Human cytotoxic lymphocyte tryptase. Its purification from granules and the characterization of inhibitor and substrate specificity

A trypsin-like enzyme (tryptase) has been purified to homogeneity from the granules of a human cytolytic lymphocyte (CTL) line, Q31, by a three-step procedure. By including 0.3% (v/v) Triton X-100 and 1 mg/ml heparin in purification buffers, near total yields of tryptase activity were obtained during the purification. The enzyme, referred to as Q31 tryptase, migrated in polyacrylamide gels with sodium dodecyl sulfate at a position corresponding to 28 kDa with and to 45 kDa without 2-mercaptoethanol. It had an amino-terminal sequence identical to a previously reported human CTL tryptase at 20 of 22 positions identified. It hydrolyzed N alpha-carbobenzyloxy-L-lysyl-thiobenzyl ester (BLT), and this BLT esterase activity was most efficient at slightly alkaline pH and was relatively more active near neutral pH than mouse CTL tryptase. Human alpha 1-protease inhibitor, human antithrombin III, phenylmethanesulfonyl fluoride, and p-aminobenzamidine inhibited the Q31 tryptase. The inhibition by human antithrombin III was rapid enough to be of physiological significance. A survey of oligopeptide p-nitroanilides found that the best substrate for human Q31 tryptase is H-D-(epsilon-carbobenzyloxy)Lys-L-Pro-L-Arg-p-nitroanilide. The Q31 tryptase appears to have broad specificity for amino acid residues at P2 and P3, i.e. at 2 and 3 residues amino-terminal to the scissile bond.     

Purification of a membrane-associated serine esterase from murine cytotoxic T lymphocytes by a single reverse-phase column

The plasma and organelle membranes of a cytotoxic T lymphocyte line, CTLL-R8, were isolated by subcellular fractionation. After dissolving in detergent-containing buffer, the membrane proteins were isolated by high-performance liquid chromatography on a single reverse-phase column. The serine esterase activity in the fractions was detected by measuring hydrolysis of the ester compound N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester. A major band was revealed in the fraction with highest serine esterase activity. Under sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this band assumes a molecular weight of about 30 kDa. The amino-terminal sequence of the protein was analyzed and shows 100% identity with that of MCSP-3/granzyme F, a soluble serine esterase previously identified in the cytoplasmic granules of cytotoxic T lymphocytes. Modifications of this reverse-phase column method would thus represent a simple, convenient strategy for obtaining high yields of all the lymphocyte surface proteases, which could then be further characterized for function.     

Human and murine cytotoxic T lymphocyte serine proteases: subsite mapping with peptide thioester substrates and inhibition of enzyme activity and cytolysis by isocoumarins

The active site structures of human Q31 granzyme A, murine granzymes (A, B, C, D, E, and F), and human granzymes (A, B, and 3) isolated from cytotoxic T lymphocytes (CTL) were studied with peptide thioester substrates, peptide chloromethyl ketone, and isocoumarin inhibitors. Human Q31, murine, and human granzyme A hydrolyzed Arg- or Lys-containing thioesters very efficiently with kcat/KM of 10(4)-10(5) M-1 s-1. Murine granzyme B was found to have Asp-ase activity and hydrolyzed Boc-Ala-Ala-Asp-SBzl with a kcat/KM value of 2.3 X 10(5) M-1 s-1. The rate was accelerated 1.4-fold when the 0.05 M NaCl in the assay was replaced with CaCl2. The preparation of granzyme B also had significant activity toward Boc-Ala-Ala-AA-SBzl substrates, where AA was Asn, Met, or Ser [kcat/KM = (4-5) X 10(4) M-1 s-1]. Murine granzymes C, D, and E did not hydrolyze any thioester substrate but contained minor contaminating activity toward Arg- or Lys-containing thioesters. Murine granzyme F had small activity toward Suc-Phe-Leu-Phe-SBzl, along with some contaminating trypsin-like activity. Human Q31 granzyme A, murine, and human granzyme A were inhibited quite efficiently by mechanism-based isocoumarin inhibitors substituted with basic groups (guanidino or isothiureidopropoxy). Although the general serine protease inhibitor 3,4-dichloroisocoumarin (DCI) inactivated these tryptases poorly, it was the best isocoumarin inhibitor for murine granzyme B (kobs/[I] = 3700-4200 M-1 s-1). Murine and human granzyme B were also inhibited by Boc-Ala-Ala-Asp-CH2Cl; however, the inhibition was less potent than that with DCI. DCI, 3-(3-amino-propoxy)-4-chloroisocoumarin, 4-chloro-3-(3-isothiureidopropoxy)isocoumarin, and 7-amino-4-chloro-3-(3-isothiureidopropoxy)isocoumarin inhibited Q31 cytotoxic T lymphocyte mediated lysis of human JY lymphoblasts (ED50 = 0.5-5.0 microM).     

