Submaxillary Gland Extracts Research Articles

Application of cross-flow filtration to the purification of biologically active peptides in human plasma after incubation with a protease-rich extract

The aim of this study was to find an experimental procedure to purify biologically active peptides from a complex biological matrix (plasma), which was incubated with a protease-rich extract (submandibular gland extract). Special interest was focused on the practicability of cross-flow filtration for this purpose. Therefore, peptides in the incubation mixture were purified with a combination of high-performance liquid chromatographic steps. Purification of biologically active peptides was monitored by a sensitive bioassay and by laser desorption/ionization mass spectrometry. This permitted not only purity control at each purification step but also identification of one of the peptides with vasoconstrictor properties as angiotensin II. This result demonstrates the practicability of cross-flow filtration for extracting enzymatic reaction products from complex substrate—enzyme mixtures during the incubation.

Epidermal growth factor concentrations in pig tissues and body fluids measured using a homologous radioimmunoassay.

A homologous radioimmunoassay for the measurement of epidermal growth factor (EGF) levels in pig tissues and body fluids has been developed using an antiserum to recombinant porcine EGF. The assay is highly specific, showing no cross-reactivity with a variety of other polypeptides including the structurally related protein, transforming growth factor-alpha. Furthermore, < 1% cross-reactivity was observed with mouse EGF emphasizing the necessity for homologous assays for EGF measurement. Immunoreactive EGF was present in extracts of pig kidney and pancreas (3.44 +/- 0.43 and 0.76 +/- 0.13 (S.E.M.) pmol/g wet weight respectively), but was not detected in extracts of submaxillary gland or liver. Although immunoreactive EGF was not detectable in uterine, allantoic or ovarian follicular fluids, colostrum contained EGF at biologically active concentrations (0.84 +/- 0.15 nmol/l). Immunoreactive EGF was also present in pig urine, with similar concentrations in samples from male or female animals. In addition, pig urine inhibited the binding of 125I-labelled EGF to 3T3 fibroblasts and stimulated DNA synthesis in quiescent monolayers of these cells, indicating that the immunoreactive material in urine is biologically active. Quantitative comparisons of the data presented here with that published previously indicate considerable species variation in the EGF levels of various tissues and body fluids.

Purification of mucin glycoproteins by density gradientcentrifugation in cesium trifluoroacetatel

A procedure for the rapid isolation of mucin glycoprotein by density gradient centrifugation in cesium trifluoroacetate (CsTFA) is described. The separation of mixtures of rat tracheobronchial mucin, DNA, hyaluronic acid, and bovine serum albumin in CsTFA gradients was superior to that in cesium bromide gradients. Inclusion of guanidinium chloride or urea in the gradient had no influence on the separation obtained. The mutins isolated from sputum samples of cystic fibrosis patients by this procedure are largely free of nucleic acid, nonglycosylated proteins, and glycosaminoglycans. The results of the use of CsTFA gradient centrifugation for the isolation of mucin from extracts of bovine submaxillary gland are also presented. The CsTFA method is particularly suitable for the high yield isolation of mucin from individual samples which are available in limited quantities.

Purification and characterization of a UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase specific for glycosylation of threonine residues.

A UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase from porcine submaxillary glands was purified to electrophoretic homogeneity. IgG prepared from antisera against the pure enzyme immunoprecipitated the transferase in Triton X-100 extracts of submaxillary glands. The submaxillary transferase is a membrane-bound enzyme in contrast to the pure bovine colostrum enzyme, which is soluble in the absence of detergents. Both transferases have similar properties but also differ significantly. Examination of the acceptor substrate specificity of the submaxillary gland transferase showed that it specifically transferred N-acetylgalactosamine from UDP-GalNAc to the hydroxyl group of threonine and was devoid of transferase activity toward serine-containing peptides. These results imply that more than one transferase is involved in forming the GalNAc-threonine and the GalNAc-serine linkages found in O-linked oligosaccharides in glycoproteins. The amino acid sequence adjacent to glycosylated threonine residues may influence the rate of glycosylation by the pure transferase. For example, the second threonine residue in the sequence, Thr-Thr, appears to be glycosylated about twice as fast as the first and more rapidly than single, isolated threonine residues. However, no unique consensus sequence for glycosylation of threonine residues is evident, and any accessible threonine residue appears to be a potential acceptor substrate.

