Enzyme-and immuno-histochemical localization of kallikrein

Summary Localization of kallikrein in the human parotid gland was investigated simultaneously by two markers: kallikrein-like (enzyme) activity and kallikrein antigenicity. Kallikrein-like activity was histochemically demonstrated by using a synthetic substrate, pro-phe-arg-naphthylester. Kallikrein antigenicity was demonstrated by an unlabelled antibody peroxidase-antiperoxidase method, where monospecific antiserum against highly purified urinary kallikrein was used as the primary antiserum. The results showed that kallikrein-like activity and kallikrein antigenicity were identical in their locations in the ductal cells, being localized in the luminal part of the striated ducts and to a lesser degree in the excretory ducts. This indicates the presence of active kallikrein in these regions. No enzyme activity nor antigenicity was observed either in acini or in intercalated ducts. Moreover, the peroxidase-antiperoxidase method reveated kallikrein antigenicity for the first time extracellularly in the basement-membrane region of acini and of ducts as well as in the interstitium surrounding ducts and major vessels.     

Isolation of tissue kallikrein in rat spleen by monoclonal antibody-affinity chromatography

Rat spleen kallikrein was identified and purified by DEAE-cellulose and monoclonal antibody-affinity chromatography. The purified enzyme has Tos-Arg-OMe esterase activity and kinin-releasing activity from a purified low-molecular-weight kininogen substrate. In the direct radioimmunoassay for tissue kallikrein, the splenic enzyme displays parallelism with standard curves of rat urinary kallikrein. The pH profiles of the Tos-Arg-OMe esterase activities of spleen and urinary kallikrein were identical with optima at 9.0 Rat spleen kallikrein was inhibited strongly by aprotinin and affinity-purified kallikrein antibody and weakly by soybean trypsin inhibitor. The IC₅₀ values were similar to those observed against rat urinary kallikrein. Neither the urinary nor the splenic enzyme was inhibited by lima bean trypsin inhibitor or preimmune serum immunoglobulins. Spleen kallikrein was labeled with [¹⁴]diisopropylphosphorofluoridate and visualized by fluorography on a sodium dodecyl sulfate-polyacrylamide gel. The electrophoretic mobility of the splenic enzyme was indistinguishable from that of urinary kallikrein A with an estimated Mr of approx. 38 000. With Western blot analyses using a rabbit anti-kallikrein antibody followed by ¹²⁵I-labeled protein A binding, the spleen and urinary kallikreins were again visualized at identical positions by autoradiography. The data show that there is a rat splenic tissue kallikrein which is indistinguishable from a renal kallikrein with respect to physicochemical properties, immunological character and susceptibility to inhibitors.     

Purification and characterization of rat urinary esterase A1

An enzyme, esterase A1, which hydrolyzes tosyl-arginine methyl ester (Tos-Arg-OMe) was separated from esterase A2 and kallikrein of male rat urine and purified by a procedure involving ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography and gel filtration. The resulting preparation was apparently homogeneous, as assessed by polyacrylamide gel electrophoresis. The molecular weight of the preparation was estimated to be 27,000 by SDS-polyacrylamide gel electrophoresis and 30,000 by gel filtration. The enzyme was more specific for arginine methyl esters than for lysine methyl esters. The optimum pH determined with Tos-Arg-OMe as a substrate was 8.0 and the Km was 11.8 mM. The Tos-Arg-OMe esterolytic activity of esterase A1 was inhibited by soybean trypsin inhibitor, but not by aprotinin. In immunodiffusion analysis, the antiserum to esterase A1 formed immunoprecipitin arcs with this enzyme and the urine collected from rat bladder, but not with esterase A2, kallikrein, plasma and the urine collected from ureters. These results indicate that rat urinary esterase A1 differs from esterase A2 and kallikrein. The esterase A1 appears to be produced by accessory sex glands and excreted via the spermiduct into the urine.     

