Distinct chromatographic forms of human hemi-myeloperoxidase obtained by reductive cleavage of the dimeric enzyme. Evidence for subunit heterogeneity

The enzyme myeloperoxidase (MPO) is a functionally important glycoprotein of neutrophilic granulocytes and occurs in three major isoforms (forms 1, 2, and 3) that are dimeric structures composed of two heavy subunit-light subunit protomers, each of which is associated with a chlorine-like prosthetic group. In the present study, highly purified MPO isoforms were obtained from the cells of a single normal donor, and each protein was subjected to reductive alkylation under nondenaturing conditions. The resulting enzymatically active protomers were separated from unreacted dimer using gel filtration chromatography. Use of a fast protein liquid chromatography cation exchange system with a Mono S matrix revealed heterogeneity of the protomers, and allowed essentially complete resolution of the protomers of MPO form 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the two resolved protomeric species under reducing conditions revealed small but reproducible differences in the Mr of their heavy subunits (59,000 and 57,000). Treatment with either endo-beta-N-acetylglucosaminidase or peptide N-glycohydrolase F reduced the Mr of each heavy subunit by approximately 3000 but did not change their relative electrophoretic mobilities. Heavy and light subunits were prepared from each of the MPO isoforms by reductive alkylation under conditions that allowed full retention of the prosthetic group with the heavy subunit. Reverse-phase chromatography and amino-terminal sequencing showed that each MPO isoform contained one major species of light subunit and several minor species. No differences in peroxidatic activity or inhibition by salicylhydroxamic acid were observed among any of the MPO isoforms or resolved protomers, but the latter were considerably more heat labile than dimeric forms of the enzyme and a monomeric form isolated from HL-60 cells. This is the first report of the isolation and partial characterization of distinct protomers from a single isoform of human MPO and suggests that the structure of MPO is more complex than considered previously.     

Molecular characterization of the sex steroid binding protein (SBP) of plasma. Re-examination of rabbit SBP and comparison with the human, macaque and baboon proteins

Physico-chemical characterization of the sex steroid-binding protein, SBP, of rabbit plasma reveals that it is a dimer of mol. wt 85,800 composed of similar subunits of mol. wt 43,000. These data confirm our original proposal for a dimeric structure. The protein contains 9% carbohydrate, comprised of mannose, galactose, N-acetylglucosamine and sialic acid. It is devoid of N-acetylgalactosamine and fucose. The protein binds one molecule of 5 alpha-dihydrotestosterone per dimer with a Kd of 0.89 nM (12 degrees C). Comparison with the human, monkey and baboon SBPs indicates that all these proteins have the same dimeric molecular organization and exhibit microheterogeneity in SDS-PAGE and isoelectricfocusing. Rabbit SBP, however, contains less carbohydrate and has a higher polypeptide molecular weight than all the other SBPs. Spectrophotometric data also indicate that some tryptophan residues are in a different chemical environment than those in other SBPs. The observed microheterogeneity in all four SBP species is due for the most part to variable glycosylation of the subunit and variability at the amino-terminal region of the subunit. Combination of these and other phenomena will generate a significant number of isomeric forms of the SBP subunit which will then interact stoichiometrically to yield active dimeric SBP molecules. These differ slightly from each other depending upon the charge and size of the subunit comprising the dimeric structure, and will result in the observed microheterogeneity of pure SBP preparations. Based on these results along with more recent amino acid sequence data, we conclude that all four SBPs are dimers composed of identical polypeptide chains.     

Direct evidence for the localization of the steroid-binding site of the plasma sex steroid-binding protein (SBP or SHBG) at the interface between the subunits.

