Roles of Arg- and Lys-gingipains in coaggregation of Porphyromonas gingivalis: identification of its responsible molecules in translation products of rgpA, kgp, and hagA genes

Arg- (Rgp) and Lys-gingipains (Kgp) are two individual cysteine proteinases produced by Porphyromonas gingivalis , an oral anaerobic bacterium, and are implicated as major virulence factors in a wide range of pathologies of adult periodontitis. Coaggregation of this bacterium with other oral bacteria is an initial and critical step in infectious processes, yet the factors and mechanisms responsible for this process remain elusive. Here we show that the initial translation products of the rgpA , kgp and hemagglutinin hagA genes are responsible for coaggregation of P. gingivalis and that the proteolytic activity of Rgp and Kgp is indispensable in this process. The rgpA rgpB kgp- and rgpA kgp hagA -deficient triple mutants exhibited no coaggregation activity with Actinomyces viscosus , whereas the kgp -null and rgpA rgpB -deficient double mutants significantly retained this activity. Consistently, the combined action of Rgp- and Kgp-specific inhibitors strongly inhibited the coaggregation activity of the bacterium, although single use of Rgp- or Kgp-specific inhibitor significantly retained this activity. We also demonstrate that the 47- and 43-kDa proteins produced from the translation products of the rgpA , kgp , and hagA genes by proteolytic activity of both Rgp and Kgp are responsible for the coaggregation of P. gingivalis.     

Hemagglutinin/Adhesin domains of Porphyromonas gingivalis play key roles in coaggregation with Treponema denticola

Porphyromonas gingivalis and Treponema denticola are major pathogens of periodontal disease. Coaggregation between microorganisms plays a key role in the colonization of the gingival crevice and the organization of periodontopathic biofilms. We investigated the involvement of surface ligands of P. gingivalis in coaggregation. Two triple mutants of P. gingivalis lacking Arg-gingipain A (RgpA), Lys-gingipain (Kgp) and Hemagglutinin A (HagA) or RgpA, Arg-gingipain B (RgpB) and Kgp showed significantly decreased coaggregation with T. denticola, whereas coaggregation with a major fimbriae (FimA)-deficient mutant was the same as that with the P. gingivalis wild-type parent strain. rgpA, kgp and hagA code for proteins that contain 44 kDa Hgp44 adhesin domains. The coaggregation activity of an rgpA kgp mutant was significantly higher than that of the rgpA kgp hagA mutant. Furthermore, anti-Hgp44 immunoglobulin G reduced coaggregation between P. gingivalis wild type and T. denticola. Treponema denticola sonicates adhered to recombinant Rgp domains. Coaggregation following co-culture of the rgpA kgp hagA mutant expressing the RgpB protease with the rgpA rgpB kgp mutant expressing the unprocessed HagA protein was enhanced compared with that of each triple mutant with T. denticola. These results indicate that the processed P. gingivalis surface Hgp44 domains are key adhesion factors for coaggregation with T. denticola.     

Coaggregation of Porphyromonas gingivalis and Prevotella intermedia.

Porphyromonas gingivalis cells coaggregated with Prevotella intermedia cells. The coaggregation was inhibited with L-arginine, L-lysine, Nalpha-p-tosyl-L-lysine chloromethyl ketone, trypsin inhibitor, and leupeptin. Heat- and proteinase K-treated P. gingivalis cells showed no coaggregation with P. intermedia cells, whereas heat and proteinase K treatments of P. intermedia cells did not affect the coaggregation. The vesicles from P. gingivalis culture supernatant aggregated with P. intermedia cells, and this aggregation was also inhibited by addition of L-arginine or L-lysine and by heat treatment of the vesicles. The rgpA rgpB, rgpA kgp, rgpA rgpB kgp, and rgpA kgp hagA mutants of P. gingivalis did not coaggregate with P. intermedia. On the other hand, the fimA mutant lacking the FimA fimbriae showed coaggregation with P. intermedia as well as the wild type parent. These results strongly imply that a heat-labile and proteinous factor on the cell surface of P gingivalis, most likely the gingipain-adhesin complex, is involved in coaggregation of P. gingivalis and P. intermedia.     

Involvement of arginine-specific cysteine proteinase (Arg-gingipain) in fimbriation of Porphyromonas gingivalis.

