Isolation, characterization, and localization of antigen gamma, a serine proteinase of the "kallikrein-family" in the rat submandibular gland

A trypsin-like serine proteinase, antigen gamma, immunologically partially identical to glandular kallikrein when run against anti-rat glandular kallikrein antiserum in immunoelectrophoresis, was purified from the rat submandibular gland. The enzyme was purified by a two-step chromatography procedure, ionexchange chromatography followed by gel filtration. The criteria for purity were one band in SDS-polyacrylamide gel electrophoresis and in immunoelectrophoresis, respectively. Antigen gamma had a molecular mass of 25,000 Da and consisted of two polypeptide chains with molecular masses of 14,000 and 11,000 Da. The preparation contained several isoenzymes with pI ranging from 4.1 to 4.5. The enzyme showed high specific enzyme activity against the substrate D-valyl-L-leucyl-L-arginine-4-nitroanilide (S-2266), some trypsin-like and kininogenase activity, but no angiotensin converting enzyme, kininase, or tonin activity. Amidolytic activity was increased and stabilized by the presence of detergent in the assay buffer. The pH-optimum of antigen gamma amidolytic activity was about 10. Antigen gamma was inhibited by SBTI and PMSF, whereas aprotinin had to be added in a more than 100 times higher concentration than for glandular kallikrein. The binding pattern of antigen gamma to plasma proteins was different from that of tonin and glandular kallikrein. Antiserum against antigen gamma was raised in rabbits and characterized against rat submandibular gland homogenate. Immunohistochemistry showed antigen gamma in the secretory granules of the submandibular gland granular tubular cells but only adhering to the luminal cell wall in the striated and main excretory ducts. Antigen gamma was not detected in the sublingual or parotid gland or in the kidney. Antigen gamma was demonstrated by immunoelectrophoresis in rat submandibular gland saliva. The concentration was higher in sympathetically than in parasympathetically induced secretion.     

Biochemical properties of epidermal growth factor in the mouse kidney

Epidermal growth factor (EGF) concentration in the mouse kidney was exceedingly low when compared with the submandibular gland level. Gel filtration of kidney extract showed that kidney EGF had the same molecular weight as the submandibular gland peptide. The isoelectric point of kidney EGF was between pH 4.3 and 4.6. From reversed phase HPLC, two species of EGF, alpha-EGF and beta-EGF, were clearly detected in the kidney sample.     

大鼠体内视网膜神经节细胞诱向因子(RGNTF)及其受体的免疫组织化学定位

本文用ＲＧＮＴＦ单克隆抗体及抗独特型单克隆抗体的免疫组织化学反应,对ＲＧＮＴＦ及其受体在 大鼠体内的分布进行了研究．结果显示,大鼠的肾脏、肾上腺、下颌下腺、胃底腺,以及睾丸的生精细胞对 ＲＧＮＴＩＦ均呈现强阳性免疫反应,并对ＲＧＮＴＦ抗独特型单克隆抗体也呈现阳性免疫反应,表明 ＲＧＮＴＦ及其受体有较广泛的分布,这种情况与神经生长因子（ＮＧＦ）及睫状节神经诱向（营养）因子 （ＣＮＴＦ）相类似．但是,ＲＧＮＴＦ及其受体的分布特点和ＮＧＦ、ＣＮＴＦ的分布是不完全相同的,提示作者 分离的ＲＧＮＴＦ与ＮＧＦ和ＣＮＴＦ不是同源物。这样肾上腺皮质、下颌下腺的浆液腺泡及导管上皮细胞、 胃底腺上皮细胞和生精细胞不仅能够产生ＲＧＮＴＦ,也能合成ＲＧＮＴＦ受体。因此,它们对ＲＧＮＴＦ可能 有自分泌的功能,ＲＧＮＴＦ对这些细胞可能有自身调节的效应。     

Identification of an endogenous activator of calpain in rat skeletal muscle

An additional component of the regulatory system of rat skeletal muscle calpain has been identified. It exerts a potent activating effect on calpain activity and is a heat stable small molecular weight protein. Of the two calpain isozymes present in muscle, the activator is specific for calpain II, being uneffective with calpain I. It promotes activation of the proteinase by reducing 50 fold, from 1 mM to of 20 microM, the requirement of Ca2+ for maximum catalytic activity of the proteinase. However in the presence of the activator calpain II expresses a consistent fraction of the maximum activity even at significantly lower concentrations of Ca2+ (below 5 microM Ca2+). The activator effect follows kinetics that are consistent with the presence of specific binding sites on the calpain molecules. The activator not only removes in a dose dependent fashion the inhibition of calpain by calpastatin, but also prevents inhibition of the proteinase upon the addition of calpastatin. Competition experiments revealed that the proteinase contains distinct sites for the activator and the inhibitor, and that both ligands can bind to calpain with the formation of an almost fully active ternary complex.     

