Pallidin. Purification and characterization of a carbohydrate-binding protein from Polysphondylium pallidum implicated in intercellular adhesion.

A carbohydrate-binding protein from Polysphondylium pallidum, a species of cellular slime mold, was purified to homogeneity by adsorption to formalinized erythrocytes and elution with D-galactose. The protein, for which we propose the name PALLIDIN, is assayed by its activity as an agglutinin of erythrocytes. It was previously shown to have different carbohydrate-binding specificities than discoidin, a carbohydrate-binding protein from Dictyostelium discoideum, another species of slime mold. Evidence has been presented previously that each of these proteins is detectable on the cell surface. In the present report we show that the physico-chemical properties of pallidin are different from discoidin. Pallidin has a subunit molecular weight of 24 800 +/- 1100 determined by polyacrylamide electrophoresis in the presence of dodecyl sulfate and 2-mercaptoethanol, compared to 26 100 +/- 1000 for discoidin. The weight-average molecular weight of pallidin is 250 000 +/- 50 000 determined by equilibrium sedimentation in the presence of D-galactose compared to 100 000 +/- 2000 for discoidin. In equilibrium sedimentation studies, pallidin exhibited some heterogeneity at equilibrium while discoidin was homogeneous. The amino acid composition of pallidin is generally similar but clearly different from the composition of discoidin. The isoelectric point of pallidin is 7.0 compared to 6.1 for discoidin. Like discoidin, pallidin contains no detectable hexosamine or neutral sugar. These results establish that agglutinins from two species of cellular slime molds are distinct. The different properties of the cell-surface agglutinins, pallidin and discoidin, are consistent with their suggested role in species-specific cellular recognition and adhesion in the species of slime mold from which they are derived.     

Purification and characterization of a sialic acid specific lectin from the hemolymph of the freshwater crab Paratelphusa jacquemontii.

A naturally occurring hemagglutinin was detected in the serum of the freshwater crab, Paratelphusa jacquemontii (Rathbun). Hemagglutination activity with different mammalian erythrocytes suggested a strong affinity of the serum agglutinin for horse and rabbit erythrocytes. The most potent inhibitor of hemagglutination proved to be bovine submaxillary mucin. The lectin was purified by affinity chromatography using bovine submaxillary mucin-coupled agarose. The molecular mass of the purified lectin was 34 kDa as determined by SDS/PAGE. The hemagglutination of purified lectin was inhibited by N-acetylneuraminic acid but not by N-glycolylneuraminic acid, even at a concentration of 100 mm. Bovine submaxillary mucin, which contains mainly 9-O-acetyl- and 8,9 di-O-acety-N-acetyl neuraminic acid was the most potent inhibitor of the lectin. Sialidase treatment and de-O-acetylation of bovine submaxillary mucin abolished its inhibitory capacity completely. Also, asialo-rabbit erythrocytes lost there binding specificity towards the lectin. The findings indicated an O-acetyl neuraminic acid specificity of the lectin.     

Gracilaria tikvahiae agglutinin. Partial purification and preliminary characterization of its carbohydrate specificity

A potent agglutinin of rabbit and sheep red blood cells, obtained from the red alga Gracilaria tikvahiae, was purified by ammonium sulfate fractionation, ion exchange, gel filtration, and hydroxylapatite chromatography. Human A and B blood group erythrocytes were also agglutinated, whereas human O blood group erythrocytes were not agglutinated. The hemagglutination titer was not significantly affected by the addition of EDTA or the divalent cations Ca2+, Mg2+, or Mn2+. The carbohydrate specificity was characterized by hemagglutination inhibition using various monosaccharides, glycoproteins, and glycopeptides. The results suggested that the agglutinin has affinity for N-acetylneuraminic acid as well as glycoconjugates containing N-acetylneuraminic acid.     