Granzyme M is a regulatory protease that inactivates proteinase inhibitor 9, an endogenous inhibitor of granzyme B

Granzyme M is a trypsin-fold serine protease that is specifically found in the granules of natural killer cells. This enzyme has been implicated recently in the induction of target cell death by cytotoxic lymphocytes, but unlike granzymes A and B, the molecular mechanism of action of granzyme M is unknown. We have characterized the extended substrate specificity of human granzyme M by using purified recombinant enzyme, several positional scanning libraries of coumarin substrates, and a panel of individual p-nitroanilide and coumarin substrates. In contrast to previous studies conducted using thiobenzyl ester substrates (Smyth, M. J., O'Connor, M. D., Trapani, J. A., Kershaw, M. H., and Brinkworth, R. I. (1996) J. Immunol. 156, 4174-4181), a strong preference for leucine at P1 over methionine was demonstrated. The extended substrate specificity was determined to be lysine = norleucine at P4, broad at P3, proline > alanine at P2, and leucine > norleucine > methionine at P1. The enzyme activity was found to be highly dependent on the length and sequence of substrates, indicative of a regulatory function for human granzyme M. Finally, the interaction between granzyme M and the serpins alpha(1)-antichymotrypsin, alpha(1)-proteinase inhibitor, and proteinase inhibitor 9 was characterized by using a candidate-based approach to identify potential endogenous inhibitors. Proteinase inhibitor 9 was effectively hydrolyzed and inactivated by human granzyme M, raising the possibility that this orphan granzyme bypasses proteinase inhibitor 9 inhibition of granzyme B.     

Active site mapping of the serine proteases human leukocyte elastase, cathepsin G, porcine pancreatic elastase, rat mast cell proteases I and II. Bovine chymotrypsin A alpha, and Staphylococcus aureus protease V-8 using tripeptide thiobenzyl ester substrates

The primary subsite specificities of human leukocyte elastase, cathepsin G, porcine pancreatic elastase, rat mast cell proteases I and II, bovine chymotrypsin A alpha, and the protease from strain V-8 of Staphylococcus aureus have been mapped with a series of tripeptide thiobenzyl ester substrates of the general formula Boc-Ala-Ala-AA-SBzl, where AA represents one of 13 amino acids. In addition, the effects of a P2 Pro and P4 methoxysuccinyl and succinyl groups were investigated. In an attempt to introduce specificity and/or reactivity into the substrate Boc-Ala-Ala-Leu-SBzl(X), the 4-chloro-, 4-nitro-, and 4-methoxythiobenzyl ester derivatives were studied. Enzymatic hydrolyses of the substrates were measured in the presence of 4,4'-dithiobis(pyridine) or 5,5'-dithiobis(2-nitrobenzoic acid), which provided a highly sensitive assay method for free thiol. The thio esters were excellent substrates for the enzymes tested, and in many cases, the best substrates reported here have kcat/KM values higher than those reported previously. The best substrate for human leukocyte elastase was Boc-Ala-Pro-Nva-SBzl(Cl), which has a kcat/KM of 130 X 10(6) M-1 s-1. A very reactive rat mast cell protease substrate, Boc-Ala-Ala-Leu-SBzl(NO2), was also found. The S. aureus V-8 protease was the most specific enzyme tested since it hydrolyzed only Boc-Ala-Ala-Glu-SBzl. Substituents on the thiobenzyl ester moiety of Boc-Ala-Ala-Leu-SBzl resulted in decreased KM values with human leukocyte elastase and rat mast cell protease I when compared to the unsubstituted derivative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Generation of catalytically active granzyme K from Escherichia coli inclusion bodies and identification of efficient granzyme K inhibitors in human plasma.

