The rates of dissociation and reassociation of the subunits of human chorionic gonadotropin

A large change in quantum yield of the fluorescent probe 1,8-anilinonaphthalene sulfonate is produced when it combines with the glycoprotein hormone, human chorionic gonadotropin. A method of analyzing for the hormone in the presence of its subunits has been developed based on the finding that the subunits have no effect on 1,8-anilinonaphthalene sulfonate fluorescence. Quantitative rates of dissociation and recombination can be obtained with very small concentrations of hormone since fluorescence measurements are fast and sensitive. The effects of temperature, pH, and urea concentration on the rate of human chorionic gonadotropin dissociation have been measured. The rates of recombination of subunits have been studied as a function of temperature, pH, and KCl concentration. Human chorionic gonadotropin is stable in water to pH 12 and pH 4.5 at 37 °C.     

The reaction of the histidine residues of luteinizing hormone with ethoxyformyl anhydride.

Bovine and porcine luteinizing hormones (B-LH, P-LH) and their subunits were treated by ethoxyformyl anhydride. The acylation of the histidine residues was followed by examination of the absorbance spectrum. All the histidine residues of the luteinizing hormone molecule can be modified at pH5. However 2 His in B-LH and 1 in P-LH appear to be much less reactive at pH 5 than the others and their acylated imidazols more labile at the same pH. At neutral pH, 2 histidines in B-LH (and 1 in P-LH) become unreactive. In the case of the subunits, 1 histidine becomes unreactive in each subunit at neutral pH. These unreactive histidine residues at neutral pH are probably those which appear to be poorly reactive at pH 5. Comparison of the results obtained with B-LH and P-LH suggests that of the 2 histidine residues present in B-LH and absent in P-LH (beta 60, beta 112), only one exhibits a low reactivity. Acylation of 4 His in B-LH do not cause dissociation into subunits of the molecule but supress 95 per cent of the biological activity.     

Immunosuppressive activity of human chorionic gonadotrophin preparations in vivo: evidence for gonadal dependence

Human chorionic gonadotrophin preparations (hCG), when injected ip daily for 4 days, suppress the delayed-type hypersensitivity (DTH) response of mice to sheep red blood cells. Preparations of crude hCG, purified hCG subunits, and hCG that was formed by recombining the purified subunits showed immunosuppressive activity in accord with their gonadotrophic activity. The immunosuppressive effects in male and female mice were comparable. However, removal of the gonads completely abrogated the immunosuppressive activity of hCG in both males and females, suggesting that the effect of hCG is mediated by a factor released from the gonads. We conclude that the hCG molecule itself exhibits immunosuppressive activity in vivo in both male and female mice and that the gonads are required for the expression of this activity.     

The interaction of chemically modified human plasma alpha1-antitrypsin with porcine pancreatic elastase.

The elastase inhibitory capacity of human plasma α₁-antitrypsin was determined following chemical modification of lysyl and arginyl residues. Modification of the guanidino group had no effect upon the inhibitory activity, while acetylation, citraconylation, and trinitrophenylation of the lysyl ?-amino group brought about a loss of elastase inhibitory capacity.     

Labeling of bovine corpus luteal plasma membrane human chorionic gonadotropin or luteinizing hormone (hCG/LH) receptor and its purification and properties.

A method has been developed for labeling receptors for human chorionic gonadotropin or luteinizing hormone ($\text{hCG}\text{LH}$) present on bovine corpus luteal plasma membranes. It consists of four steps: (a) protection of the receptor by treating the plasma membranes with hCG; (b) iodination of the membranes with KI using glucose, glucose oxidase, and lactoperoxidase; (c) unmasking the receptor with either 2 m NaCl, 1 m guanidine hydrochloride, or rabbit anti-hCG; and (d) reiodination of the membranes using Na¹³¹I. After solubilization by successive treatments with Sepharose-concanavalin A and Sepharose-hCG and finally by preparative disc electrophoresis, the resulting purified receptor after electrophoresis in polyacrylamide gel showed a single radioactive band containing receptor activity. This highly purified receptor is fairly stable and retains its hormonal specificity, binding affinity, and pH optimum. It was observed that the receptor alone or as a complex with the hormone tends to aggregate. The receptorhormone complex does not dissociate during polyacrylamide-gel electrophoresis.     

Ca++ binding properties of human prothrombin.

The binding of Ca++ to human prothrombin has been investigated by equilibrium dialysis. The protein exhibited a positive cooperativity phenomenon for the first three Ca++ bound. Eleven to twelve Ca++ binding sites have been found. They could be differentiated in terms of two classes of sites with respect to their Ca++ affinity: 5 strong binding sites (log Kassoc = 3.9) and 7 weak binding sites (log Kassoc = 2.9). We attempted to determine the Hill coefficient of the strong binding sites responsible for cooperativity. Results have been compared to data previously reported for bovine prothrombin.     