Complete dissociation of dimeric plasma sex steroid-binding protein (SBP or SHBG) was obtained in 6 M urea at 10 degrees C. Removal of urea resulted in the refolding of monomers, followed by reformation of dimeric SBP, which migrates with the same mobility as the native protein. Dimerization does not require Ca+2 or steroid. Renatured monomers yield dimers with dissociation constants for 5 alpha-dihydrotesterone (DHT) and 17 beta-estradiol (E2) indistinguishable from those of native human SBP. This phenomenon was also demonstrated by mixing human and rabbit SBPs that, upon renaturation, form a hybrid dimer composed of one human subunit and one rabbit subunit. The hybrid binds both DHT and E2 in contrast to rSBP, which only binds the androgen. Therefore, we conclude that (1) docking of the two subunits creates an asymmetric steroid-binding site located at the interface between the subunits, and (2) only one face of the dimer defines the specificity for binding E2 by encompassing portion of a structural motif that recognizes the flat ring A of E2. The remaining portion, which recognizes the saturated ring A of DHT, is shared by both faces of the dimer. Because native monomers do not exist alone, the often-asked question of whether the SBP monomer binds steroid can be considered meaningless; steroid-binding activity is expressed only in the dimeric state. Finally, formation of the hybrid indicates that SBP dimerization represents a conserved event during the molecular evolution of SBP, suggesting that the structural elements responsible for dimerization will be homologous in SBPs from other species.     

Light chains of mammalian cytoplasmic dynein: identification and characterization of a family of LC8 light chains.

Cytoplasmic dynein is a large multisubunit motor protein that moves various cargoes toward the minus ends of microtubules. In addition to the previously identified heavy, intermediate, and light intermediate chains, it has recently been recognized that cytoplasmic dynein also has several light chain subunits with apparent molecular weights between 8-20 kDa. To systematically identify the light chains of purified rat brain cytoplasmic dynein, peptide sequences were obtained from each light chain band resolved by gel electrophoresis. Both members of the tctex1 light chain family, tctex1 and rp3, were identified in a single band. Only one member of the roadblock family, roadblock-2, was found. Two members of the LC8 family were resolved as separate bands, the previously identified LC8 subunit, and a second novel cytoplasmic dynein family member, LC8b. The tissue distribution of these two dynein LC8 subunits differed, although LC8b was the major family member in brain. Database searches found that both LC8a and LC8b were also present in several mammalian species, and a third mammalian LC8 sequence, LC8c was found in the human database. The amino acid sequences of both LC8a and LC8b were completely conserved in mammals. LC8a and LC8b differ in only six of the 89 amino acids. The amino acid differences between LC8a and LC8b were located near the N-terminus of the molecules, and most were in the outward facing alpha-helices of the LC8 dimer. When the mammalian LC8a sequence was compared to the LC8 sequences found in six other animal species including Xenopus and Drosophila, there was, on average, 94% sequence identity. More variation was found in LC8 sequences obtained from plants, fungi, and parasites. LC8c differed from the other two human LC8 sequences in that it has amino acid substitutions in the intermediate chain binding domain at the C-terminal of the molecule. The position of amino acid substitutions of the three mammalian LC8 family members is consistent with the hypothesis that they bind to different proteins.     

Subunit structure of the galactose and N-acetyl-D-galactosamine-inhibitable adherence lectin of Entamoeba histolytica

The galactose and N-acetyl-D-galactosamine-inhibitable adherence lectin of Entamoeba histolytica is a cell surface protein which mediates parasite adherence to human colonic mucus, colonic epithelial cells, and other target cells. The amebic lectin was purified in 100-micrograms quantities from detergent-solubilized trophozoites by monoclonal antibody affinity chromatography. The adherence lectin was purified 500-fold as judged by radioimmunoassay. The nonreduced lectin had a molecular mass of 260 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point of pH 6.2. The amebic lectin reduced with beta-mercaptoethanol consisted of 170- and 35-kDa subunits. Both subunits could be labeled on the cell surface with 125I, and both were metabolically labeled with [3H]glucosamine. The amino termini of the subunits had unique amino acid sequences, and polyclonal antisera to the heavy subunit did not cross-react with the light subunit. The yield of phenylthiohydantoin derivatives from the second and third positions in the sequence of the heavy and light subunits gave a molar ratio of one 170- to one 35-kDa subunit. Antibodies directed to the heavy subunit inhibited amebic adherence to Chinese hamster ovary cells by 100%, suggesting that the heavy subunit is predominantly responsible for mediating amebic adherence.     

