Human tissue kallikrein. I. Isolation and characterization of human pancreatic kallikrein from duodenal juice

Human pancreatic kallikrein was purified from duodenal juice by ion exchange chromatography on DEAE-Sepharose and immunoaffinity chromatography. Thus, an enzyme preparation with a specific activity (using Ac-Phe-Arg-OEt as substrate) of 1 000 U/mg protein was obtained. A specific biological activity of 1310 KE/mg protein was measured in the dog blood pressure assay and of 0.361 HMW kininogen-U/mg, corresponding to the liberation of 383 micrograms bradykinin-equivalents per mg enzyme per min from HMW kininogen in the rat uterus assay. In dodecyl sulfate gel electrophoresis one protein band corresponding to a molecular mass of 27 kDa was obtained. Using gel filtration on Ultrogel AcA-44 a molecular mass of 40 kDa was measured. The amino-acid composition was determined and isoleucine and alanine were identified as the only N-terminal amino-acid residues. On isoelectric focusing four protein bands with isoelectric points of 5.60, 5.65, 5.70 and 5.85 were separated. The bimolecular velocity constant for the inhibition by diisopropyl fluoro phosphate was determined as 10.5 l x mol-1 x min-1. The dissociation constant Ki of the human pancreatic kallikrein-aprotinin complex was calculated to be 1.5 x 10(-10)M. The kinetic constants for the kallikrein-catalysed hydrolysis of Ac-Phe-Arg-OEt and D Val-Leu-Arg-Nan were determined. Immunological studies showed a close relationship between the human pancreatic kallikrein and other human tissue kallikreins, especially with human urinary kallikrein. Detergents such as Triton X-100, Tween 20 and lysolecithin, as well as human serum albumin, activated the human pancreatic kallikrein preparation.     

Characterization of pancreatic lipase-related protein 2 isolated from human pancreatic juice

Human pancreatic lipase-related protein 2 (HPLRP2) was identified for the first time in pancreatic juice using specific anti-peptide antibodies and purified to homogeneity. Antibodies were raised in the rabbit using a synthetic peptide from the HPLRP2 protein sequence deduced from cDNA. Western blotting analysis showed that these antibodies did not react with classical human pancreatic lipase (HPL) or human pancreatic lipase-related protein 1 (HPLRP1) but cross-reacted with native rat PLRP2 (RPLRP2), as well as with recombinant rat and guinea-pig PLRP2 (GPLRP2). Immunoaffinity chromatography was performed on immobilized anti-recombinant HPLRP2 polyclonal antibodies to purify native HPLRP2 after conventional chromatographic steps including gel filtration and chromatrography on an anion-exchanger. The substrate specificity of HPLRP2 was investigated using various triglycerides, phospholipids and galactolipids as substrates. The lipase activity on triglycerides was inhibited by bile salts and weakly restored by colipase. The phospholipase activity of HPLRP2 on phospholipid micelles was very low. A significant level of galactolipase activity was measured using monogalactosyldiglyceride monomolecular films. These data suggest that the main physiological function of HPLRP2 is the hydrolysis of galactolipids, which are the main lipids present in vegetable food.     

Characterization of pig pancreatic kallikreins A and B.

Pig pancreatic kallikreins A and B are both composed of the same 229 amino acids, a figure resembling the number of amino acid residues found in other serine proteinases of pancreas. Both forms of the enzyme contain N-terminal isoleucine and alanine and C-terminal leucine/serine (about half a mol each per mol kallikrein) and proline. Values for the glucosamine content of the kallikreins obtained on the amino acid analyzer after hydrolysis with p-toluenesulfonic acid, a procedure also used for the determination of amide ammonia, agreed with those determined by a gas-chromatographic method. Neuraminidasetreated kallikrein B differs from the A form only in containing roughly double the amount (on the average a total of 11.5 vs. 5.6% by weight) of carbohydrate (glucosamine, mannose, galactose, and fucose) and possibly by a higher content (20 vs. 17 residues) of amide ammonia. From the composition, molecular weights of 26800 and 28600 are calculated for sialic-acid-free kallikreins A and B, respectively, and of 25300 for the protein part of kallikrein. The molar absorbance of both forms of the enzyme has been determined as (50.6 +/- 1.3) X 10(3)M-1 cm-1 at 280 nm. A comparison of kallikreins A and B with kallikreins d1 and d2 described by Zuber and Sache reveals as principal difference a much lower specific activity of the latter preparations with all reagents tested. Conceivably, the reported lower carbohydrate contents of kallikreins d1 and d2 and their separation into three instead of two major subunits are related to this finding.     