New specific assays for tonin and tissue kallikrein activities in rat submandibular glands: Assays reveal differences in the effects of sympathetic and parasympathetic stimulation on proteinases in saliva

At least fourteen separate bands of proteinase activity, labelled A-N, were identified by an enzyme overlay membrane technique, using oligopeptide-7-amino-4-trifluoromethylcoumarin (AFC) substrates in rat submandibular gland extracts fractionated on pH4-6.5 isoelectric focusing gels. The proteinases were eluted into an ammonium bicarbonate buffer pH9.8 containing 0.1% Triton X-100 and the relative contribution of each band to total activity evaluated using d-Val-Leu-Arg-AFC ( dVLR-AFC) and Z-Val-Lys-Lys-Arg-AFC (ZVKKR-AFC) as substrates. Immunoblotting of band eluants run on sodium dodecyl sulphate gels with antibodies showed that band A was identical with tonin and bands K-N contained tissue kallikrein. Tonin was found to hydrolyse ZVKKR-AFC but not dvlr-AFC. Estimates of the K m values of tissue kallikrein for dvlr-afc and tonin for ZVKKR-AFC were found to be similar (approx. 20 μM) yet the former enzyme hydrolysed its substrate five times faster. Tonin was inhibited by soybean trypsin inhibitor (SBTI) but not by aprotinin. Tissue kallikrein, on the other hand, was inhibited by aprotinin but was considerably more resistant to inhibition by SBTI. In tissue extracts 95% of the ZVKKR-AFC lytic activity in the presence of 1 μm aprotinin is due to tonin and a similar percentage of the dvlr-afc hydrolysing activity in the presence of 10 μM SBTI is due to tissue kallikrein. These findings were used for the specific measurement of these two proteinases in submandibular gland extracts and in saliva without prior purification. Using these inhibitor based assays we revealed qualitative differences in the composition of proteinases secreted into saliva during parasympathetic versus sympathetic stimulation of the submandibular gland. The distribution of proteinases in sympathetic saliva is very similar to that found in submandibular extracts but on parasympathetic stimulation, although much less proteinase is released, the contributions of the more acidic isomers of tissue kallikrein are increased and that of tonin and other proteinases dramatically decreased. The data suggest that parasympathetic and sympathetic nerves induce proteinase secretion via different pathways.

A potent vasoconstrictor in the rat submandibular gland.

We detected a novel vasoconstrictor in an arginine esterase fraction separated from fractions containing tonin and other esterases that were obtained from a rat submandibular gland extract. When tested on isolated rabbit aorta rings, the substance caused dose-related contractions that were slow in onset, long-lasting, and difficult to reverse by rinsing. The substance acts directly on vascular smooth muscle, since preincubation with plasma or intact endothelium is not required. The fact that the constrictor was destroyed by heat and incubation with pronase suggests that it is a protein. Molecular sieving indicates an estimated molecular weight of 24,000 Da. It has a neutral isoelectric point that is higher than the pI of tonin, from which it can be separated by anion exchange chromatography. A small amount of the vasoconstrictor was obtained by gel filtration and eluted from isoelectric focusing polyacrylamide gels. The purified substance showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was a potent vasoconstrictor; an estimated concentration of 2.5 nM induced contraction of isolated rabbit aorta rings ranging from 15% to 40% of the maximum contraction obtained by 60 mM KCl. Contraction was completely blocked by 1 mM (p-amidinophenyl)methanesulfonyl fluoride, a serine protease inhibitor. Contractile activity was not affected by hirudin, a thrombin inhibitor, but was completely inhibited by soybean trypsin inhibitor and blunted by aprotinin; thus it may be a trypsin-like serine protease. Purified vasoconstrictor preparation showed hydrolyzing activity on Pro-Phe-Arg-methyl-coumarin amide, a kallikrein substrate. We conclude that a novel vasoconstrictor serine protease is present in the rat submandibular gland.

Purification and characterization of a kallikrein-like T-kininogenase.

