Inhibition of human neutrophil elastase by erythromycin and flurythromycin, two macrolide antibiotics.

Fourteen-member-ring macrolides are antibiotics with a variety of anti-inflammatory activities, and have repeatedly been reported to reduce mucus hypersecretion in conditions such as cystic fibrosis and bronchiectasis. Their structure is characterized by a macrocyclic lactone ring. Because human neutrophil elastase (HNE) plays a crucial role in the vicious circle leading to mucus hypersecretion, and lactones are known to be elastase inhibitors, we hypothesized that macrolides might directly inhibit elastase. To investigate this hypothesis we designed a series of spectrophotometric experiments using a chromogenic substrate with two macrolides, erythromycin (Er) and flurythromycin (FE). We determined the 1st order rate constant (k(obs)) by inhibition and competitive substrate assays, the latter allowing us to calculate the substrate binding constant or inhibition constant and the acylation rate constant (k(a)). A proflavine displacement assay was used to determine the deacylation rate constant (k(d)). Both Er and FE are good HNE inhibitors, showing a high k(a) and a low k(d). Because the number of turnovers per inactivation of Er was congruent with 20-fold higher than that of FE, we supposed that the lower reactivation of HNE-FE was due to the formation of a more stable inactivated enzyme. This hypothesis was confirmed by the hydrazine reactivation of the acyl enzyme. For Er we identified a k(d) only, whereas for FE, in addition to the k(d), an alkylation constant (k(2)) was calculated, correlated to a fully inactivated enzyme. From our kinetics data, we therefore conclude that Er acts as an alternate substrate HNE inhibitor, whereas FE acts as an inactivator.

Evaluation of diastolic function.

Abnormalities of diastolic function are increasingly recognized as important components of the abnormal physiology in many patients with heart failure. In order to better understand the role of abnormalities of individual parameters or diastolic function affecting filling of the left ventricular a broader understanding of the relationship of systolic and diastolic performance on overall left ventricular pump performance should be considered. While measurement of diastolic function noninvasively has become the predominant way of assessing diastolic performance, invasive evaluation remains important. Moreover, understanding the physiology of diastolic performance remains essential to proper diagnosis and management.

Dipeptide vinyl sulfones suitable for intracellular inhibition of dipeptidyl peptidase I.

In granules of hematopoetic cells, dipeptidyl peptidase I (DPPI) processes inactive proenzymes into active enzymes, e.g., lymphocyte progranzyme A. Our goal was to develop irreversible inhibitors of intracellular DPPI. First, we identified inhibitors with aqueous stability. Then we determined which inhibitors were nontoxic, could enter cells and inactivate intracellular DPPI. We screened nine dipeptide vinyl sulfone (VS) inhibitors (kobs/[I] > 72 M-1 s-1) and found six that were nontoxic. Four affected intracellular DPPI at < 25 microM. These compounds contained only uncharged amino acid residues; the two less reactive compounds contained charged Glu residues. The best one, Leu-Phe-VS-CH3, inactivated DPPI in cells with an ID50 of approximately 5 microM. This inhibitor was not the best inhibitor of purified DPPI. Longer aqueous stabilities were important predictors of cellular efficacy. Leu-Phe-VS-CH3 had a half life of 97 min at the pH of the extracellular medium (7.5) and 1302 min at pH 5.5 (the intracellular environment of DPPI). This VS had no direct effect on granzyme activities. In contrast, the diazomethyl ketone inhibitor Gly-Phe-CHN2 inhibited chymase activity. Several good intracellular DPPI VS inhibitors lacked reactivity with cathepsins B, H and L. In conclusion, we have identified DPPI inhibitors suitable for cellular applications.

The pathogenesis of acute pulmonary edema associated with hypertension.