Distribution of post-proline cleaving enzyme in human brain and the peripheral tissues

Summary We have studied the distribution of post-propline cleaving enzyme activity in the various tissues in humans using 7-(succinyl-Gly-Pro)-4-methylcoumarinamide as substrate. The post-propline cleaving enzyme activity was high in muscle, testes, kidney and submandibular gland, but was low in the heart, mesenterium and aorta. In the brain, relatively high post-propline cleaving enzyme activity was observed in the cerebral cortex, but other brain regions showed a very low enzyme activity.On Sephadex G-100 column chromatography, enzyme activity in human kidney showed a major peak and a minor peak. The major peak coincided with the enzyme in human cerebral cortex, but was different from human serum enzyme. Diisopropylfluorophosphate, a serine protease inhibitor, strongly inhibited the enzyme activity of each active fraction. The enzyme in the cerebral cortex and kidney was inhibited by heavy metals and thiol blocking agents. However, inhibition of enzyme activity in the serum was not observed with such inhibitors. Therefore, we suppose that post-proline cleaving enzyme activity in the brain is similar, if not identical, to that in the kidney.     

Characterization of an alkaline proteinase of fish muscle

1. The alkaline proteinase showing pH optimum 8.0 from white croaker (Sciaena schlegeli) skeletal muscle was purified electrophoretically homogeneously (2000-fold) using a combination of DEAE-cellulose chromatography, hydroxylapatite chromatography and Ultrogel AcA 34 gel filtration. 2. It was stable for 1 hr at 50 degrees C. The molecular weight of the enzyme was estimated to be 430,000 by gel filtration, with the enzyme composed of four kinds of subunits, the chain molecular weights of which were 45,000, 48,000, 51,000 and 57,000. 3. From the effects of inhibitors, the enzyme was identified as cysteine proteinase. ATP and Cu2+ inhibited the activity 50% at 10 mM and 70% at 0.1 mM, respectively. 4. Thus the enzyme was characterized as a high molecular weight, heat-stable, alkaline cysteine proteinase (HAP). 5. The enzyme showed hardly any activity below 50 degrees C but considerable activity at around 60 degrees C against myofibrils, digesting myosin heavy chain, actin and tropomyosin. With the addition of 5 M urea the enzyme hydrolyzed myofibrils well at around 30 degrees C.     

Immunochemical study and acinar localization of AM1, a murine submandibular glycoprotein with esterolytic activity

Glycoprotein AM1, a glycoprotein from the submandibular glands of the mouse was isolated from the 100 000 X g tissue extract by polyacrylamide gel electrophoresis. An antiserum to purified glycoprotein AM1 was prepared, and its specificity was tested by immunodiffusion and immunoelectrophoresis. Glycoprotein AM1 could be detected in large quantity only in the submandibular glands of the mouse and in very small amounts in the parotid and sublingual glands and in serum. No glycoprotein AM1 was found in the murine brain, heart, lung, liver, spleen, kidney, pancreas, spinal cord and testis. In addition, glycoprotein AM1 was not detectable in the submandibular glands of the rat and rabbit, and in whole human saliva. No cross-reactivity was found with murine submandibular proteinase A and porcine pancreatic kallikrein. The cellular localization of glycoprotein AM1 was determined by the indirect immunofluorescence technique. In the submandibular glands bright fluorescence was only present in the acinar cells, throughout the whole gland. In the sublingual glands faint fluorescence was detectable as a diffuse network around the acini and possibly in the serous acinar demilune cells. On a subcellular level, glycoprotein AM1 could be demonstrated in the extract of the SMC secretory granular fraction, which originates largely from the acinar cells. On the other hand, glycoprotein AM1 was hardly detectable in the SMB secretory granular fraction, which originates predominantly from the granular convoluted tubular cells. Concomitantly, glycoprotein AM1 was secreted in vivo and could be detected in whole saliva, particularly after stimulation with isoproterenol and carbamylcholine, and also with phenylephrine, but to a much lesser extent.     