A T-kininogenase has been purified to homogeneity from rat submandibular gland extracts by DEAE-Sepharose chromatography and preparative gel electrophoresis. The purified protein has an apparent Mr of 28,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and splits into heavy and light chains with Mr of 22,000 and 6,000 in the presence of dithiothreitol. It is an acidic glycoprotein with pI of 4.65-4.75. The carbohydrate moiety is located on the light chain and binds concanavalin A and wheat germ agglutinin. The active site serine residue of the heavy chain is labeled with [14C]diisopropylfluorophosphate and visualized by fluorography. NH2-terminal amino acid sequences of the light and heavy chains reveal 74-84% identity to rat tissue kallikrein, tonin, and other kallikrein-related enzymes. The enzyme cleaves T-kininogen to release T-kinin which was separated by high performance liquid chromatography on a reverse phase C18 column and identified by a kinin radioimmunoassay. Its T-kininogenase but not N-tosyl-L-arginine methyl ester esterase activity can be enhanced 10-fold in the presence of dithiothreitol. The esterolytic activity of the enzyme is inhibited by soybean trypsin inhibitor, aprotinin, leupeptin, and antipain; whereas lima bean and ovomucoid trypsin inhibitors stimulate its activity. The enzyme is localized at the granular convoluted tubule and striated duct cells in rat submandibular glands by immunohistochemistry. The results indicate that T-kininogenase belongs to the group of structurally similar yet distinct kallikrein-like serine proteases.

Release Mechanisms of Inactive Renin from Rat Renal Cortical Slices: Role of the Submandibular Gland

In our previous studies, we showed an in vivo stimulating effect of the extract of the rat submandibular gland on plasma inactive renin release. In this study, we evaluated the effects of the rat submandibular gland extract and of some plasma active renin stimulants on inactive renin release from rat renal cortical slices. Adult male Wistar rats (250-350g) were kept on a regular diet (Na 260mg/100g) and nephrectomized under pentobarbital anesthesia (50mg/kg, i.p.). Five thin renal cortical slices were obtained from each kidney by using a razor blade. These renal cortical slices were incubated in Earle's buffer (pH7.4, Difco) at 37 degrees C for 30 min (preincubation), then transferred into 10ml fresh Earle's buffer with or without some agents and incubated at 37 degrees C for 1 hour (experimental incubation). For each experiment, 6 groups of 5 renal cortical slices were employed. The agents used in this study were as follows: isoproterenol (10(-5)M), furosemide (50 micrograms/ml), prostaglandin E1 (10(-5)M), prostaglandin I2 (10(-5)M) and the rat submandibular gland extract (100 microliters) which was obtained after homogenation with 10 x (w/v) 0.01M pyrophosphate buffer (pH6.5) including 0.1M NaCl. One ml of samples of this Earle's buffer were withdrawn every 20 min. Active renin in the samples was assayed by the commercial RIA-kit (Dainabot), and total renin was assayed after trypsin (Worthington) treatment (30 micrograms/300 microliters sample) at 4 degrees C for 10 min. Inactive renin was determined as the difference between total renin and active renin. Active and inactive renins increased linearly in the buffer without any agents (control) during the observation period (60 min). Isoproterenol (10(-5)M) stimulated the release of active renin significantly (p less than 0.01 vs. control) but did not affect the release of inactive renin. Furosemide (50 micrograms/ml) stimulated the release of active and inactive renins significantly at 20 and 40 min (p less than 0.05 vs. control) but did not affect the release of either renin at 60 min. Both prostaglandins E1 and I2 (10(-5)M) stimulated the release of active renin significantly (p less than 0.01 vs. control) but inhibited, on the other hand, the release of inactive renin significantly (p less than 0.01 vs. control). The rat submandibular gland extract (100 microliters) did not affect the release of active renin but stimulated the release of inactive renin significantly (p less than 0.05 vs. control).(ABSTRACT TRUNCATED AT 400 WORDS)

Proteinic prorenin-releasing-stimulator (PRS) in the rat submandibular gland

The release of prorenin as well as renin from rat renal slices was confirmed by a rat prorenin-prosegment ELISA system and an assay system for determining the renin activity. A significant increase of the prorenin release was found by adding rat submandibular gland extract to the slice medium, indicating the existence of a prorenin-releasing stimulator (PRS) in the extract. The p I and molecular mass of PRS were 8.5–8.7 and 28–30 kDa, respectively. The PRS was completely inactivated by boiling or a proteinase treatment. Renin releasing stimulator, pro-; Cortical slice; Submandibular gland; ELISA, prorenin-prosegment; (Rat kidney)