Characterization in rat pancreatic juice of a protein homologous to the human pancreatic stone protein

1. The pancreatic stone protein (PSP, Mr 15,000) which has been discovered in human calculi derives from the native glycosylated forms of the protein (Mrs 17,500-22,000) which are present in human pancreatic juice through tryptic cleavage of the Arg 11-Ile 12 bond. 2. In the present study, a homologous native form of the protein (Mr 17,000) was purified from rat pancreatic juice. 3. Its N-terminal amino acid sequence was found to display a high degree of homology with that of the human native protein forms, apart from the fact that it was not glycosylated. 4. In rat as in human, tryptic cleavage of the Arg 11-Ile 12 bond transforms a soluble protein into one which is practically insoluble at neutral pH.     

Characterization of the human kallikrein locus.

The human kallikrein gene family is composed of three members: tissue kallikrein (KLK1), prostate-specific antigen (PA or APS), and human glandular kallikrein-1 (hGK-1 or KLK2). The three genes have previously been isolated and mapped to chromosome 19q13.2-q13.4. Further analysis of an area of 110 kb surrounding the kallikrein genes by CHEF electrophoresis and chromosome walking showed clustering of the three genes. The KLK1 gene is positioned in the opposite orientation of the APS and KLK2 genes in the order KLK1-APS-KLK2. The APS and KLK2 gene are separated by 12 kb; the distance between KLK1 and APS is 31 kb. A CpG island was detected in the region between KLK1 and APS. Preliminary data indicate that this CpG island is located directly adjacent to a gene that is unrelated to the kallikreins and seems to be ubiquitously expressed.     

The immunological similarity of rat glandular kallikreins.

The immunological properties of the submandibular kallikrein, urinary kallikrein, pancreatic kallikrein and pancreatic prekallikrein of the rat were studied by immunodiffusion, immunoelectrophoresis and radioimmunoassay. Although they behaved differently electrophoretically, all the antigens showed identical immunological behaviour. The implications of this are discussed.     

Purification of human lysozyme from milk and pancreatic juice

Human milk lysozyme was purified by heparin-Sepharose affinity chromatography and Sepharose 4B gel-permeation chromatography. This procedure was also found applicable to the purification of human pancreatic juice lysozyme. Double-diffusion analyses indicated that human milk lysozyme was immunochemically identical to human saliva and human pancreatic juice lysozyme. Based on the identity of the N-terminal 10-amino-acid-residue sequence analyzed, it was suggested that human milk lysozyme and human pancreatic juice lysozyme are identical molecular entities.     

alpha-Amylase from human pancreatic juice as an electrophoretically pure isozyme.

Human pancreatic juice collected antiseptically and strictly free from other body fluids and juices, was examined for the isozyme composition of alpha-amylase. Unlike those of the extract of and exudate from pancreatic tissue, the composition of alpha-amylase isozyme in the secreted juice was simple and consisted of only two isozymes, a major one and a minor one, and the reason for difference in the isozyme composition is discussed. The major alpha-amylase isozyme was obtained in a crystalline state with a molecular weight of 5.4-5.5 X 10(4).     

Similarity between a kininogenase (kallikrein) from human large intestine and human urinary kallikrein.