Arginine-specific cysteine proteinase (Arg-gingipain [RGP], a major proteinase secreted from the oral anaerobic bacterium Porphyromonas gingivalis, is encoded by two separate genes (rgpA and rgpB) on the P. gingivalis chromosome and widely implicated as an important virulence factor in the pathogenesis of periodontal disease (K. Nakayama, T. Kadowaki, K. Okamoto, and K. Yamamoto, J. Biol. Chem. 270:23619-23626, 1995). In this study, we investigated the role of RGP in the formation of P. gingivalis fimbriae which are thought to mediate adhesion of the organism to the oral surface by use of the rgp mutants. Electron microscopic observation revealed that the rgpA rgpB double (RGP-null) mutant possessed very few fimbriae on the cell surface, whereas the number of fimbriae of the rgpA or rgpB mutant was similar to that of the wild-type parent strain. The rgpB+ revertants that were isolated from the double mutant and recovered 20 to 40% of RGP activity of the wild-type parent possessed as many fimbriae as the wild-type parent, indicating that RGP significantly contributes to the fimbriation of P. gingivalis as well as to the degradation of various host proteins, disturbance of host defense mechanisms, and hemagglutination. Immunoblot analysis of cell extracts of these mutants with antifimbrilin antiserum revealed that the rgpA rgpB double mutant produced small amounts of two immunoreactive proteins with molecular masses of 45 and 43 kDa, corresponding to those of the precursor and mature forms of fimbrilin, respectively. The result suggests that RGP may function as a processing proteinase for fimbrilin maturation. In addition, a precursor form of the 75-kDa protein, one of the major outer membrane proteins of P. gingivalis, was accumulated in the rgpA rgpB double mutant but not in the single mutants and the revertants, suggesting an extensive role for RGP in the maturation of some of the cell surface proteins.     

Identification of a new membrane-associated protein that influences transport/maturation of gingipains and adhesins of Porphyromonas gingivalis

The dual membrane envelopes of Gram-negative bacteria provide two barriers of unlike nature that regulate the transport of molecules into and out of organisms. Organisms have developed several systems for transport across the inner and outer membranes. The Gram-negative periodontopathogenic bacterium Porphyromonas gingivalis produces proteinase and adhesin complexes, gingipains/adhesins, on the cell surface and in the extracellular milieu as one of the major virulence factors. Gingipains and/or adhesins are encoded by kgp, rgpA, rgpB, and hagA on the chromosome. In this study, we isolated a P. gingivalis mutant (porT), which showed very weak activities of gingipains in the cell lysates and culture supernatants. Subcellular fractionation and immunoblot analysis demonstrated that precursor forms of gingipains and adhesins were accumulated in the periplasmic space of the porT mutant cells. Peptide mass fingerprinting and N-terminal amino acid sequencing of the precursor proteins and the kgp'-'rgpB chimera gene product in the porT mutant indicated that these proteins lacked the signal peptide regions, consistent with their accumulation in the periplasm. The PorT protein seemed to be membrane-associated and exposed to the periplasmic space, as revealed by subcellular fractionation and immunoblot analysis using anti-PorT antiserum. These results suggest that the membrane-associated protein PorT is essential for transport of the kgp, rgpA, rgpB, and hagA gene products across the outer membrane from the periplasm to the cell surface, where they are processed and matured.     

Human lactoferrin binds and removes the hemoglobin receptor protein of the periodontopathogen Porphyromonas gingivalis

Porphyromonas gingivalis possesses a hemoglobin receptor (HbR) protein on the cell surface as one of the major components of the hemoglobin utilization system in this periodontopathogenic bacterium. HbR is intragenically encoded by the genes of an arginine-specific cysteine proteinase (rgpA), lysine-specific cysteine proteinase (kgp), and a hemagglutinin (hagA). Here, we have demonstrated that human lactoferrin as well as hemoglobin have the abilities to bind purified HbR and the cell surface of P. gingivalis through HbR. The interaction of lactoferrin with HbR led to the release of HbR from the cell surface of P. gingivalis. This lactoferrin-mediated HbR release was inhibited by the cysteine proteinase inhibitors effective to the cysteine proteinases of P. gingivalis. P. gingivalis could not utilize lactoferrin for its growth as an iron source and, in contrast, lactoferrin inhibited the growth of the bacterium in a rich medium containing hemoglobin as the sole iron source. Lactoferricin B, a 25-amino acid-long peptide located at the N-lobe of bovine lactoferrin, caused the same effects on P. gingivalis cells as human lactoferrin, indicating that the effects of lactoferrin might be attributable to the lactoferricin region. These results suggest that lactoferrin has a bacteriostatic action on P. gingivalis by binding HbR, removing it from the cell surface, and consequently disrupting the iron uptake system from hemoglobin.     