Granzymes are granule-stored lymphocyte serine proteases that are implicated in T- and natural killer cell-mediated cytotoxic defense reactions after target cell recognition. A fifth human granzyme (granzyme 3, lymphocyte tryptase-2), renamed as granzyme K (gene name GZMK), has recently been cloned from lymphocyte tissue. For its further characterization we successfully generated catalytically active enzyme in milligram quantities per liter of Escherichia coli culture. The natural proform of granzyme K with the amino-terminal propeptide Met-Glu was expressed as inclusion bodies and converted to its active enzyme by cathepsin C after refolding of precursor molecules. Recombinant granzyme K cleaves synthetic thiobenzyl ester substrates after Lys and Arg with k(cat)/K(m) values of 3.7 x 10(4) and 4.4 x 10(4) M(-1) s(-1), respectively. Granzyme K activity was shown to be inhibited by the synthetic compounds Phe-Pro-Arg-chloromethyl ketone, phenylmethylsulfonyl fluoride, PefablocSC, and benzamidine, by the Kunitz-type inhibitor aprotinin and by human blood plasma. The plasma-derived inter-alpha-trypsin inhibitor complex, its bikunin subunit, and the second carboxyl-terminal Kunitz-type domain of bikunin were identified as genuine physiologic inhibitors with K(i) values of 64, 50, and 22 nM, respectively. Inter-alpha-trypsin inhibitor and free bikunin have the potential to neutralize extracellular granzyme K activity after T cell degranulation and may thus control unspecific damage of bystander cells at sites of inflammatory reactions.     

Structural relationship between alpha 1-microglobulin from man, guinea-pig, rat and rabbit

Rabbit alpha 1-microglobulin was purified from the urine of sodium-chromate-treated animals by the use of gel chromatography on Sephadex G-100, affinity chromatography on concanavalin-A--Sepharose and ion-exchange chromatography on DEAE-Sephadex. Rabbit alpha 1-microglobulin had a molecular mass of 25.6 kDa on SDS/polyacrylamide gel electrophoresis. Alpha 1-microglobulin has previously been purified from the urine of humans, guinea-pigs and rats by similar methods, and the molecular masses of the four homologues were compared by SDS/polyacrylamide gel electrophoresis and gel chromatography in a denaturing medium. By these two methods the human homologue was 6 kDa and 3 kDa larger, respectively, than the other three proteins. Endoglycosidase F digestion of alpha 1-microglobulin, followed by SDS/polyacrylamide gel electrophoresis, revealed three protein bands in the human alpha 1-microglobulin sample, and only two bands in guinea-pig, rat and rabbit alpha 1-microglobulin, with a gap between each band of 2.6--2.9 kDa. The amino-terminal amino acid sequences of the four homologues were determined and between 72% and 81% homology was seen. The five amino-terminal amino acids present in the other species were missing in guinea-pig alpha 1-microglobulin. Our results indicate that human alpha 1-microglobulin is substituted with two N-linked oligosaccharides, while only one is attached to each of the other alpha 1-microglobulins, and that the extra glycosylamine-linked oligosaccharide in the human protein is attached to asparagine in position 17. Finally it is shown that all four homologues inhibit antigen stimulation of human lymphocytes, a finding which is consistent with our previous suggestion that the N-linked oligosaccharides carry the immunosuppressive activity of alpha 1-microglobulin.     

Molecular weight forms of inhibin a and inhibin B in the bovine testis change with age