A human colon kininogenase (kallikrein) was isolated by gel filtration on Sephacryl S-200 and affinity chromatography on Trasylolbound Sepharose, yielding a material with a specific activity of 1.3 U/mg (substrate: AcPheArgOEt). The molecular weight of the enzyme as estimated by gel filtration is approximately 70 000. After reduction with mercaptoethanol two bands were obtained in dodecyl sulfate eletrophoresis with molecular weights of 27 000 and 70 000. The bimolecular velocity constant for the inhibition by diisopropyl fluorophosphate was determined as 4 l x mol-1 x min-1. The preparation was characterized by immunological and enzymatic methods. Using the radioimmumoassay for human urinary kallikrein cross-reactivity and parallel binding curves were obtained. Kinin liberation from human high Mr-kininogen was totally inhibited by antibodies directed against human urinary kallikrein. Trasylol and diisopropyl fluorophosphate, but not by antibodies directed against human trypsin and plasma kallikrein. The effect on dog blood pressure was comparable to that obtained with human urinary kallikrein. The amino acid composition of human large intestine kallikrein is very similar to that of human urinary kallikrein.     

Immunohistochemical localization of human kallikreins 6 and 10 in pancreatic islets

Tissue kallikreins are thought to be present in the pancreatic islets of Langerhans and to aid in the conversion of proinsulin to insulin. In recent immunohistochemical studies, we observed strong staining of the newly identified human kallikreins 6 and 10 (hK6 and hK10) in the islets of Langerhans. Here, we examine hK6 and hK10 immunoexpression in different types of islet cells of the endocrine pancreas, in order to obtain clues for hK6 and hK10 function in these cells. Ten cases of normal pancreatic tissue, two cases of nesidioblastosis, five insulin-producing tumours and one case of multiple endocrine neoplasia 1 syndrome, containing an insulin-, a somatostatin- and several glucagon-producing tumours, as well as tiny foci of endocrine dysplasia with different predominance of the secreted hormones (mainly glucagon and pancreatic polypeptide) were included in the study. A streptavidin–biotin–peroxidase and an alkaline phosphatase protocol, as well as a sequential immunoenzymatic double staining method were performed, using specific antibodies against hK6, hK10, insulin, glucagon, somatostatin, pancreatic polypeptide, and serotonin. hK6 and hK10 immunoexpression was observed in the islets of Langerhans, including the pancreatic polypeptide-rich islets, in the normal pancreas. Scattered hK6 and hK10 positive cells were localized in relationship with pancreatic acinar cells. In the exocrine pancreas, a cytoplasmic and/or brush border hK6 and hK10 immunoexpression was observed in the median and small sized pancreatic ducts, while the acinar cells were negative. Foci of nesidioblastosis and endocrine dysplasia expressed both kallikreins. hK6 and hK10 were also strongly and diffusely expressed throughout all insulin-, glucagon- and somatostatin-producing tumours. The double staining method revealed co-localization of each hormone and hK6/hK10 respectively, in the same cellular population, in the normal as well as in the diseased pancreas. Our results support the view that hK6 and hK10 may be involved in insulin and other pancreatic hormone processing and/or secretion, as well as in physiological functions related to the endocrine pancreas.     

Effect of covalent binding of aprotinin with a polysaccharide carrier on its inhibition of kallikrein from human plasma, porcine pancreatic kallikrein and trypsin

The inhibition of trypsin, human blood plasma kallikrein and porcine pancreatic kallikrein by aprotinin (native and immobilized on carboxymethyl ester of dextran) was investigated. The experimental values of Ki of native and immobilized aprotinin--enzyme complexes are equal to 0.037 and 0.045 nM for trypsin, 0.38 and 112.3 nM for pancreatic kallikrein and 34.4 and 454.5 nM for plasma kallikrein with N alpha-benzoyl-L-arginine ethyl ester as substrate, and to 82.6 and 231.7 nM for plasma kallikrein with a natural substrate--kininogen. These data suggest that covalent binding of aprotinin to the water-soluble polysaccharide carrier does not interfere with its interaction with trypsin, whereas the inhibition of kallikreins decreases, especially that of pancreatic kallikrein. The experimental results indicate the marked differences in the structure of the binding site of the active center (or its environment) of plasma and pancreatic kallikreins, on one hand, and trypsin, on the other, as well as the differences between the plasma and pancreatic kallikreins. A high requirement of kallikreins to the maintenance of the native conformation of aprotinin during immobilization is postulated.     

Characterization and partial purification of acid lipase from human leucocytes.