Construction of recombinant hemagglutinin derived from the gingipain-encoding gene of Porphyromonas gingivalis, identification of its target protein on erythrocytes, and inhibition of hemagglutination by an interdomain regional peptide

Porphyromonas gingivalis, an anaerobic gram-negative bacterium associated with chronic periodontitis, can agglutinate human erythrocytes. In general, hemagglutination can be considered the ability to adhere to host cells; however, P. gingivalis-mediated hemagglutination has special significance because heme markedly accelerates growth of this bacterium. Although a number of studies have indicated that a major hemagglutinin of P. gingivalis is intragenically encoded by rgpA, kgp, and hagA, direct evidence has not been obtained. We demonstrated in this study that recombinant HGP44(720-1081), a fully processed HGP44 domain protein, had hemagglutinating activity but that an unprocessed form, HGP44(720-1138), did not. A peptide corresponding to residues 1083 to 1102, which was included in HGP44(720-1138) but not in HGP44(720-1081), could bind HGP44(720-1081) in a dose-dependent manner and effectively inhibited HGP44(720-1081)-mediated hemagglutination, indicating that the interdomain regional amino acid sequence may function as an intramolecular suppressor of hemagglutinating activity. Analyses by solid-phase binding and chemical cross-linking suggested that HGP44 interacted with glycophorin A on the erythrocyte membrane. Glycophorin A and, more effectively, asialoglycophorin, which were added exogenously, inhibited HGP44(720-1081)-mediated hemagglutination. Treatment of erythrocytes with RgpB proteinase resulted in degradation of glycophorin A on the membrane and a decrease in HGP44(720-1081)-mediated hemagglutination. Surface plasmon resonance detection analysis revealed that HGP44(720-1081) could bind to asialoglycophorin with a dissociation constant of 3.0 x 10(-7) M. These results indicate that the target of HGP44 on the erythrocyte membrane appears to be glycophorin A.     

Haemoglobin receptor protein is intragenically encoded by the cysteine proteinase-encoding genes and the haemagglutinin-encoding gene of Porphyromonas gingivalis

The obligately anaerobic bacterium Porphyromonas gingivalis produces characteristic black-pigmented colonies on blood agar. It is thought that the black pigmentation is caused by haem accumulation and is related to virulence of the microorganism. P. gingivalis cells expressed a prominent 19 kDa protein when grown on blood agar plates. Analysis of its N-terminal amino acid sequence indicated that the 19 kDa protein was encoded by an internal region (HGP15 domain) of an arginine-specific cysteine proteinase (Arg-gingipain, RGP)-encoding gene ( rgp1 ) and was also present in genes for lysine-specific cysteine proteinases ( prtP and kgp ) and a haemagglutinin ( hagA ) of P. gingivalis . The HGP15 domain protein was purified from an HGP15-overproducing Escherichia coli and was found to have the ability to bind to haemoglobin in a pH-dependent manner. The anti-HGP15 antiserum reacted with the 19 kDa haemoglobin-binding protein in the envelope of P. gingivalis. P. gingivalis wild-type strain showed pH-dependent haemoglobin adsorption, whereas its non-pigmented mutants that produced no HGP15-related proteins showed deficiency in haemoglobin adsorption. These results strongly indicate a close relationship among HGP15 production, haemoglobin adsorption and haem accumulation of P. gingivalis .     

