The Location of Prekallikrein in the Rat Pancreas - Semantic Scholar

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receptors was investigated. IgG from Graves’ sera with high thyroid-stimulating antibody activity were diluted in IgG from a pool of normal human serum (so that each sample contained the same amount of total IgG) and incubated overnight at 0°C with labelled thyrotropin and membranes solubilized with 0.5 % Triton. The mixtures were then analysed by chromatography on Sepharose or by poly(ethy1ene glycol) precipitation. The binding of labelled thyrotropin to the soluble-receptor preparations was inhibited in a dose-dependent manner by thyroid-stimulating antibodies (Fig. 2). Gel filtration of mixtures of antibodies, labelled thyrotropin and soluble thyrotropin-receptor preparations indicated that a trimolecular complex consisting of the three components was not formed. Further, analysis of mixtures of antibodies and labelled thyrotropin only by gel filtration and poly(ethy1ene glycol) precipitation showed that no direct interaction between them occurred, i.e. the antibodies did not demonstrate thyrotropin-binding activity. These data suggested that the binding of thyrotropin and thyroid-stimulating antibodies to the thyrotropin receptors was mutually exclusive, and that thyrotropin and the antibodies interacted with the same receptor molecule. Lowry, 0.H., Rosebrough,N. .I. Farr, , A. L. &Randall, R. J. (1951)J. Biol. Chem. 193,265-275 Manley, S . W., Bourke, J. R. & Hawker, R. W. (1974) J . Endocrinol. 61, 437-445 Smith, B. R. & Hall, R. (1974) Lancet ii, 427-431

The Location of Prekallikrein in the Rat Pancreas DAVID PROUD,* GRAHAM S. BAILEY,* TORILL BERG 0RSTAVIKt and KJELL NUSTADS *Department of Chemistry, University of Essex, Wivenhoe Park, Colchester C04 3SQ, Essex, U . K . , tlnstitirte of Microbiology, Dental Faculty, University of Oslo, Blindern, Oslo 3, Norway, and $ Department of Clinical Chemistry, The Norwegian Radium Hospital, Montebello, Oslo 3, Norway

Glandular kallikreins are peptidyl hydrolases (EC 3.4.21 3)that produce small peptide hormones called kinins. The kinins are believed to be involved in several pathological and physiological processes including regulation of local blood flow and vascular permeability (Reichgott & Melmon, 1973). Glandular kallikreins are found in tissue extracts and secretions of salivary glands, kidneys and pancreas (Webster et al., 1970). In the case of the latter organ the enzyme occurs as the inactive zymogen, prekallikrein. In previous studies kallikrein has been located in the striated duct cells of the rat sublingual gland, to the granular tubules of the rat submandibular gland (0rstavik et al., 1975) and also to the distal tubular cells of the rat kidney (0rstavik e f al., 1976). The purpose of the present investigation was to locate prekallikrein in the rat pancreas by using a direct immunofluorescence technique. Rat pancreas (6g), which had been extirpated immediately after death and stored at -80°C until use, was homogenized in 60ml of 0.01~-phosphatebuffer, pH6.0, containing 0.1 M-NaCI and 0.3mg of soya-bean trypsin inhibitor/ml in a motorized Potter-Elvehjem-type homogenizer at 2°C with 25 strokes at 1800rev./rnin. The homogenate was centrifuged for 5min at 500g (ray. 10.5cm) to sediment unbroken cells. The supernatant was filtered through gauze and stirred for 1 h at 2°C after the addition of 0.5% (w/v) sodium deoxycholate. The preparation was centrifuged in an MSE High-speed 25 centrifuge for 20min at 30000g (rav. 10.5cm). The supernatant was then filtered to remove fat and stored at 2°C. The sediments from the two centrifugations were resuspended in 20ml of the phosphate buffer and rehomogenized with 20 strokes at 1800rev./min. This homogenate was treated with sodium deoxycholate as before and centrifuged to produce a second supernatant. Finally, the sediment was re-treated to give a third supernatant. The three supernatants were then combined and the protein content of the preparation was determined by the method of Warburg & Christian (1942). 1977

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A preliminary experiment on ion-exchange chromatography was carried out essentially according to the details given in the legend to Fig. 1 except that fractions were pooled according to the absorbance profile. Those fractions were then tested for esterase activity against 20 mM-N-a-benzoyl-L-arginineethyl ester hydrochloride by using a titrimetric assay at pH8.0 and 25"C, both before and after suitable activation. Active fractions were then tested on immunodiffusion and immunoelectrophoresis against an antibody to rat submandibular kallikrein. Fractions corresponding to prekallikrein activity were shown to be electrophoretically different to rat submandibular kallikrein on immunoelectrophoresis, but behaved identidally with the submandibular enzyme on immunodiffusion. Having shown this a radioimmunoassay was then used to monitor a further ion-exchange run. A sample of pure rat submandibular kallikrein was labelled with 1251 by the method of Hunter & Greenwood (1962) with chloramine-T as oxidant. The labelled enzyme was separated from free I- by gel filtration on Sephadex G-75, and shown to be pure by using ion-exchange chromatography. Antibody to rat submandibular kallikrein was raised in two rabbits by injecting 150,ug of the pure enzyme at 2-week intervals until bleeding produced an antibody with sufficiently high titre and avidity for use. The antibody was normally used at a final dilution of 1 :160000. Pure rat submandibular kallikrein was used to construct standard curves with a range of concentrations from 0 to 10ng/0.2ml. Samples were assayed as follows: 200pl of standard or unknown, at suitable dilution, was incubated with 1OOp1 of labelled enzyme and 10Opl of antibody for 16-18h at 2°C. The antibody-antigen complex was then separated from free antigen by immunoprecipitation. DASP (Organon) sheep anti-(rabbit y-globulin) immunosorbent (0.5ml) was added to each tube and incubated in a shaker bath at 25°C for 2h. Then 3ml of 0.07~-barbitonebuffer, pH8.5, was added to each mixture. The samples were then centrifuged on an MSE Mistral 4L centrifuge for lOmin at 2500g (rav.22.1 cm). The supernatant was removed by aspiration and the precipitated antigen-antibody

