gastric mucus glycoprotein synthesized by tissue culture in the presence and absence of 0.1 M ethanol was investigated. The glycoprotein product of ethanol-supplemented culture was found to contain 68% less associated lipids and 81% less covalently bound fatty acids, but exhibited unaltered content of carbohydrate and protein. The lipid and fatty acyl deficient glycoprotein was 5-times more rapidly and 2-3-times more extensively degraded by pepsin than the glycoprotein synthesized in the absence of ethanol. Following delipidation with organic solvents and deacylation with hydroxylamine both glycoproteins were digested at the same rate and degraded to the same extent. The lower content of fatty acyl residues markedly affected the overall pattern of the proteolytic fragments identified by SDS gel electrophoresis. The peptides corresponding to the acylated fragments of control were degraded and an increase in the amount of smaller peptides was observed. The in vitro assays of the fatty acyltransferase activity towards the substrates obtained from control and alcohol-containing cultures revealed that the enzyme activity was similar and increased proportionally with increased concentration of both glycoprotein substrates and enzyme. However, addition of 0.1 M ethanol to the assay tubes containing complete incubation mixture decreased the acylation of either glycoprotein by 40%. Based on the results presented here, and on previous studies of mucus glycoprotein synthesis in the presence of ethanol, we conclude that ethanol interferes with the process of acylation of mucus glycoprotein with fatty acids.     

Isolation and partial characterization of serine- and threonine-rich porcine gastric mucus glycopeptides.

1. Two subfractions from purified porcine gastric mucus glycopeptide were found to separate from each other by cesium chloride equilibrium centrifugation. The highest density fraction and two lower density fractions separated were designated VHD, HD and LD, respectively. A comparative study of these components was made. 2. The high and low density fractions, HD and LD, appeared almost the same or identical, while VHD differed completely from either of them in the following respects: (1) VHD exhibited strong alcian blue binding activity. (2) 57% of VHD bound to the DEAE-Toyopearl column equilibrated with 0.2 M NaCl. (3) VHD eluted from the Sephacryl S-400 column as a lower molecular subunit. (4) One third of the sialic acid as a minor component in VHD was constituted by N-glycolylneuraminic acid. (5) Carbohydrate composition showed typical mucus glycoprotein with slightly higher fucose content. (6) Amino acid compositions of the anionic components prepared from VHD showed the highest Ser/Thr ratio, 1.92 compared to 0.46 for LD and 0.62 for HD. (7) Oligosaccharide released from VHD by alkaline-sodium borohydride treatment was larger than that from HD or LD. 3. The above results indicate the minor component, VHD, separated from the major components, to be a quite similar but not identical component to the so-called sulfated mucus glycoprotein reported previously [Slomiany et al. (1972) J. biol. Chem. 247, 5062-5070].     

Functional incorporation and preferred orientation of phytohemagglutinin receptor glycoproteins in phospholipid vesicles

Porcine lymphocyte Phaseolus vulgaris phytohemagglutinin (PHA) receptor glycoproteins purified by affinity chromatography have been reassembled into vesicles made of phosphatidylcholine and phosphatidylserine by detergent (dodecyltrimethylammonium bromide) dialysis. The receptor glycoproteins were incorporated into the lipid vesicles in a nonselective manner with a yield of 65-70%. Vesicles containing the glycoproteins were sealed as evidenced by their impermeability to calcium ions, using quin 2 trapped inside the vesicles. The vesicles were agglutinated by PHA, suggesting that the saccharidic moiety of the reconstituted glycoproteins was, at least in part, oriented towards the extravesicular medium. This observation was further supported by the fact that the vesicles bound 125I-labeled PHA in a specific and saturable manner. At maximum amount of lectin bound, a ratio of 1.01 +/- 0.05 microgram of PHA per microgram glycoprotein incorporated was measured. When the binding data were analyzed by Scatchard plot, a downward concave profile was observed, suggestive of a positive cooperativity at low concentrations of lectin. The orientation of the reconstituted lectin receptor glycoproteins was determined by proteolytic treatments of labeled glycoproteins. The combined action of trypsin and chymotrypsin released, in the 120,000 X g supernatant, approximately 80% of label when 125I-tagged PHA receptor glycoproteins were incorporated into the vesicles. When the oligosaccharidic moieties of the receptor glycoproteins were specifically labeled, the simultaneous action of the two enzymes released approximately 70% of tritium labeling present in the reconstituted system. Taken together, these results suggest that the reconstituted PHA receptors are preferentially oriented into the phospholipid vesicles. The reconstituted PHA receptor glycoproteins competed effectively with cellular receptors in the assay of PHA-induced porcine lymphocyte activation. A 50% inhibition of [3H]thymidine incorporation was observed when 1 microgram of glycoproteins in vesicles was added to the cultured cells, whereas vesicles alone had no effect at this (equivalent) concentration.     

