Role of sialic acid in the microheterogeneity of serum thyroxine-binding globulin. Study by two-dimensional isoelectric focusing

A new technique combining a neuraminidase treatment of thyroxine-binding globulin after serum isoelectric focusing and a second-dimensional isoelectric focusing was developed to study the role of sialic acid in the microheterogeneity of native thyroxine-binding globulin. By showing the change of PI occasioned by the desialylation for each of the bands constituting the thyroxine-binding globulin pattern separately, this procedure clearly demonstrated that the microheterogeneity of the native protein could not be imputed to the varying sialic acid content of the bands only. We suggest that at least three molecules of thyroxine-binding globulin, with probably slight differences in their amino acid composition, are present in the serum, and that different degrees of sialylation ensure greater microheterogeneity of this protein.     

Microheterogeneity of mammalian haptoglobins in isoelectric focusing.

1. Human haptoglobin type 1-1, porcine haptoglobin, and equine haptoglobin were isolated and purified. 2. These haptoglobins were similar in polyacrylamide gel electrophoresis and in subunit structure but showed microheterogeneity in isoelectric focusing. 3. Isoelectric points of human haptoglobin as determined with photopolymerized gels were found to be 4.03-4.24, of porcine haptoglobin 4.0-4.30, and of horse haptoglobin 3.80-4.15, respectively. 4. Results obtained with chemically polymerized gels were 0.08-0.3 pH units higher. 5. Examined haptoglobins differed also in the ability of complex formation with hemoglobin, in sialic acid content and in antigenic specificity.     

SDS-PAGE electrophoretic property of human chorionic gonadotropin (hCG) and its beta-subunit

The microheterogeneity property of hCG with regards to its sialic acid contents resulted in variable mobility of the glycoprotein in SDS-PAGE. The intact hCG molecule is composed of two dissimilar subunits, namely alpha- and beta-subunits. The identification of hCG bands in SDS-PAGE was accomplished by the immunoblotting experiment, whereby the antibody directed toward the specific region of beta-subunit of hCG was used. The data shows that the different mobility of intact hCG was attributed to the different degree of desialylation of the glycoprotein. Nevertheless, unlike the intact hCG, the mobility of its beta-subunit was not affected by its variety sialic acid content. This characteristic of beta-hCG is beneficial when semi-quantification of total hCG is required. Quantification of hCG using the HPLC-reversed phase C18 analytical column is not possible as the glycoprotein was eluted in multiple fractions at different retention times. The identification of denatured hCG (HPLC eluted fractions) was carried out by immunoblotting experiment whilst immunoassay technique failed to detect its presence in any fraction.     

N-linked oligosaccharide analysis of glycoprotein bands from isoelectric focusing gels

Glycoproteins often display a complex isoelectric focusing profile because of the presence of negatively charged carbohydrates, such as sialic acid, phosphorylated mannose, and sulfated GalNAc. Until now, understanding the role of these charged carbohydrates in determining the isoelectric focusing profile has been limited to observing pattern shifts following complete removal of the sugars in question. We have developed a simple and sensitive method for analyzing N-linked oligosaccharides from the individual isoelectric focusing bands of a glycoprotein using recombinant human thyroid-stimulating hormone as a model system. N-linked oligosaccharides were released and profiled from individual bands following electroblotting of isoelectric focusing gels. As might be predicted, high-pH anion-exchange chromatography-pulsed amperometric detection and matrix-assisted laser desorption/ionization-time of flight analyses indicated that the bands that migrated closer to the positive electrode contained more sialylated N-linked oligosaccharides. The sialic acid content of these bands correlated with that predicted from the corresponding oligosaccharide analyses.     

Transferrin microheterogeneity as a probe in normal and disease states

Isoelectric focusing of iron saturated serum has been established as a convenient method for showing transferrin glycan microheterogeneity. In a clinical setting, the method is used in the detection of cerebrospinal fluid leakage, the screening for surreptitious alcohol abuse and in the diagnosis of the carbohydrate deficient glycoprotein syndrome. In normal physiological states it can also be used as a tool to probe for changes in N-glycosylation.     

Purification and properties of normal human alpha 1-antitrypsin

A relatively gentle purification method has been devised for the major serine-protease inhibitor in human plasma. The product, a single chain glycoprotein of 50,000 molecular weight, is obtained in 25% yield. It contains 11.5% carbohydrate by weight, a single residue of cysteine and two of tryptophan. N-terminal glutamate or glutamine was found by dansylation. Isoelectric focusing at high resolution in acrylamide gels revealed three major and several minor protein species. Several possible mechanisms to account for this isoelectric microheterogeneity are discussed.     