Structural studies and two-dimensional gel electrophoresis of gamma-glutamyl transpeptidase

The heterodimeric enzyme gamma-glutamyl transpeptidase (EC 2.3.2.2) was isolated from adult rat kidney and purified to homogeneity for structural studies using papain solubilization and multiple chromatographies. Two-dimensional gel electrophoresis was found to resolve the active papain-purified enzyme into at least 18 components. Seven components with apparent molecular masses of 23,000-26,000 and isoelectric point range of 5.4-7.0 constitute the light subunit, and 11 components with apparent molecular mass of 51,000-53,000 and isoelectric point range of 5.8-7.1 constitute the heavy subunit. Immunoblot analysis of two-dimensional gels showed that all of these components are immunoreactive with a mixture of the two antibodies generated separately against the light and heavy subunits. Preparative subunit separation was achieved using reverse-phase HPLC under acidic but nonreducing conditions. N-Terminal amino acid sequencing of the separated subunits of the papain-purified enzyme yielded sequence information for the first 32 residues of the heavy chain with the N-terminal starting sequence Gly-Lys-Pro-Asp-His-Val-Tyr-Ser-Arg-Ala, and for the first 36 residues of the light subunit with the N-terminal starting sequence Thr-Ala-His-Leu-Ser-Val-Val-Ser-Glu-Asp.     

RAPID CONCURRENT AUTOMATED FLUORIMETRIC ASSAY OF NORADRENALINE, DOPAMINE, 3, 4-DIHYDROXYPHENYLACETIC ACID, HOMOVANILLIC ACID AND 3-METHOXYTYRAMINE IN MILLIGRAM AMOUNTS OF NERVOUS TISSUE AFTER ISOLATION ON SEPHADEX G10

Abstract— Brain tubulin subunits were separated by a combination of isoelectric focusing and electrophoresis in the presence of sodium dodecyl sulfate (SDS) using a two-dimensional polyacrylamide slab gel technique. Isoelectric focusing separated tubulin subunits into two major groups of bands, such that the more acidic group corresponded to the α subunit and the less acidic group corresponded to the β subunit. In addition, isoelectric focusing resolved the β subunit into two subspecies which differed slightly in isoelectric properties but were the same apparent molecular weight. The a subunit was resolved into many subspecies that appear to differ from each other by both apparent molecular weight and isoelectric properties.     

The microheterogeneity of androgen-binding protein in rat serum and epididymis is due to differences in glycosylation of their subunits

The microheterogeneity of androgen-binding protein (ABP) from rat serum and epididymis was examined by subjecting purified native or deglycosylated preparations to analysis by one- or two-dimensional polyacrylamide gel electrophoresis (PAGE) followed by electrophoretic transfer to nitrocellulose and immunochemical localization. Analysis of native ABP by one-dimensional sodium dodecyl sulfate-PAGE confirmed earlier observations that it is composed of subunits and that the subunits of serum ABP had higher apparent molecular weights than those of epididymal ABP. Treatment with neuraminidase, N-glycanase, or O-glycanase, alone or in combination, resulted in decreases in the apparent molecular weight of the subunits. These analyses indicated that terminal sialic acid residues and Asn-linked oligosaccharides were present on both subunits of ABP from the two sources. The fact that the greatest reduction in the Mr of the heavy subunit occurred following treatment with all three enzymes provides evidence that O-linked sugars are present on it. While enzyme treatment did not result in the appearance of a single subunit, chemical deglycosylation did (Mr 39,600). The carbohydrate composition of the heavy and light subunits of intact serum and epididymal ABP was 22 and 9% and 19 and 8%, respectively. Analysis by two-dimensional PAGE indicated that both subunits of the ABPs were composed of isoelectric variants. Although ABP from the two sources had several variants in common, differences were also observed. Treatment of the ABPs with the enzymes resulted in a shift of the pI values to a more basic pH range, indicating that carbohydrate removal also removed charged moieties. The most dramatic shift in the pI values of the isoforms occurred when O-glycanase was present in the enzyme mixture, providing further evidence for the presence of O-linked oligosaccharides on ABP. Isoelectric variants were present even after chemical deglycosylation of ABP.     

