Further characterization of a lectin and its in vivo receptor from Geodia cydonium

From the results of two-dimensional isoelectric focusing, SDS-gel electrophoresis and from immunochemical data it became evident that lectin I and lectin II (corresponding to fractions Geodia I and Geodia II isolated on immobilized lactose) from the sponge Geodia cydonium are apparently identical mixtures of several isolectins, the pI values of their subunits ranging, in contrast to our previous report, from 4.8–7.5. The hypothetical concept of sugar-mediated, specific lectin-lectin interactions (self-recognition) could not be verified by binding of FITC-labelled isolectins (Geodia I) to the lectin subunits, which had been purified by SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. The concept should also be dismissed on the basis of carbohydrate analyses revealing in contradiction with previous results the exclusive presence of alkali-labile bound tetraglucose on the purified isolectins (1 mol/mol lectin protein). The combining site of the isolectins was shown by a quantitative microprecipitation inhibition assay to be most complementar to oligosaccharides of the β-galactoside series and to interact specifically with particular structural elements of the subterminal sugar(s). Carbohydrates of the anti aggregation receptor, which are assumed to represent the functional ligand of the Geodia-isolectins in vivo, could be demonstrated to have a high affinity for the lectin combining site, exceeding that of the best disaccharide inhibitor, lactose, by five orders of magnitude. A preliminary chemical characterization of the receptor carbohydrate revealed that D-galactose and D-glucose (each approx. 200 mol/mol receptor) are organized in an oligosaccharide, which could be cleaved from the protein by trifluoroacetolysis.     

Purification and characterization of multiple species (isolectins) of a slime mold lectin implicated in intercellular adhesion.

An improved purification procedure for the carbohydrate-binding proteins (lectins) of cohesive Polysphondylium pallidum cells has been devised. The procedure uses extraction of cells with lactose-containing buffer followed by ammonium sulfate precipitation and affinity chromatography of the redissolved precipitate on a column of acid-treated Sepharose 6B. All hemagglutination activity is adsorbed to the column and recoveries are about 70% of the activity of the starting cell lysate. Sodium dodecyl sulfate-gel electrophoresis of the protein obtained with this procedure resolved three subunits with molecular weights of 26,500 (A), 26,000 (B), and 25,000 (C). Three species are resolved by isoelectric focusing with apparent pI values of 6.4 (I), 7.3 (II), and 7.5 (III) which contain Subunits A, B, and C in the following ratios: I, B:C at 2:1; II, A:B at 2:1, and III, A:B at 1:2. All three isoforms agglutinate rabbit and human type O erythrocytes and are thus isolectins. Isoforms II and III are separated from Isoform I by galactose-gradient elution of the Sepharose 6B column. Isoforms II and III aggregate extensively (nonamers and multiples thereof), but reduction with 2-mercaptoethanol reverses this process yielding a single species of Mr = 73,000 (trimer). Isoform I exists as trimers and hexamers and reduction has no effect on this distribution. Amino acid compositions and tryptic peptide maps of S-[14C]carboxymethyl-isolectins indicate that Subunits A and B are very similar and may represent the same peptide chain, while Subunit C is a peptide quite distinct from A and B.     

Five alpha-D-galactopyranosyl-binding isolectins from Bandeiraea simplicifolia seeds.

The alpha-D-galactopyranosyl-binding lectin previously purified from Bandeiraea simplicifolia seeds (Hayes, C.H., and Goldstein, I.J. (1974) J. Biol. Chem. 249, 1904) is shown to consist of five isolectins separable on polyacrylamide gel electrophoresis at pH 9.5. The isolectins are tetrameric structures composed of various combinations of two different glycoprotein subunits designated A and B. The A and B subunits appear to be immunochemically indistinguishable against rabbit antisera prepared from the isolectin mixture. The A subunit contains no methionine, whereas the B subunit contains 1 residue. The subunits migrate differently on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and, although each subunit contains 1 residue of cysteine, they react differently toward 5,5'-dithiobis(2-nitrobenzoic acid). The carbohydrate binding specificity of the two subunits differs significantly: the A subunit exhibits a primary specificity for alpha-D-GalNAcp but also reacts with alpha-D-Galp units, whereas the B subunit shows a sharp specificity toward alpha-D-Galp residues. The differences in carbohydrate binding specificity were exploited in separating the isolectins. B. simplicifolia I isolectins (A4) and (A3B) were purified on a Bio-Gel melibionate column, and (A2B2), (AB3), and (B4) were separated on a column of insolubilized blood group A substance.     