To investigate alterations in the molecular weight forms of inhibin in bull testis from the infantile (4-5 wk of age) to postpubertal (49-56 wk of age) periods, testicular homogenates were obtained from animals of various ages and fractionated by a combination of immunoaffinity chromatography and SDS-PAGE. Subsequently, the fractions eluted from the SDS gels were assayed for total inhibin, inhibin A, and inhibin B by fluoroimmunoassay or immunofluorometric assays (IFMAs) and for inhibin bioactivity by an in vitro bioassay. The molecular mass patterns of inhibin A and inhibin B in the testis, as determined by the dimer-specific IFMAs, showed the presence of a peak of approximate 47 kDa until 21-26 wk of age. However, the peak disappeared after 31-32 wk of age. As bulls aged, especially after 31-32 wk of age, inhibin A and inhibin B levels increased in the molecular mass region of 27-34 kDa. Total inhibin showed two peaks, of between 20 and 26 kDa and at approximately 47 kDa, until 21-26 wk of age and a single peak between 20 and 30 kDa after 31-32 wk of age. The eluted fractions corresponding to 29, 31, or 47 kDa gave a dose-response curve that was parallel to the curve generated with 32-kDa inhibin A or 29-kDa inhibin B standard in the IFMA for inhibin A or inhibin B. The fractions corresponding to 29 and 31 kDa suppressed basal release of FSH from rat pituitary cells, but the 47-kDa fraction had a lower FSH-suppressing activity. In the testes of older bulls, immunoblot analysis revealed the presence of a 29-kDa band cross-reacting with inhibin alpha and inhibin betaB antibodies and of a 31-kDa band cross-reacting with inhibin alpha and inhibin betaA antibodies. The 47-kDa band was recognized by the alpha, betaA, and betaB antibodies. Immunohistochemisty of the testis at each age showed that inhibin alpha subunits were found exclusively in Sertoli cells, but the intensity of immunostaining diminished in older bulls, in parallel with the decrease in the testicular concentrations of total inhibin. We conclude that 1) bovine Sertoli cells produce both inhibin A and inhibin B, 2) inhibin production in Sertoli cells during the prepubertal period is characterized by the 47 kDa inhibin-related material that contains precursor forms of inhibin A and inhibin B, and 3) the proportion of the mature forms of inhibin A and inhibin B increases as bulls age, although total inhibin production in Setroli cells decreases.     

The enzymatic activity of human cytotoxic T-lymphocyte granzyme A and cytolysis mediated by cytotoxic T-lymphocytes are potently inhibited by a synthetic antiprotease, FUT-175

The synthetic antiprotease, FUT-175 (6-amidino-2-naphthyl-4-guanidinobenzoate), was found to be an extraordinarily potent and rapid inhibitor of human Q31 cytotoxic T-lymphocyte granzyme A. The granzyme A was inhibited in a time-dependent manner with kobs/i = 430,000 +/- 80,000 M-1 s-1. Four other FUT-175 analogs were also found to be potent, rapid Q31 granzyme A inhibitors. All five compounds inhibited Q31 cytotoxic T-lymphocyte-mediated cytolysis of human JY lymphoma cells, but at concentrations far in excess of those needed for granzyme A inhibition. The data presented suggest that postmarketing surveillance of FUT-175 should include a review of possible immunosuppressive side-effects, such as increased susceptibility to viral infections and to neoplastic transformations.     

Polymorphic serum prealbumin (Pa) of pig, identified as an a!-protease inhibitor

Pig serum proteins were analysed by horizontal polyacrylamide gel electrophoresis, with a discontinuous buffer system (pH 9.0). A 12 % acrylamide concentration in the separation gel was used. Each of the two prealbumin (Pa) alleles gave rise to two closely migrating fractions. The polymorhic Pa was identified as an a,-protease inhibitor as the Pa fractions inhibited the esterolytic activity of both bovine trypsin and chymotrypsin. Therefore, it has been proposed that the locus symbol for this prealbumin be changed to Pi-1. The protease inhibitory spectra and electrophoretic mobility of the Pa (Pi-1) fractions suggested that this protein was probably the same as the pig serum a,-protease inhibitor described in some earlier studies and that it corresponds to human serum a,-protease inhibitor (Pi).     

Filamin (280-kDa actin-binding protein) is a caspase substrate and is also cleaved directly by the cytotoxic T lymphocyte protease granzyme B during apoptosis

We used yeast two-hybrid screening to identify the cytoskeletal protein filamin as a ligand for the proapoptotic protease granzyme B, produced by cytotoxic T lymphocytes. Filamin was directly cleaved by granzyme B when target cells were exposed to granzyme B and the lytic protein perforin, but it was also cleaved in a caspase-dependent manner following the ligation of Fas receptors. A similar pattern of filamin cleavage to polypeptides of approximately 110 and 95 kDa was observed in Jurkat cells killed by either mechanism. However, filamin cleavage in response to granzyme B was not inhibited by the caspase inhibitor z-Val-Ala-Asp-fluoromethylketone at concentrations that abolished DNA fragmentation. Filamin staining was redistributed from the cell membrane into the cytoplasm of Jurkat cells exposed to granzyme B and perforin and following ligation of Fas receptors, coincident with the morphological changes of apoptosis. Filamin-deficient human melanoma cells were significantly (although not completely) protected from granzyme B-mediated death compared with isogenic filamin-expressing cells, both in clonogenic survival and (51)Cr release assays, whereas death from multiple other stimuli was not affected by filamin deficiency. Thus, filamin is a functionally important substrate for granzyme B, as its cleavage may account at least partly for caspase-independent cell death mediated by the granzyme.     