BACKGROUND: Patients with acute pulmonary edema often have marked hypertension but, after reduction of the blood pressure, have a normal left ventricular ejection fraction (> or =0.50). However, the pulmonary edema may not have resulted from isolated diastolic dysfunction but, instead, may be due to transient systolic dysfunction, acute mitral regurgitation, or both. METHODS: We studied 38 patients (14 men and 24 women; mean [+/-SD] age, 67+/-13 years) with acute pulmonary edema and systolic blood pressure greater than 160 mm Hg. We evaluated the ejection fraction and regional function by two-dimensional Doppler echocardiography, both during the acute episode and one to three days after treatment. RESULTS: The mean systolic blood pressure was 200+/-26 mm Hg during the initial echocardiographic examination and was reduced to 139+/-17 mm Hg (P< 0.01) at the time of the follow-up examination. Despite the marked difference in blood pressure, the ejection fraction was similar during the acute episode (0.50+/-0.15) and after treatment (0.50+/-0.13). The left ventricular regional wall-motion index (the mean value for 16 segments) was also the same during the acute episode (1.6+/-0.6) and after treatment (1.6+/-0.6). No patient had severe mitral regurgitation during the acute episode. Eighteen patients had a normal ejection fraction (at least 0.50) after treatment. In 16 of these 18 patients, the ejection fraction was at least 0.50 during the acute episode. CONCLUSIONS: In patients with hypertensive pulmonary edema, a normal ejection fraction after treatment suggests that the edema was due to the exacerbation of diastolic dysfunction by hypertension--not to transient systolic dysfunction or mitral regurgitation.

Characterisation of the antitrypanosomal activity of peptidyl alpha-aminoalkyl phosphonate diphenyl esters.

Two groups of irreversible serine peptidase inhibitors, peptidyl chloromethyl ketones and peptidyl phosphonate diphenyl esters, were examined for antitrypanosomal activity against the bloodstream form of Trypanosoma brucei brucei. Both peptidyl chloromethyl ketones and peptidyl phosphonate diphenyl esters inhibited trypsin-like peptidases of the parasites and exhibited antitrypanosomal activity at micromolar concentrations. In live T. b. brucei, labelled analogues of both of these groups of inhibitors primarily targeted an 80-kDa peptidase, possibly a serine oligopeptidase known as oligopeptidase B. In an in vivo mouse model of infection, one of these inhibitors, carbobenzyloxyglycyl-4-amidinophenylglycine phosphonate diphenyl ester, was curative at 5 mg kg(-1) day(-1) but appeared toxic at higher doses. There was no significant correlation between the inhibitory potency (as evaluated against purified T. b. brucei oligopeptidase B) and the in vitro antitrypanosomal efficacy of either group of inhibitors, suggesting that these inhibitors were acting on multiple targets within the parasites, or had different cell permeability properties. These findings suggest that serine peptidases may represent novel chemotherapeutic targets in African trypanosomes.

Efficacy of novel calpain inhibitors in preventing renal cell death.

Inhibitors of calpains, calcium-activated neutral proteases, protect against cell death produced by anoxia and a variety of toxicants both in vitro and in vivo. The problems with known calpain inhibitors are a lack of specificity, low membrane permeability, and/or low potency. The goal of this study was to determine the effects of seven novel dipeptide and tripeptide calpain inhibitors on calpain activity and antimycin A-induced cell death in rabbit renal proximal tubule (RPT) suspensions. We chose the compounds based on their inhibitory constants for mu- versus m-calpain, specificity of the inhibitors for calpain, and membrane permeability. Only three of the compounds inhibited calpain in RPT and were cytoprotective (Z-Leu-Phe-COOH, Z-Leu-Abu-CONH-CH(2)-CH(OH)-Ph, and Z-Leu-Phe-CONH-Et). Interestingly, Z-Leu-Phe-COOEt, Z-Leu-Abu-CONH-CH(2)-CH(OH)-C(6)F(5), and Z-Leu-Abu-CONH-CH(2)-2-quinolinyl were greater than 60% cytoprotective but did not inhibit calpain in RPT. Z-Leu-Abu-CONH(CH(2))(3)-morpholine was neither cytoprotective nor inhibited calpain. Although these results suggest that six of the seven peptide calpain inhibitors are cell permeable, only three of them inhibited calpain activity in RPT and were cytoprotective. Their ability to inhibit calpain or produce cytoprotection did not correlate with their ability to selectively inhibit purified mu- or m-calpain. Thus it remains to be determined whether they inhibit mu-calpain, m-calpain, or both in RPT. These results also suggest that inhibition of other protease(s) in addition to calpains may be responsible for the cytoprotective actions of some compounds.

Granzymes (lymphocyte serine proteases): characterization with natural and synthetic substrates and inhibitors.

