Jack V. Pierce

The nature of the kallidins released from human plasma by kallikreins and other enzymes.

The kallikreins are hypotensive enzymes of endogenous origin whose injection intravenously in very minute quantities causes a rapid lowering of the blood pressure of the dog. Urinary, pancreatic and serum kallikreins were originally thought to be one substance. Since the pharmacologically active material was found in large amounts in the pancreas, Kraut, Frey and Werlel in 1930 named it kallikrein from the Greek word for pancreas, kallikreas. However, more recent e v i d e n ~ e ~ ~ , has made it clear that kallikreins derived from different sources or different species vary in their response to proteolytic inhibitors or to antibody (TABLE 1).

Hageman factor fragment inhibitor in corn seeds: Purification and characterization

Abstract A specific, potent inhibitor of Hageman factor fragment (HFf) was isolated from sweet corn kernels. Three variants with pIs of 5.1, 6.3, and 7.7, and Mr 11,000–14,000 were found. The corn HFf inhibitor (CHFI) forms a 1:1 molar complex with HFf or trypsin, and prolongs the activated partial thromboplastin time of human plasma. The Ki of CHFI 6.3 with HFf was 2.4 × 10-8 M. CHFI does not inhibit human plasma or urinary kallikreins, plasmin, α-thrombin, hog pancreatic kallikrein, bovine Factor Xa or α-chymotrypsin. CHFI appears to be identical with the corn trypsin inhibitor (CTI) previously reported (Swartz et al., J. Biol. Chem. 252 , 8105, 1977). The corn inhibitor is unique in its strong inhibition of HFf and very narrow spectrum of inhibition of other serine proteinases.

Human plasma kallidins: Isolation and chemical studies

Abstract The action of the kallikreins on plasma to form a hypotensive and smooth muscle stimulating peptide, substance DK, was first reported by Werle and coworkers ( Werle, Gőtze and Keppler, 1937 ; Werle and Grunz, 1939 ). Subsequent studies ( Werle and Hambuechen, 1943 ; Werle and Berek, 1950 ; Werle, 1955 ) showed that the kallikreins act enzymatically on a plasma α 2 -globulin, called kallidinogen, to release the peptide, renamed kallidin (Werle and Berek, 1948) . Simultaneously with these investigations, Rocha e Silva, Beraldo and Rosenfeld (1949) reported that trypsin and some snake venoms are capable of releasing from plasma a hypotensive and smooth muscle stimulating peptide which they called bradykinin. Bradykinin formed by the action of trypsin on a bovine plasma fraction (Elliott, Horton and Lewis, 1961) has been shown to be the nonapeptide H.Arg.Pro.Pro.Gly.Phe.Ser.Pro.Phe.Arg.OH by degradation (Elliott, Lewis and Horton, 1960) and by synthesis (Boissonnas, Guttmann and Jaquenoud, 1960) . Crude preparations of bradykinin and kallidin were found to be chemically and pharmacologically indistinguishable by several investigators ( Werle, kehl and Koebke, 1950 ; Goldstock, Mathias and Schachter, 1957 ; Mathias and Schachter 1958; Gaddum and Horton, 1959 ; Schachter, 1960 ). Similar results were obtained by Van Arman and Miller (1961) , with the notable exception that two bradykinins and probably two kallidins could be detected. The present report describes the isolation of two kallidins from the incubation of human urinary kallikrein with acid-treated human plasma.

Rat submandibular gland kallikreins: purification and cellular localization.

1 Four submandibular gland kallikreins (E.C. 3.4.21.8) were isolated by chromatography on DEAE‐Sephadex A‐50 and hydroxyapatite, followed by gel filtration and electrofocusing. The pI values were 3.87, 3.96, 4.07 and 4.16, and a common molecular weight of 34,000 was found. 2 The kallikreins were localized by direct immunofluorescence with an antibody to rat urinary kallikrein, to the granular tubules, striated duct cells and some main duct cells in the submandibular gland, and to striated duct cells in the sublingual gland. Kallikrein was not found in acini and stroma. 3 Several non‐kallikrein esterases present in the submandibular gland reacted with the antibody to rat urinary kallikrein. The antibody was made monospecific for kallikrein by absorption with the crossreacting esterases. 4 We suggest that kallikrein is produced in striated duct cells. Granular tubules, which are differentiated from striated duct cells, have preserved the ability to produce kallikrein. These cells also store large quantities of kallikrein.