Characterization of a seventh different subunit of beef heart cytochrome c oxidase. Similarities between the beef heart enzyme and that from other species.

Beef heart cytochrome c oxidase has been resolved into seven subunits by electrophoresis in highly cross-linked gels containing urea and sodium dodecyl sulfate. The molecular weights of the polypeptides are estimated to be I, 35 400; II, 24 100; III, 21 000; IV, 16 800; V, 12 400; VI, 8200; and VII, 4400. It has been shown that subunits II and III can coelectrophorese on standard sodium dodecyl sulfate-polyacrylamide gels and appear as a single component with an apparent molecular weight of 22 500. This accounts for previous reports that the beef heart enzyme contains only six subunits. Amino acid analysis of the isolated subunits I, II, and III revealed that they have polarities of 35.5, 44.7, and 39.9%, respectively. All three subunits have an extremely high leucine content and a low percentage of basic amino acids relative to subunits IV-VII. The size, number, and properties of subunits in the beef heart cytochrome c oxidase complex suggest that it has essentially the same subunit structure as the complexes isolated from Saccharomyces cerevisiae and Neurospora crassa.     

Monoclonal antibodies against a beta-galactoside-binding lectin of chick embryo

Monoclonal antibodies against an endogenous beta-galactoside-binding lectin (monomer molecular weight 14,000, 14K lectin) of chick embryo were prepared and characterized. The inhibitory activities against hemagglutination, antigenic determinants and binding specificities were examined. Monoclonal antibody S1A4-5 strongly inhibited the hemagglutination activity of this lectin. This antibody did not bind to any cyanogen bromide (BrCN) fragment of the lectin. Another monoclonal antibody, S1A4-3, bound to one of the BrCN fragments (residues 34-66). However, this antibody inhibited hemagglutination only weakly. The bindings to isolectins of beta-galactoside-binding lectin, namely 14K lectin (monomer molecular weight 16,000) and a third species which is assumed to be a hybrid molecule consisting of 14K and 16K lectin subunits, were examined. The antibody SIA4-5 bound to 14K lectin but not to 16K lectin. In the case of the third species, intermediate binding was observed.     

Isolation and characterization of a group of isolectins with galactose/N-acetylgalactosamine specificity from hemolymph of the giant silk moth Hyalophora cecropia

A series of isolectins from Hyalophora cecropia was purified by affinity chromatography on d-GalNAc-Sepharose. The yield of the lectin was 0.4 mg/ml of hemolymph and the concentration was about the same in larvae and pupae. The molecular weight of the lectin was around 160,000 and the sizes of the subunits were 41 and 38 kD for the A and the B chains, respectively. The isolectins are believed to be tetramers with varying proportions between the two subunits. Inhibition studies indicate that the A chain has a binding site with specificity for d-Gal/d-GalNAc while the B chain has specificity for an unknown structure on rabbit erythrocytes not necessarily of carbohydrate nature. A d-Gal/d-GalNAc specific lectin was found also in Anthereae pernyi but the yield was only 0.03 mg/ml of hemolymph.     