Epidermal Growth Factor Immunoreactive Material in the Rat Brain: Localization and identification of multiple species

Abstract Brains of adult male rats were dissected into five distinct regions: brainstem, cerebellum, hippocampus, diencephalon, and telencephalon. Epidermal growth factor-like immunoreactivity was isolated and characterized by radioimmunoassay and nondenaturing polyacrylamide gel electrophoresis. Radioimmunoassay indicated levels of standard rat epidermal growth factor equivalents ranging from 0.99 to 0.33 ng/g wet weight of brain tissue. Competition curves were not parallel to those generated with standard rat epidermal growth factor, indicating a lack of structural identity between the immunoreactive material in the brain and standard rat epidermal growth factor. Extracts of submandibular gland and blood did, however, produce parallel competition curves. Electrophoresis indicated the presence of multiple bands of immunoreactive material in each of the regions of the brain. The major bands of activity migrated to positions distinct from that of standard rat epidermal growth factor. This is the first demonstration of multiple forms of epidermal growth factor-like immunoreactive material in the central nervous system.

T-kinin release from T-kininogen by rat-submaxillary-gland endopeptidase K.

Submaxillary gland extracts have been fractionated to characterize the enzyme responsible for the T-kininogenase activity previously reported in this tissue [Damas, J. & Adam, A. (1985) Mol. Physiol 8, 307-316] and to know whether this activity could be of physiological relevance, since no enzyme reacting in catalytic amounts has been described so far to be able to release a vasoactive peptide from T-kininogen. The purified enzyme, provisionally called endopeptidase K, has an apparent Mr of 27,000 when not reduced prior to analysis but 21,000 after reduction and an acidic pI of 4.3 +/- 0.1. Antigenically, it is not related to tissue kallikrein. Upon incubation with purified T-kininogen it may induce a complete liberation of T-kinin from the precursor provided it is added in stoichiometric amounts. However, in parallel with the liberation of immunoreactive kinin, a proteolysis of T-kininogen is observed which is not restricted to the site of insertion of T-kinin as would be expected using a specific kininogenase. In agreement with these results, no change of the mean blood pressure was observed upon injection of endopeptidase K into the circulation of normal rats even if the amount of injected enzyme was up to ten times that required for tissue kallikrein to induce a significant fall in blood pressure. However, in spite of the large proteolysis induced by incubation with stoichiometric amounts of endopeptidase K, the total papain inhibiting capacity of T-kininogen as well as the value of the apparent inhibition constant, Ki, with this proteinase remained unchanged. Proteolytic fragments which retain cysteine-proteinase-inhibiting activity may therefore be released from T-kininogen by endopeptidase K more easily than immunoreactive kinin, thus emphasizing a prominent function of proteinase inhibitor or of proteinase inhibitor precursor for this molecule.

The role of epidermal growth factor in normal and neoplastic growth of mouse mammary epithelial cells.

Epidermal growth factor (EGF) was discovered by Cohen in 1962 [1] as the principal in extracts of male mouse submandibular gland which produced acceleration of incisor eruption and eyelid opening when administered to newborn mice. It has since been purified to homogeneity and sequenced [2]. Mouse EGF is a single-chain polypeptide (mol. wt. 6045) containing 53 amino acid residues and 3 disulfide linkages. EGF is also present in biological fluids such as plasma, saliva, milk, urine, amniotic fluid, sweat, pancreatic juice, and cerebrospinal fluid [3–5]. EGF exhibits diverse biological actions, influencing proliferation, differentiation, and functional activities of various types of mammalian cells [3–6]. EGF has also been a useful tool for the analysis of receptor hormone interactions and receptor mediated endocytosis of polypeptide hormones. Several excellent reviews of the chemistry and biology of EGF have been published [3–7].

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.