Molecular cloning and sequencing of the cDNA of rat alpha 1-protease inhibitor and its expression in COS-1 cells

A cDNA clone for alpha 1-protease inhibitor (pc alpha 1P1212) was isolated from a lambda ZAP rat liver cDNA library. The 1.4 kb cDNA insert of pc alpha 1P1212 contained an open reading frame that encodes a 411-residue polypeptide (46,125 Da), in which a signal peptide of 24 residues was identified by comparison with the NH2-terminal sequence of the purified protein. Three potential sites for N-linked glycosylation were found in the molecule, accounting for the difference in molecular mass between the predicted form and the purified protein (56 kDa). The deduced primary structure of rat alpha 1-protease inhibitor showed 68.5% homology to that of the human inhibitor. We then constructed the expression plasmid pSV2 alpha 1PI from pSV2-gpt and pc alpha 1P1212, and transfected it into COS-1 cells. The transfected cells synthesized a molecule which was precipitated with anti-(rat alpha 1-protease inhibitor)-IgG and had the same molecular size as that of the inhibitor produced by rat hepatocytes.     

Partial characterization of the 30 kD Ig-binding protein from Pseudomonas maltophilia.

We have previously demonstrated that Pseudomonas maltophilia (ATCC 13637) possess a 30 kDa cell wall protein which binds various subclasses of IgG's and IgA by their Fc region. The protein was solubilized by papain and purified by affinity chromatography on cyanogen bromide activated sepharose beads conjugated with human IgG. The eluent was electrophoresed on a 12% polyacrylamide gel under denaturing conditions, and the immunoactive bands identified by Western blot analysis, a second gel was stained with Coomassie blue. The affinity purified eluent was electrophoresed on a one-dimensional 15% polyacrylamide gel and stained with Coomassie blue. The protein band of interest was cut. The protein band was then digested in situ with Staphylococcus aureus V-8 protease. The peptide bands were separated by electrophoresis on a second one dimensional 15% polyacrylamide gel and then electroblotted into a polyvinylidine difluoride membrane. The bands were visualized by staining with Coomassie blue, cut out, and sequenced using an automated gas phase sequencer. Minimal amino acid composition was determined in a similar fashion. We have thus obtained partial N-terminal amino acid sequence data from the above method.     

15 kDa granulysin causes differentiation of monocytes to dendritic cells but lacks cytotoxic activity

Granulysin is expressed as two isoforms by human cytotoxic cells: a single mRNA gives rise to 15 kDa granulysin, a portion of which is cleaved to a 9 kDa protein. Studies with recombinant 9 kDa granulysin have demonstrated its cytolytic and proinflammatory properties, but much less is known about the biologic function of the 15 kDa isoform. In this study, we show that the subcellular localization and functions of 9 and 15 kDa granulysin are largely distinct. Nine kilodalton granulysin is confined to cytolytic granules that are directionally released following target cell recognition. In contrast, 15 kDa granulysin is located in distinct granules that lack perforin and granzyme B and that are released by activated cytolytic cells. Although recombinant 9 kDa granulysin is cytolytic against a variety of tumors and microbes, recombinant 15 kDa granulysin is not. The 15 kDa isoform is a potent inducer of monocytic differentiation to dendritic cells, but the 9 kDa isoform is not. In vivo, mice expressing granulysin show markedly improved antitumor responses, with increased numbers of activated dendritic cells and cytokine-producing T cells. Thus, the distinct functions of granulysin isoforms have major implications for diagnosis and potential new therapies for human disease.     