Hydrolysis of glycerol trioleate by human leucocytes was characterized and the enzymes responsible for this activity were obtained in a purified form by means of gel chromatography on Sephadex G-100 as well as by zonal ultracentrifugation followed by gel chromatography. The activity is localized in the granule fraction of leucocytes (15 000 X g, 20 min) and shows a sharp pH optimum at pH 5.25. As judged from the elution profile obtained by gel chromatography, two proteins are likely to contribute to the hydrolysis of glycerol trioleate. The approximate molecular weights of the two enzymes are 74 100 and 60 300, respectively. The activity is reduced in the presence of NaCl, KCl, CaCl2 as well as of p-hydroxymercuribenzoate. The enzymes are stable at -25 degrees C but loose about 50% of their activity within 48 h at 4 degrees C.     

The kinetics of hydrolysis of some extended N-aminoacyl-L-arginine methyl esters by porcine pancreatic kallikrein. A comparison with human plasma Kallikrein.

The effects of subsite interactions in the S2-S4 region [Schechter & Berger (1967) Biochem. Biophys. Res. Commun. 27, 157-162] of porcine pancreatic kallikrein (EC 3.4.21.8) on its catalytic efficiency have been investigated. Kinetic constants (Kcat, Km) have been determined for a series of seven extended N-aminoacyl-L-arginine methyl esters whose sequence is based on either the C-terminal sequence of kallidin (-Pro-Phe-Arg) or (-Gly-)nArg. With these substrates it has been found that neither acylation nor deacylation of the enzyme is rate-limiting. Values of Kcat. range from 21.5 to 2320s-1, indicating that there are interactions with different residues in the N-aminoacyl chain and enzyme subsites in the S2-S4 region. It is shown that possible hydrogen-bonded interactions with the enzyme in the S3-S4 region have a significant effect on catalysis. The presence of L-phenylalanine at P2 has a very large effect on both Kcat, and Km, giving a greatly enhanced catalytic efficiency. Substrates with L-proline at P3 also have a marked effect, but in this case the overall effect is one of lowered catalytic efficiency. By comparison with the results of a similar study with human plasma kallikrein I (EC 3.4.21.8), it has been possible to demonstrate that there are considerable differences in kinetic behaviour between the two enzymes. These are related to relative differences in the rates of acylation and deacylation with ester substrates and also the roles of subsites S2 and S3 of the two enzymes.     

Characterization of a high-molecular-weight form of human acrosin. Comparison with human pancreatic trypsin.

A high-molecular-weight form of acrosin (alpha-acrosin, EC 3.4.21.10) was extracted from spermatozoa obtained from frozen semen and purified over 300-fold. Purification was effected by sequential use of Sephadex G-150, CM-cellulose and DEAE-cellulose chromatography. Properties of human acrosin were compared with those of human pancreatic trypsin. The molecular weight (Mr) of acrosin (70000) was greater than that of trypsin (Mr 21000). Isoelectric points for acrosin (pI = 9.0) and trypsin (pI = 8.2) were also different. alpha-N-Benzoyl-L-arginine ethyl ester was hydrolysed 50% more rapidly by acrosin than by trypsin. Acrosin had similar kcat. values for the hydrolysis of esters with different acylating groups (i.e. benzoyl-L-arginine and p-tosyl-L-arginine esters). In contrast, trypsin had dissimilar kcat. values for the hydrolysis of esters with different acylating groups. Kinetic data argue against deacylation as the rate-limiting step in ester hydrolysis by acrosin. Acrosin was less sensitive than trypsin to inhibition by 7-amino-1-chloro-3-L-tosylamidoheptan-2-one ('TLCK'), di-isopropyl fluorophosphate and soya-bean trypsin inhibitor. D-Fructose and D-arabinose inhibited acrosin, but had no effect on trypsin. The data indicate that definite differences exist between human acrosin and trypsin.     

Purification of pancreatic phospholipase A2 from human duodenal juice

Phospholipase A2 (EC 3.1.1.4) was purified from delipidated human duodenal juice by hydrophobic and cation exchange chromatography, followed by molecular sieving on an HPLC column. The resulting enzyme preparation of phospholipase A2 had a molecular weight of 14 kDa, a specific activity of 2000 U/mg protein, and an N-terminal amino acid sequence which was characteristic for human pancreatic phospholipase A2.