Identification and expression of kallikrein gene family in rat submandibular and prostate glands using monoclonal antibodies as specific probes

Panels of monoclonal antibodies to three vasoactive peptide-producing enzymes: tissue kallikrein, tonin and arginine esterase A were developed, characterized and used as probes for identification of tissue-specific expression. In addition, immunoblot analyses were performed, using monospecific monoclonal antibodies which did not show cross-reactivity to related-purified enzymes in enzyme-linked immunosorbant assay (ELISA), and radioimmunoassay. We obtained the following results. In rat submandibular gland extract, the expression of 38 kDa kallikrein, 32 kDa tonin, and 18 kDa heavy chain of esterase A was identified by monoclonal antibodies to kallikrein (V 4D 11), tonin (1F 11), and esterase A (5A 10, 6C 11, and 4B 12), respectively. In the prostate gland, a 32 kDa kallikrein-like protein was identified by monoclonal antibodies to esterase A (5A 10, 6C 11 and 4B 12) and by antibodies recognizing both tonin and esterase A (5A 5), but not by antibody to kallikrein (V 4D 11) or to tonin (1F 11, 1G 6) in Western blot analysis. The esterase A-like enzyme in the prostate gland was found within the cytoplasm of ductal epithelial cells by using monoclonal anti-esterase A antibody (5A 10) but not by employing anti-tonin antibody (1F 11). These results indicate that tissue kallikrein, tonin, and esterase A are all expressed in the submandibular gland, while only esterase A or an esterase A-like enzyme is expressed in the prostate gland. The specific monoclonal antibodies can be used as probes for the identification and expression of the kallikrein gene-family enzymes.

Growth-promoting activity in serum-free medium of kallikreinlike arginylesteropeptidases from rat submaxillary gland.

The characterization and purification of the growth-promoting activity present in rat submaxillary gland extracts, known to be required for the proliferation of adipose precursor cells in serum-free medium, have been undertaken. Fractionation of the extracts by ion-exchange chromatography, gel filtration, and affinity chromatography on immobilized benzamidine allowed the copurification of the mitogenic activity with two distinct arginylesteropeptidases of apparent molecular weight 25,000; one of these enzymes has been purified to homogeneity and shown to be immunologically related to tonin, a well-characterized kallikreinlike protease from submaxillary gland. The specificity of both enzymes was similar to that of plasma and glandular kallikreins, as indicated by the relative rates of hydrolysis of peptide p-nitroanilide substrates. Prior treatment of the kallikreinlike proteases with phenylmethylsulfonylfluoride or aprotinin abolished completely both mitogenic and arginylesteropeptidase activities, indicating that enzymatic activity was essential for the manifestation of their growth-promoting ability. The kallikreinlike proteases from rat submaxillary gland were able to replace thrombin to support the proliferation of Chinese hamster lung fibroblasts in serum-free medium. These results underline the role of proteases in controlling cell growth and are discussed in light of adipose tissue development.

Synthesis of nerve growth factor mRNA and precursor protein in the thyroid and parathyroid glands of the rat.

Nerve growth factor (NGF) mRNAs were detected in the thyroid and parathyroid glands by hybridization to a preproNGF and a NGF-beta cDNA probe. The thyroid NGF transcript was identical in size to that found in the mouse submaxillary gland. Mature NGF-beta was not detected in the thyroid tissue either by immunoprecipitation or by immunohistochemical methods. In contrast, an antiserum directed against the predicted precursor protein sequence immunoprecipitated a protein of Mr 30,000-31,000 in thyroid extracts. A protein of identical molecular weight was also detected by this serum in the mouse submaxillary gland extracts. In vitro translation of hybrid-selected NGF RNAs detected two peptides of Mrs 35,000 and 28,000. These peptides closely correspond to the proposed translation products of the preproNGF mRNA sequence, suggesting that two different initiation codons can be utilized, at least in vitro. The proNGF antiserum also cross-reacted with the in vitro synthesized Mr 35,000 peptide. The antigenic determinant recognized by the precursor-specific antibody was localized to the convoluted granular tubules of the mouse submaxillary gland, known to be the site of synthesis of the NGF-beta. We present evidence for the localization of unprocessed proNGF in the parathyroid gland and in some parafollicular cells of the thyroid.

Age and hormonal dependence of tonin levels in rat submandibular gland as determined by a new direct radioimmunoassay.