Immunologically related multimeric forms of 30-40 kDa peptides associated with lung surfactant in various mammalian species

A comparative study of lung surfactant associated proteins was undertaken to determine which mammalian species would best serve as models for investigating alterations of the human lung surfactant system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified surfactants in the presence of dithiothreitol revealed that surfactant invariably contains at least one peptide with molecular weight of 30 000-40 000. In the absence of disulfide reducing agents, the above peptides were in the form of high-molecular-weight proteins (greater than 400 kDa) in primates and cat, whereas in dog, rat and rabbit, the protein was a 72 kDa dimer. The 30-40 kDa peptide subunits were isolated from human, rat and dog surfactants and found to contain four or five residues of hydroxyproline. Antisera to either the human 34 kDa peptide or high-molecular-weight proteins reacted with the high-molecular-weight bands, the 34 kDa subunit and at least six intermediate disulfide-linked forms separated from purified human surfactant by electrophoresis under nonreducing conditions. Following electrophoresis in the presence of dithiothreitol, both antisera detected the 34 kDa peptide as well as other peptides ranging in molecular weight from 23 000 to 160 000. The isolated 34 kDa peptide readily reaggregated into disulfide-linked forms including 68 and 100 kDa complexes which were not reduced by 40 mM dithiothreitol. We conclude that the 34 kDa surfactant-associated peptide forms a complex system of monomeric and multimeric proteins, which varies among the species and could conceivably vary in distribution during lung development or disease.     

Immunologically related multimeric forms of 30–40 kDa peptides associated with lung surfactant in various mammalian species

A comparative study of lung surfactant associated proteins was undertaken to determine which mammalian species would best serve as models for investigating alterations of the human lung surfactant system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified surfactants in the presence of dithiothreitol revealed that surfactant invariably contains at least one peptide with molecular weight of 30 000–40 000. In the absence of disulfide reducing agents, the above peptides were in the form of high molecular-weight proteins (> 400 kDa) in primates and cat, whereas in dog, rat and rabbit, the protein was a 72 kDa dimer. The 30–40 kDa peptide subunits were isolated from human, rat and dog surfactants and found to contain four or five residues of hydroxyproline. Antisera to either the human 34 kDa peptide or high-molecular-weight proteins reacted with the high-molecular-weight bands, the 34 kDa subunit and at least six intermediate disulfide-linked forms separated from purified human surfactant by electrophoresis under nonreducing conditions. Following electrophoresis in the presence of dithiothreitol, both antisera detected the 34 kDa peptide as well as other peptides ranging in molecular weight from 23 000 to 160 000. The isolated 34 kDa peptide readily reaggregated into disulfide-linked forms including 68 and 100 kDa complexes which were not reduced by 40 mM dithiothreitol. We conclude that the 34 kDa surfactant-associated peptide forms a complex system of monomeric and multimeric proteins, which varies among the species and could conceivably vary in distribution during lung development or disease.     