A sensitive isotopic procedure eor the assay oe esterase activity: measurement oe human urinary kaeeikrein

Abstract A sensitive assay has been developed for measurement of the esterase activity of kallikrein using p-tosyl- l -arginine-[ 3 H]methyl ester ([ 3 H]TAMe) as substrate. [ 3 H]TAMe is hydrolyzed to yield [ 3 H]methanol which is separated from unhydrolyzed [ 3 H]TAMe by passage of the incubation mixture over a column of carboxylic acid ion exchange resin, which retains the [ 3 H]TAMe while the [ 3 H]methanol passes through and is measured in the effluent. The optimal conditions for the assay and its application to measurement of kallikrein esterase activity in normal human urine are described.

Action of the Kallikreins on Synthetic Ester Substrates

Summary Evidence is presented which suggests that the kallikreins derived from hog pancreas, human pancreas, human urine and human plasma are capable of digesting those synthetic esters which contain arginine as the specific amino acid residue. Highly purified hog pancreatic kallikrein also digests acetyl-L-tyrosine ethyl ester. Arginine esters and benzoyl-L-arginine amide prevented both inhibition of human urinary and pancreatic kallikreins by human plasma and inhibition of human plasma kallikrein by diisopropyl-fluorophosphate. Acetone activates not only plasma kallikrein but another proteolytic enzyme(s) which attack arginine esters and are not inhibited by SBTI.

Characterization and origin of immunoreactive glandular kallikrein in rat plasma

Abstract A radioimmunoassay (RIA) for rat urinary kallikrein (RUK), capable of detecting 200 pg of antigen, has been developed and used to help characterize the nature and origin of immunoreactive glandular kallikrein in rat plasma. Although rat plasma did not cross-react in a parallel manner in the RIA, a highly significant (P M r , 29,500) on sodium dodecylsulfate-polyacrylamide gel electrophoresis than that of RUK (32,000). Hence, glandular kallikrein circulates in rat plasma in an inactive form. The submandibular and sublingual glands appear to be a major source of the plasma antigen, and the kidney plays an important role in the clearance and/or metabolism of glandular kallikrein from plasma.

Purification of dihydrofolic reductase from chicken liver by affinity chromatography.

Summary The procedure for coupling methotrexate (4-amino-10-methylpteroylglutamic acid) to Sepharose via a six carbon chain is described. An affinity column prepared from this material quantitatively adsorbs the dihydrofolic reductase activity from a partially purified extract of chicken liver. Elution of the enzyme readily occurs with dilute K 2 HPO 4 in the presence of dihydrofolate. Approximately 250-fold purification occurs by affinity chromatography yielding a preparation which appears to be homogeneous.

The Kallikrein-Kinin System in the Kidney

To understand the role of the kallikrein-kinin system in the kidney all components of the system and their localization need to be considered. About half the kallikrein in urine occurs as the proenzyme which arises in the distal tubule. Kinins are formed in the distal tubule and collecting duct from urokinnogen which is found throughout the tubule. Urine contains about twice as much lysyl-brandykinin as bradykinin. A third kinin, methionyl-lysyl-bradykinin, also can occur in urine. It is probably produced by uropepsin as the kinin is largely formed in acidified urine and its formation is inhibited by pepstatin. The significance of the three kinins is unknown. Kinins are normally slowly (few hours) destroyed in urine. The importance of kallikrein, urokinogen and kininases in regulating the level of kinins needs to be determined.

Enzymatic properties of human hageman factor fragment with plasma prekallikrein and synthetic substrates

Hageman factor fragment (HFf) was isolated from acetone-treated human plasma. The Km and kcat of HFf were determined with a natural substrate, plasma prekallikrein, and 34 synthetic amides and esters. The highest “catalytic efficiency”, kcat/Km, was found with prekallikrein (2,980 mM−1sec−1). Of the synthetic substrates D-Pro-Phe-Arg-pNA, Ac-Phe-Arg-OEt, Cbz-Arg-OMe, and Bz-Ile-Glu-Gly-Arg-pNA had the four largest kcat/Km values (130–670 mM−1s−1).