Natural killer (NK) and cytotoxic T-lymphocytes (CTLs) kill cells within an organism to defend it against viral infections and the growth of tumors. One mechanism of killing involves exocytosis of lymphocyte granules which causes pores to form in the membranes of the attacked cells, fragments nuclear DNA and leads to cell death. The cytotoxic granules contain perforin, a pore-forming protein, and a family of at least 11 serine proteases termed granzymes. Both perforin and granzymes are involved in the lytic activity. Although the biological functions of most granzymes remain to be resolved, granzyme B clearly promotes DNA fragmentation and is directly involved in cell death. Potential natural substrates for Gr B include procaspases and other proteins involved in cell death. Activated caspases are involved in apoptosis. The search continues for natural substrates for the other granzymes. The first granzyme crystal structure remains to be resolved, but in the interim, molecular models of granzymes have provided valuable structural information about their substrate binding sites. The information has been useful to predict the amino acid sequences that immediately flank each side of the scissile peptide bond of peptide and protein substrates. Synthetic substrates, such as peptide thioesters, nitroanilides and aminomethylcoumarins, have also been used to study the substrate specificity of granzymes. The different granzymes have one of four primary substrate specificities: tryptase (cleaving after Arg or Lys), Asp-ase (cleaving after Asp), Met-ase (cleaving after Met or Leu), and chymase (cleaving after Phe, Tyr, or Trp). Natural serpins and synthetic inhibitors (including isocoumarins, peptide chloromethyl ketones, and peptide phosphonates) inhibit granzymes. Studies of substrate and inhibitor kinetics are providing valuable information to identify the most likely natural granzyme substrates and provide tools for the study of key reactions in the cytolytic mechanism.

Cutaneous protease activity in the mouse ear vesicant model.

Tissue homogenates from mouse ear skin exposed to sulfur mustard (HD, which is a military designation and probably originated from a World War I slang term 'Hun Stuff') were assayed for serine and cysteine protease activities. Enzyme activity was measured using synthetic chromogenic thioester and fluorogenic 7-amino-4-methylcoumarin (AMC) substrates. The tissue samples were obtained from animals (n = 6) at 3, 6, 12 and 24 h post-exposure from the right ear (HD exposed), whereas control samples were obtained from the left ear (treated only with dichloromethane vehicle). The samples of naive control (left and right ear) were obtained from animals that received no HD treatment (n = 3). Elastase activity was assayed with t-butyloxycarbonyl-Ala-Ala-Ala-thiobenzylester, tryptase activity with benzyloxycarbonyl-Arg-AMC and benzyloxycarbonyl-Arg-thiobenzylester, chymase activity with succinylAla-Ala-Pro-Phe-thiobenzylester and succinyl-Ala-Ala-Pro-Phe-AMC, cathepsin B activity with benzyloxycarbonyl-Arg-Arg-AMC, cathepsin H activity with Arg-AMC and calpain activity with succinyl-Leu-Tyr-AMC. The HD-exposed skin homogenates obtained at 12 and 24 h post-exposure had higher elastase activity (670% and 1900% increase) than control samples. For tryptase and calpain activities, only HD-exposed skin homogenates at 24h post-exposure showed higher activities (220% and 170% increase) when compared to the control. No differences from control were observed for HD-exposed skin obtained at 3 and 6 h post-exposure for elastase, tryptase and calpain activities. Generally, both unexposed and HD-exposed skin had distinct cathepsin B and cathepsin H enzyme activities and small chymase activity. Enzymatic assays were also performed for other serine, cysteine and metalloproteases. These data document that proteases are involved in HD skin injury and continued assessment of proteolytic activity should be useful for identifying effective antiproteases with therapeutic use in reducing or eliminating tissue injury caused by HD cutaneous exposure.

The human cytotoxic T cell granule serine protease granzyme H has chymotrypsin-like (chymase) activity and is taken up into cytoplasmic vesicles reminiscent of granzyme B-containing endosomes.

Serine proteases (granzymes) contained within the cytoplasmic granules of cytotoxic T cells and natural killer cells play a variety of roles including the induction of target cell apoptosis, breakdown of extracellular matrix proteins and induction of cytokine secretion by bystander leukocytes. Different granzymes display proteolytic specificities that mimic the activities of trypsin or chymotrypsin, or may cleave substrates at acidic ("Asp-ase") or at long unbranched amino acids such as Met ("Met-ase"). Here, we report that recombinant granzyme H has chymotrypsin-like (chymase) activity, the first report of a human granzyme with this proteolytic specificity. Recombinant 32-kDa granzyme H expressed in the baculovirus vector pBacPAK8 was secreted from Sf21 cells and recovered by Ni-affinity chromatography, using a poly-His tag encoded at the predicted carboxyl terminus of full-length granzyme H cDNA. The granzyme H efficiently cleaved Suc-Phe-Leu-Phe-SBzl (v = 185 nM/s at [S] = 0.217 mM) and also hydrolyzed Boc-Ala-Ala-X-SBzl (X = Phe, Tyr, Met, Nle, or Nva) with slower rates but had little tryptase or Asp-ase activity. Enzymatic activity was inhibited completely by 0.1 mM 3,4-dichloroisocoumarin and 84% by 1.0 mM phenylmethylsulfonyl fluoride. Fluoresceinated granzyme H was internalized in a temperature-dependent manner by Jurkat cells into endosome-like vesicles, suggesting that it can bind to cell surface receptors similar to those that bind granzyme B. This suggests a hitherto unsuspected intracellular function for granzyme H.