Release of alkaline phosphodiesterase I from rat kidney plasma membrane produced by the phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis

The release of plasma-membrane-bound enzymes by phosphatidylinositol-specific phospholipase C obtained from Bacillus thuringiensis was investigated. Among the ectoenzymes of plasma membrane tested, alkaline phosphodiesterase I was released markedly from rat kidney cortex slices, in addition to alkaline phosphatase and 5'-nucleotidase. Other membrane-bound enzymes; alanine aminopeptidase, leucine aminopeptidase, dipeptidyl peptidase, leucine aminopeptidase, dipeptidyl peptidase IV, esterase and gamma-glutamyl transpeptidase could not be liberated from the treated slices. Alkaline phosphodiesterase I was released linearly from rat kidney slices with the concentration of phosphatidylinositol-specific phospholipase C, but little enzyme was released from rat liver slices. Alkaline phosphodiesterase I separated from kidney tissue with n-butanol still retained phosphatidylinositol and was transformed into a lower molecular weight form by phosphatidylinositol-specific phospholipase C. This suggests an important function for phosphatidylinositol in the binding of alkaline phosphodiesterase I to the plasma membrane of rat kidney cells. The alkaline phosphodiesterase I released from rat kidney had a molecular weight of about 240,000 and an isoelectric point (pI) of 5.4. The enzyme hydrolyzed the phosphodiester linkage of p-nitrophenyl-thymidine 5'-monophosphate at pH 8.9 and had a Km value of 0.3 mM. The enzyme was activated by Mg2+ and Ca2+, but was inhibited by EDTA. Strong inhibition took place on the addition of adenosine 5'-phosphosulfate or the nucleotide pyrophosphates, i.e., UDP-galactose and alpha, beta-methylene ATP.     

Components of human split ejaculates. II. Enzymes and proteinase inhibitors.

Lysozyme, alpha-amylase, neutral proteinase and plasminogen activator were most concentrated in the initial portion of the ejaculate that consists mostly of Cowper's gland and prostate gland fluids as well as spermatozoa. The concentration of the high molecular weight proteinase inhibitors, alpha1-antitrypsin and alpha1X-antichymotrypsin, was essentially unaltered throughout the ejaculate fractions, although their absolute amounts showed an increase towards the final fraction. By contrast, the total inhibitory activity towards pancreatic trypsin was highest both in concentration and amount in the last fraction, thus indicating that the seminal vesicles are its primary source. Plasminogen, prothrombin, Factor XIII, and the proteinase inhibitors antithrombin III, alpha2-macroglobulin, inter-alpha-trypsin inhibitor and C1S-inactivator could not be detected immunochemically in whole ejaculates, and indicates the dissimilarity between the coagulation/liquefaction processes of semen and blood.     

Purification and characterization of a trypsin-like protease in the submandibular gland of rats

A trypsin-like protease (named RSP-V) was purified to homogeneity from rat submandibular glands by isoelectric focusing and high-performance liquid chromatography. The purified enzyme had an isoelectric point of 5.3 and an apparent molecular weight of 25,000, and consisted of two subunits with molecular weights of 19,500 and 6,000. RSP-V hydrolyzed BAEE, BAPA, and TAME, but not ATEE or BTPA. It had an optimum pH at around 10.0. RSP-V was strongly inhibited by soybean trypsin inhibitor, aprotinin, leupeptin, antipain, and benzamidine, but not by ovomucoid trypsin inhibitor, p-CMB, or iodoacetic acid. This enzyme partly resembled, but was not identical with, tonin. It was also different from kallikrein, salivain, and glandulain in rat submandibular gland. Although the physiological role of RSP-V has not yet been clarified, this enzyme inactivated dopa decarboxylase alone among catecholamine-synthesizing enzymes.     

