High Sialic Acid Content Research Articles

Sialic acid levels and lag time of growth in chemically defined medium containing 200 mM phosphate among strains of various serotypes of Streptococcus agalactiae.

The type-specific capsular polysaccharide antigen of Streptococcus agalactiae has in previous experimental studies been considered a significant antiphagocytic factor, whereas the lipoteichoic acid moiety has been suggested to be a factor in adherence to human fetal cell lines. Since epidemiological data concerning these cell constituents in strains from the genital tract are lacking, we attempted serotyping and analysis of these constituents of 100 vaginal isolates. The capsular polysaccharide level was shown to be the amount of sialic acid that occupied the terminal side chains of the polysaccharide. We carried out a study to ascertain whether strains exhibited a lag time of growth in a chemically defined medium containing 200 mM phosphate, which has been suggested to be characteristic of strains with high lipoteichoic acid levels. Strains were classified, on the basis of the results of distribution of sialic acid levels, into three categories: (i) strains with a low sialic acid content of equal to or less than 9 micrograms/mg of cell dry weight; (ii) strains with a moderate sialic acid content of more than 9 but less than 12 micrograms/mg of cell dry weight; and (iii) strains with a high sialic acid content of equal to or more than 12 micrograms/mg of cell dry weight. Strains that belonged to the last category, which, as previous experimental data indicate, are potentially virulent strains, were significantly distributed among isolates of types Ia (P less than 0.001) and III (P less than 0.05). On the other hand, strains exhibiting a lag time of growth in the above-mentioned medium were detected to a significant extent in type III isolates (P <0.02). These results may be related to the epidemiological finding that isolates from neonates with late-onset infection were more frequently serotype Ia and III isolates.

Affinity purification of the voltage-sensitive sodium channel from electroplax with resins selective for sialic acid

The voltage-sensitive sodium channel present in the eel (Electrophorus electricus) has an unusually high content of sialic acid, including alpha-(2----8)-linked polysialic acid, not found in other electroplax membrane glycopeptides. Lectins from Limax flavus (LFA) and wheat germ (WGA) proved the most effective of 11 lectin resins tried. The most selective resin was prepared from IgM antibodies against Neisseria meningitidis alpha-(2----8)-polysialic acid which were affinity purified and coupled to Sepharose 4B. The sodium channel was found to bind to WGA, LFA, and IgM resins and was readily eluted with the appropriate soluble carbohydrates. Experiments with LFA and IgM resins demonstrated binding and unbinding rates and displacement kinetics, which suggest highly specific binding at multiple sites on the sodium channel protein. In preparative-scale purification of protein previously fractionated by anion-exchange chromatography, without stabilizing TTX, high yields were reproducibly obtained. Further, when detergent extracts were prepared from electroplax membranes fractionated by low-speed sedimentation, a single step over the IgM resin provided a 70-fold purification, yielding specific activities of 3200 pmol of [3H]TTX-binding sites/mg of protein and a single polypeptide of approximately 285,000 Da on SDS-acrylamide gels. No small peptides were observed after this 5-h isolation. We further describe a cation-dependent stabilization with millimolar levels of monovalent and micromolar levels of divalent species.

Posttranslational modifications distinguish the envelope glycoprotein of the immunodeficiency disease-inducing feline leukemia virus retrovirus