Purification and characterization of an agglutinin from mucus of the snail Achatina fulica

The mucus of the snail Achatina fulica shows the presence of an agglutinin that nonspecifically agglutinates human erythrocytes. The agglutinin has been purified by affinity chromatography using Sepharose 4B-hog gastric mucin as the affinity matrix. Homogeneity was checked by polyacrylamide gel electrophoresis, immunodiffusion, immunoelectrophoresis, and gel filtration. The agglutinin is a glycoprotein of native molecular weight 70,000. The isoelectric point of the protein was found to be 8.0. The predominant amino acids are aspartic acid and glutamic acid (or amides) and serine, which account for 32% of the total amino acid residues. The agglutinin has 10% carbohydrate (wt/wt) and the most abundant sugar is N-acetylglucosamine. The cd spectra of the agglutinin show the presence of random coil conformation. The inhibition of hemagglutination data indicates that the agglutinin is specific for beta glycosides of D-Gal and D-GalNAc.     

alpha-D-galactosidase from soybeans destroying blood-group B antigens. Purification by affinity chromatography and properties.

alpha-D-Galactosidase was isolated from untoasted soybean meal and purified to homogeneity by affinity chromatography on N-epsilon-aminoacaproyl alpha-D-galactopyranosylamine-Sepharose. The purified enzyme destroyed the B-specificity of human ovarian cyst B-glycoprotein with an accompanying increase in H-specificity, and converted human type-B erythrocytes to type O. The enzyme consists primarily of a tetramer, molecular weight 150 000 +/- 5 000 at pH 4.0 and of a monomer, molecular weight 40 000 +/- 3 000 at pH 8.0. Polyacrylamide gel electrophoresis in dodecyl sulfate at pH 7.2 distinguished between two types of monomeric unit of similar molecular weight. N-terminal alanine was identified as the sole N-terminal amino acid residue. The enzyme was shown to be devoid of carbohydrate.     

Isolation and characterization of a hemagglutinin from Amphitrite ornata, a polychaetous annelid.

Extracts of the marine polychaetous annelid, Amphitrite ornata, agglutinate rat, rabbit, chicken and human erythrocytes and in other work have been shown to inhibit the growth of Ehrlich ascites tumors in mice. Fractionation of extracts on Sephadex G-100 gave three active fractions with molecular weights of 30 000, 54 000 and 100 000. The 30 000 dalton fraction (B) was purified 72-fold by ammonium sulfate precipitation, gel filtration and preparative disc gel electrophoresis. The purified hemagglutinin, amphitritin, was homogenous on analytical disc gel electrophoresis at four different pH values and gave a sharp boundary in sedimentation velocity ultracentrifugation. The three fractions showed paralled specificity toward rat and chicken erythrocytes, the former giving the higher titer. The purified agglutinin was active toward human blood groups A, B and O and exhibited 4-fold higher activity toward group A. The hemagglutinin titer against rat red blood cells was lowered only by N-acetylgalactosamine, the terminal sugar residue of the group A determinant. None of the saccharides tested inhibited agglutination of chicken erythrocytes. Hemagglutinin activity was insensitive to dialysis or treatment with EDTA. The activity was not affected by digestion with trypsin or pronase, but was destroyed by phenol extraction. Analytical disc gel electrophoresis showed one protein band with high anodal mobility at pH 8.5, which was not affected by proteolytic enzymes but was removed by phenol. Activity was unaffected by heating at 70 degrees C for 30 min but was destroyed by similar treatemtn at 85 degrees C. Activity was at a maximum at pH 7-9 and decreased reversibly down to pH 4 at which point it was irreversibly inactivated. The higher molecular weight agglutinin (A1) could be dissociated to give amphitritin by treatment with 6M urea of precipitation in 55% (NH4)2SO4. This dissociation was not reversed by dialysis. Amphitritin is a glycoprotein with a molecular weight determined by gel filtration of 30 000 and by approach to equilibrium sedimentation of 32 000. Amino acid analysis showed a preponderance of aspartic and glutamic acids and relatively large amounts of glycine, proline, alanine, valine and cysteine. The carbohydrate moeity which represented 12.8% of the molecule, contained mannose, galactose, glucosamine and sialic acid. Amphitritin is the first hemagglutinin to be isolated from a polychaetous annelid.     