Tissue specificities of protease A-like esteroprotease in male mice

An esteroprotease hydrolyzing p-tosyl-L-arginine methyl ester (TAME) has been purified to homogeneity from male mice submandibular glands by the ammonium sulphate precipitation, Sephadex gel chromatography and DEAE-cellulose chromatography. The enzyme was shown as a single chain acidic protein (pI = 5.7) with the molecular weight of 27.5 K and evidence was obtained to reveal that it was similar to protease A. Using this enzyme as antigen we prepared anti-TAMEase antibody. The immunoblotting studies on tissue specificity using 20 different tissues from male mice revealed that cross-reactivities with anti-TAMEase antibody were observed in the crude extract from the sublingual gland, parotid gland and pancreas. The species specificity studies with the submandibular glands of 7 different species indicated that only the crude extract from rat submandibular glands reacted against anti-TAMEase antibody but it exerted a low TAMEase activity.     

JEAP, a novel component of tight junctions in exocrine cells.

Tight junctions (TJs) consist of transmembrane proteins and many peripheral membrane proteins. To further characterize the molecular organization of TJs, we attempted here to screen for novel TJ proteins by the fluorescence localization-based expression cloning method. We identified a novel peripheral membrane protein at TJs and named it junction-enriched and -associated protein (JEAP). JEAP consists of 882 amino acids with a calculated molecular weight of 98,444. JEAP contained a polyglutamic acid repeat at the N-terminal region, a coiled-coil domain at the middle region, and a consensus motif for binding to PDZ domains at the C-terminal region. Exogenously expressed JEAP co-localized with ZO-1 and occludin at TJs in polarized Madin-Darby canine kidney cells, but not with claudin-1, JAM, or ZO-1 in L cells. Endogenous JEAP localized at TJs of exocrine cells including pancreas, submandibular gland, lacrimal gland, parotid gland, and sublingual gland, but not at TJs of epithelial cells of small intestine or endothelial cells of blood vessels. The present results indicate that JEAP is a novel component of TJs, which is specifically expressed in exocrine cells.     

Isolation and biochemical characterization of ten-nanometer filaments from cultured ovarian granulosa cells

Ten-nm filaments have been isolated from control and colchicine-treated primary cultures of rat ovarian granulosa cells. Negative stain electron microscopy indicates an average filament diameter of 10.3 nm in the isolated fiber bundles, which, upon sodium dodecyl sulfate polyacrylamide gel electrophoresis, are found to contain a major polypeptide with a molecular weight of 57,000 and several minor components including actin. One-dimensional peptide mapping and two-dimensional gel electrophoresis demonstrate similarity between the granulosa cell and baby hamster kidney cell 10-nm filament subunit protein, both of which are distinguishable from keratin, desmin, actin, and tubulin. Quantitative gel densitometry experiments demonstrate little difference in the total amount of the 10-nm filament protein in control cells or cells treated with colchicine, accounting for 12 or 15% of the total cellular protein, respectively. The purification procedure, which involves extraction in Triton X-100 and KCl followed by DNase I treatment, yields 709% of the total granulosa cell intermediate filament protein, and 70% of the newly synthesized 57,000 molecular weight component. Two-dimensional gel electrophoresis of cultures labeled with [32P]phosphate show by autoradiography that the 57,000-dalton polypeptide, actin, and a 130,000-dalton protein are the most readily phosphorylated polypeptides in granulosa cell cultures. These studies identify the major intermediate filament subunit protein of granulosa cells as a 57,000-dalton phosphorylatable polypeptide which comprises a substantial portion of the granulosa cell cytoskeleton.     

Characterization of human alpha 2-macroglobulin monomers obtained by reduction with dithiothreitol.