Genetic studies of low-abundance human plasma proteins. I. Microheterogeneity of zinc-α2-glycoprotein in biological fluids

A high-resolution isoelectric focusing technique followed by immunoblotting has been utilized to determine the microheterogeneity of zinc-alpha 2-glycoprotein in a large number of plasma samples from U.S. Caucasians, Blacks, and Eskimos. With the exception of one Black individual, all samples were found to contain an invariant multiple-banded pattern which, after desialylation, was reduced to a single band, suggesting that the microheterogeneity observed is due to differences in the sialic acid content of a single protein product. The asialo forms of the variant sample consist of two distinct bands, consistent with the occurrence of a rare genetic variant at the zinc-alpha 2-glycoprotein structural locus. Unfortunately family studies were not feasible. In addition to plasma, the present technique has been applied to detection of zinc-alpha 2-glycoprotein microheterogeneity in amniotic fluid, saliva, and tears. The amniotic fluid pattern is identical to that present in plasma. However, the patterns observed in saliva and tears are different from each other as well as from that in plasma and could be controlled by separate loci.     

Free alpha subunits of glycoprotein hormone with dissimilar carbohydrates produced by pathologically different carcinomas

The two kinds of glycoprotein hormone alpha subunit ectopically produced by an undifferentiated carcinoma of the left femoral region (TM-alpha) and an adenocarcinoma of the right external genitalia (FS-alpha) were examined for amino acid composition, isoelectric focusing, molecular weight, the ability to combine with standard hCG beta and affinity with lectins (Con A, Ricin and PNA). Both TM-alpha and FS-alpha exhibited immunoantigenicity similar to standard hCG alpha. Furthermore, there were no significant differences in the amino acid compositions of TM-alpha, FS-alpha or standard hCG alpha. In isoelectric focusing, while standard hCG alpha exhibited a neutral charge, both TM-alpha and FS-alpha exhibited strong negative charges. FS-alpha was as sensitive to sialidase as standard hCG alpha, whereas most of the TM-alpha exhibited resistance to sialidase. TM-alpha contains sialidase-insensitive peripheral material with a negative charge. The affinity with Ricin-Sepharose indicated that most of the FS-alpha and some of the TM-alpha may contain terminal sialic acid and the penultimate structure, Gal beta 1----4G1cNAc; the affinity with PNA-Sepharose indicated that both may also contain terminal sialic acid and the penultimate structure, Gal beta 1----3GalNAc. These observations suggest that dissimilar glycosylation processes are present in the carcinoma ectopic biosynthesis of glycoprotein hormone alpha subunit.     

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.     

Chemical and physicochemical characterization of the sialic acid-specific lectin from Cepaea hortensis

A sialic acid-specific lectin was isolated from the albumin glands of the garden snail Cepaea hortensis by affinity chromatography on fetuin-Sepharose following gel filtration on Superdex 200. The purified native lectin showed a molecular mass of about 95 kDa by gel filtration and 100 kDa by SDS electrophoresis. It was cleaved by boiling in buffer containing SDS in three serological identical bands corresponding to molecular masses of about 24, 20 and 16 kDa, respectively. From these three fragments, only the 24- and the 20-kDa bands were found to be glycosylated. Only the three sugars mannose, galactose and N-acetylglucosamine could be detected in a molar ratio of 3:8.6:2. The oligosaccharide moieties seem to be N- and partially O-glycosidic bound. Isoelectric focusing (IEF) of the purified lectin revealed a heterogeneous pattern with bands in the pH range of 4.3-5.0. Isolated bands of different isoelectric points showed in SDS electrophoresis the same three fragments with molecular masses of 24, 20 or 16 kDa. The heterogeneity of the lectin was revealed either by IEF or amino acid sequencing of internal tryptic peptides.     

α1-Acute-phase globulins of rats. Microheterogeneity after isoelectric focusing

1. Improved resolution of mixtures of alpha(1)-globulins was obtained by the use of isoelectric focusing. 2. Because material recovered after isoelectric focusing in polyacrylamide gels behaved in a manner which suggested interaction with components derived from the gel, isoelectric focusing when used for preparative purposes was done in a matrix of Sephadex G-75. 3. By this means material from the individual bands formed by isoelectric focusing in 6m-urea could be isolated. The stability of these substances was examined by further isoelectric focusing. 4. Analysis of material that had been shown to be homogenous by isoelectric focusing in the absence of urea and of that from several individual bands derived from the same sample by isoelectric focusing in 6m-urea showed different proportions of sialic acid but no change in amino acid composition. 5. In the presence of 6m-urea the isoelectric points found were increased by 0.14-0.25 pH unit. After removal of most of the sialic acid with neuraminidase the increase was 0.36-0.72 pH unit. After treatment with 0.025m-H(2)SO(4) at 80 degrees C for 1h, which removed all the sialic acid, the increase was 0.40-0.87 pH unit. 6. Because removal of all the sialic acid did not decrease the number of bands formed by isoelectric focusing the observed heterogeneity could not be caused entirely by the presence of various proportions of sialic acid.     