Amino sugars in the glycoprotein toxin from Bacillus thuringiensis subsp. israelensis.

The carbohydrate content of purified Bacillus thuriniensis subsp. israelensis crystal toxin was determined by six biochemical tests, column chromatography on an amino acid analyzer, and the binding of 11 fluorescent lectins. The crystals contained approximately 1.0% neutral sugars and 1.7% amino sugars. The amino sugars consisted of 70% glucosamine and 30% galactosamine. No N-acetylneuraminic acid (sialic acid) was detected. The presence of amino sugars was confirmed by the strong binding of fluorescent wheat germ agglutinin and the weak binding of fluorescent soybean agglutinin. These lectins recognize N-acetyl-D-glucosamine and N-acetyl-D-galactosamine, respectively. The lectin-binding sites appeared evenly distributed among the protein subunits of the crystal. The sugars were covalently attached to the crystal toxin because wheat germ agglutinin still bound alkali-solubilized toxin which had been boiled in sodium dodecyl sulfate, separate by polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. This study demonstrates the covalent attachment of amino sugars and indicates that the B. thuringiensis subsp. israelensis protein toxins should be viewed as glycoprotein toxins. The crystals used in the present study were purified on sodium bromide density gradients. Studies employing crystals purified on Renografin density gradients can give artificially high values for the anthrone test for neutral sugars.     

Purification and characterisation of alpha-L-fucosidase from human placenta. pH-dependent changes in molecular size.

alpha-L-Fucosidase has been purified 12 000 fold from human placenta. The enzyme is a glycoprotein containing, by weight: 0.9% galactose; 1.9% mannose, 1.9% N-acetylglucosamine and 1.9% N-acetylneuraminic acid. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate separated proteins with molecular weights ot 55 000, 51 400 and 25 000. Resolution of the two larger protein bands varied with the gel system and these proteins may differ only in carbohydrate content. Gel filtration of te purified enzyme failed to separate the three proteins. Treatments with the cross-linking reagent dimethyl suberimidate prior to electrophoresis, resulted in a diminution of the original protein bands and the formation of oligomers with molecular weights of 80 000, 100 000, 130 000, and 144 000. These results suggest that the heavy (55 000 and 51 400) and light (25 000) proteins are structurally associated. The molecular weight of the native enzyme, measured by gel filtration, was dependent on the pH of the eluting buffer. At pH 5.0 or 6.0 a catalytically active peak was observed, with a molecular weight of 305 000. At pH 7.5 this peak was completely absent and the enzyme eluted as an asymmetrical peak with an apparent molecular weight of about 60 000. The reduction in apparent molecular weight at pH 7.5 was reversible by dialysis of isolated fractions at pH 6.0. In agreement with these findings the sedimentation coefficient was 8.5 S at pH 5.0 but only 3.6 S at pH 7.5. The results can be accounted for by the existence of a pH-dependent equilibrium between aggregated and dissociated forms of the enzyme or by pH-depedent conformational changes.     

High-molecular-weight kininogen from horse plasma. Isolation, characterization and comparison with bovine high-Mr kininogen