We compared the physicochemical characteristics of alpha 2-macroglobulin (alpha 2M) monomers produced by limited reduction and carboxamidomethylation to those of the naturally occurring monomeric alpha-macroglobulin homologue rat alpha 1-inhibitor 3 (alpha 1 I3). Unlike alpha 1 I3, alpha 2 M monomers fail to inhibit proteolysis of the high molecular weight substrate hide powder azure by trypsin. In contrast to alpha 1 I3, which remains monomeric after reacting with proteinase, alpha 2 M monomers reassociate to higher molecular weight species (dimers, trimers, and tetramers) after reacting with proteinase. Reaction of alpha 2 M monomers at molar ratios of proteinase to alpha 2M monomers as low as 0.3:1 leads to extensive reassociation and is accompanied by complete bait-region and thiolester bond cleavage. During the reaction of alpha 2M monomers with proteinases, the proteinase binds to the reassociating alpha 2M subunits but is not inhibited. Of significance, all the bound proteinase was covalently linked to the reassociated alpha 2M species. Treatment of alpha 2M monomers with methylamine results in thiolester bond cleavage but minimal reassociation. Treatment of alpha 2M monomers with methylamine followed by proteinase results in complete bait-region cleavage and is accompanied by marked reassociation of alpha 2M monomers to higher molecular weight species. However, no proteinase is associated with these higher molecular weight forms. We infer that bait-region cleavage is more important than thiolester bond cleavage in driving alpha 2M monomers to reassociate. Despite many similarities between alpha 1I3 and alpha 2M monomers, significant differences must exist with respect to proteinase orientation within the inhibitor to account for the failure of alpha 2M monomers to protect large molecular weight substrates from proteolysis by bound proteinase, in contrast to the naturally occurring monomeric homologue rat alpha 1 I3.     

Kallikrein localization in rodent salivary glands and kidney with the immunoglobulin-enzyme bridge technique.

Kallikrein has been localized in rodent kidney and salivary glands by means of an immunoglobulin-enzyme bridge technique. In sections of kidney, anti-kallikrein antibodies bound to the apical region of certain distal tubule segments in the cortex, to reabsorption droplets of proximal convoluted tubules, and to certain duct segments in the papilla. In salivary glands of both male and female rats and mice, and apical rim of most striated duct cells of submandibular, parotid and sublingual glands and granular tubules of submandibular glands exhibited immunoreactivity. Granular intercalated duct cells in female submandibular glands also displayed immunostaining for kallikrein. Phenylephrine administration resulted in loss of immunoreactive granules from the granular convoluted tubule cells of male mouse submandibular gland. This response was paralleled by a biochemically demonstrable decrease in kallikrein-like tosylarginine methyl ester (TAME) esterase activity.     

Low molecular weight renin as a storage form in renin granules of the dog

The molecular weight of renin extracted from isolated renin granules of the dog was estimated by gel filtration, using tetradecapeptide as substrate, and was approximately 43,000 daltons. Neither big renin nor big big renin was demonstrable. On the other hand, crude extract of kidney cortex showed angiotensin I generating enzymes other than 43,000 dalton form of renin, whose molecular weight were over 100,000 and around 70,000 daltons. They seemed nonspecific proteases, since they hydrolyzed tetradecapeptide but not plasma angiotensinogen. Therefore renin is stored in the renin granules as a low molecular weight form.     

Multiple cyclic nucleotide phosphodiesterase activities from rat tissues and occurrence of a calcium-plus-magnesium-ion-dependent phosphodiesterase and its protein activator.

1. Supernatant fluids from rat cerebral cortex, cerebellum, kidney, heart and liver contained more phosphodiesterase activity hydrolysing cyclic GMP than that hydrolysing cyclic AMP when assayed with sub-saturating concentrations of substrate. 2. These activities were resolved into several fractions by Sephadex G-200 gel filtration; no two tissues had similar activity profiles. 3. With every tissue examined, a fraction (fraction II) with a molecular weight of about 150,000 was obtained which hydrolysed cyclic GMP preferentially at sub-saturating substrate concentrations in the presence of micromolar concentration of Ca2+, millimolar concentration of Mg2+ and a protein activator. 4. The activity of fraction II accounted for about 60 percent in liver, more than 80 percent in heart and cerebellum, and almost 100 percent in cerebral cortex of the total activity for cyclic GMP hydrolysis, calculated from the activity profiles. 5. Km values of fraction II samples from kidney, heart and liver for cyclic GMP were 1.3, 1.7 and 5 muM respectively. 6. 3-Isobutyl-1-methylxanthine inhibited hydrolysis of cyclic GMP by fraction II with an I50 value of 3muM for heart and liver and 50 muM for cerebrum. 7. The activator protein, with an estimated molecular weight of about 30,000 was isolated from all the tissues listed in 1.8. The concentrations of activator protein and of the isolated enzyme, fraction II, did not correspond exactly.     

Isolation and characterisation of peptide hydrolases from the maize root.