Cell Wall Antigens of Pneumocystis carinii Trophozoites and Cysts: Purification and Carbohydrate Analysis of these Glycoproteins

We have purified and biochemically analyzed individual cell wall glycoproteins of Pneumocystis carinii. Our results show that corresponding core glycoproteins constitute the cell wall antigens in both trophozoites and cysts, and glycosylation of these glycoproteins does not appear to be significantly altered during development. Cysts and trophozoites in rat-derived organism preparations were separated from each other by counterflow centrifugal elutriation, then treated with Zymolyase to obtain the cell wall fractions. Gel electrophoresis patterns of these fractions from both life-cycle stages were qualitatively similar. Ten major antigenic glycoproteins in these fractions were purified by preparative continuous elution gel electrophoresis. All ten glycoproteins from cysts and trophozoites contained mannose, glucose, galactose. and N-acetylglucosamine, and some contained traces of fucose. The glycoproteins of cysts had more mannose than their trophozoite counterparts. The trophozoite glycoproteins differed from those of the cyst by the presence of xylose. To examine the species-specificity of glycoprotein glycosylation, preparations of human-derived P. carinii (comprised of mixed life-cycle stages) were also examined and found to contain the same sugars as those found in rat-derived organisms. Most of the purified rat-derived glycoproteins bound Concanavalin A, which was abolished by treatment with N-glycanase. This suggested that the majority of the oligosaccharides were N-linked to the proteins, but attempts to identify carbohydrate linkage sites by amino acid sequencing were hampered by apparent modifications of residues. The peptides derived by cyanogen bromide cleavage revealed distinct size patterns for each glycoprotein, suggesting that they were distinct proteins. Most of the glycoproteins reacted with monoclonal antibodies which recognize a highly conserved epitope on rat P. carinii. Four of the individually purified glycoprotein preparations elicited in vitro cellular immune responses, implicating their involvement in the recognition of P. carinii by host T cells. The identification and characterization of P. carinii cell wall proteins will be helpful in analyzing the relationship of the organism to its mammalian host. Supplementary key words. Biochemical analysis, developmental stages, opportunistic pathogen, structure.     

Prostaglandin effect on the physical properties of gastric mucus glycoprotein and its susceptibility to pepsin

The effect of 16,16-dimethyl prostaglandin E2 (DMPGE2) on gastric mucus glycoprotein viscosity, permeability to hydrogen ion and degradation by pepsin was investigated. Preincubation with DMPGE2 produced a marked enhancement in the glycoprotein viscosity. The increase was concentration dependent and at 2.6 X 10(-5)M DMPGE2 reached a value of 178%. Permeability measurements revealed that 2.6 X 10(-7)M DMPGE2 increased the retardation ability of the glycoprotein to hydrogen ion by 10%, while 22% increase was obtained with 2.6 X 10(-4)M DMPGE2. The results of peptic activity assay showed that DMPGE2 had no inhibitory effect on the rate of glycoprotein proteolysis, and actually a small stimulatory influence was consistently observed. The results suggest that prostaglandins beneficially affect the physical properties of mucus glycoprotein which are considered to be essential for the protective function of gastric mucus.     