Glutamate dehydrogenase from the thermoacidophilic archaebacterium Sulfolobus solfataricus

An NAD(P)-dependent glutamate dehydrogenase was purified to homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus. The enzyme is a hexamer (subunit mass 45 kDa) which dissociates into lower states of association when submitted to gel filtration. Isoelectric focusing analysis of the purified enzyme showed a pI of 5.7 and occasionally revealed microheterogeneity. The enzyme is strictly specific for the natural substrates 2-oxoglutarate and L-glutamate, but is active with both NADH and NADPH. S. solfataricus glutamate dehydrogenase revealed a high degree of thermal stability (at 80 C the half-life was 15 h) which was strictly dependent on the protein concentration. Very high levels of glutamate dehydrogenase were found in this archaebacterium which suggests that the conversion of 2-oxoglutarate and ammonia to glutamate is of central importance to the nitrogen metabolism in this bacterium.     

Genetic studies of low-abundance human plasma proteins. VI. Polymorphism of hemopexin.

An analytical isoelectric focusing method in 3 M urea followed by immunoblotting has been devised to detect genetic and biochemical variation in the glycoprotein hemopexin (HPX) in human plasma or serum. HPX reveals extensive microheterogeneity with multiple major and minor components that are susceptible to neuraminidase treatment, suggesting that the observed biochemical variation is due to differences in sialic acid content between HPX isoproteins. However, charge differences that persist in HPX isoproteins following neuraminidase treatment suggest the presence of genetically determined HPX variation, and this is confirmed by population and family studies. HPX was found to be monomorphic, with an invariant pattern, in U.S. whites; but it is polymorphic in U.S. blacks, with three alleles controlled by a single locus, a situation that demonstrates an autosomal codominant pattern of inheritance. The HPX 1, HPX 2, and HPX 3 allele frequencies in U.S. blacks are .941, .018, and .041, respectively.     

Physicochemical and immunological homogeneity of spinin, the subunit-protein of bacterial spinae.

Bacterial spinae from marine bacterium D71 are multi-subunit structures of a single protein. This protein, called spinin, is homogeneous by immunodiffusion and immunoelectrophoresis, amino acid composition, polyacrylamide gel electrophoresis with a number of buffer systems, sedimentation velocity and diffusion boundary analysis. Sedimentation equilibrium gives Mr = 19,000, while phosphate polyacryl-amide gel electrophoresis in presence of dodecyl sulfate gives Mr = 32,000. The lower Mr estimate for spinin is supported by sedimentation equilibrium in 6 M guanidine . HCl, and covalent cross-linking with dimethyl suberimidate or glutaraldehyde. The higher Mr value probably arises from an anomalous spinin-dodecyl sulfate interaction. Isoelectric focusing in polyacrylamide gel gives pI = 3.45; however, the focusing pattern also contains three distinct bands that may arise from hydrolysis of the spinin protomer during anodic migration. This study presents the first extensive physicochemical characterization of spinin and provides the basis for investigating the subunit assembly of spinae.     

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.     

Cellular localization and substrate specificity of isoelectric forms of human liver neuraminidase activity.

The four major isoelectric forms of human liver neuraminidase (with pI values between 3.4 and 4.8) have been isolated by preparative isoelectric focusing and characterized with regard to their substrate specificity using glycoprotein, glycopeptide, oligosaccharide and ganglioside natural substrates. All forms exhibited a rather broad linkage specificity and were capable of hydrolyzing sialic acid glycosidically linked alpha 2-3, alpha 2-6 and alpha 2-8, although differential rates of hydrolysis of the substrates were found for each form. The most acidic form 1 (pI 3.4) was most active on sialyl-lactose, whereas form 2 (pI 3.9) and 3 (pI 4.4) were most active on the more hydrophobic ganglioside substrates. Form 4 (pI 4.8) was most active on the low-Mr hydrophilic substrates (fetuin glycopeptide, sialyl-lactose). Each form was less active on the glycoprotein fetuin than on a glycopeptide derived from fetuin. Organelle-enriched fractions were prepared from fresh human liver tissue and neuraminidase activity on 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid was recovered in plasma membrane, microsomal, lysosomal and cytosolic preparations. Isoelectric focusing of the neuraminidase activity recovered in each of these preparations resulted in significantly different isoelectric profiles (number, relative amounts and pI values of forms) for each preparation. The differential substrate specificity of the isoelectric forms and the different isoelectric focusing profiles of neuraminidase activity recovered in subcellular-enriched fractions suggest that specific isoelectric forms with broad but defined substrate specificity are enriched at separate sites within the cell.     