High-molecular-weight (high-Mr) kininogen was purified from horse plasma by chromatography on columns of DEAE-Sephadex A-50, CM-Sephadex C-50, p-chlorobenzylamine-Sepharose and Sephadex G-150. The yield was about 150 mg from 81 of fresh plasma. The purified material gave a single band on sodium dodecylsulfate/polyacrylamide gel electrophoresis and a single precipitin line on immunodiffusion and immunoelectrophoresis. The molecular weight of horse high-Mr kininogen was estimated to be 78000 by dodecylsulfate gel electrophoresis using the Ferguson plot. Its polypeptide content was determined to be 86% by amino acid analysis and there was a total of 581 amino acid residues/molecule of protein. The kininogen contained a total of 13.9% carbohydrates, consisting of hexoses (7.8%), glucosamine (1.9%), galactosamine (0.6%) and sialic acid (3.6%). On incubation of horse high-Mr kininogen with bovine and horse plasma kallikreins, several fragments which contained extremely high levels of histidine, were liberated, in addition to kinin. After the liberation of kinin and histidine-rich fragments, a protein free of kinin and its fragments was isolated. This protein consisted of two polypeptide chains, heavy chain and light chain, which are bridged by disulfide bonds. The molecular weight and amino acid composition of the heavy chain and the light chain from horse high-Mr kininogen were very similar to those of the heavy and light chains from bovine high-Mr kininogen, respectively. From these results, it was revealed that horse high-Mr kininogen is quite similar to bovine high-Mr kininogen in terms of their physicochemical and chemical properties, although they are immunologically distinguishable.     

The isolation and partial characterization of diphosphoglycerate mutase from human erythrocytes.

Diphosphoglycerate mutase has been purified to homogeneity from outdated human erythrocytes. The native enzyme has a molecular weight of 57 000 as determined by equilibrium centrifugation and exclusion chromatography. Disc gel electrophoresis in the presence of sodium dodecyl sulfate yields a single protein band with a molecular weight of about 26 500, indicating that diphosphoglycerate mutase is comprised of two subunits of similar mass. The enzyme exhibits the following intrinsic activities: diphosphoglyceratemutase, monophosphoglycerate mutase, and 2,3-diphosphoglycerate phosphatase. The latter activity is enhanced in the presence of either organic or inorganic anions. Glycolate-2-P, particularly, has a profound activating effect. Nonspecific phosphatase and enolase activities are absent. The enzyme has an extinction coefficient at 280 nm of 1.65 cm2/mg. The amino acid composition of the homogeneous protein has been determined.     

Purification and partial characterization of the myelin-associated glycoprotein from adult rat brain

The myelin-associated glycoprotein was purified from rat central nervous system myelin by selective extraction with lithium diiodosalicylate-phenol followed by gel filtration on a column of Sepharose CL-6B. Amino acid analysis of the purified glycoprotein revealed an excess of acidic over basic amino acids and a relatively high content of nonpolar residues. On the basis of weight, the molecule is about one-third carbohydrate consisting of 5% fucose, 23% mannose, 20% galactose, 34% N-acetylglucosamine, and 18% N-acetylneuraminic acid.     

Diversities in animal vitronectins. Differences in molecular weight, immunoreactivity and carbohydrate chains.

Six animal plasma vitronectins, human, horse, porcine, bovine, rabbit and chicken vitronectins purified by a novel method using two successive heparin affinity columns, showed marked diversity in molecular weight, immunoreactivity and carbohydrate composition. Chicken vitronectin had a distinctly different amino acid composition from the mammalian vitronectins; and bovine vitronectin was the only one to contain N-glycolylneuraminic acid as well as N-acetylneuraminic acid. Binding studies with horseradish peroxidase-labelled lectins indicated that all the vitronectins contained complex-type, sialylated N-linked sugar chains and that only porcine vitronectin had a fucosylated sugar chain. D-Galactosamine determinations and binding studies with horseradish peroxidase-peanut lectin on native and asialovitronectins revealed that the mammalian vitronectins other than human vitronectin contained O-linked sugar chains with sialic acid, chicken vitronectin contained unsialylated chains, and human vitronectin contained neither. The results indicate that diversities in vitronectins are apparent in their molecular weights and glycosylations, especially in the number and structure of O-linked sugar chains.     