Isolation and characterization of angiotensin I-converting enzyme inhibitory peptides derived from porcine hemoglobin

Animal blood is potentially an untapped source of drugs and value-added food production. More than 400 million pigs are slaughtered each year but porcine blood is usually discarded in China. This study describes the isolation and characterization of angiotensin I-converting enzyme (ACE) inhibitory peptides derived from porcine hemoglobin. The most active hydrolysate was obtained from the peptic digestion of porcine hemoglobin. After the purification of ACE-inhibitory peptides with Sephadex LH-20 gel chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC) on C(18) column, two active fractions were obtained. They were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). They were LGFPTTKTYFPHF and VVYPWT, corresponding to the 34-46 fragment of the alpha chain and the 34-39 fragment of the beta chain of porcine hemoglobin, with IC(50) values of 4.92 and 6.02 microM, respectively. They were the first found from porcine hemoglobin; in particular, LGFPTTKTYFPHF was a novel ACE-inhibitory peptide. In addition, the purified ACE inhibitors both competitively inhibited ACE, and maintained inhibitory activity even after incubation with gastrointestinal proteases. This suggests that these peptides might have a potential antihypertensive effect.     

Partial purification and characterization of an endo-alpha-N-acetylgalactosaminidase from the culture of medium of Diplococcus pneumoniae.

The culture medium of Diplococcus pneumoniae contains enzymic activity that cleaves Galbeta1 leads to 3GalNAc from desialized human erythrocyte membrane glycoprotein. The enzyme was purified 180-fold by ammonium sulfate fractionation, gel filtration through a Sephadex G-200 column, and DEAE A-25 Sephadex chromatography. The purified enzyme liberates Galbeta1 leads to 3GalNAc from glycopeptides and glycoproteins with Galbeta1 leads to 3GalNAcalpha1 leads to Ser and Thr moieties. The optimum pH of this enzyme is 6.0. Using glycopeptides obtained by trypsin digestion of human erythrocyte membrane glycoprotein as a substrate, a Km of 0.20 mM (on the basis of the amount of Galbeta1 leads to 3GalNAc residues) was obtained. So far, the enzyme appears to have a strict specificity for Galbeta1 leads to 3GalNAcalpha1 leads to Ser and Thr structures, because no oligosaccharides larger than trisaccharides were liberated from porcine submaxillary mucin.     

Inhibition of the reconstitution of the haemolytic activity of the first component of human complement by a pepsin-derived fragment of subcomponent C1q.

1. A fragment of subcomponent C1q, which contained all the collagen-like features present in the intact molecule, was isolated by pepsin digestion as described by Reid [Biochem. J. (1976) 155, 5-17]. 2. The pepsin-derived fragment of subcomponent C1q did not bind to antibody-coated erythrocytes under conditions where complete binding of sub-component C1q took place. 3. The peptic fragment blocked the reconstitution of C1 haemolytic activity by competing with intact subcomponent C1q in the utilization of a mixture of the other two subcomponents, C1r and C1s. 4. Reduction and alkylation of the interchain disulphide bonds in the pepsin fragment did not markedly affect its inhibitory effect, whereas heating at 56 degrees C for 30min completely abolished the effect. 5. Lathyritic rat skin collagen and CNBr-derived peptides of pig type II collagen showed no ability to mimic the inhibitory effect of the pepsin fragment when tested over the same concentration range as used for the peptic fragment. 6. The peptic fragment was unable to block efficiently the reconstitution of C1 haemolytic activity unless it was added to the mixture of subcomponents C1r and C1s before the attempt to reconstitute C1 haemolytic activity, in solution, or on the surface of antibody-coated erythrocytes. 7. Evidence was obtained that suggested that subcomponent C1q bound the subcomponent C1r-C1s complex more efficiently when the subcomponent C1q was bound to antibody than when it was free in solution.     

酶标白桂木凝集素糖蛋白结合特性的分析

采用辣根过氧化物酶 ( HRP)标记白桂木凝集素 ( AHL) ,应用酶联夹心法及糖竞争抑制实验 ,研究 AHL的糖蛋白结合特性 .研究表明 ,AHL能与两种不同类型的糖蛋白结合 ,一类以胃蛋白酶为代表 ,AHL能以高亲和力与胃蛋白酶结合 .其次能与β-乳球蛋白、牛血清清蛋白结合 ,但结合力依次递减 .AHL也能与透明质酸以较高亲和力相结合 .AHL与胃蛋白酶、β-乳球蛋白、牛血清清蛋白、透明质酸的结合受 Me- Gal的强烈竞争抑制 ,亦受 Me- Man\D- Gal\Raf的抑制 .另一类为Con A,AHL与 Con A的结合受 Me- Man的强烈竞争抑制 ,并受 Me- Glc\D- Man\D- Fru\D- Glc的较强抑制 .各种糖的封闭性抑制实验结果与竞争性抑制实验相似 .提示 AHL上存在 O-糖苷键结合位点 .     