Purification and characterization of the glycoprotein hormone alpha-subunit-like material secreted by HeLa cells

The protein secreted by HeLa cells that cross-reacts with antiserum developed against the alpha-subunit of human chorionic gonadotropin (hCG) has been purified approximately 30,000-fold from concentrated culture medium by organic solvent fractionation followed by ion exchange, gel filtration, and lectin affinity chromatography. The final preparation had a specific activity (by RIA) of 6.8 x 10(5) ng of alpha/mg of protein and appeared homogeneous by electrophoresis on reducing/denaturing polyacrylamide gels (SDS-PAGE). Amino acid analysis indicated that HeLa-alpha had a composition very similar to that of the urinary hCG alpha-subunit. Peptide fingerprints of the HeLa protein and hCG-alpha revealed that several of the Tyr-, Met-, and Cys-containing tryptic peptides were held in common, thus identifying the tumor protein as a glycoprotein hormone alpha-subunit with a primary structure similar to that of hCG-alpha. However, comparison of hCG-alpha and HeLa-alpha demonstrated that the tumor-associated subunit was not identical with its normal counterpart. Only two of the three Tyr-containing tryptic peptides present in hCG-alpha could be detected in HeLa-alpha after iodination with 125I. HeLa-alpha eluted prior to hCG-alpha during Sephadex G-75 chromatography, but the subunits coeluted when the tumor protein was first subjected to mild acid hydrolysis. The purified tumor protein had an apparent molecular weight greater than that of the urinary alpha-subunit when analyzed by SDS-PAGE (Coomassie blue staining), and this difference was even greater when a partially purified preparation was examined by an immunoblot technique (Western). Isoelectric focusing of the HeLa and hCG subunits demonstrated that the tumor protein had a lower pI (4.7-5.5 compared to 6.5-7.8), and removal of sialic acid by mild acid hydrolysis did not entirely eliminate this difference. Immunoprecipitation and electrophoresis of alpha-subunit from HeLa cultures labeled with [3H]fucose indicated that the tumor subunit was fucosylated, whereas analysis of hCG-alpha hydrolysates by HPLC confirmed previous reports that the placental subunit does not contain fucose. HeLa alpha-subunit was unable to combine with hCG beta-subunit to form holo-hCG under conditions where the hCG alpha-subunit was able to do so. The results indicate that, regardless of whether or not a single alpha-subunit gene is being expressed in both normal and neoplastic tissues, posttranslational modifications lead to a highly altered subunit in the tumor. The differences observed may be useful in diagnosing neoplastic vs hyperplastic conditions and may lend insight into the mechanism of ectopic hormone production by tumors     

Purification and characterization of plasma membrane-associated human sperm alpha-L-fucosidase

Detergent and salt extraction studies, as well as cytochemical localization with fluorescein isothiocyanate-bovine serum albumin-L-fucose, have provided further evidence for the plasma membrane association of a novel human sperm, alpha-L-fucosidase. This alpha-L-fucosidase has been solubilized and purified 8600-fold to high specific activity (35 000 U/mg protein) by affinity chromatography on agarose-C(24)-fucosylamine. To our knowledge, this is the first report concerning the purification and characterization of a mammalian plasma membrane-associated alpha-L-fucosidase. Both SDS-PAGE and Western blot analysis indicated the alpha-L-fucosidase is highly purified and contains a single subunit with a molecular mass of 51 kDa. N-glycanase studies indicated the subunit contains N-glycans, and lectin blot analysis detected the presence of mannose, but no terminal galactose or sialic acid residues. Isoelectric focusing indicated the presence of two major alpha-L-fucosidase isoforms (pIs 6.5 and 6.7) and a possible minor isoform (pI 6.3). Treatment of alpha-L-fucosidase with neuraminidase did not change its isoform profile, providing further evidence for the enzyme's lack of sialic acid residues. Kinetic analysis with 4-methylumbelliferyl alpha-L-fucopyranoside indicated that sperm alpha-L-fucosidase has a pH optimum near 7, an apparent K(m) of 0.08 mM, and a V(max) of 6.8 micro mol/min/mg protein. The unusual properties of human sperm alpha-L-fucosidase argue in support of a potentially important, but presently unknown, role for this enzyme in human reproduction.     

Micro isoelectric point heterogeneity of a murine monoclonal antibody (L6) originating from cell cultivation conditions

Summary Isoelectric point (pI) microheterogeneity of an IgG monoclonal antibody (L6) was studied by rapid affinity chromatography, isoelectric focusing on polyacrylamide gels, and rapid ion-exchange chromatography. An abiotic or strictly chemical basis for observed changes is postulated. The described methods are useful for monoclonal antibody process monitoring since they are rapid, automated assay procedures.