Isolation and partial characterization of rat urinary esterase A2

An enzyme, esterase A2, which hydrolyzes tosyl-arginine methyl ester was isolated from the urine of female, inbred, Dahl-salt-resistant rats using DEAE-Sephadex ion-exchange, aprotinin-agarose affinity and molecular sieve column chromatography. The purest preparation obtained showed four closely migrating bands on polyacrylamide gel electrophoresis. All four bands of the esterase A2 preparation had enzyme activity since all were stainable on zymograms using N-acetyl-L-methionine alpha-naphthyl ester as substrate. Three of these four bands showed decreased electrophoretic mobility following treatment with neuraminidase, indicating that variable sialic acid content accounts for part of the microheterogeneity. The preparation of esterase A2 used was free of rat urinary kallikrein as shown by radioimmunoassay, electrophoretic and isoelectric focusing experiments. The relative kinin-generating ability of rat urinary kallikrein and esterase A2 was highly dependent on the assay used. Using canine plasma as a source of kininogen and the rat uterus to bioassay kinins, esterase A2 was 47% as active as kallikrein; using pure bovine low-molecular-weight kininogen and a radioimmunoassay to measure generated kinins, esterase A2 was only 6% as active as kallikrein. Esterase activity of A2 was activated non-specifically by proteins and detergents. Esterase A2 was 50% inhibited by an 8-fold molar excess of aprotinin and by a 26.5-fold molar excess of soybean trypsin inhibitor, but ovomucoid inhibitor was not inhibitory.     

Purification and characterization of a serine protease (esterase B) from rat submandibular glands

A new protease has been purified to homogeneity from rat submandibular gland homogenate by using DEAE-Sephadex chromatography, chromatofocusing, aprotinin-Sepharose affinity chromatography, and high-performance liquid chromatography. The enzyme has been named esterase B, since it represents the second major esterolytic peak on DEAE-Sephadex chromatography of submandibular gland homogenate. It is an acidic protein (pI = 4.45) with an apparent molecular weight of 27 000. It is heat-stable and has an optimum pH of 9.5. Esterase B hydrolyzed the synthetic substrates tosyl-L-arginine methyl ester and Val-Leu-Arg-p-nitroanilide (S2266). It also cleaved dog plasma kininogen to produce a kinin, identified as bradykinin on reverse-phase high-performance liquid chromatography. Esterase B, however, is only a weak kininogenase, since it had only 5% of the kininogenase activity of equimolar concentrations of glandular kallikrein and had no effect on rat mean blood pressure or on the isolated rat uterus. Esterase B activated plasminogen and had caseinolytic activity. It was inhibited by aprotinin, soybean trypsin inhibitor, lima bean trypsin inhibitor, phenylmethanesulfonyl fluoride, antipain, leupeptin, and p-tosyl-L-lysine chloromethyl ketone. On double immunodiffusion, when reacted with kallikrein and tonin antisera, esterase B showed partial identity with kallikrein but not with tonin. On immunoelectrophoresis against kallikrein antisera, esterase B formed a precipitin arc at a position different from that of kallikrein. Esterase B appears to be a trypsin-like serine protease having some homology with glandular kallikrein.     

Purification and characterization of a non-kallikrein arginine esterase from dog urine