Epitopes in human growth hormone and chorionic somatomammotropin studied with monoclonal antibodies

The immune complexes formed by human growth hormone or human chorionic somatomammotropin and various monoclonal antibodies have been studied by gel filtration and polyacrylamide gel electrophoresis. Two of the monoclonal antibodies gave rise to complexes with molecular weights suggesting an antigen:antibody 1:1 ratio. When both antibodies were simultaneously incubated with human growth hormone the ratio estimated for the new complex was 1:2, indicating the existence of two nonoverlapping epitopes in the antigen. The other monoclonal antibodies exhibited a more intricate behavior: incubated separately with human growth hormone they gave rise to both types of the aforementioned complexes. A similar phenomenon could be demonstrated with human chorionic somatomammotropin. The study of the immunoreactivity of a synthetic peptide indicates that the involved epitopes are localized within the region limited by amino acid residues 44 and 128 of human growth hormone.     

A homogeneous isoenzyme of human liver acid phosphatase.

An isoenzyme of human liver acid phosphatase (orthophosphoric monoester phosphohydrolase (acid optimum), EC 3.1.3.2) has been purified 4560-fold to homogeneity. The purification procedure includes ammonium sulfate fractionation, acid treatment, ion exchange chromatography on O-(carboxymethyl)-cellulose and DEAE-cellulose, Sephacryl S-200 chromatography, and affinity chromatography on Concanavalin A-Sepharose 4B. The homogeneous enzyme is a glycoprotein having 4% carbohydrate by weight in the form of mannose and glucosamine. In polyacrylamide gel electrophoresis under varied conditions of pH and cross-linking, the purified enzyme displays a single protein band coincident with activity. The native enzyme has a molecular weight of 93,000 as determined by gel elution chromatography and consists of two equivalent polypeptide chains. The subunit weight is 50,000–52,000 by sodium dodecyl sulfate gel electrophoresis. l-(+)-Tartrate is a strong competitive inhibitor of the enzyme; K_i is 4.3 × 10^?7m at pH 4.8 in 50 mm sodium acetate/100 mm sodium chloride. K_i values for a number of other inhibitors are given. Although it catalyzes the hydrolysis of a variety of phosphomonoesters, this isoenzyme of human liver acid phosphatase does not hydrolyze adenosine 5′-diphosphate, adenosine 5′-triphosphate, pyrophosphate, or choline phosphate at a detectable rate. The values of V differ with different alcohol or phenol leaving groups. The pH dependence of K_m and V values for the hydrolysis of p-nitrophenyl phosphate have been determined together with the pH dependence of K_i for l-(+)-tartrate. The pH dependence of both K_m and V indicate the effect of substrate ionization (pK ~ 5.2) and the involvement of a group in the EScomplex having a pKa value of approximately 6–7 which is ascribed either to a phosphoryl-enzyme intermediate or to the ionization of substrate in the ES-complex. An irreversible modification of the enzyme and a rapid loss of enzymic activity occurs upon treatment of the enzyme with Woodward's reagent K. The enzyme is protected against inactivation by the presence of competitive inhibitors. These and other data suggest that at least one carboxylic acid group plays an important role in catalysis.     

Glyconamides as inhibitors of human beta-glucosidases and beta-galactosidases.

d-Gluconamide, d-gluconyl hydrazide, and N-(6-aminohexyl)-d-gluconamide were prepared from d-glucono-1,5-lactone by treatment with ammonia, hydrazine, and 1,6-diaminohexane, respectively. These d-gluconamide derivatives were tested for their inhibitory action on human liver lysosomal glucocerebrosidase and human spleen neutral aryl β-glucosidase. Analogous d-galactonamide derivatives were evaluated for their inhibition of human spleen galactocerebrosidase and G_M1-ganglioside β-galactosidase. d-Gluconyl hydrazide and d-gluconamide were effective inhibitors of the lysosomal glucocerebrosidase, attaining 50% inhibition at 5 and 12 mm, respectively. In contrast, N-(6-aminohexyl)-d-gluconamide did not inhibit the glucocerebrosidase. d-Gluconyl hydrazide was also the most effective inhibitor of human liver and spleen aryl β-glucosidase, 50% inhibition being achieved at 4 mm concentration (competitive inhibition, K_i = 0.4–0.9 mM). d-Galactonamide was the most effective inhibitor of spleen galactocerebrosidase; 4 mm d-galactonamide caused 50% inhibition of the enzyme activity (noncompetitive inhibition). N-(6-Aminohexyl)-d-galactonamide is a potent inhibitor (90% inhibition, 5 mm) of G_M1-ganglioside β-galactosidase but is without effect on galactocerebrosidase. It has, therefore, the potential usefulness in distinguishing between two of the galactosphingolipid β-galactosidases.     

Locomotion of human lymphoid cells. I. Effect of culture and con A on T and non-T lymphocytes.

Human peripheral lymphocytes were separated from whole blood on a Ficoll-Hypaque gradient. They were then depleted of monocytes, separated into T and non-T fractions, and assayed for locomotor responses toward casein and endotoxin-activated serum in Boyden chambers. Non-T cells showed higher random motility than did T cells. Culture prior to assay was necessary in order to demonstrate locomotor activity of T cells, but this requirement, although desirable, was not essential for non-T lymphocytes. It was not necessary for Con A to be present in the culture medium or for either T or non-T lymphocytes to be in blast form to show locomotion.     