Chemical and immunochemical characteristics of tropomyosins from striated and smooth muscle

1. On electrophoresis in dissociating conditions the tropomyosins isolated from skeletal muscles of mammalian, avian and amphibian species migrated as two components. These were comparable with the alpha and beta subunits of tropomyosin present in rabbit skeletal muscle. 2. The alpha and beta components of all skeletal-muscle tropomyosins contained 1 and 2 residues of cysteine per 34000g respectively. 3. The ratio of the amounts of alpha and beta subunit present in skeletal muscle tropomyosins was characteristic for the muscle type. Muscle consisting of slow red fibres contained a greater proportion of beta-tropomyosin than muscles consisting predominantly of white fast fibres. 4. Mammalian and avian cardiac muscle tropomyosins consisted of alpha-tropomyosin only. 5. Mammalian and avian smooth-muscle tropomyosins differed both chemically and immunologically from striated-muscle tropomyosins. 6. Antibody raised against rabbit skeletal alpha-tropomyosin was species non-specific, reacting with all other striated muscle alpha-tropomyosin subunits tested. 7. Antibody raised against rabbit skeletal beta-tropomyosin subunit was species-specific.     

Purification, properties, and kinetics of D-ribulokinase from Aerobacter aerogenes.

The enzyme D-ribulokinase from Aerobacter aerogenes was purified to near homogeneity. The molecular weight, as determined by Sephacryl gel chromatography, is 116,000. The subunit molecular weight, determined by sodium dodecyl sulfate-gel electrophoresis, is 59,000, suggesting that D-ribulokinase is a dimer of identical subunits. Initial rate kinetic studies, involving substrate analogs and products, were carried out. These investigations support a kinetic mechanism of the Random Bi Bi type. Isotope partitioning, utilizing D-[3H]ribulose, indicates that the mechanism is steady state Random Bi Bi.     

Variability of the Low Molecular Weight Globulin,Conglutin δ, Within Lupin Species

Conglutin δ, a 2S globulin, was purified and compared in six species or varieties of lupin seeds. A common pattern is suggested, present in all species, corresponding to a protein which could exist as a monomer or a dimer. The first form contains one subunit, from 11 to 16.2 kDa, according to the species. It possesses a quaternary structure closely related to conglutin δ1 and was previously described in the narrow-leaved lupin. The second form contains two similar subunits (23 to 26 kDa) and could be the conglutin δ2. These two subunits are associated even when SDS is used and are probably disulfide-linked subunits. Each subunit is composed of two disulfide-linked polypeptides. One is acidic with molecular weight from 14 to 17.3 kDa and the second is acidic to neutral, from 2.4 to 4.5 kDa. Three species (L. luteus, L. arboreus and L. pilosus) present a supplementary subunit, with different molecular weight and p than that previously described and which never associates in a dimer form. It has been purified in L. luteus. When native, this protein is oligomeric. The subunit of 12 kDa in this species is composed of a polypeptide of 9 kDa (pl 4.5) disulfide-linked to one of 3 kDa (pl 6.5). This supplementary protein remains partly associated with the first in the yellow lupin (L. luteus). It probably corresponds to a new protein, different from conglutin δ.     

Superoxide dismutase in the housefly,Musca domestica (L.)

Four superoxide dismutase (SOD) (E.C. 1.15.1.1) isozymes were present in whole tissue homogenates of Musca domestica when examined by polyacrylamide gel electrophoresis. One of the isozymes contained manganese, and the other three contained copper and zinc. All were observed in each of the body tagma (head, abdomen, and thorax) and at each developmental stage (egg to adult). The copper- and zinc-containing isozymes purified from newly emerged, adult M. domestica had a relative molecular weight of 34,800 as determined by gel filtration chromatography but consisted of two equal-size subunits of 16,000 as measured by sodium dodecylsulfate polyacrylamide gel electrophoresis. An isoelectric point between 4.8 and 5.1 was measured. Approximately 2 mol each of copper and zinc were present per dimer. The three copper, zinc isozymes were identified as charge variants. The amino acid composition of the enzyme was similar to that of copper, zinc-containing superoxide dismutases from other sources. Purified housefly copper, zinc superoxide dismutase was neither deactivated nor able to protect lactic dehydrogenase against deactivation in the presence of light and rose bengal, a known generator of singlet oxygen. The role of SOD in the phototoxic reaction involving rose bengal is discussed.     