A non-kallikrein arginine esterase (esterase I) has been purified from dog urine and characterized. The enzyme was purified by a three-step procedure, including ion exchange chromatography on DEAE-Sephacel, affinity chromatography on p-aminobenzamidine-Sepharose, and final gel filtration on Ultrogel AcA-54. The purified preparation gave three protein bands on polyacrylamide gel electrophoresis, all of which had esterolytic activity. The enzyme has a specific activity of 601 esterase units/mg protein. It has negligible kininogenase activity. Esterase I gave two closely migrating protein bands on reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis with molecular weights of 34,000 and 33,300. Esterase I is a glycoprotein with a pH optimum of 9.5 and a pI of 4.62. The enzyme is strongly inhibited by a host of inhibitors including aprotinin, leupeptin, antipain, soybean trypsin inhibitor, lima bean trypsin inhibitor, and DPhe-Phe-Arg-chloromethyl ketone (I50 in the 10(-9)-10(-8) M range). However, p-aminobenzamidine, N alpha-p-tosyl-lysyl chloromethyl ketone and phenylmethylsulfonyl fluoride were weak inhibitors, with I50 values in the 10(-5)-10(-7) M range. The enzyme preferentially hydrolyzes Pro-Arg bonds. Among fluorogenic substrates used in this study, butyloxycarbonyl-Val-Pro-Arg-methylcoumarinamide (alpha-thrombin substrate) was found to be the best, with a Km of 1.7 microM and a kcat/Km of 6.3 s.microM-1. However, esterase I does not convert fibrinogen to fibrin nor activate plasminogen to plasmin. Esterase I is immunologically distinct from dog urinary kallikrein, having no cross-reactivity with antibodies against dog kallikrein.     

Identification and characterization of a tissue kallikrein in rat skeletal muscles.

A tissue kallikrein was purified from rat skeletal muscle. Characterization of the enzyme showed that it has alpha-N-tosyl-L-arginine methylesterase activity and releases kinin from purified bovine low-Mr kininogen substrate. The pH optimum (9.0) of its esterase activity and the profile of inhibition by serine-proteinase inhibitors are identical with those of purified RUK (rat urinary kallikrein). Skeletal-muscle kallikrein also behaved identically with urinary kallikrein in a radioimmunoassay using a polyclonal anti-RUK antiserum. On Western-blot analysis, rat muscle kallikrein was recognized by affinity-purified monoclonal anti-kallikrein antibody at a position similar to that of RUK (Mr 38,000). Immunoreactive-kallikrein levels were measured in skeletal muscles which have different fibre types. The soleus, a slow-contracting muscle with high mitochondrial oxidative-enzyme activity, had higher kallikrein content than did the extensor digitorum longus or gastrocnemius, both fast-contracting muscles with low oxidative-enzyme activity. Streptozotocin-induced diabetes reduced muscle weights, but did not alter the level of kallikrein (pg/mg of protein) in skeletal muscle, suggesting that insulin is not a regulator of kallikrein in this tissue. Although the role of kallikrein in skeletal muscle is unknown, its localization and activity in relation to muscle functions and disease can now be studied.     

Urinary kallikrein and non-kallikrein arginine esterase activities in beagle dogs

The activity of urinary kallikrein and non-kallikrein arginine esterase was measured in the 16hrs urine of 16 beagle dogs. Enzyme activity was assayed using D-valyl-L-leucyl-L-arginine-p-nitroanilide as a substrate at 30 degrees C, pH 8.0, and the unit of measurement was defined as the activity which catalyzes the hydrolysis of 1 mumol of the substrate per minute. Kallikrein activity ranged from 3.87 to 48.92 (28.48 average) mU/ml of urine, or from 1.05 to 12.51 (4.56 average) U/16 hrs; whereas that of non-kallikrein arginine esterase ranged from 0.06 to 24.28 (7.11 average) mU/ml, or from 0.01 to 9.78 (1.43 average) U/16 hrs. The ratio between the activity of these two enzymes was 1: 0.002-1: 2.98 (1: 0.42 average). Male dogs had a tendency to show a higher enzyme activity than females for urinary kallikrein and non-kallikrein arginine esterase.     

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 (V₄D₁₁), tonin (1F₁₁), and esterase A (5A₁₀, 6C₁₁, and 4B₁₂), respectively. In the prostate gland, a 32 kDa kallikrein-like protein was identified by monoclonal antibodies to esterase A (5A₁₀, 6C₁₁ and 4B₁₂) and by antibodies recognizing both tonin and esterase A (5A₅), but not by antibody to kallikrein (V₄D₁₁) or to tonin (1F₁₁, 1G₆) 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 (5A₁₀) but not by employing anti-tonin antibody (1F₁₁). 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.     