Purification and characterization of five forms of glutathione transferase from human uterus

Five glutathione transferase (GST) forms were purified from human uterus by glutathione-affinity chromatography followed by chromatofocusing, and their structural, kinetic and immunological properties were investigated. Upon SDS/polyacrylamide slab gel electrophoresis all forms resulted composed of two subunits of identical molecular size. GST V (pI 4.5) is a dimer of 23-kDa subunits. GST I (pI 6.8) and GST IV (pI 4.9) are dimers of 24-kDa subunits whereas GST II (pI 6.1) and GST III (pI 5.5) are dimers of 26.5-kDa subunits. GST V accounts for about 85-90% of the activity whereas the other isoenzymes are present in trace quantities. On the basis of the molecular mass of the subunits, amino acid composition, substrate specificities, sensitivities to inhibitors, CD spectra and immunological studies, GST V appeared very similar to transferase pi. Structural and immunological studies provide evidence that GST IV is closely related to the less 'basic' transferase (GST pI 8.5) of human skin. Extensive similarities have been found between GST II and GST III. The comparison includes amino acid compositions, subunits molecular size and immunological properties. The two enzymes, however, are kinetically distinguishable. The data presented also indicate that GST II and GST III are related to transferase mu and to transferase psi of human liver. Even though GST I has a subunit molecular mass identical to GST IV, several lines of evidence, including catalytic and immunological properties, indicate that they are different from each other. GST I seems not to be related to any of known human transferases, suggesting that it may be specific for the uterus.     

Subunit arrangement in beef heart complex III

Beef heart mitochondrial complex III was separated into 12 polypeptide bands representing 11 different subunits by using the electrophoresis conditions described by Sch?gger et al. [(1986) Methods Enzymol. 126, 224-237]. Eight of the 12 polypeptide bands were identified from their NH2-terminal sequences as obtained by electroblotting directly from the NaDodSO4-polyacrylamide gel onto a solid support. The topology of the subunits in complex III was explored by three different approaches. (1) Protease digestion experiments of submitochrondrial particles in the presence and absence of detergent showed that subunits II and VI are on the M side of the inner membrane and subunits V and XI on the C side. (2) Labeling experiments with the membrane-intercalated probes [125I]TID and arylazidoPE indicated that cytochrome b is the predominant bilayer embedded subunit of complex III, while the non-heme iron protein appears to be peripherally located. (3) Cross-linking studies with carbodiimides and homobifunctional cleavable reagents demonstrated that near-neighbor pairs include subunits I+II, II+VI, III+VI, IV+V, V+X, and reagents demonstrated that near-neighbor pairs include subunits I+II, II+VI, III+VI, IV+V, V+X, and VI+VII. The cytochrome c binding site was found to include subunits IV, VIII, and X. The combined data are used to provide an updated model for the topology of beef heart complex III.     

Purification and characterization of four isolectins of mushroom (Agaricus bisporus)

Four lectins were purified from a mushroom (Agaricus bisporus) by ammonium sulfate fractionation, anion-exchange chromatography, affinity chromatography on bovine submaxillary mucin-Sepharose 4B and preparative isoelectric focusing. They were designated as ABA-I (pI 6.70), II (pI 5.98), III (pI 5.69) and IV (pI 5.53). Polyacrylamide gel electrophoresis of each lectin in the presence of sodium dodecyl sulfate gave a single band with an apparent molecular mass of 16 000 Da. Sedimentation equilibrium analysis suggested that each lectin is a tetramer of subunits. The four lectins were found to have quite similar carbohydrate-binding specificities. The hemagglutination activities of the lectins were effectively inhibited by bovine and porcine submaxillary mucins (BSM and PSM), and NH2-terminal glyco-octapeptides obtained by cyanogen bromide cleavage of human erythrocyte glycophorin A. In addition, desialylated PSM-glycopeptides were more potent inhibitors than untreated PSM-glycopeptides. Among monosaccharides and their glycosides, only methyl N-acetyl-alpha-galactosaminide inhibited lectin binding at a high concentration, but a synthetic oligosaccharide, O-beta-galactopyranosyl-(1----3)-O-(2-acetamido-2-deoxy-alpha-D- galactopyranosyl)-N-tosyl-L-serine, was a strong inhibitor.     