The envelope glycoprotein (gp70) of a molecularly cloned, replication-defective feline leukemia virus (FeLV-FAIDS clone 61C) carries determinants for induction of fatal immunodeficiency disease, whereas the gp70 of its companion replication-competent, probably parent virus (clone 61E) does not. Immunoprecipitation analysis of the extracellular glycoproteins of 61E and EECC, a replication-competent viral construct composed of the 61C env and 3' long terminal repeat fused to the 61E gag-pol genes, demonstrated that the gp70 of EECC could be distinguished from that of 61E by both feline immune serum and a murine monoclonal antibody. Molecular weights of both the envelope precursor polyprotein (gp80) and the mature extracellular glycoprotein (gp70) of 61E were smaller than the corresponding proteins from the pathogenic EECC. Both the molecular weight disparity and monoclonal antibody discrimination of the two gp80s were abolished by inhibition of envelope protein glycosylation with tunicamycin, whereas the apparent gp70 size differences were resolved by enzymatic removal of N-linked oligosaccharides. Pulse-chase studies in EECC-infected cells demonstrated that processing of gp80 to gp70 was delayed and that this retardation of envelope glycoprotein processing could be simulated in 61E-infected cells by treatment with the glucosidase inhibitor N-methyldeoxynojirimycin, a compound that causes retention of oligosaccharides in the high-mannose form. The resultant 61E gp70 then could be recognized by sera from EECC-immunized cats. The presence of a higher content of sialic acid on the apathogenic 61E gp70 indicated that oligosaccharides of 61E and EECC gp70 were processed differently. These data suggested that the unique biochemical properties which distinguish the envelope glycoproteins of the FeLV-FAIDS variant from its companion apathogenic parent virus were responsible for T-cell cytopathicity and induction of immunodeficiency disease. Further biochemical characterization of these glycoproteins should be useful in understanding the pathogenic mechanisms of immunodeficiency disease induced by retroviruses.

Design of a Long-Lived Thyrotropin Antagonist from Derivatives of Human Chorionic Gonadotropin*

Previous studies have revealed that desialylated forms of hCG have a high potency to inhibit both the binding of bovine TSH (bTSH) to human thyroid membranes and the bTSH-stimulated adenylate cyclase activity therein. The biological activities of these desialylated forms, however, are greatly reduced in vivo due to the high affinity of asialo-glycoproteins for hepatic receptors and their consequent rapid clearance from the circulation. Intact purified hCG (hCGp), on the other hand, has little affinity for hepatic receptors and has a relatively long half-life in vivo, but displays only negligible inhibitory potency in the human thyroid. In the present studies, we have sought to obtain one or more compounds derived from hCG that are able to inhibit binding to TSH to the high affinity receptor in human thyroid membranes, and, consequently, could inhibit the stimulatory effect of TSH and Graves' immunoglobulin G and which also display a relatively low affinity for the hepatic asialoglycoprotein receptor and, as a consequence, have a reasonable survival in the circulation. Two hCG forms isolated by sequential chromatography of crude hCG on DEAE-52 and Sephadex G-100 were of interest, since they displayed some degree of selectivity in binding to thyroid and liver receptors. The first form (hCGv), whose isolation we described previously, was comprised of partially desialylated variant forms of intact hCG. The second material, as judged from its immunoreactivity, elution behavior on Bio-Gel P-100, and migration during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, contained a fragment of the beta-subunit (BF), similar to the beta-core fragment described by others. However, BF differed from the beta-core fragment in having a higher sialic acid content. Interestingly, we found that BF as well as the enzymatically desialylated form of BF displayed a much lower affinity for mouse liver receptors than did asialo-hCGp or the free asialo-beta- and asialo-alpha-subunits. Further, the activity of BF to inhibit the binding of [125I]bTSH to human thyroid membranes exceeded that of the desialylated subunits of hCGp as well as that of intact hCGp, but was only exerted at the low affinity binding site and was not accompanied by inhibition of TSH-stimulated adenylate cyclase. In an attempt to shift the locus of BF action from the low to the high affinity TSH receptor, we recombined BF with either an intact alpha-subunit of hCG or an asialo-alpha-subunit. This led to the creation of two novel forms of hCG with properties of the type that we were seeking.(ABSTRACT TRUNCATED AT 400 WORDS)

Concentration differences in isoforms of transferrin in blood from alcoholics during abuse and abstinence

At least five different forms of iron-saturated transferrin (Tf) from blood plasma can be separated by isoelectric focusing. These transferrin forms differ in the carbohydrate parts, especially the amount of sialic acid. The increased relative concentration of the form with isoelectric point 5.7 (Tf 5.7) has been shown to be a good indicator for liver effects caused by alcohol abuse However, in alcoholics during abstinence the newly formed transferrin has a higher sialic acid content than most of the transferrin already present in the blood. This indicates that the elevated concentration of Tf 5.7 with a low sialic acid content, found in alcoholics is not due to a defect at sialylation, but most probably caused by an impaired uptake of sialic acid-deficient transferrin by the hepatocytes due to membrane dysfunction.