Endocytic delivery of intramolecularly quenched substrates and inhibitors to the intracellular yeast Kex2 protease1.

Kex2 in the yeast Saccharomyces cerevisiae is a transmembrane, Ca2+-dependent serine protease of the subtilisin-like pro-protein convertase (SPC) family with specificity for cleavage after paired basic amino acids. At steady state, Kex2 is predominantly localized in late Golgi compartments and initiates the proteolytic maturation of pro-protein precursors that transit the distal secretory pathway. However, Kex2 localization is not static, and its itinerary apparently involves transiting out of the late Golgi and cycling back from post-Golgi endosomal compartments during its lifetime. We tested whether the endocytic pathway could deliver small molecules to Kex2 from the extracellular medium. Here we report that intramolecularly quenched fluorogenic substrates taken up into intact yeast revealed fluorescence due to specific cleavage by Kex2 protease in endosomal compartments. Furthermore, the endocytic delivery of protease inhibitors interfered with Kex2 activity for precursor protein processing. These observations reveal that the endocytic pathway does intersect with the cycling itinerary of active Kex2 protease. This strategy of endocytic drug delivery has implications for modulating SPC protease activity needed for hormone, toxin and viral glycoprotein precursor processing in human cells.

Electrostatic reversal of serine proteinase substrate specificity.

Mouse granzyme B is a member of the chymotrypsin family of serine proteinases that has an unusual preference for cleavage of substrates following aspartate residues. We show here that granzyme B can be redesigned by a single amino acid substitution in one wall of the specificity pocket, arginine-226 to glutamate, to hydrolyze preferentially thioester substrates following basic amino acids. Amide substrates, however, were not hydrolyzed by the variant granzyme B. These results show that residue 226 is a primary determinant of granzyme B specificity and imply that additional structural components are required for catalysis of amide bonds. Molecular modeling indicated subtle variation in glutamate-226 orientation depending upon the state of protonation of the gamma-carboxylate, which may account for the secondary specificity of this enzyme for substrates containing phenylalanine. This represents the first example of electrostatic reversal of serine proteinase substrate specificity and suggests that residue 226 is a primary substrate specificity determinant in the granzyme B lineage of serine proteinases.

Oxyanion-mediated inhibition of serine proteases.

Novel aryl derivatives of benzamidine were synthesized and tested for their inhibitory potency against bovine trypsin, rat skin tryptase, human recombinant granzyme A, human thrombin, and human plasma kallikrein. All compounds show competitive inhibition against these proteases with Ki values in the micromolar range. X-ray structures were determined to 1.8 A resolution for trypsin complexed with two of the para-substituted benzamidine derivatives, 1-(4-amidinophenyl)-3-(4-chlorophenyl)urea (ACPU) and 1-(4-amidinophenyl)-3-(4-phenoxyphenyl)urea (APPU). Although the inhibitors do not engage in direct and specific interactions outside the S1 pocket, they do form intimate indirect contacts with the active site of trypsin. The inhibitors are linked to the enzyme by a sulfate ion that forms an intricate network of three-centered hydrogen bonds. Comparison of these structures with other serine protease structures with noncovalently bound oxyanions reveals a pair of highly conserved oxyanion-binding sites in the active site. The positions of noncovalently bound oxyanions, such as the oxygen atoms of sulfate, are distinct from the positions of covalent oxyanions of tetrahedral intermediates. Noncovalent oxyanion positions are outside the "oxyanion hole." Kinetics data suggest that protonation stabilizes the ternary inhibitor/oxyanion/protease complex. In sum, both cations and anions can mediate Ki. Cation mediation of potency of competitive inhibitors of serine proteases was previously reported by Stroud and co-workers [Katz, B. A., Clark, J. M., Finer-Moore, J. S., Jenkins, T. E., Johnson, C. R., Ross, M. J., Luong, C., Moore, W. R., and Stroud, R. M. (1998) Nature 391, 608-612].

