Development and characterization of a radioimmunoassay to measure human tissue kallikrein in biological fluids.

A direct radioimmunoassay has been developed to measure tissue kallikrein in human biological fluids. These fluids include serum, plasma, urine, pancreatic juice and saliva. Purified kallikreins from human urine and human saliva were used to raise rabbit antibody and each was labelled with Na125I for use in the radioimmunoassay. A comparison of the different antigen-antibody systems was then made. Bound and free enzyme were separated by a double-antibody technique. The usable range of the standard curve was from 2.5 to 100 micrograms kallikrein/l. The intra-assay coefficient of variation was 4.7%, the interassay coefficient of variation 8.9% and the recoveries of purified kallikrein added to the samples were 99.3, 96.0, 110.8 and 81.2% for urine, saliva, serum and plasma respectively. Parallel dilution curves were obtained for serum and plasma, as well as for urine, saliva and pancreatic juice. However, plasma anticoagulated with EDTA or heparin gave consistently lower values than serum, when measured in the radioimmunoassay. From eight different subjects plasma (EDTA) values were on average 50% lower than those of serum. Experiments designed to determine the cause of this difference revealed that treatment of blood with some anticoagulants, in particular heparin and EDTA, resulted in a marked reduction in the amount of measurable tissue kallikrein.

Purification and properties of guinea-pig submandibular-gland kallikrein.

Guinea-pig submandibular kallikrein has been purified from the glands to electrophoretic homogeneity by conventional procedures. The enzyme is active as a kininogenase, releasing kallidin at a rate of 462 micrograms/min per mg of protein from bovine kininogen, and proved potently hypotensive in the guinea pig and in the dog, properties which indicate its tissue kallikrein nature. The specific activity determined on the substrate N-alpha-benzoyl-L-arginine ethyl ester (11.1 mumol/min per mg of protein) is much lower than that measured with N-acetyl-L-phenylalanyl-L-arginine ethyl ester (483 mumol/min per mg of protein). The latter value is of an order of magnitude comparable with the specific activities of other tissue kallikreins determined with this sensitive kallikrein substrate. The enzyme is a glycoprotein consisting of 237 amino acid residues and containing three to four glucosamine molecules. Its amino acid composition is not identical with that reported for guinea-pig coagulating-gland kallikrein, but is remarkably similar to that of the porcine tissue kallikreins. Apparent Mr values are 29000 (sodium dodecyl sulphate/polyacrylamide-gel electrophoresis) or 34000 (gel filtration). The amino acid sequence of the first 31 N-terminal residues was determined and was found to be closely homologous with that of other tissue kallikreins.

Immunogenicity of guinea-pig submandibular and coagulating gland kininogenases.

The antigenic relationship between submandibular and coagulating gland kininogenases of the guinea-pig has been examined using Ouchterlony double diffusion, crossed and rocket immunoelectrophoresis and immunofluorescent techniques. The evidence suggests a partial immunological identity between submandibular and coagulating gland kininogenases of the guinea-pig. In this respect these enzymes seem to differ from the glandular kallikreins of other species (porcine, human and rat) which apparently share a complete immunological identity.

Stimulus-secretion coupling: role of cyclic AMP, cyclic GMP and calcium in mediating enzyme (kallikrein) secretion in the submandibular gland.

1. The role of adenosine 3':5'-phosphate (cyclic AMP) and guanosine 3':5'-phosphate (cyclic GMP) as second messengers for the enzyme secretory response evoked by the autonomic neurotransmitters, noradrenaline and acetylcholine, is examined in this in vitro study on the guinea-pig submandibular gland. 2. Noradrenaline increased enzyme (kallikrein) secretion. The initial stimulation of enzyme release appeared to be dose-dependent. The time course of cumulative kallikrein secretion revealed a complex pattern. Isoprenaline and phenylephrine were almost as potent as noradrenaline in releasing kallikrein. Both propranolol and phentolamine were required to fully inhibit the noradrenaline-stimulated enzyme secretion. 3. The cumulative secretion of kallikrein evoked by acetylcholine was dose-dependent. The onset of secretion showed a significantly greater time-lag than that observed with noradrenaline. Atropine effectively blocked the release of kallikrein by acetylcholine. 4. Dibutyryl cyclic AMP stimulated enzyme secretion. Dibutyryl cyclic GMP caused an initial increase which was not maintained. 5. The cyclic nucleotide phosphodiesterase inhibitors, theophylline and papaverine, increased basal kallikrein secretion. The action of the cyclic phosphodiesterase inhibitors on the secretory response to noradrenaline, acetylcholine, dibutyryl cyclic AMP and dibutyryl cyclic GMP was complex. In general, the increase in enzyme release produced by the secretagogues was additively enhanced by both inhibitors. 6. Omission of calcium inhibited both acetylcholine and dibutyryl cyclic GMP stimulated kallikrein release, but to a lesser degree than that of noradrenaline and dibutyryl cyclic AMP. High concentrations of extracellular calcium (10 mM) appeared to enhance the action of acetylcholine. 7. Noradrenaline produced a rise in the intracellular level of cyclic AMP. The increase preceded the stimulated secretion of kallikrein. Of the various adrenergic agonists, noradrenaline and isoprenaline were the most potent, whereas phenylephrine was significantly less effective in raising basal cyclic AMP values. Acetylcholine was without effect, even in the presence of a cyclic phosphodiesterase inhibitor. 8. Acetylcholine and noradrenaline raised intracellular levels of cyclic GMP only when the tissue incubations were performed in the presence of a cyclic phosphodiesterase inhibitor. The increase in cyclic GMP produced by acetylcholine preceded enzyme secretion. 9. Morphological data substantiated the finding that the in vitro release of kallikrein evoked by the secretagogues was associated with the depletion of secretory granules and vacuolations in acinar cells of the gland slices. 10. The molecular mechanisms which control enzyme secretion in the exocrine submandibular gland are discussed. Models are presented for the role of transmitter-specific cyclic nucleotides and calcium in stimulus-secretion coupling.