Characterization of the 1,4-dihydropyridine receptor using subunit-specific polyclonal antibodies. Evidence for a 32,000-Da subunit

The purified receptor for the 1,4-dihydropyridine Ca2+ channel blockers from rabbit skeletal muscle contains protein components of 170,000 Da (alpha 1), 175,000 Da (alpha 2), 52,000 Da (beta), and 32,000 Da (gamma) when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Subunit-specific polyclonal antibodies have now been prepared and used to characterize the association of the 32,000-Da polypeptide (gamma subunit) with other subunits of the dihydropyridine receptor. Immunoblot analysis of fractions collected during purification of the dihydropyridine receptor shows that the 32,000-Da polypeptide copurified with alpha 1 and alpha 2 subunits at each step of the purification. In addition, monoclonal antibodies against the alpha 1 and beta subunits immunoprecipitate the digitonin-solubilized dihydropyridine receptor as a multisubunit complex which includes the 32,000-Da polypeptide. Polyclonal antibodies generated against both the nonreduced and reduced forms of the alpha 2 subunit and the gamma subunit have been used to show that the 32,000-Da polypeptide is not a proteolytic fragment of a larger component of the dihydropyridine receptor and not disulfide linked to the alpha 2 subunit. In addition, polyclonal antibodies against the rabbit skeletal muscle 32,000-Da polypeptide specifically react with similar proteins in skeletal muscle of other species including avian and amphibian species. Thus, our results demonstrate that the 32,000-Da polypeptide (gamma subunit) is an integral and distinct component of the dihydropyridine receptor.     

Quantitation of myosin in muscle

The amount of myosin per gram of cardiac and skeletal muscle was determined in sodium dodecyl sulfate-solubilized tissue homogenates by radioimmunoassay and by isotope dilution. In the rabbit ventricle, there was an average of 27 mg myosin/g wet wt of tissue. In chickens, the myosin content of typical "red" (anterior latissimus dorsi) and "white" (posterior latissimus dorsi) skeletal muscles was higher than that of ventricular muscle, averaging 36 and 48 mg/g of tissue, respectively. The stoichiometry of the heavy and light chains in cardiac myosin was also determined from the quantitative binding of 125I-labeled Coomassie blue to each subunit after separation of the subunits by sodium dodecyl sulfate-gel electrophoresis. With this procedure, we found that the combined light-chain subunits contributed 19% of the myosin mass. After adjustment for the light-chain contribution, the myosin heavy-chain content of the rabbit ventricle averaged 22 mg/g wet wt of tissue.     

Characterization of vitellin,egg-specific protein and 30 kDa protein from Bombyx eggs,and their fates during oogenesis and embryogenesis

Three major yolk proteins, vitellin, egg-specific protein and 30 kDa proteins, were purified from the same extracts of Bombyx mori eggs by high-performance liquid chromatography on a molecular sieving column. Each preparation was judged to be homogeneous by polyacrylamide gel electrophoresis. The subunit structure was estimated to be as follows: vitellin is a tetramer with a molecular mass of 420 kDa, consisting of two heavy subunits (178 kDa) and two light subunits (43 kDa); egg-specific protein is a trimer (225 kDa) of two heavy subunits (72 kDa) and one light subunit (64 kDa); 30 kDa proteins are a mixture of three monomers (1, 2 and 3) consisting of respective subunit molecular masses of 32.0, 31.0 and 29.5 kDa. The three yolk proteins contained the usual amino acids together with various lipids and carbohydrates. Antisera to each protein did not cross-react. The titration of vitellin, egg-specific protein and 30 kDa proteins on rocket immunoelectrophoresis showed a differential accumulation pattern during the course of oogenesis. In newly laid eggs, vitellin, egg-specific protein and 30 kDa proteins accounted for approx. 40%, 25% and 35%, respectively, in weight. The eggs developed in male hosts after implantation of ovary discs were deficient in vitellin but contained egg-specific protein and 30 kDa proteins at comparable levels to the normal female eggs. During embryogenesis, egg-specific protein was rapidly and completely utilized. Approx. 35% vitellin and 50% 30 kDa proteins remained unused and were carried over to the hatched larvae. Such accumulation and utilization of yolk proteins are correlated with the fates of the proteins during oogenesis and embryogenesis of B. mori.     