Immunological studies on cytochrome c oxidase: arrangements of protein subunits in the solubilized and membrane-bound enzyme.

Seven protein subunits of cytochrome c oxidase from bovine heart were isolated by gel filtration in the presence of sodium dodecyl sulphate (subunits I, II and III) and guanidine hydrochloride (subunits V, VI and VII), and ion-exchange chromatography in 6 M urea (subunit IV) after the enzyme had been dissociated in 6 M guanidine hydrochloride. When analysed by highly cross-linked sodium dodecyl sulphate/polyacrylamide gel electrophoresis in the presence of urea, the apparent molecular weights were = I, 36700; II, 24300; III, 20400; IV, 17300; V, 12300; VI, 8700: and VII, 5100. Monospecific rabbit antisera were obtained against subunits I, IV, V, VI and VII and a mixture of subunits II and III. These subunit-specific antisera with the exception of anti-I serum all cross-reacted with the detergent-solubilized native oxidase. Enzymatic studies on purified oxidase indicated that immunoglobulins against subunits II + III, IV, V, VI and VII respectively caused 25, 65, 20, 30 and 25% inhibition while anti-I immunoglobulin did not inhibit the activity. The subunit-specific antisera were used to examine the arrangements of the subunits in the membrane. Enzymatic studies using bovine heart mitochondria and rat liver mitochondrial digitonin particles showed that anti-(II + III) serum, anti-V serum and anti-VII serum all inhibited the oxidase activity while the other antisera did not. On the other hand, results of using 125I-labelled immunoglobulins showed that anti-IV, anti-V and anti-VII sera were bound to the surface of inverted vesicles (matrix side) while all other antisera were not. These results indicate that cytochrome oxidase subunits II and III are situated on the outer surface, and subunit IV is exclusively on the matrix surface while subunits V and VII are exposed on both surfaces of the mitochondrial membrane. Subunits I and VI are buried within the membrane, not exposed on either side.     

Large scale isolation and properties of subunits from bovine heart cytochrome oxidase

The subunits of the cytochrome oxidase from bovine heart were isolated in large quantities suitable for amino acid sequence studies. The preparation of subunits III, IV, V, VI, and VII for sequence determination can be achieved without employing sodium dodecyl sulfate. The method presented essentially involves pyridine extraction, pH fractionation, ammonium sulfate fractionation, and various types of column chromatography. However, subunits I and II can be prepared only in the presence of sodium dodecyl sulfate by molecular sieve chromatography; subunit III can also be isolated in this manner. The separation of subunits is found to be hindered by phospholipids associated with the enzyme and therefore the phospholipid-depleted preparation is used as the starting material. The molecular weights of subunits I, II, III, IV, V, VI, and VII are 40,000, 21,000, 14,800, 13,500, 11,600, 9,500, and 7,600, respectively. These values are based on the results of the conventional Weber and Osborn method of gel electrophoresis in the presence of sodium dodecyl sulfate. The amino termini of subunits I and II have been determined as N-formylmethionine, and those of subunits III, IV, V, VI, and VII are alanine, alanine, serine, alanine, and an N-acetyl-blocked residue, respectively. The carboxyl termini for subunits I to VII are lysine, leucine, lysine, histidine, valine, isoleucine, and valine, respectively. The complete amino acid sequence of some subunits has been published and that of other subunits will be reported elsewhere in collaboration with the Amino Acid Sequence Group of Cytochrome Oxidase at the University of Hawaii.     