Purification, Biological Activities, and Molecular Cloning of a Novel Mannose-Binding Lectin from Bulbs of Zephyranthes candida Herb （Amaryllidaceae）

A novel mannose-bindlng aggiutinln was purified from bulbs of Zephyranthes candida Herb by extraction, precipitation with 80% （NH4）2SO4, and ion-exchange chromatography on DEAE-Sepharose followed by gel flitration on Sephscryl S-100. The purified Z. candida agglutlnln （ZCA） migrated as a single band of 12 kDa on sodium dodecyi suifate-poiyecryiamide gel electrophoresis under reducing and non-reducing conditions. The apparent molecular mass of the iectln, as datermlned by gel filtration chromatography, was 48 kDa. The results Indicated that ZCA was composed of four Identical subunlts of 12 kDa each （homotetramerlc nature）. The ZCA agglutlhated rabbit erythrocytes, Escherichla coil and Saccharomyces cerevislae ceils at concentrations of 0.95, 1.90, and 31.30 μg/mL, respectively. Bloassays Indicated that ZCA has a significant effect on wheat aphid survival. Mortality after 7 d was 〉 90% at 0.26%. A degenerate primer was designed In accordance with the N-terminal partial sequence of purified ZCA. The full-length cDNA was cloned by 3＇- and 5＇-rapid amplification of cDNA ends. The full-length cDNA had 661 bp and the sequence encoded an open reading frame of 168 amino acids. The mature protein of ZCA Includes 109 amino acid residues and the molecular weight of the protein was 12.1 kDa. The result show that the zca gene encodes a protein precursor with a signal peptlde, a mature protein, and a C-terminal cleavage amino acids sequence. Molecular modeling of ZCA Indicated that Its three-dimensional atructure strongly resembies that of the snowdrop aggiutinin. Blocks＇ analysis revealed that the deduced amino acid sequence of ZCA has three functional domains specific for agglutination and three carbohydrate binding boxes （QDNY）.     

Further characterization of the cold agglutinin from the snail Achatina fulica.

The cold agglutinin from the albumin gland of the snail Achatina fulica was purified to homogeneity by using sheep gastric mucin-Sepharose 4B as affinity column followed by gel filtration on Bio-Gel P-300. The homogeneity was checked by alkaline gel electrophoresis, immunodiffusion and immunoelectrophoresis. The purified cold agglutinin is a glycoprotein of native M2 220,000 consisting of three non-covalently bound subunits of Mr 84,000, 74,000 and 62,000 and having a pI value of 4.5. The predominant amino acids are aspartic acid and glutamic acid (or amides) and serine, which account for 39% of the residues. About 3% of the residues are half-cystine. The lectin is a glycoprotein with about 30.7% carbohydrate, the most abundant sugars being galactose, N-acetylgalactosamine and N-acetylglucosamine. Mannose, xylose and fucose are also present. The inhibition of agglutination of human umbilical-cord erythrocytes by the cold agglutinin is specific for methyl beta-D-galactoside and also for glycolipids present on cord erythrocytes. The c.d. data show only negative ellipticity values in the far-u.v. region for the protein at various concentrations and temperatures and also in the presence of the hapten lactose (at different concentrations), indicating the presence of a random-coil conformation in the agglutinin that varies according to temperature.     

Purification and properties of Arthrobacter neuraminidase

Neuraminidase (EC 3.2.1.18) from an Arthrobacter species was purified homogeneity by conventional procedures (yield approx. 1 mg/1) and was judged to be homogeneous by sodium dodecyl sulfate gel electrophoresis. Gel electrofocusing of neuraminidase revealed 1 major band (85-90%), pI 5.35 +/- 0.05, and 6 minor bands, whose pI ranged from 5.25 to 5.70, and each of which had catalytic activity. Arthrobacter neuraminidase is a monomeric glycoprotein of molecular weight 88 000, has an apparent Km of 7.8-10(-4) M for N-acetylneuraminlactose, is insensitive to inhibition by N-acetylneuraminic acid, and is about 2% carbohydrate by weight. The amino acid composition as well as the galactosamine and glucosamine content was determined. The enzyme can hydrolyze (alpha, 2-3), (alpha, 2-6), (alpha, 2-8) linkages. The active size of the enzyme appears to be inaccessible since no inhibition was observed by reagents known to modify sulfhydryl, lysyl, carboxyl, histidinyl, and argininyl residues. In contrast, N-bromosuccinimide at a 60-fold molar ratio to enzyme, gave complete inhibition. These results suggest that a tryptophan residue is essential for catalysis.     