The subunit interface of the Escherichia coli ribosome. Crosslinking of 30 S protein S9 to proteins of the 50 S subunit.

Purified 50 S ribosomal subunits were found to contain significant amounts of protein coincident with the 30 S proteins S9 and/or S11 on two-dimensional polyacrylamide/urea electropherographs. Peptide mapping established that the protein was largely S9 with smaller amounts of S11. Proteins S5 and L6 were nearly coincident on the two-dimensional polyacrylamide/urea electropherographs. Peptide maps of material from the L6 spot obtained from purified 50 S subunits showed the presence of significant amounts of the peptides corresponding to S5. Experiments in which ³⁵S-labelled 30 S subunits and non-radioactive 50 S subunits were reassociated to form 70 S ribosomes showed that some radioactive 30 S protein was transferred to the 50 S subunit. Most of the transferred radioactivity was associated with two proteins, S9 and S5. Sulfhydryl groups were added to the 50 S subunit by amidination with 2-iminothiolane (methyl 4-mercaptobutyrimidate). These were oxidized to form disulfide linkages, some of which crosslinked different proteins of the intact 50 S ribosomal subunit. Protein dimers were partially fractionated by sequential salt extraction and then by electrophoresis of each fraction in polyacrylamide gels containing urea. Slices of the gel were analysed by two-dimensional polyacrylamide/sodium dodecyl sulfate diagonal gel electrophoresis. Final identification of the constituent proteins in each dimer by two-dimensional polyacrylamide/urea gel electrophoresis showed that 50 S proteins L5 and L27 were crosslinked to S9. The evidence suggests that proteins S5, S9, S11, L5 and L27 are located at the interface region of the 70 S ribosome.     

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.     

The microheterogeneity of rabbit testosterone-binding globulin is due to differential glycosylation of its single protomer

Affinity-purified rabbit testosterone-binding globulin (rbTeBG) is a homodimer with a molecular weight (Mr) of about 92,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the chemically cross-linked protein. When noncross-linked rbTeBG is subjected to SDS-PAGE, individual protomers (Mr approximately equal to 44,400 +/- 400 and Mr approximately equal to 42,000 +/- 1300) are resolved. The protomers are present in a ratio of approximately 2 (heavy):1 (light). Enzymatic deglycosylation of native rbTeBG or of rbTeBG that had been photoaffinity-labeled with [1,2-3H]17 beta-hydroxy-4,6-androstadien-3-one was conducted. The products were then identified on immunoblots using a monoclonal antibody that cross-reacts with rbTeBG, or by fluorography. These analyses indicated that rbTeBG contained sialic acid and asparagine (Asn)-linked oligosaccharides and provided evidence for the presence of serine/threonine (O)-linked glycans on the molecule. The presumptive removal of all oligosaccharides by enzymatic or chemical means resulted in the appearance of a single subunit (Mr approximately equal to 37,150 +/- 1200). On the basis of this monomeric molecular weight, carbohydrate would contribute 16% and 11% to the relative molecular mass of the nondeglycosylated heavy and light subunits, respectively. Therefore, the size heterogeneity of the nondeglycosylated rbTeBG subunits is a result of their differential glycosylation. In addition to size heterogeneity, the rbTeBG subunits are composed of multiple-charge variants. Although enzymatic and chemical methods of glycan removal altered the isoelectric points of the isoforms, none of the treatments yielded a single isoform. Thus, it is possible that moieties other than oligosaccharides are contributing charge to the isoelectric variants of rbTeBG.