A simple and sensitive direct radioimmunoassay for tonin (EC.3.4.99.-) has been developed. This assay incorporates a modified and convenient poly(ethylene glycol) technique for separation of free from bound tonin. A rabbit antiserum in a final dilution of 1:160,000 was used and the purified tonin was labelled with 125I by using a lactoperoxidase method. It detects 20 pg of immunoreactive tonin per tube. Serial dilutions of rat submandibular gland extracts showed complete parallelism with tonin standard curves. No cross-reactivity with rat tissue kallikrein was seen. Intra- and inter-assay errors were 3.2 and 5.6%, respectively. Using this assay, immunoreactive tonin was detected in the rat submandibular gland as early as 3 weeks after birth (body wt. approximately 50-60 g). Tonin levels are shown to be dependent on age and sex with significantly higher levels in male than in female rats. Castration results in decrease of tonin levels and 17 alpha-methyltestosterone replacement reversed the level to higher than the sham-operated control rats. Cortisol treatment increased, but thyroxine or oestradiol had no effect, on tonin levels in the submandibular gland of castrated rats. This newly developed radioimmunoassay can now be used to measure low levels of tonin in various tissues and body fluids to address questions about its regulation and functional significance.

Thyroxine increases neonatal mouse submandibular gland mRNA-directed synthesis of epidermal growth factor

Thyroid hormones are known to modulate the concentrations of epidermal growth factor (EGF) in the mouse submandibular gland (SMG); this action is presumably mediated by the nuclear triiodothyronine receptor. To test the hypothesis that thyroid hormones act to increase SMG EGF concentrations by increasing the number of poly(A)+ -specific mRNA, poly(A)+ RNA was isolated from SMGs of neonatal mice which had been treated daily from birth through to 21 days of age with thyroxine (T4,0.4 microgram/g body weight). Poly(A)+ RNA also was extracted from SMGs of intact 21-day-old mice which had received vehicle alone. No significant differences in total nucleic acid, total RNA, or poly(A)+ RNA yields were noted between the two groups of animals. The isolated poly(A)+ RNAs from T4-treated and control mice were translated in an in vitro wheat germ system. Although no significant differences in efficiency of [35S]cysteine incorporation into trichloracetic acid precipitable material were noted between the two poly(A)+ RNA preparations, a significantly greater proportion of radioactivity was immunoprecipitable by anti-EGF antiserum in the translation medium derived from T4-treated mice (17.2 +/- 0.9%, mean +/- SEM) than in that of control mice (7.3 +/- 0.5%, P less than 0.001). Polyacrylamide gel electrophoresis of the immunoprecipitates (IMMP) revealed the presence of three radioactive bands with apparent relative masses (MrS) of 12,000, 9000, and 6000. The latter species comigrated with purified EGF, [125I]EGF, and an IMMP of a SMG extract. The translation product IMMPs following polyacrylamide gel electrophoresis were iodinated and digested with alpha-chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)

A monoclonal antibody to rat tissue kallikrein: use in biochemical and immunohistochemical studies.

A monoclonal antibody (V4G6) to rat tissue kallikrein (EC 3.4.21.35) has been developed and characterized. This clone showed no cross-reactivity with rat tonin, rat esterase A or human urinary kallikrein in either radioimmunoassay or enzyme-linked immunosorbent assay. The monoclonal antibody used in the direct radioimmunoassay detects purified rat urinary kallikrein in a range of 0.32 to 40 ng per tube. The displacement curves for rat submandibular gland, pancreatic and kidney extracts and urine were parallel with the standard curve of purified rat urinary kallikrein. Analysis of immunoprecipitates from [14C] DFP labeled submandibular gland extract with SDS-polyacrylamide gel electrophoresis, demonstrates that this antibody recognizes only one 38,000 dalton serine protease while polyclonal antiserum identifies multiple species. Using this specific monoclonal reagent, tissue kallikrein was localized immunohistochemically in the granular convoluted tubules and striated ducts of the rat submandibular gland and in the acinar cells of the rat pancreas. The results showed that the monoclonal antibody (V4G6) can specifically recognize a single kallikrein in the tissue extracts without cross-reacting with other kallikrein-related serine proteases. This monoclonal antibody can be used as a specific reagent for quantitation, identification and immunohistochemical studies of tissue kallikrein.

A fluorimetric kinetic method for the determination of tonin activity in rat submaxillary glands

A method for the kinetic determination of tonin activity in rat submaxillary gland extracts is described. The method is based on the fluorogenic reaction of o-phthalaldehyde with the dipeptide histidyl-leucine generated in the enzymatic hydrolysis of angiotensin I. The reaction is monitored by fluorescence at 440 nm. At pH 7.5, the method allows the determination of 2–15 nmol/ml histidyl-leucine with relative standard deviation of 6%.