What is the cause of benign transient hyperphosphatasemia? A study of 35 cases.

In a study of 35 children with benign transient hyperphosphatasemia, I found a marked seasonal clustering of cases after the summer months. Furthermore, plasma 25-hydroxyvitamin D concentrations were almost twice those of controls matched for age and time of year. Many children had evidence of weight loss and one had idiopathic hypercalcemia of infancy. Activities both of liver and bone isoenzymes of alkaline phosphatase (EC 3.1.3.1) in plasma were increased. The liver and (to a lesser extent) bone isoenzymes had enhanced electrophoretic mobility, and both showed increased binding to wheat-germ lectin by affinity electrophoresis. For the liver (and probably also the bone) isoenzyme, these changes were due to an increased content of sialic acid. A possible etiology for the condition is proposed involving (a) increased synthesis of alkaline phosphatase, mediated by vitamin D metabolites, and (b) decreased hepatic clearance caused by the high sialic acid content and exacerbated in some cases by the effects of some drugs on the liver.

The structure of glycophorins of animal erythrocytes

Glycophorins are red cell membrane sialoglycoproteins, which contain multipleO-linked oligosacchride chains and carry most of the cell surface sialic acid. Due to this high content of sialic acid the glycophorins are strongly stained with periodic acid-Schiff (PAS) reagent after sodium sodicylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The term “glycophorin” was proposed initially for human red cell sialoglycoproteins [1,2] and now it is also used for sialoglycoproteins in animal red cell membranes. Furthermore, similar glycoproteins of non-erythrocyte origin have also been identified and given the same name [3], although the terms “leukosialin” and “sialophorin” were proposed for a major sialoglycoproteins of human leukocytes [4,5]. In this article the term “glycophorin” will be used only for sialoglycoproteins existing in the erythrocyte membrane. Glycophorins of human erythrocytes, carrying blood group MN, Ss and other determinants, have been thoroughly studied and their properties described in several review articles [3,6,7,8]. The aim of this article is to summarize studies carriedout on the structure of non-human glycophorins, although some data concerning human glycophorins are included for comparative purposes.

Lectin binding of subunits of placental and serum oxytocinase after electrophoresis and transblotting to nitrocellulose

1. 1. Oxytocinase enzymes were purified from maternal serum and human placenta, run by SDS-PAGE and transferred onto nitrocellulose. Both enzymes were homogeneous in protein staining with M r of 145,000. 2. 2. Both serum and placental oxytocinases bound concanavalin A (Con A), limax flavus agglutinin (LFA) and wheat germ agglutinin (WGA). The WGA-binding of the placental enzyme was more strongly inhibited by 0.2 M N-acetylglucosamine than that of the serum enzyme which may indicate a higher sialic acid content in the serum enzyme. 3. 3. Neuraminidase treatment did not affect the binding of Con A but decreased the bincling of WGA to serum and placental enzymes. Serum enzyme showed a pl 4.7 on isoelectric focusing.

Reduced clearance rate of thyroxine-binding globulin (TBG) with increased sialylation: a mechanism for estrogen-induced elevation of serum TBG concentration.