Granule-mediated killing by granzyme B and perforin requires a mannose 6-phosphate receptor and is augmented by cell surface heparan sulfate

During granule-mediated killing by cytotoxic T lymphocytes or natural killer cells, the serine protease granzyme B enters the target cell by endocytosis and induces apoptosis. Previous studies suggested a role for the mannose 6-phosphate receptor, but further experiments with purified granzyme B indicated this was not essential. Additionally, it is now clear that grB is exocytosed from killer cells in a high-molecular-weight complex with the proteoglycan serglycin. Here granzyme B was delivered as a purified monomer, or in complex with either glycosaminoglycans or serglycin, and killing was evaluated. When granzyme B was a monomer, soluble mannose 6-phosphate had a limited impact, whereas apoptosis induced by the complexed grB was effectively inhibited by mannose 6-phosphate. Most importantly, when granzyme B and perforin were delivered together from granules, inhibition by mannose 6-phosphate was also observed. In pulldown assays mediated by the cation-independent mannose 6-phosphate receptor, granzyme B bound to the receptor more intensely in the presence of immobilized heparan sulfate. We therefore propose the model that under physiological conditions serglycin-bound granzyme B is critically endocytosed by a mannose 6-phosphate receptor, and receptor binding is enhanced by cell surface heparan sulfate.     

Dependence of granzyme B-mediated cell death on a pathway regulated by Bcl-2 or its viral homolog, BHRF1

The molecular pathways responsible for apoptosis in response to granzyme B have remained unresolved. Here we present data supporting the notion that granzyme B-mediated cell death is largely dependent on a pathway that is inhibitable by Bcl-2 or its viral analog BHRF1. We used a panel of stably transfected FDC-P1 mouse myeloid cell lines to show that overexpression of functional, wild-type Bcl-2 or BHRF1 rescued cells from granzyme B-mediated apoptosis, whereas mutated (Gly145-->Glu) Bcl-2, or wild-type Bcl-2 directed to the plasma membrane conferred no protection. Overexpression of Bcl-2 resulted in inhibition of multiple parameters of apoptosis in response to purified perforin and granzyme B, including DNA fragmentation, changes in light scatter profile indicating cell shrinkage and increased refractivity, loss of mitochondrial membrane potential and inhibited colony formation in clonogenic assays. Nevertheless, when exposed to cytotoxic lymphocytes, FDC-P1 and YAC-1 cells overexpressing Bcl-2 remained susceptible to death imparted by cytolytic granules, irrespective of whether the granules contained granzyme B. Thus, alternative granzyme B-independent pathways can be activated by intact lymphocytes to overcome Bcl-2-like inhibitors of apoptosis, enabling CTLs to overcome potential viral blocks to granzyme B-mediated cell death.     

Leukophysin: a 28-kDa granule membrane protein of leukocytes.

A membrane glycoprotein of human platelet dense granules, called granulophysin, with serologic homology to synaptophysin has recently been identified. To determine if this protein was present in granulated leukocytes, we examined several cell types for the presence of the protein by indirect immunofluorescence. Antigranulophysin mAb staining was detected in a granular pattern in the cytoplasm of permeabilized IL-2-stimulated CD3+ peripheral lymphocytes, neutrophils, U937 monocytes, and mast cells. Immunohistochemistry of human lymph nodes showed cytoplasmic staining of macrophages, neutrophils, and some dendritic cells. Induction of granule exocytosis in granulated CD3+ lymphocytes after stimulation with PMA and calcium ionophore A23187 resulted in a redistribution of the reactive epitope from the cytoplasm to the plasma membrane. Subcellular fractions contained two peaks of reactivity; the first peak coincided with N-benzyloxycarbonyl-L-lysine thiobenzyl ester-esterase activity in dense granules whereas the second peak was present in lighter fractions. The affinity purified protein from both peaks was identical in Western blot analysis and had a molecular mass of 28 kDa under reducing conditions. The protein could only be solubilized in detergent suggesting that it was an integral membrane protein. We have named this protein leukophysin to differentiate it from the 40-kDa granulophysin of platelets. Monocytes contained a protein with identical m.w. to leukophysin, whereas a protein of a slightly higher m.w. was detected in neutrophils. We propose that leukophysin is a common granule membrane protein of leukocytes.     

Use of N-benzoyl-L-tyrosine thiobenzyl ester as a protease substrate. Hydrolysis by alpha-chymotrypsin and subtilisin BPN.