Purification and characterization of a coagulant protein from the venom of Russell's viper.

The coagulant protein from the venom of Russell's viper was purified by means of successive chromatography on Sephadex G-50, DEAE-cellulose and Sephadex G-200. The purified coagulant protein was homogeneous by polyacrylamide gel electrophoresis and ultracentrifugation. The molecular weight was estimated to be about 100 000 by ultracentrifuge analysis and 130 000 by gel filtration. The coagulant protein contains 11.1% carbohydrate which includes 5.1% hexose (galactose: mannose = 1:1), 5% hexosamine (glucosamine), and 1% neuraminic acid (N-acetylneuraminic acid and N-glycolyneuraminic acid). The isoelectric point is pH 6.3. The results of both sodium dodecyl sulfate electrophoresis and gel filtration in 6 M guanidium chloride suggest that it consists of four polypeptide chains. The coagulant protein functions as an enzyme in activating blood coagulation factor X in the presence of Ca2+. N-a-p-Toluenesulfonyl-L-arginine methyl ester hydrolyzing activity in the preparation definitely decreased during purification and it suggests that the clotting activity is not associated with the esterase activity. The clotting activity is inhibited by diisopropyl phosphorofluoridate and by phenylmethylsulfonyl fluoride, suggesting that the coagulant protein is a serine protease. The optimum pH is between pH 7.0 and pH 8.0. At neutral pH the coagulant protein is stable below 50 degrees C, but is rapidly inactivated above 55 degrees C.     

Isolation, molecular and biological properties of a lectin from rice embryo: relationship with wheat germ agglutinin properties

Rice lectin (Oryza sativa, var. Balilla 28) was purified from defatted embryos by aqueous acid extraction at pH 1.3 followed by ammonium sulfate precipitation between 2 and 4 M, affinity chromatography on agarose-p-aminophenyl-beta-D-N-acetylglucosamine, and gel filtration on AcA 54. The homogeneity of the lectin was checked by polyacrylamide gel electrophoresis, gel filtration, and immunodiffusion. The amino acid analysis revealed a high half-cystine content (9%) and a low aromatic and hydrophobic amino acid content. The lectin contained neither neutral carbohydrates nor amino sugars. The isoelectric point was estimated to be 8.1. The molecular weight of rice lectin was estimated to be 38,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions showed two polypeptides with Mr 19,000 and 15,000. The circular dichroism spectrum of rice lectin in far ultraviolet was characterized by a positive maximum at 228 nm and a negative band at 203 nm suggesting the presence of a beta-pleated sheet and the absence of alpha-helix. Rice lectin had no human blood group specificity and agglutinated rabbit erythrocytes more efficiently than erythrocytes from other animal species. Furthermore, agglutination was enhanced by trypsin treatment of erythrocytes. The erythroagglutinating activity was very high since the minimal concentration needed to agglutinate erythrocytes was 0.05 micrograms/ml. Although [methyl-3H]thymidine incorporation was stimulated in human lymphocytes, rice lectin could not be considered as a mitogenic lectin since it stimulated neither blast transformation nor lymphocyte proliferation. The saccharide specificity of rice lectin was related to N-acetylglucosamine and its oligomers: N,N',N"-triacetylchitotriose was the most powerful inhibitor. Furthermore, the N-acetylneuraminic acid was not a specific rice determinant. Finally, the double immunodiffusion method revealed a cross-reactivity between rice lectin and wheat germ agglutinin, indicating that these lectins were closely antigenically related. The analogies and differences between biological and immunological properties of rice lectin and wheat germ agglutinin are discussed and the possibility of their evolution from a common ancestor is put forward.     