Hyperestrogenemic states, including pregnancy, cause an increase in serum T4-binding globulin (TBG) concentrations and an increase in the proportion of TBG molecules with greater anodal mobility on isoelectric focusing, indicating greater sialic acid content. The possible causal relationship between the degree of sialylation and accumulation of TBG in serum was explored by measuring the in vivo half-lives (t1/2) of TBGs with different isoelectric points. TBG in unfractionated serum and its major peaks, isolated by chromatofocusing and defined by their isoelectric points on isoelectric focusing were each injected iv into rats. The resulting TBG concentrations, measured by specific RIA in serum samples obtained at intervals after injection, were used for the calculation of the t1/2. TBG in serum from a pregnant woman had a significantly longer t1/2 of 17.2 +/- 1.2 h (mean +/- SD) compared to those of 13.3 +/- 1.5 and 12.9 +/- 0.9 h for TBG in serum from a man and a nonpregnant woman, respectively. TBG peaks II, III, IV, and V, with increasing anodal mobility, had progressively longer t1/2 values of 11, 13, 15, and 33 h, respectively. However, TBG peaks of the same mobility on IEF isolated from serum of pregnant or nonpregnant subjects had similar t1/2 values. Neither the TBG concentration nor estrogen had a direct effect on the rate of TBG clearance. Indeed, the t1/2 of TBG from a subject with inherited TBG excess was not different from that of TBG from a nonpregnant woman or a man. Chronic treatment of rats with estradiol did not alter the rate of clearance of injected human TBG. Finally, the more heavily sialylated anodal bands of purified but unfractionated serum TBG, analyzed by Western blots, survived longer in the circulation of a rat. These results indicate that the rate of in vivo metabolism of TBG is dependent on its sialic acid content. The increased proportion of TBG molecules with higher sialic acid content thus contributes to the increase in the serum TBG concentration in hyperestrogenemic states.

Isolation and characterization of the two major apoproteins in human lipoprotein [a

Human Lp[a] was isolated in preparative amounts from two donors; the native lipoprotein and its constituent apoproteins, apo[a] and apoB, were characterized extensively. Based on differences in apparent molecular weight, four different isoforms of apo[a], a1-a4, were observed between the two donors. The number and relative distribution of these isoforms varied between donors but were constant for each donor. Each apo[a] isoform was shown to be derived from a discrete apo[a]-B100 disulfide-linked complex present before reduction. Complete delipidation of Lp[a] was followed by solubilization, reduction, and carboxamidomethylation of the constituent apoproteins. These apoproteins were then separated by immunoaffinity chromatography using anti-apo[a]- or anti-apoB-Sepharose; their purity and structural integrity were demonstrated by Western blot analysis. ApoB isolated by this procedure was essentially identical to apoB from autologous LDL with respect to molecular weight, secondary structure, amino acid composition, and sialic acid content. However, apo[a] differed from apoB in that it exhibited: a much less alpha-helical, less beta, but much more disordered structure; a lower proportion of aspartate, isoleucine, leucine, phenylalanine, and lysine, but a higher proportion of proline, glycine, and threonine; and a much higher content of sialic acid. These results indicate that apo[a] is not a superglycosylated form of apoB but is distinctly different in its composition and structure.

Mucus-glycoproteins (mucins) of the cat trachea: Characterisation and control of secretion

Glycoproteins produced by the tracheae of anaesthetized cats were radiolabelled biosynthetically by a pulse administration of Na 2 35SO 4 and [ 3H]glucose into the tracheal lumen. Subsequently, radiolabelled secretions were washed from the tracheal lumen. Repeated doses of pilocarpine and then ammonia vapour were given to stimulate secretion. Pilocarpine-stimulated glycoproteins, which came mainly from the submucosal glands, were particularly enriched with 35S. Ammonia-stimulated secretions, which probably came mostly from the microvillous border of the surface epithelium, contained mainly 3H radioactivity but little 35S. Two negatively-charged glycoproteins of different molecular size were identified in the secretions: the larger component was excluded on Sepharose CL-4B and it had a higher 3H 35S ratio than the smaller component which was retarded on Sepharose CL-4B. The relative amount of the smaller component decreased progressively with repeated pilocarpine stimulation and it was not detected in secretions induced by ammonia. Pilocarpine stimulation caused little alteration in carbohydrate composition of the secreted glycoproteins. In response to ammonia, glycoproteins were secreted with a high sialic acid content but quantitatively they represented a small amount of material compared with that induced by pilocarpine. These findings suggest that tracheal glycoproteins from different epithelial-cell sources have distinctive chemical compositions and that their secretions may be independently regulated. The 35S-rich high-molecular-weight glycoproteins from the submucosal glands were of the mucin-type but those derived from the microvillus border may represent a different class of airway glycoproteins from typical epithelial mucins.

Changes of gastric mucus glycoproteins with aspirin administration in rats.