The maize root has two main proteinase and carboxypeptidase components. Proteinase I and carboxypeptidase I, which predominate in older plants, appear to have a serine group at their active sites and have been estimated to have molecular weights of approximately 54000 and 77000 respectively. Proteinase I, which has been purified up to 500-fold, degrades haemoglobin and azocasein with maximum activity at pH 4 and 9--10 respectively, while on maize root protein it gives most hydrolysis in the neutral pH range. The main portion of the nitrate-reductase-inactivating activity in the maize root extract is due to proteinase I. Carboxypeptidase I, like several other plant carboxypeptidases such as carboxypeptidase C which have now (IUB Recommendations 1978) been classified as serine carboxypeptidases (EC 3.4.16.1), has maximum activity around pH 5 and has esterase activity. A second group of proteases, proteinase II and carboxypeptidase II, separated from the above on carboxymethyl-cellulose, were shown to have different molecular weight properties and be equally sensitive to serine and thiol group inhibitors. Proteinase II degrades haemoglobin, but not azocasein and does not mediate nitrate reductase inactivation. Associated with this second group of proteases was a macromolecular component which inactivated nitrate reductase but, unlike the action of proteinase I, was not inhibited by phenylmethylsulphonyl fluoride or casein. It was inhibited by metal chelating agents which were without effect on nitrate reductase inactivation due to proteinase I.     

Identification of tonin in brain and exocrine tissues and in the cell-free translation products encoded by the mRNA of these tissues.

Tissue-specific expression of the esteropeptidase tonin [EC 3.4.99.-] was investigated in rat brain, submandibular gland, pancreas and kidney. Specific polyclonal and monoclonal antibodies to purified rat tonin from the submandibular gland have been developed and characterized and have been purified via a tonin-agarose affinity column. Immunoreactive tonin was measured by a recently developed tonin direct radioimmunoassay using a rabbit tonin antiserum. Resulting tonin levels were found to be 105.27 +/- 2.71 micrograms/mg (of protein) in submandibular gland, 3.18 +/- 0.32 ng/mg in pancreas, 1.35 +/- 0.08 ng/mg in kidney and 0.12 +/- 0.01 ng/mg in brain (means +/- S.E.M.). Western-blot analysis shows that affinity-purified anti-tonin antibody binds to a 32,000-Mr protein from brain and submandibular-gland extracts. The protein, a tonin precursor, was identified from cell-free translation products directly by polyadenylated [Poly(A)+]mRNA species in a wheat-germ system. After the translation products were subjected to immunoprecipitation with affinity-purified tonin antibody, SDS/polyacrylamide-gel electrophoresis of these precipitates revealed two precursors of tonin, with Mr values of 30,000 and 29,000, which are encoded by brain and submandibular-gland mRNA; however, only the 30,000-Mr preprotonin was encoded by pancreas and kidney mRNA. Collectively, the data show that tonin exists in brain, submandibular gland, pancreas and kidney, and can be synthesized by the mRNA of these tissues.     

Effect of chronic capsaicin treatment on tachykinin NK1 binding sites in the rat.

Binding sites for [125I]-Bolton-Hunter substance P (BHSP) were investigated in homogenates of rat submandibular gland, colon smooth muscle, and urinary bladder. In vehicle-treated animals, the equilibrium dissociation constant (KD) was similar for both submandibular gland (0.46 +/- 0.03 nM) and colon (0.57 +/- 0.04 nM), although the maximum density of binding sites (Bmax) was about six-fold higher in submandibular gland compared with colon. These binding parameters remained unchanged in capsaicin-pretreated animals (140 mg/kg IP). In contrast, capsaicin pretreatment reduced (p less than 0.05) the Bmax in urinary bladder by twenty-five percent (0.56 fmol/mg wet weight) when compared to vehicle-treated controls (0.73 fmol/mg wet weight), although the KD was unchanged (vehicle, 0.29 +/- 0.08 nM; capsaicin, 0.24 +/- 0.04 nM). These data demonstrate that the NK1 receptors in submandibular gland and colon smooth muscle are not associated with or dependent upon intact primary afferent sensory neurons. However, a minority of NK1 receptors in the urinary bladder were lost after capsaicin, indicating that these receptors are located on sensory terminals, or may be dependent on growth factors or other chemicals released from these nerves.