Synthesis and kinetic studies of an amidine-containing phosphonofluoridate: a novel potent inhibitor of trypsin-like enzymes.

The amidine-containing alpha-aminoalkyl phosphonofluoridate 3 (Cbz-(4-AmPhGly)P(OPh)(F)) is a very potent inhibitor of trypsin-like enzymes. It was prepared by hydrolyzing the corresponding phosphonate diphenyl ester 4 followed by reaction of fluoride with the phosphonochloridate prepared from the intermediate phosphonic acid monoester 5. Compound 3 is the most potent amidine-containing organophosphorus inhibitor yet reported for trypsin-like enzymes. It inhibits trypsin and thrombin with second-order rate constants (Kobs/[I]) of 2.6 x 10(5) M-1 s-1 and 1.0 x 10(5) M-1 s-1, respectively, showing a 130-fold and a 1250-fold rate enhancement over the corresponding diphenyl ester (4). It also inactivates trypsin 2 orders of magnitude more potently than simple phosphonofluoridates such as DFP,1 Sarin and Soman. The phosphonofluoridate 3 does not inhibit other serine proteases such as porcine pancreatic elastase (PPE) and the esterase acetylcholinesterase (AChE). The phosphonofluoridate 3 is hydrolyzed rapidly in buffer solution and has a t1/2 of 4.5 s at pH 7.5.

Lower extremity purpura associated with new-onset autoimmune hepatitis: an unusual presentation.

Autoimmune hepatitis is a chronic necroinflammatory disorder of unknown cause. The morbidity and mortality of this potentially lethal disorder can be minimized by timely diagnosis and treatment. Patients can present with a wide variety of clinical and laboratory manifestations, including high titers of antinuclear antibody and other autoantibodies commonly associated with dermatologic disorders. Overt evidence of hepatic injury can be a late finding. An unusual presentation of autoimmune hepatitis and a brief review of its clinical features are presented, with an emphasis on those findings most relevant to a practicing dermatologist.

A serine proteinase inactivator inhibits chondrocyte-mediated cartilage proteoglycan breakdown occurring in response to proinflammatory cytokines.

The role played by serine proteinases with trypsin-like specificity in chondrocyte-mediated cartilage proteoglycan breakdown was investigated by use of a selective proteinase inactivator, 7-amino-4-chloro-3-(-3-isothiureidopropoxy)isocoumarin, in explant culture systems. This compound was a rapid inactivator of urokinase-type plasminogen activator. It potently inhibited interleukin 1- and tumor necrosis factor-stimulated proteoglycan release from both nasal and articular cartilage. Its less potent inhibition of basal and retinoic acid-stimulated release appeared to be due to cytotoxic effects. The functional half-life of the inactivator in culture medium was 95 min, and its concentration in cartilage was 2.5-fold higher than in the surrounding medium. Following spontaneous hydrolysis the breakdown products of the inactivator were unable to inhibit proteoglycan release. Trypsin-like activity was demonstrated by enzyme histochemistry to be chondrocyte-associated and inhibited by the serine proteinase inactivator. Cell-associated and secreted plasminogen activator activity was detected by zymography. These results suggest the involvement of a serine proteinase(s) with trypsin-like specificity, possibly urokinase-type plasminogen activator, in chondrocyte-mediated cartilage proteoglycan breakdown occurring as a result of stimulation with proinflammatory cytokines. Basal proteoglycan breakdown may occur via a different pathway. Our findings point to a pathological role for serine proteinase(s) in the development of cartilage diseases such as arthritis, possibly in a cascade which results in the activation of the enzyme(s) directly responsible for proteoglycan breakdown. It remains to be shown whether the target serine proteinase is urokinase-type plasminogen activator.

Synthesis and evaluation of diphenyl phosphonate esters as inhibitors of the trypsin-like granzymes A and K and mast cell tryptase.