Active-site labeling of rat and human urinary kallikrein by chloro(N alpha-p-tosyllysyl)methane

This is the first report to demonstrate that chloro(N alpha-p-tosyllysyl)methane (TosLys-CH2Cl) inhibits mammalian glandular kallikrein activities. The inhibitory effect of TosLysCH2Cl on purified rat urinary kallikrein was carried out with three assay methods: 1) Tos-Arg-OMe hydrolysis activity measured by a radiochemical method; 2) kininogenase activity using purified bovine low molecular weight kininogen as substrate and the released kinins subsequently measured by radioimmunoassay; 3) bioassay using isolated rat uterus preparation. Purified rat urinary kallikrein was inhibited by TolLysCH2Cl in a dose and time-dependent manner with all three methods used. The inhibition of purified human urinary kallikrein esterase and kinin-releasing activities were also demonstrated. The results indicate that TosLysCH2Cl inactivates kallikrein activity and support the notion that reactive histidine residue(s) participates in the active center of Kallikrein for catalysis.     

Comparison of genetic and endocrine control of renin and kallikrein in mouse submandibular gland

The effects of strain, sex, hypophysectomy and hormone treatment on mouse submandibular gland renin, kallikrein, S2266 hydrolase, and BAEe esterase activities have been examined. Renin activity is determined by the $\text{Rnr}$ locus on mouse Chromosome 1. Female SWR/J mice ($\text{Rnr}$^$\text{s}$/$\text{Rnr}$^$\text{s}$) have 1000-fold higher submandibular gland renin activity than C57BL/6J mice ($\text{Rnr}$^$\text{b}$/$\text{Rnr}$^$\text{b}$). Both strains have similar kallikrein activity. Renin, BAEe esterase, and S2266 hydrolase are substantially higher in male mice compared to females of the same strain whereas kallikrein is not. Dihydrotestosterone and/or thyroxine treatment induces renin, BAEe esterase, and S2266 hydrolase in female mice with little effect on kallikrein. All four enzyme activities are profoundly reduced by hypophysectomy. Dihydrotestosterone and thyroxine are both required to restore renin, BAEe esterase, and S2266 hydrolase to induced levels. Dihydrotestosterone and.or thyroxine restores kallikrein to control levels. We conclude that renin and kallikrein in the mouse submandibular gland are under different genetic and endocrine control. In addition, the synthetic substrate S2266 is not a specific substrate for kallikrein activity in mouse submandibular gland cytosol.     

Studies on rat renal cortical cell kallikrein. I. Separation and measurement.

A technique has been developed to separate and measure kallikrein in a heterogeneous population of rat renal cortical cells in suspension. After rat kidneys were perfused in situ in anaesthetized rats, viable, counted cortical cell suspensions were obtained. Cells were suspended in a sucrose/Tris buffer containing 0.5% deoxycholate, homogenized, centrifuged, dialyzed, and gel filtered on Sephadex G-25. Column chromatography on DEAE-cellulose resulted in a single peak of esterase activity between 0.20 to 0.25 M NaCl/sodium phosphate buffer. Subsequent elution yielded an alkaline esterase which was identical to kallikrein isolated from rat urine, insofar as pH optimum, effects of inhibitors, bioassay activity and immunological properties were concerned. Calculated yields were about 70% of the total esterase activity present in the parent cell homogenates. Recoveries of a purified rat urinary kallikrein added to the cell homogenates, the DEAE-cellulose columns, or the eluates from the columns ranged from 83-108% (mean 96%). Using this technique, it was found that the amount of kallikrein activity present in non-incubated renal cortical cells ranged from 0.6-10(-2) to 4.6 - 10(-2) alpha-N-tosyl-L-arginine methyl ester (Tos-Arg-OMe) esterase units per 10(8) cells. However, cells incubated in a nutrient medium at 37 degrees C for 3-8 h contained no measurable kallikrein activity, whereas the surrounding medium had kallikrein activity which could be significantly increased by aldosterone and decreased by spironolactone.     