The qualitative changes of gastric mucus glycoproteins occurring after aspirin dosing in rats were further investigated. Mucus glycoprotein contents of test animals were 55 and 53% of the control value at 3 and 7 h after dosing, respectively. The glycoproteins were further fractionated by CM-Sepharose CL-6B chromatography. 5 fractions were separated, 1 unadsorbed (fraction 1) and 4 adsorbed (fractions 2-5). Ratios of the major fractions 1:2:4 were 36:47:17 for control, 40:35:25 3 h after aspirin dosing, and 47:33:20 7 h after aspirin administration. Fractions 1, 2, and 4 corresponded to sulfomucin and sialomucin (relatively high content of sialic acid), sialomucin, and neutral mucin, respectively. While the absolute amount of each mucus glycoprotein fraction decreased with aspirin administration, the diminution of fraction 2 was distinct.

Kinetics of homophilic binding by embryonic and adult forms of the neural cell adhesion molecule.

The neural cell adhesion molecule, N-CAM, is a cell surface glycoprotein found on embryonic and adult neurons and on a variety of ectodermal and mesodermal tissues in very early embryos. During development, it shows local variations in prevalence at the cell surface as well as conversion from an embryonic form (E form) with high sialic acid content to an adult form (A form) with lesser amounts of this sugar. This E leads to A conversion occurs on different schedules in different brain regions, and it has been hypothesized that both the conversion and the prevalence changes are related to early regulation of pattern formation and connectivity. In order to identify precisely the consequences of these mechanisms of local cell surface modulation of N-CAM, an assay was developed to measure the rate of aggregation either of vesicles reconstituted from lipid and purified N-CAM or of native brain membrane vesicles. In both preparations, aggregation was greater than 95% inhibitable by specific anti-(N-CAM) Fab' fragments. The rates of aggregation of reconstituted N-CAM vesicles and native brain vesicles were found to be inversely related to the sialic acid content of their N-CAM molecules, with full desialylation resulting in about a 4-fold increase in rate over E-form N-CAM. Intermediate rates were obtained both with A-form N-CAM (which contains only one-third of the sialic acid content of E-form N-CAM) and with partially desialylated E-form N-CAM. The rate of coaggregation of reconstituted vesicles containing E-form N-CAM with reconstituted vesicles containing A-form N-CAM was also intermediate, implying that desialylation did not change the nature of (N-CAM)-(N-CAM) binding but only its rate. Even larger alterations in vesicle aggregation rate were seen when the amount of N-CAM per vesicle was altered. A 2-fold increase in the N-CAM-to-lipid ratio of reconstituted vesicles resulted in a greater than 30-fold increase in their rate of aggregation. Moreover, desialylation did not cause a further increase in the rate of aggregation of these already rapidly aggregating vesicles. These results in a model system demonstrate the large range of binding rates that are obtainable by various forms of local surface modulation of N-CAM. They are consistent with the proposal that similar alterations affecting (N-CAM)-mediated cell adhesion in vivo may be major factors in pattern formation during development of the nervous system.

Biological and binding activities of pituitary hormones from the ostrich, Struthio camelus