Thirty-six new amino acid and peptidyl diphenyl phosphonate esters were synthesized and evaluated to identify potent and selective inhibitors for four trypsin-like proteases: lymphocyte granzymes A and K, human mast cell tryptase, and pancreatic trypsin. Among five Cbz derivatives of Lys and Arg homologues, Z-(4-AmPhe)P(OPh)2 is the most potent inhibitor for granzyme A, and Z-LysP(OPh)2 is the best inhibitor for granzyme K, mast tryptase, and trypsin. The amidino P1 residue D,L-(4-AmPhGly)P(OPh)2 was utilized in a series of compounds with several different N-protecting groups and systematic substitutions at P2 in Cbz-AA derivatives and at P3 in Cbz-AA-Ala derivatives. Generally, these phosphonates inhibit granzyme A and trypsin more potently than granzyme K and tryptase. The P2 Thr and Ala dipeptide phosphonates, Cbz-AA-(4-AmPhGly)P(OPh)2, are the most potent inhibitors for granzyme A, and Cbz-Thr-(4-AmPhGly)P(OPh)2 (kobs/[I] = 2220 M-1 s-1) was quite specific with much lower inhibition rates for granzyme K and trypsin (kobs/[I] = 3 and 97 M-1 s-1, respectively) and no inhibition with tryptase. The most effective inhibitor of granzyme A was Ph-SO2-Gly-Pro-(4-AmPhGly)P(OPh)2 with a second-order rate constant of 3650 M-1 s-1. The most potent inhibitor for granzyme K was 3, 3-diphenylpropanoyl-Pro-(4-AmPhGly)P(OPh)2 with a kobs/[I] = 1830 M-1 s-1; all other phosphonates inhibited granzyme K weakly (kobs/[I] < 60 M-1 s-1). Human mast cell tryptase was inhibited slowly by these phosphonates with Cbz-LysP(OPh)2 as the best inhibitor (kobs/[I] = 89 M-1 s-1). The overall results suggest that scaffolds of Phe-Thr-(4-AmPhe) and Phe-Pro-Lys will be useful to create selective phosphonate inhibitors for granzymes A and K, respectively, and that P4 substituents offer opportunities to further enhance selectivity and reactivity.

Purification and characterization of lymphocyte chymase I, a granzyme implicated in perforin-mediated lysis.

One mechanism of killing by cytotoxic lymphocytes involves the exocytosis of specialized granules. The released granules contain perforin, which assembles into pores in the membranes of cells targeted for death. Serine proteases termed granzymes are present in the cytotoxic granules and include several chymases (with chymotrypsin-like specificity of cleavage). One chymase is selectively reactive with an inhibitor, Biotinyl-Aca-Aca-Phe-Leu-PheP(OPh)2, that blocks perforin lysis. We report the purification and characterization of this chymase, lymphocyte chymase I, from rat natural killer cell (RNK)-16 granules. Lymphocyte chymase I is 30 kDa with a pH 7.5 to 9 optimum and primary substrate preference for tryptophan, a preference distinct from rat mast cell chymases. This chymase also reacts with other selective serine protease inhibitors that block perforin pore formation. It elutes by Cu2+-immobilized metal affinity chromatography with other granzymes and has the N-terminal protein sequence conserved among granzymes. Chymase I reduces pore formation when preincubated with perforin at 37 degrees C. In contrast, addition of the chymase without preincubation had little effect on lysis. It should be noted that the perforin preparation contained sufficient residual chymase activity to support lysis. Thus, the reduction of lysis may represent an effect of excess prolytic chymase I or a means to limit perforin lysis of bystander cells. In contrast, other chymases and granzyme K were without effect when added to perforin during similar preincubation. Identification of the natural substrate of chymase I will help resolve how it regulates perforin-mediated pore formation.

Expression and purification of enzymatically active recombinant granzyme B in a baculovirus system.

Granzyme B (GranB), a serine protease stored in the granules of cytotoxic T lymphocytes and natural killer cells, can initiate target cell apoptosis. To produce large amounts of purified active enzyme, recombinant murine granzyme B (rGranB) was expressed from baculovirus in insect cells. The expressed rGranB is secreted into the culture medium and can be readily purified to homogeneity by one-step affinity chromatography to yield 1.5 mg enzyme per liter insect cell medium. RGranB is recognized by a GranB-specific anti-peptide antibody and is active against synthetic substrate Boc-Ala-Ala-Asp-SBzl with kinetic constant (kcat/Km 45,000 M-1s-1) comparable to purified human GranB, RGranB processes the caspase pro-CPP32 into its enzymatically active form and induces DNA fragmentation in isolated nuclei in the presence of cytosolic factors. The ability to express enzymatically active rGranB using the baculovirus system will help elucidate the role of this granzyme in the immune response.