Purification and chemical studies on rat urinary kallikrein.

A highly purified kallikrein was obtained from rat urine by chromatography on DE-32 cellulose, affinity chromatography on Bio-gel P-200-Aprotinin and gel filtration over Sephadex G-100 coarse and superfine. A molecular weight of 32,000 by sodium dodecyl sulfate polyacrylamide disc gel electrophoresis was estimated. The aminoacid composition and the esterase activity of the purified material were determined. Biological characterization of the purified kallikrein was tested by liberation of a kinin from rat plasma kininogen, by direct action on the isolated rat uterus and by the lowering of rat arterial pressure after intravenous injection of the enzyme. The preparation of insoluble derivative of Aprotinin is described herein. The polymer used as insoluble support (Bio-gel P-200) was before changed to its corresponding azide, which reacts with Aprotinin; the product maintained the binding property of the Aprotinin with urinary kallikrein.     

A novel lipolytic enzyme located in the outer membrane of Pseudomonas aeruginosa

A lipase-negative deletion mutant of Pseudomonas aeruginosa PAO1 still showed extracellular lipolytic activity toward short-chain p-nitrophenylesters. By screening a genomic DNA library of P. aeruginosa PAO1, an esterase gene, estA, was identified, cloned, and sequenced, revealing an open reading frame of 1,941 bp. The product of estA is a 69.5-kDa protein, which is probably processed by removal of an N-terminal signal peptide to yield a 67-kDa mature protein. A molecular mass of 66 kDa was determined for (35)S-labeled EstA by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The amino acid sequence of EstA indicated that the esterase is a member of a novel GDSL family of lipolytic enzymes. The estA gene showed high similarity to an open reading frame of unknown function located in the trpE-trpG region of P. putida and to a gene encoding an outer membrane esterase of Salmonella typhimurium. Amino acid sequence alignments led us to predict that this esterase is an autotransporter protein which possesses a carboxy-terminal beta-barrel domain, allowing the secretion of the amino-terminal passenger domain harboring the catalytic activity. Expression of estA in P. aeruginosa and Escherichia coli and subsequent cell fractionation revealed that the enzyme was associated with the cellular membranes. Trypsin treatment of whole cells released a significant amount of esterase, indicating that the enzyme was located in the outer membrane with the catalytic domain exposed to the surface. To our knowledge, this esterase is unique in that it exemplifies in P. aeruginosa (i) the first enzyme identified in the outer membrane and (ii) the first example of a type IV secretion mechanism.     

Concentrations of glandular kallikrein in human nasal secretions increase during experimentally induced allergic rhinitis

We have previously demonstrated that a mixture of bradykinin and lysylbradykinin is generated in nasal secretions during the immediate allergic response to allergen. The present studies were performed to determine whether glandular kallikrein plays a role in kinin formation during the allergic reaction. Allergic individuals (n = 7) and nonallergic controls (n = 7) were challenged intranasally with appropriate allergen, and nasal lavages obtained before and after challenge were assayed for immunoreactive glandular kallikrein as well as for histamine, kinins, and N-alpha-tosyl-L-arginine methyl esterase (TAME-esterase) activity. The increase in postchallenge immunoreactive glandular kallikrein levels above baseline was significantly greater (p less than 0.01) for the allergic group (16.3 +/- 14 ng/ml; means +/- SD) than for the nonallergic controls (1.0 +/- 1.9 ng/ml). Increased levels of immunoreactive glandular kallikrein correlated with increases in kinins, histamine, and TAME-esterase activity and with the onset of clinical symptoms. Characterization of immunoreactive glandular kallikrein purified from postchallenge lavages by immunoaffinity chromatography confirmed the identity of this material as an authentic glandular kallikrein on the basis of its inhibition by protease inhibitors and by monospecific antibody to tissue kallikrein, its chromatographic behavior on gel filtration, and its ability to generate lysylbradykinin from highly purified human low m.w. kininogen. The specific activity of this purified material, in terms of kinin generation from kininogen, was very similar to that for authentic glandular kallikrein, suggesting that most if not all of the immunoreactive material purified from nasal lavages represented active enzyme. Inhibition studies by using pooled postchallenge lavages suggest that the majority of the kinin generating activity in these samples was due to glandular kallikrein. We conclude, therefore, that glandular kallikrein is secreted during the allergic response and can contribute to the formation of the lysylbradykinin produced during the allergic reaction.     