Biological and binding activities of adenohypophysial hormones purified from the ostrich (ost), Struthio camelus, were compared to those of the corresponding hormones derived from mammalian and other avian species. The potency of ostrich prolactin was comparable to those of other avian preparations and slightly less active than the ovine hormone when tested in the pigeon crop-sac assay, but ostrich growth hormone (GH) was more potent than several other avian preparations and was comparable to mammalian GH in the rat tibia bioassays. Marked discrepancies were evident in the activities of both ostrich gonadotropins (ostGn) when they were tested in a variety of in vivo and in vitro bioassays and radioreceptor assays (RRAs). Both the ostrich follicle-stimulating hormone (ostFSH) and luteinizing hormone (ostLH) were among the most potent tested thus far in in vivo bioassays for total gonadotropin in a lizard and a cockerel; a high sialic acid content may account for these high potencies. OstFSH was also the most potent nonmammalian Gn tested in two FSH specific mammalian bioassays, an in vivo (ovarian augmentation) and an in vitro (cAMP production) rat bioassay; in fact, ostFSH behaved more like a mammalian than an avian hormone in these assays. However, the binding activity of ostFSH was not unlike that of other avian FSH preparations when tested in either mammalian or nonmammalian FSH-RRA systems. OstLH was more potent than other avian preparations in an in vitro mammalian bioassay, but not in avian or amphibian LH bioassays; species specificity was pronounced among these LH assays. Binding activities of ostLH in mammalian and avian LH-RRAs were generally consistent with potencies in the two species of bioassays. However, a marked discrepancy was apparent in the behavior of ostLH in FSH-RRAs. Although ostLH had very low FSH activity when tested by radioimmunoassay, by bioassay, or by FSH-RRA with avian gonads, it was equipotent to ostFSH in competing for FSH-binding sites on the mammalian gonad; in this respect it was more like turkey than chicken LH. The ability of ostLH to antagonize the biological activities of ostFSH in the stimulation of cAMP by rat seminiferous tubules confirms that ostLH binds to the same functional receptors as FSH on the rat testis, even though it does not induce the characteristic physiological response associated with FSH.

Separation and characterization of two different species of rat angiotensinogen

Two distinct species of rat angiotensinogen (A-1 and A-2) were purified from plasma of nephrectomized rats by combining ammonium sulfate fractionation, chromatography on Blue Sepharose and SP-Sephadex, gel filtration and preparative polyacrylamide gel electrophoresis. Separation of the two species was accomplished in the SP-Sephadex chromatography step, A-1 eluting before A-2. The two angiotensinogen species had identical electrophoretic mobilities on analytical polyacrylamide gel electrophoresis, but differed in their apparent molecular weights as obtained by SDS-gel electrophoresis (A-1, M r 60000; A-2, M r 56400). In analytical isoelectric focusing each species displayed a characteristic double band with isoelectric points of 4.54 and 4.60 for A-1, and 4.69 and 4.76 for A-2. These physicochemical differences can be accounted for by the difference in carbohydrate content: A-1, when compared to A-2, had a higher content of sialic acid (5.0 and 2.1 mol/mol), neutral hexoses (10.2 and 5.9 mol/mol) and aminobexoses (10.5 and 7.0 mol/mol, respectively). Antiserum raised against rat angiotensinogen crossreacted completely with both angiotensinogens. Both species could also be isolated from plasma of non-nephrectomized rats, which indicates that they may be present under physiological conditions. The physiological significance of the occurrence of these species of angiotensinogen is still unknown.

The loss of sialic acid and its prevention in stored human platelets

The loss of sialic acid was determined in human platelets stored during a seven day period in their homologous plasma. Approximately 30% of the sialic acid was lost during the first three days of incubation at room temperature and a total of 73% was lost after seven days. The rapid in vitro loss of sialic acid may mimic a slower in vivo loss. It was found that platelets with a greater density had a higher sialic acid content than the less dense platelets. The loss of sialic acid from stored platelets could be completely inhibited by the addition of sialyl compounds to the incubation plasma. The trisaccharide, N-acetylneuramin-lactose, gave a greater degree of protection than fetuin at comparable concentrations.

Purification and partial characterization of a protein from cancer ascites fluid which stimulates the resorption of bone explants in vitro.

A protein capable of stimulating bone resorption in vitro has been purified approximately 1250-fold from cancer ascites fluid. Purification was accomplished employing successive fractionation with ammonium sulfate, ion exchange, and Cibacron blue affinity chromatography, isoelectric focusing, and selective adsorption on hydroxylapatite. The bone-resorptive protein obtained by this procedure appeared homogeneous in polyacrylamide gels at pH 9.5, migrating with the mobility of an alpha 2-globulin, and in sodium dodecyl sulfate polyacrylamide gels from which an apparent molecular weight of 43,000 was calculated. The amino acid composition of the bone-resorptive protein distinguished itself by the absence of methionine and by its relatively high content of glycine (17%) and proline (11%). Furthermore, the protein possesses a single NH2-terminal amino acid residue (glycine). The ascites protein was found to contain 19% carbohydrate by weight including a high content of sialic acid (15 residues/mol) as compared to the other sugars (27 residues/mol). As to its biological properties, the homogeneous ascites glycoprotein proved to be as potent as parathyroid hormone in its ability to stimulate bone resorption in vitro.