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Fraction no. Fig. 1. ton-exchange chromatography of rat pancreatic homogenate A sample of supernatant (approx. 500mg of protein) was applied to a column (5cmx IOcm) of DEAE-Sephadex A-50 ion-exchange resin. The column was first washed withstartingbuffer(0.01 M-phosphatebuffer/O.I M-NaCI, pH6.0),and theneluted with a linear salt gradient (starting buffer 0.01 M-phosphate buffer/l .OM-NaCI, pH6.0). A flow rate of 45ml/h was used and 7.5ml fractions were collected. The fractions were monitored for their AZB0,conductivity and kallikrein content as determined by radioimmunoassay. A , AZ8";0 , conductivity; kallikrein concentration. --f

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complex was counted on a Tracerlab Corumatic 200 scintillation counter. The results in the case of the ion-exchange run are shown in Fig. I . The fraction that contained most of the antigenic activity was then used to locate the zymogen in the pancreas by using a direct immunofluorescence technique on tissue sections. Antibody to rat submandibular kallikrein was labelled with tetramethylRhodamine isothiocyanate. The conjugate was purified by gel filtration on Sephadex G-50 and by anionic-exchange chromatography by using the method of Brandtzaeg (1973~).The selected fractions had an absorbance ratio ( A 2 8 0 / A 5 , 5of ) 2.4 and were used at an immunoglobulin concentration of 0.6mg/ml as established by performance testing. Samples of the conjugate were adsorbed with mouse liver powder to decrease non-specific background staining. For the same purpose dilutions of the conjugate were made in 12% bovine serum albumin, and the tissue sections were preincubated with bovine serum albumin for 30min (Brandtzaeg, 19736). Conjugate incubation, washing and mounting of tissue sections were performed as described by Brandtzaeg (1973h). Immunological specificity of the fluorescence reaction was controlled by adsorption of the conjugate with purified rat submandibular kallikrein o r the partially pure rat pancreatic prekallikrein. Fluorescence microscopy was carried out with a Leitz Ortholux microscope equipped with a Ploem-type vertical illuminator. Conditions for narrow-band excitation and selective filtration of Rhodamine fluorescence (Brandtzaeg, 19736) were used. Prekallikrein was located in the acini of the rat pancreas. It may therefore be hypothesized that pancreatic kallikrein has its main function outside the gland and, upon secretion into the duodenum, may act upon the gut epithelium. Brandtzaeg, P. (1973~)Scand. J. Immunol. 2, 273-290 Brandtzaeg, P. (19736) Scand. J. Immunol. 2, 333-348 Hunter, W. M. & Greenwood, F. C. (1962) Nature (London) 194,495-496 0rstavik, T. B., Brandtzaeg, P., Nustad, K. & Halvorsen, K. M. (1975) Acta Histochem. 54, 183-192

0rstavik, T. B., Nustad, K., Brandtzaeg, P. & Pierce, J. V. (1976) J. Histochem. Cytochem. 24, 1037-1039

Reichgott, M. J. & Melmon, K. L. (1973) Methods Invest. Diagn. Endocrinol. 2B, 1190-1 199 Warburg, 0. & Christian, W. (1942) Biochem. Z. 310, 384-421 Webster, M. E., Mares-Guia, M. &Diniz, C. R. (1970) Hand. Exp. Pharmakol. 25,131-155

Effect of Glutathione on the Activity of Bilirubin-Binding Proteins from Rat Liver Cytosol JULES A. T. P. MEUWISSEN,* MARCEL ZEEGERS,* KAILA S l N G H S R A I t and BRIAN KETTERERT *Laboratory of Hepatology, Department of Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium, and t Courtauld Institute of Biochemistry, Middlesex Hospital Medical School, London W I P 7PN, U . K .

Liver cytosol of rats contains two protein fractions that bind bilirubin with high affinity. One, of mol.wt. 46000, is apparently identical with ligandin (Litwack et al., 1971) a protein that has also been known as aminoazodye-binding protein B (Ketterer et al., 1967) or Y-protein (Levi et al., 1969), and that is identical with glutathione S-transferase B (Habig et al., 1974). The other, of mol.wt. 14000, is probably identical with aminoazodye-binding protein A (protein A) (Ketterer et al., 1967, 1976) o r Z-protein (Levi et al., 1969). Besides bilirubin (Meuwissen et al., 1972; Tipping et al., 1976a), a number of other substances both endogenous and exogenous have been shown to possess varying affinities for either one o r both of these proteins (Tipping et al., 1976b,c; Ketterer et al., 1976). The binding proteins are of importance for the cellular transport of their ligands. In the case of bilirubin these proteins may be 1977