Purification and some physico-chemical and enzymatic properties of tissue kallikrein from human urine

A procedure for obtaining tissue kallikrein (EC 3.4.21.35) from large specimens of human urea (100 l) has been developed. The isolation procedure included primary extraction of the protein with chitosan (a crustacean chitin deacylated by alkaline treatment), desorption from chitosan with 1 M NH3, affinity chromatography on contrical-Sepharose, ion-exchange chromatography on DEAE-Sepharose and gel filtration on Sephadex G-100. This method permits to obtain tissue kallikrein preparations purified 1080-fold (with respect to AcPheArg-OEt esterase) and 1360-fold (with respect to kininogenase) with 33 and 40% yields, respectively. Tissue kallikrein preparations were homogeneous as could be judged from the results of electrophoresis performed in 12% PAAG in the presence of 0.1% SDS as well as from the presence of one N-terminal amino acid identified as isoleucine. Purified tissue kallikrein had specific activities of 133 mumol/min/mg protein (with respect to AcPheArg-OEt hydrolysis) and 8.8 mumol/min/mg protein (with respect to D-Val-Leu-Arg-pNa hydrolysis) and liberated 462 micrograms equiv. of bradykinin/min/mg protein from heated human blood plasma used as a kininogen source. The protein exhibited the highest stability at pH 8.0-9.0; the pH optimum is at pH 8.0 with AcPheArg-OMe as substrate. The enzyme revealed a high thermostability and was fully inactivated only after 1-hour heating in a boiling water bath. The identity of the urine enzyme to tissue kallikrein could be confirmed by the resistance of the enzyme activity to SIT, high sensitivity to the inhibiting effect of aprotinin (Ki = 0.94 x 10(-10) M) and by an exceedingly low value of the second order inhibition constant for DPP (4.6 M-1 min-1). The fact that this value differs drastically from that for human blood plasma kallikrein (EC 3.4.21.34) which is equal to 360 M-1 min-1 points to marked differences in the structure of the active centers of the both kallikreins as well as to the uniqueness of the tissue kallikrein active center.     

Immunohistochemical localization of rat submandibular gland esterase B (homologous to the RSKG-7 kallikrein gene) in relation to other serine proteases of the kallikrein family.

The rat submandibular gland contains several members of the kallikrein family. In the present study we purified and raised an antiserum against one of these enzymes, i.e., esterase B, which was first described by Khullar et al. in 1986. N-terminal amino acid analysis revealed complete homology between esterase B and the kallikrein family gene RSKG-7. For characterization of the antiserum, flat-bed isoelectrofocusing with immunoblotting was superior to immunoelectrophoresis and double immunodiffusion in detecting and identifying crossreacting proteins. This was due to the fact that kallikrein-like enzymes were readily separated by isoelectrofocusing, and immunoreactivity was easily detected by the sensitive peroxidase-anti-peroxidase staining after blotting onto nitrocellulose membrane. Immunohistochemical controls were carried out accordingly, including homologous as well as crossreacting antigens. In the submandibular gland, esterase B was detected exclusively in all granular convoluted tubular cells, co-localized with tissue kallikrein and tonin. Some staining was also observed in striated duct cells; however, this staining reaction was induced by cross-reactivity with kallikrein, since staining was abolished by addition of kallikrein as well as esterase B to the primary antiserum. It was therefore concluded that like tonin and antigen gamma, but unlike kallikrein, esterase B was not detected in the striated ducts of the submandibular, parotid, or sublingual glands. This separation in anatomic distribution between esterase B and kallikrein may indicate that prokallikrein activation is not the only biological function of esterase B.     