重组组织型纤溶酶原激活剂大规模纯化及部分性质的研究

收集产重组组织型纤溶酶原激活剂（ｒｔ－ＰＡ）的ＣＨＯ工程细胞灌流培养上清,经过Ｓｔｒｅａｍｌｉｎｅ扩张柱床和赖氨酸－Ｓｅｐｈａｒｏｓｅ４Ｂ柱亲和吸附色谱纯化后,最后产品的比性达到６０００００ＩＵ／ｍｇ蛋白,ｒｔ－ＰＡ总活性回收率在９８％左右,经还原ＳＤＳ－ＰＡＧＥ分析主要为高相对分了质量ｒｔ－ＰＡ蛋白,ＨＰＬＣ分析达到色谱纯,Ｎ端１５个氨基酸序列和ｐＩ与献报道的一致。蛋白质印迹实验证实具有ｔ－Ｐ     

A new multimeric hemagglutinin from the coelomic fluid of the sea urchin Anthocidaris crassispina

A hemagglutinin was purified from the coelomic fluid of the sea urchin Anthocidaris crassispina by ion-exchange chromatography on DEAE-cellulose and affinity adsorption to glutaraldehyde-fixed ghosts of human erythrocytes, followed by elution with 10 mM EDTA. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it showed a single protein band with a molecular weight of 13 000 and 26 000 in the presence and absence of 2-mercaptoethanol, respectively. The molecular weight of the native protein with a hemagglutinating activity was determined to be 300 000 by sedimentation equilibrium analysis. Its sedimentation coefficient, S0(20),w, and Stokes radius were 13.7 S and 5.5 nm, respectively. The hemagglutinating activity of this protein required calcium ions. When calcium ions were depleted, no activity was observed and its sedimentation coefficient, S0(20),w, decreased to 11.4 S while its Stokes radius increased to 6.7 nm without a change in its molecular weight. The purified hemagglutinin agglutinated human erythrocytes regardless of their ABO and MN blood types. The hemagglutination reaction was not affected appreciably by various simple sugars but was inhibited by tryptic fragments released from human erythrocyte membranes. The results of alkaline borohydride treatment of the inhibitory tryptic fragments showed that the receptor sites for this hemagglutinin were mainly composed of alkali-labile carbohydrate chains with the structure AcNeu alpha 2----3Gal beta 1----3(AcNeu alpha 2----6)GalNAc----serine (or threonine).(ABSTRACT TRUNCATED AT 250 WORDS)     

The measurement of membrane potential during photosynthesis and during respiration in intact cells of Rhodopseudomonas capsulata by both electrochromism and by permeant ion redistribution.

Adenosine deaminase was purified 3038-fold to apparent homogeneity from human leukaemic granulocytes by adenosine affinity chromatography. The purified enzyme has a specific activity of 486 mumol/min per mg of protein at 35 degrees C. It exhibits a single band when subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, non-denaturing polyacrylamide-gel electrophoresis and isoelectric focusing. The pI is 4.4. The enzyme is a monomeric protein of molecular weight 44000. Both electrophoretic behaviour and molecular weight differ from those of the low-molecular-weight adenosine deaminase purified from human erythrocytes. Its amino acid composition is reported. Tests with periodic acid-Schiff reagent for associated carbohydrate are negative. Of the large group of physiological compounds tested as potential effectors, none has a significant effect. The enzyme is specific for adenosine and deoxyadenosine, with Km values of 48 microM and 34 microM respectively. There are no significant differences in enzyme function on the two substrates. erythro-9-(2-Hydroxy non-3-yl) adenine is a competitive inhibitor, with Ki 15 nM. Deoxycoformycin inhibits deamination of both adenosine and deoxyadenosine, with an apparent Ki of 60-90 pM. A specific antibody was developed against the purified enzyme, and a sensitive radioimmunoassay for adenosine deaminase protein is described.