Loss of assembly of the main basement membrane collagen, type IV, but not fibril-forming collagens and embryonic death in collagen prolyl 4-hydroxylase I null mice

Collagen prolyl 4-hydroxylases (C-P4Hs) catalyze the formation of the 4-hydroxyproline residues that are essential for the generation of triple helical collagen molecules. The vertebrate C-P4Hs I, II, and III are [alpha(I)]2beta2, [alpha(II)]2beta2, and [alpha(III)]2beta2 tetramers with identical beta subunits. We generated mice with targeted inactivation of the P4ha1 gene encoding the catalytic alpha subunit of C-P4H I to analyze its specific functions. The null mice died after E10.5, showing an overall developmental delay and a dilated endoplasmic reticulum in their cells. The capillary walls were frequently ruptured, but the capillary density remained unchanged. The C-P4H activity level in the null embryos and fibroblasts cultured from them was 20% of that in the wild type, being evidently due to the other two isoenzymes. Collagen IV immunofluorescence was almost absent in the basement membranes of the null embryos, and electron microscopy revealed disrupted basement membranes, while immunoelectron microscopy showed a lack of collagen IV in them. The amount of soluble collagen IV was increased in the null embryos and cultured null fibroblasts, indicating a lack of assembly of collagen IV molecules into insoluble structures, probably due to their underhydroxylation and hence abnormal conformation. In contrast, the null embryos had collagen I and III fibrils with a typical cross-striation pattern but slightly increased diameters, and the null fibroblasts secreted fibril-forming collagens, although less efficiently than wild-type cells. The primary cause of death of the null embryos was thus most likely an abnormal assembly of collagen IV.     

Chemical and biological characterization of the galactose binding lectins from Trichosanthes kirilowii root tubers

Three galactose binding isolectins have been isolated from Trichosanthes kirilowii root tubers. Two of the isolectins, TK-I and TK-II, are similar in many aspects including molecular weight, amino acid composition, NH2-terminal amino acid residue, blood group and carbohydrate specificities, immunodiffusion and immunoelectrophoretic behavior, hemagglutinating and insulinomimetic activities, and in possessing subunits with different molecular weights. Compared to TK-I and TK-II, lectin TK-III has a larger molecular weight, subunits with the same molecular weight, a single and distinctive NH2-terminal amino acid residue, a different isoelectric point and lower hemagglutinating activity. The three lectins share common antigenic determinants in their structures. beta-Linked terminal oligosaccharides containing D-galactose inhibit hemagglutination induced by the lectins with a higher potency than alpha-linked oligosaccharides. The lectins are non-mitogenic and did not inhibit the concanavalin-A induced mitogenic response of lymphocytes.     

Distribution of Lectins in the Jumbo Virginia and Spanish Varieties of the Peanut, Arachis hypogaea L

Peanut lectin was purified from seed meal of the Spanish and Jumbo Virginia varieties of peanut (Arachis hypogaea L.) by affinity chromatography on lactose coupled to Sepharose 4B. Polyacrylamide gel isoelectric focusing resolved the lectin preparation from Jumbo Virginia seeds into seven isolectins (pI 5.7, 5.9, 6.0, 6.2, 6.3, 6.5, and 6.7). Seed meal from the Spanish variety contained six isolectins which were indistinguishable from the pI 5.7, 5.9, 6.2, 6.3, 6.5, and 6.7 isolectins from Jumbo Virginia. Quantitative, lactose-specific hemagglutination was used to examine the lectins in tissues of both peanut varieties. In young (3- to 9-day-old) seedlings of each variety, more than 90% of the total amount of lectins detected in the plants was in the cotyledons. Most of the remainder was in hypocotyls, stems, and leaves; young roots contained no more than 4 micrograms of lectin per plant. Lectins were present in all nonroot tissues of 21- to 30-day-old seedlings, except 27-day-old Spanish hypocotyls. As cotyledons of each variety senesced, several of the more basic isolectins decreased to undetectable levels, but the acidic isolectins remained until at least 15 days after planting. Some of the seed isolectins and several apparently new lactose-binding lectins were also identified in affinity-purified extracts of 5-day-old roots and hypocotyls. Rabbit antibodies raised against the Jumbo Virginia seed isolectin preparation reacted with seed, cotyledon, and hypocotyl lectin preparations from both varieties. Analysis of seed lectin preparations from seven varieties of A. hypogaea and of a related species (A. villosulicarpa) indicated that isolectin composition in Arachis may be a characteristic of both the species and the subspecies (botanical type) to which the variety belongs.     