Identification of liver plasma membrane glycoproteins which bind to 125I-labelled concanavalin A following electrophoresis in sodium dodecyl sulfate.

Following electrophoresis of ovalbumin in sodium dodecyl sulfate (SDS) this glycoprotein bound 125I-labelled concanavalin A (Con A). The reaction was specific and proportional to the amount of glycoprotein present on the gel. This technique was used to study the Con-A-binding glycoproteins of liver cell surfaces. Mouse liver plasma membranes were purified and subfractionated to yield two fractions corresponding to the bile canalicular surface and the surface between adjacent hepatocytes (Evans, W.H. (1970) Biochem. J. 116, 833-842). Both fractions bound 125I-labelled Con A, the former binding two to three times more lectin than the latter. Following SDS gel electrophoresis individual membrane glycoproteins reacted with 125I-labelled Con A. Both membrane subfractions yielded qualitatively similar Con A binding profiles, seven binding proteins being present in each. The results are consistent with a generally uniform distribution of glycoproteins over the hepatocyte surface. The reaction of lectins with glycoproteins following SDS gel electrophoresis should find general application in the study of membrane composition.     

The macromolecular properties of blood-group-specific glycoproteins. Characterization of a series of fractions obtained by solvent fractionation

1. The glycoprotein components of a human ovarian-cyst fluid were isolated by a solvent [95% (w/w) phenol]-extraction procedure; the phenol-insoluble water-soluble glycoprotein was further fractionated by (NH(4))(2)SO(4) and by ethanol to yield eight fractions. 2. The fractions were analysed in terms of amino acids, fucose, galactose, N-acetylglucosamine, N-acetylgalactosamine and sialic acid. Variations occurred, particularly in the proportion of peptide; these were partly correlated with varying extent of serological activity. 3. The fractions were characterized physicochemically in terms of buoyant density and degree of spreading in a density gradient, sedimentation velocity and molecular weight; their partial specific volumes and specific refraction increments were also determined. 4. The fractions showed wide variations in their sedimentation-velocity and density-gradient patterns, and gave evidence of pauci-dispersity in density. The fraction regarded as the most typical blood-group-specific glycoprotein sedimented as a single rapidly spreading peak and was of high molecular weight. 5. Significant correlations were observed between the physical properties of the glycoprotein fractions and the amount of their peptide component. The buoyant densities and sedimentation coefficients varied in a manner that suggested the existence of two families of glycoproteins. 6. It is suggested that variability in the extent of glycosylation, or in the degree of cross-linking, might account for the two families of glycoproteins, and that the extent of cross-linkage might also be a factor determining the solubility of these glycoproteins in hot saturated (NH(4))(2)SO(4).     

Isolation and characterization of a specific enterokinase inhibitor from kidney bean (Phaseolus vulgaris)

A specific enterokinase inhibitor from kidney bean (Phaseolus vulgaris) was purified to homogeneity. It showed a single protein band on sodium dodecyl sulphate/polyacryl-amide-gel electrophoresis in the presence of mercaptoethanol, and the Mr was 31000. Aspartic acid was identified as the N-terminus of the inhibitor. The Mr by gel chromatography on Sephadex G-200 was found to be 60000, indicating the dimeric nature of the inhibitor. The inhibitor was found to be a glycoprotein. The monosaccharide moieties were glucose, mannose, glucuronic acid and glucosamine in the proportions 3.15%, 5.0%, 0.85% and 1.3% respectively. The inhibitor was most active on pig enterokinase, followed by bovine and human enterokinases. Maximal inhibitory activity was elicited by preincubation of the inhibitor with the enzyme for 15 min. Digestion with pepsin resulted in loss of inhibitory activity. The inhibitor was stable to exposure to a wide range of pH values (2-10), and exposure to pH above 10 resulted in loss of inhibitory activity. Modification of arginine residues by cyclohexane 1,2-dione and ninhydrin led to complete loss of enterokinase-inhibitory activity.