Enzyme- and immuno-histochemical localization of kallikrein. I. The human parotid gland

Localization of kallikrein in the human parotid gland was investigated simultaneously by two markers: kallikrein-like (enzyme) activity and kallikrein antigenicity. Kallikrein-like activity was histochemically demonstrated by using a synthetic substrate, pro-phe-arg- naphthylester . Kallikrein antigenicity was demonstrated by an unlabelled antibody peroxidase-antiperoxidase method, where monospecific antiserum against highly purified urinary kallikrein was used as the primary antiserum. The results showed that kallikrein-like activity and kallikrein antigenicity were identical in their locations in the ductal cells, being localized in the luminal part of the striated ducts and to a lesser degree in the excretory ducts. This indicates the presence of active kallikrein in these regions. No enzyme activity nor antigenicity was observed either in acini or in intercalated ducts. Moreover, the peroxidase-antiperoxidase method revealed kallikrein antigenicity for the first time extracellularly in the basement-membrane region of acini and of ducts as well as in the interstitium surrounding ducts and major vessels.     

Coupled oxidation of carotene by lipoxygenase requires two isoenzymes.

The esterase activity of rat urinary kallikrein is increased up to fourfold by the anionic detergent, deoxycholate and the nonionic detergents, Triton X-100, Lubrol PX, and Brij 58. The cationic detergents, benzyltriphenylphosphonium chloride and cetyltri-methylammonium bromide, inhibit kallikrein activity. Certain trypsin inhibitors stimulate kallikrein activity but this stimulation is not observed when kallikrein is preincubated with deoxycholate. In addition, deoxycholate weakens the inhibition of kallikrein activity by Trasylol. Deoxycholate-induced conformational changes of kallikrein are noted by a change in circular dichroism spectra in the far and near ultraviolet region. A maximal change of ellipticity at 275 nm suggests binding of deoxycholate to kallikrein at or around the tyrosine residue(s) or changes in the microenvironment of these residue(s).     

Studies on rat renal cortical cell kallikrein I. Separation and measurement

A technique has been developed to separate and measure kallikrein in a heterogeneous population of rat renal cortical cells in suspension. After rat kidneys were perfused in situ in anaesthetized rats, viable, counted cortical cell suspensions were obtained.Cells were suspended in a sucrose/Tris buffer containing 0.5% deoxycholate, homogenized, centrifuged, dialyzed, and gel filtered on Sephadex G-25. Column chromatography on DEAE-cellulose resulted in a single peak of esterase activity between 0.20 to 0.25 M NaCl/sodium phosphate buffer. Subsequent elution yielded an alkaline esterase which was identical to kallikrein isolated from rat urine, insofar as pH optimum, effects of inhibitors, bioassay activity and immunological properties were concerned. Calculated yields were about 70% of the total esterase activity present in the parent cell homogenates. Recoveries of a purified rat urinary kallikrein added to the cell homogenates, the DEAE-cellulose columns, or the eluates from the columns ranged from 83–108% (mean 96%). Using this technique, it was found that the amount of kallikrein activity present in non-incubated renal cortical cells ranged from 0.6 · 10⁻² to 4.6 · 10⁻²α-N-tosyl-l-arginine methyl ester (Tos-Arg-OMe) esterase units per 10⁸ cells. However, cells incubated in a nutrient medium at 37°C for 3–8 h contained no measurable kallikrein activity, whereas the surounding medium had kallikrein activity which could be significanyly decreased by aldosterone and decreased by spironolactone.