In the course of searching for specific chromogenic substrates which might be useful in screening for protease-deficient mutants of Bacillus subtilis, we have developed a method for the synthesis of N-benzoyl-L-tyrosine thiobenzyl ester (BzTyrSBzl) in good yield. Spontaneous base hydrolysis of this thiol ester is low, but several serine proteases hydrolyze it readily. Spectrophotometric measurement of the hydrolysis of the ester in the presence of 5,5'-dithiobis(2-nitrobenzoic acid) provides a continuous assay for chymotrypsin as sensitive as any assay reported in the literature. Serine proteases which hydrolyze this substrate may be detected in polyacrylamide disc gels by incubation in the presence of nitro blue tetrazolium. Apparent Km values of 0.02 and 7 mM and kcat values of 37 S-1 and 126 S-1 were observed for the hydrolysis of BzTyrSBzl by alpha-chymotrypsin and subtilisin BPN', respectively. Additionally, 5 mM indole was observed to behave as a strict competitive inhibitor of the alpha-chymotrypsin-catalyzed hydrolysis of BzTyrSBzl but was observed to increase the maximal rate of hydrolysis of p-nitrophenyl acetate by alpha-chymotrypsin by 30%, as previously described. These data, the published data of other workers, and results from studies with molecular models of trypsin and subtilisin BPN' are used as the basis for describing more fully a secondary hydrophobic binding pocket on alpha-chymotrypsin. The pocket is immediately adjacent to the active site serine and is tentatively suggested to be composed of 4 aliphatic side chain residues and 2 glycine residues.     

Reaction of peptide thiobenzyl esters with mammalian chymotrypsinlike enzymes: A sensitive assay method

Sensitive methods for the determination of rat mast cell protease I, rat mast cell protease II, human skin chymotrypsinlike enzyme, dog skin chymotrypsinlike enzyme, human leukocyte cathepsin G, and bovine chymotrypsin A_α with peptide thiobenzyl ester substrates are reported. Kinetic constants as well as the maximum sensitivity for the hydrolysis of the peptide substrates succinyl-phenylalanyl-leucyl-phenylalanine thiobenzyl ester and succinyl-alanyl-alanyl-prolyl-phenylalanine thiobenzyl ester were determined. Hydrolysis rates were followed spectrophotometrically at 324 nm by the formation of 4-thiopyridone (? = 19,800 m^?1 cm^?1), the product of the reaction between benzylthiol, released during hydrolysis of the peptide thiobenzyl esters, and 4,4′-dithiodipyridine present in the assay mixture. Peptide thiobenzyl ester substrates were shown to be very sensitive substrates, predominantly because of the large extinction coefficient of 4-thiopyridone and the high $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ values for these compounds.     

Regulated secretion of mature cathepsin B from rat exocrine pancreatic cells.

By indirect immunofluorescence and immunogold electron microscopy with an antibody that recognizes specifically the two forms of native mature rat cathepsin B (31 kDa and 5:25 kDa) but not the proenzyme, we detected cathepsin B not only in lysosomes of adult rat exocrine pancreatic cells but also in the trans Golgi condensing vacuoles, the zymogen granules and the pancreatic juice in the intralobular ducts. In contrast, immunocytochemistry with an antibody specific for rat cathepsin D showed the latter to be present in the same cells only in lysosomal compartments as expected. The same pattern of labeling with these two antibodies was found in the first zymogen granules to form in 17-day-old fetal rat pancreas. Counts of the extent of immunogold labeling of cathepsin B in the adult exocrine cells showed that the concentration of the enzyme was only two-fold higher in the lysosomal compartments than in the zymogen granules. To confirm these observations, rat pancreatic postnuclear supernatant (PNS), a fraction enriched in zymogen granules and rat pancreatic juice obtained by catheterization of the pancreatic duct, were subjected to 2D gel electrophoresis followed by immunoblotting with the cathepsin B antibody. All three samples contained a 31 kDa protein recognized by the antibody with a pI of about 4.5, the single chain mature form of cathepsin B. We then radiolabeled pancreatic PNS and zymogen granule fractions with benzyloxycarbonyl-Tyr[125I]-Ala-CHN2, an affinity label that covalently binds to the active sites of mature forms of both cathepsin B and cathepsin L. In both PNS and zymogen granule fractions this reagent labeled cathepsin B. Immunoprecipitation experiments showed that the antibody to cathepsin B recognized specifically both the single chain and the double chain mature forms of cathepsin B in the native state. Finally, Northern blots with a cDNA of rat cathepsin B showed that the concentration of cathepsin B mRNA in total pancreatic RNA increased following in vivo stimulation of the exocrine pancreatic cells with optimal doses of cerulein, a cholecystokinin analogue. We conclude that significant amounts of mature cathepsin B are secreted from exocrine pancreatic cells via the apical regulated exocytotic pathway, and we discuss this in terms of models for sorting of proteins to the cores of dense cored secretory granules.