Chemical and physical properties of human bronchial mucus glycoproteins.

Human bronchial mucus glycoproteins of different chemical types were isolated by Ecteola and gel exclusion chromatography. Chemical analysis indicated polydispersity with regard to content of sulfate and sialic acid. No blood group A, B or H activity was found in these glycoproteins. Compositions are reported for amino acid and sugar residues for several fractions obtained from both cystic fibrotic and chronic bronchitic mucus. It is noteworthy that glycoproteins extracted from a single subject contain molecules with different acid groups as well as significant differences in carbohydrate chain length.     

Human T-cell hybridomas producing lymphokines. II. Enhancement of lymphotoxin secretion from human T-cell hybridomas by phorbol myristate acetate

Human lymphotoxin (LT)-producing T-cell hybridomas were constructed by fusing concanavalin A-activated human peripheral blood lymphocytes with emetine-actinomycin D-pretreated human acute lymphatic leukemia cells. LT secretion from these hybridomas was considerably enhanced by stimulation with phorbol-12-myristate-13-acetate (PMA) and concanavalin A or PMA alone. A study using cloned hybrid lines revealed that PMA/Con A acted directly on the LT-producing clones. Furthermore, PMA/Con A stimulated A-B9-24, one of the cloned hybridomas, and secreted fourfold larger amounts of LT under serum-free conditions than under serum-containing conditions. However, MIF/MAF and LT-producing cloned hybrid line E10-20 secreted rather decreased amounts of MIF/MAF when stimulated with PMA, while the LT secretion from the same hybridoma was enhanced with PMA.     

Functional subunit structure of photosystem 1 reaction center in Synechococcus sp

Photochemical activities of six different P700-chlorophyll a-proteins (CP1-a, -b₁, -b₂, -c, -d, and -e) separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis from digitonin particles of a thermophilic cyanobacterium Synechococcus sp. were examined. CP1-a, -b₁, -b₂, and -c contain the competent reaction center of photosystem 1: They were highly active in photooxidation of cytochrome c-553, the physiological electron donor to P700 in the organism, with methyl viologen as electron acceptor and showed flash-induced absorption changes indicating the charge separation between P700 and the secondary electron acceptors, P430 and A₂. The cytochrome photooxidation and P430 and A₂ photoresponses were significantly suppressed in CP1-d. CP1-e which lacks P430 and A₂ was least active in the cytochrome photooxidation. A₁, the primary electron acceptor of P700, is present in CP1-e as well as in other CP1 complexes. Comparison of the results with the polypeptide composition of CP1 complexes (Y. Takahashi, H. Koike, and S. Katoh, 1982, Arch. Biochem. Biophys.219, 209–218). indicates that CP1-c which contains four polypeptides with molecular weights of 62,000, 60,000, 14,000, and 10,000 represents the functional core of the photosystem 1 reaction center. P700, A₁, and antenna chlorophyll are associated with 62,000- and 60,000-dalton polypeptides, whereas 14,000- and 10,000-dalton polypeptides are assumed to carry P430 and A₂. The 13,000-dalton polypeptide which is associated with CP1-a, -b₁, and -b₂ is not required for the functioning of the reaction center.     

Retinoic acid: an endogenous compound of human blood. Unequivocal demonstration of endogenous retinoic acid in normal physiological conditions.

The occurrence of retinoic acid (vitamin A acid) as a normal constituent of the vitamin A reserve from the body is demonstrated. Improved methodologies based on selected peak monitoring (spm) and high-performance liquid chromatography (hplc) are used for the detection of retinoic acid in EDTA-plasma. After extensive cleanup by double-phase extraction and chromatography on Sephadex LH-20, retinoic acid is determined as its methyl derivative by spm. A different approach using double-phase extraction combined with a preextraction step and hplc is used to confirm the findings of the spm experiments. Both techniques proved the presence of retinoic acid in human plasma at a concentration of 1 to 3 ng/ml.     

The environment of the sulfhydryl group in human plasma albumin as determined by spin labeling

A series of spin labels, varying in chain length between the maleimide attaching group and the nitroxide free radical, has been used to investigate the environment of the sulfhydryl group in human plasma albumin. From the electron spin resonance spectra, the degree of freedom of the nitroxide was determined and the location of the sulfhydryl was assessed. The effect of bound fatty acids on the sulfhydryl environment was also determined. The environment was found to be analogous to that in the bovine protein, that is, a crevice approximately 9.5 Å deep and not affected in the native state by fatty acids.     

Affinity of bronchial secretion glycoproteins and cells of human bronchial mucosa for Ricinus communis lectins.

The coupling of Ricinus communis lectins to Sephadex G 25 was used in order to study mucins and other glycoproteins from human bronchial secretion. The major part of human bronchial mucins and other glycoproteins such as immunoglobulins A, bronchotransferrin and alpha1-antichymotrypsin were isolated by this procedure. A parallel study of human bronchial mucosa was achieved with peroxidase labeled Ricinus communis lectins; this study characterized goblet cells and mucous cells which contain mucins, and serous cells which are involved in the synthesis or the secretion of the other glycoproteins.