The murine 3β-hydroxysteroid dehydrogenase multigene family: Structure, function and tissue-specific expression

The classical form of the enzyme 5-ene-3β-hydroxysteroid dehydrogenase/isomerase (3βHSD), expressed in adrenal glands and gonads, catalyzes the conversion of 5-ene-3β-hydroxysteroids to 4-ene-3-ketosteroids, an essential step in the biosynthesis of all active steroid hormones. To date, four distinct mouse 3βHSD cDNAs have been isolated and characterized. These cDNAs are expressed in a tissue-specific manner and encode proteins of two functional classes. Mouse 3βHSD I and III function as 3β-hydroxysteroid dehydrogenases and 5-en→4-en isomerases using NAD⁺ as a cofactor. The enzymatic function of 3βHSD II has not been completely characterized. Mouse 3βHSD IV functions only as a 3-ketosteroid reductase using NADPH as a cofactor. The predicted amino acid sequences of the four isoforms exhibit a high degree of identity. Forms II and III are 85 and 83% homologous to form I. Form IV is most distant from the other three with 77 and 73% sequence identity to I and III, respectively. 3βHSD I is expressed in the gonads and adrenal glands of the adult mouse. 3βHSD II and III are expressed in the kidney and liver with the expression of form II greater in kidney and form III greater in liver. Form IV is expressed exclusively in the kidney. Although the amino acid composition of forms I, III and IV predicts proteins of the same molecular weight, the proteins have different mobilities on SDS-polyacrylamide gel electrophoresis. This characteristic allows for differential identification of the expressed proteins. The four structural genes encoding the different isoforms are closely linked within a segment of mouse chromosome 3 that is conserved on human chromosome 1.     

Studies on hemagglutinins (lectins) from plasma of murrel fish, family Channidae

Hemagglutinating activity can be identified in the plasma of different species of murrel fish. This activity may be divided into four types according to their agglutinability towards erythrocytes from different sources. Type I plasma agglutinates human blood group A erythrocytes, type II can agglutinate neuraminidase treated human A B O erythrocytes, type III shows no agglutinating activity towards human erythrocytes, while type IV agglutinates human erythrocytes non-specifically. All of them bind to DEAE-cellulose but elute out by different salt concentrations. Type IV plasma is found to be a combination of three separate hemagglutinins, which are separable by sequential binding to human A B O erythrocytes. Blood group A specific lectin activity is purified from this plasma using formalinised A group erythrocytes. The apparent homogeneity of this purified lectin is established by polyacrylamide gel electrophoresis, isoelectric focusing and immunodiffusion. This agglutinin is antigenically identical with that isolated from type I plasma by affinity chromatography on N-acetyl-D-galactosamine coupled to epoxy-activated cellulose column. Their molecular weights are also found to be identical (Mr 140,000) in polyacrylamide gel electrophoresis, having two identical subunits. Forssman glycolipid (0.03 mM) was found to be the most potent inhibitor of agglutination, although Gal beta 1-3 GalNAc (0.09 mM) is also a good inhibitor. Exhaustive dialysis of the purified lectin (hemagglutinin) against EDTA denatures it irreversibly by dissociating it to its subunit structure. Thus human A group agglutinating activity isolated from type I and type IV plasma are identical.     

Rat lung glutathione S-transferases: subunit structure and the interrelationship with the liver enzymes

Six forms of glutathione S-transferases designated as GSH S-transferase I (pI 8.8), II (pI 7.2), III (pI 6.8), IV (pI 6.0), V (pI 5.3) and VI (pI 4.8) have been purified from rat lung. GSH S-transferase I (pI 8.8) is a homodimer of Mr 25,000 subunits; GSH S-transferases II (pI 7.2) and VI (pI 4.8) are homodimers of Mr 22,000 subunits; and GSH S-transferases III (pI 6.8), IV (pI 6.0) and V (pI 5.3) are dimers composed of Mr 23,500 and 22,000 subunits. Immunological properties, peptide fragmentation analysis, and substrate specificity data indicate that Mr 22,000, 23,500 and 25,000, are distinct from each other and correspond to Ya, Yb, and Yc subunits, respectively, of rat liver.     