A subfraction of human fibrinogen with high sialic acid content and elongated gamma chains.

Fibrinogen isolated from normal human single donor or pool plasma was fractionated by DEAE-cellulose chromatography. Three different fibrinogen subfractions were obtained. The most acidic fraction comprising 22% of the whole fibrinogen pool was prominent by two special features: 1) its sialic acid content was significantly higher than that of bulk fibrinogen, namely 8 mol of sialic acid/mol of fibrinogen versus 6 mol in bulk fibrinogen. 2) Two-dimensional electrophoresis of the polypeptide chains obtained after reduction revealed a preferential accumulation in this subfraction of the elongated gamma chains previously described as gamma chain heterogeneity of whole human fibrinogen.

Isolation and immunologic properties of a heterogenous antigen with the characteristics of the heavy chain of human plasma kininogen

Kininogen antigen was purified from human plasma fraction IV by ion exchange chromatography, gel filtration and affinity chromatography with antibody specific immunoadsorbents. The immunologically pure glycoprotein had a mol. wt of ~ 60,000 and only one polypeptide chain by SDS-PAGE. An extensive charge heterogeneity by isoelectric focusing and gel filtration on polyacrylamide agarose could only in part depend on a comparatively high sialic acid content, but may be caused by differences in the carbohydrate structures sustained by lectin-binding heterogeneity on Con A-Sepharose. This antigen shares a dominating determinant with native plasma kininogens shown by complete patterns of identity in immunochemical analyses and with the monospecific antisera developed in rabbits against the heterogeneous components. The similar size, amino acid composition, low histidine content, lack of N-terminal amino acid and antigenic homogeneity fit all the so far known characteristics of the human kininogen heavy chain. Notably the antigenic determinant is resistant to degradation by activated kallikrein. This antigen with unimpaired immunologie activity may be a useful tool for preparation of antiserum for immunochemical determination of human plasma kininogen.

Characteristics of hybrids of ovine LH and human glycoprotein hormone subunits in rat and chicken in vitro test systems.

Hybrids of ovine luteinizing hormone (oLH) and human chorionic gonadotropin (hCG) subunits were prepared and characterized. Human luteinizing hormone (hLH), hCG, oLH, and the hybrid gonadotropins hCGo:oLHj3 and oLHa:hCGLI were tested for biological potency using three assays: 1) rat testis radioreceptor assay, 2) rat Leydig cell steroidogenesis assay, and 3) rooster testis steroidogenesis assay. In each assay the hybrid gonadotropins behaved characteristically as the parent gonadotropins from which the beta subunits were obtained. In the rat testis radioreceptor assay, hCG and oLHa:hCGI3 were about 3 times as potent as 0LH and hCGa:0LHII. When assayed using the rat Leydig cell steroidogenesis assay, hCG and oLH :hCGj3 were about 1.25 times as potent as 0LH and hCGo:oLHj3. In the rooster testis steroidogenesis assay, 0LH and hCGo: oLHfl were about 300 times more potent than hCG or oLHa:hCG$3. The hLH behaved similarly but not identical to hCG in each assay system. This study shows that the structure(s) responsible for differences in potency between human and ovine LH resides in the beta subunit. The relatively similar behavior of hLH and hCG in these assays as contrasted to 0LH indicates that the structure responsible for differing LH potencies is not in the carboxyl terminal 26 amino acids which are unique to chorionic gonadotropins. The high sialic acid content of hCG also appears unrelated since 80% desialated hCG was slightly less potent than hCG in the chicken test system. The conclusion is that one or more point mutations have occurred in the evolution of the LH beta subunit which render human glycoprotein hormones with luteinizing hormone activity almost inactive in the chicken, or more specifically, the chicken receptor system. The glycoprotein hormone alpha subunits from the sheep and human species appear functionally identical.