The ratio of oxidative phosphorylation complexes I-V in bovine heart mitochondria and the composition of respiratory chain supercomplexes

The ratios of the oxidative phosphorylation complexes NADH:ubiquinone reductase (complex I), succinate:ubiquinone reductase (complex II), ubiquinol:cytochrome c reductase (complex III), cytochrome c oxidase (complex IV), and F1F0-ATP synthase (complex V) from bovine heart mitochondria were determined by applying three novel and independent approaches that gave consistent results: 1) a spectrophotometric-enzymatic assay making use of differential solubilization of complexes II and III and parallel assays of spectra and catalytic activities in the samples before and after ultracentrifugation were used for the determination of the ratios of complexes II, III, and IV; 2) an electrophoretic-densitometric approach using two-dimensional electrophoresis (blue native-polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis) and Coomassie blue-staining indices of subunits of complexes was used for determining the ratios of complexes I, III, IV, and V; and 3) two electrophoretic-densitometric approaches that are independent of the use of staining indices were used for determining the ratio of complexes I and III. For complexes I, II, III, IV, and V in bovine heart mitochondria, a ratio 1.1 +/- 0.2:1.3 +/- 0.1:3:6.7 +/- 0.8:3.5 +/- 0.2 was determined.     

Purification and Characterization of Griffonia simplicifolia Leaf Lectins

Leaves from mature Griffonia simplicifolia plants were examined for the presence of leaf lectins possessing sugar binding specificities similar to the four known seed lectins (GS-I, GS-II, GS-III, GS-IV). Three (GS-I, -II, -IV) of the four known G. simplicifolia seed lectins were present in the leaves. Leaf G. simplicifolia lectins I and IV were similar to the respective seed lectins. Leaf GS-II, however, was composed of two types of subunits (M_r = 33,000 and 19,000), whereas the seed lectin consists of only one type of subunit (M_r 32,500). Seed and leaf GS-II lectins also had different isoelectric points. All leaf and seed lectins were similar with respect to their hemagglutination and glycoconjugate precipitation properties and all subunits contained covalently bound carbohydrate. Leaf GS-IV appeared slightly under-glycosylated compared to seed GS-IV.

The fate of GS-I and GS-II seed lectins in aging cotyledons was investigated. GS-I isolectins usually contain isolectin subtypes associated with each main isolectin. Upon inbibition and germination, these GS-I isolectin subtypes disappeared. Over time, GS-II lectin did not change its disc gel electrophoretic properties.

     

Multiple forms of rat-liver dihydropteridine reductase identified by their differing isoelectric points

Purified rat-liver dihydropteridine reductase is homogeneous by gel filtration (Mr approximately 51,000), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr approximately 25,500), and native polyacrylamide gel electrophoresis, suggesting that the enzyme is composed of two identical subunits. However, analysis by isoelectric focusing has revealed three enzyme forms with approximate isoelectric points of 6.5, 5.9, and 5.7 (designated forms, I, II, and III, respectively). The three forms, isolated in 65% yield by preparative chromatofocusing, are stable in 0.05 M phosphate buffer, pH 6.8, containing 1 mM beta-mercaptoethanol and exhibit similar kinetic constants when the catalytic activities of the isolated forms are compared with quinonoid dihydrobiopterin as substrate. All forms generate complexes with the enzymatic cofactor NADH which are also detectable by IEF. When examined further by IEF under denaturing conditions in 6 M urea the enzyme demonstrates a differing subunit composition for its three forms. Two distinct subunits, designated alpha and beta, can be identified, and additional evidence suggests that the native enzyme forms I, II, and III represent the three differing dimeric combinations alpha alpha (form I), alpha beta (form II), and beta beta (form III).