Genotype variation and capsular serotypes of Porphyromonas gingivalis from chronic periodontitis and periodontal abscesses

Porphyromonas gingivalis is considered an important pathogen in periodontal disease. While this organism expresses a number of virulence factors, no study combining different virulence polymorphisms has, so far, been conducted. The occurrence of combined virulence (Cv) genotypes in 62 isolates of P. gingivalis was investigated from subjects displaying either chronic periodontitis or periodontal abscess. The Cv genotypes, based on gene variation of fimbriae (fimA), Lys-specific cystein proteinase (kgp) and Arg-specific cystein proteinase (prpR1/rgpA), were evaluated by PCR. The isolates were also subjected to capsular polysaccharide K-serotyping. A total of 18 Cv genotype variants based on fimA: kgp: rgpA were identified, of which II:I:A and II:II:A Cv genotypes (53.3%) were the two most frequently detected combinations. Moreover, 36% of the isolates were K-typeable, with the K6 serotype being the most prevalent (23%). Two isolates had the same genotype as the virulent strain W83. The results indicate that chronic periodontitis is not associated with a particularly virulent clonal type. A highly virulent genotype (e.g. strain W83) of P. gingivalis can be found in certain periodontitis patients.     

Characterization of the binding activities of proteinase-adhesin complexes from Porphyromonas gingivalis.

Adhesins from oral bacteria perform an important function in colonizing target tissues within the dentogingival cavity. In Porphyromonas gingivalis certain of these adhesion proteins exist as a complex with either of two major proteinases referred to as gingipain R (arginine-specific gingipain) and gingipain K (lysine-specific gingipain) (R. N. Pike, W. T. McGraw, J. Potempa, and J. Travis, J. Biol. Chem. 269:406-411, 1994). With specific proteinase inhibitors, it was shown that hemagglutination by either proteinase-adhesin complex could occur independently of proteinase activity. Significantly, low concentrations of fibrinogen, fibronectin, and laminin inhibited hemagglutination, indicating that adherence to these proteins and not the hemagglutination activity was a primary property of the adhesin activity component of complexes. Binding studies with gingipain K and gingipain R suggest that interaction with fibrinogen is a major function of the adhesin domain, with dissociation constants for binding to fibrinogen being 4 and 8.5 nM, respectively. Specific association with fibronectin and laminin was also found. All bound proteins were degraded by the functional proteinase domain, with gingipain R being more active on laminin and fibronectin and gingipain K being more effective in the digestion of fibrinogen. Cumulatively, these data suggest that gingipain R and gingipain K, acting as proteinase-adhesin complexes, progressively attach to, degrade, and detach from target proteins. Since such complexes appear to be present on the surfaces of both vesicles and membranes of P. gingivalis, they may play an important role in the attachment of this bacterium to host cell surfaces.     

Peptidase profiles from non-albicans Candida spp. isolated from the blood of a patient with chronic myeloid leukemia and another with sickle cell disease

Candida lipolytica and Candida rugosa were isolated from blood samples from a patient with chronic myeloid leukemia (31 years old) and a patient with sickle cell disease (1-year-old), respectively. Isolates were grown for 48 h at 37 degrees C in either Sabouraud or tryptone soy broth (TSB). Peptidases were tested for using substrate sodium dodecyl sulfate-polyacrylamide gels with gelatin, casein, bovine serum albumin (BSA) or hemoglobin. Enzymography analyses were made on the following substrates: human albumin, IgG and human fibrinogen, which had been incubated with the concentrated supernatants. For C. lipolytica, a approximately 60-kDa gelatin-degrading serine proteolytic activity was found in the TSB supernantant as well as a metallopeptidase activity capable of hydrolysing human albumin, IgG and human fibrinogen. With C. rugosa, albumin, IgG and human fibrinogen substrates were degraded by an aspartyl-like peptidase activity. Supernatants from C. rugosa also showed three serine proteolytic activities towards gelatin (approximately 50 kDa, TSB), casein ( approximately 94 kDa, TSB) and BSA ( approximately 120-kDa, Sabouraud), in addition to a metallopeptidase capable of degrading casein ( approximately 110 kDa, Sabouraud). Little is known about peptidases of C. rugosa and C. lipolytica and this preliminary data may prove useful for future work on host-parasite relationship and antifungal agents.     

Biochemical properties of metalloproteinases from the hemolymph of the mussel Mytilus galloprovincialis Lam

The expression of matrix metalloproteinases (MMP) with gelatinase activity was found in the whole hemolymph of the marine mussel Mytilus galloprovincialis Lam. Cleavage activity was specific for gelatin; very little activity towards human type-IV collagen, and no activity for cold fish gelatin, casein or bovine serum albumin were detected. EDTA and 1,10-phenanthroline were inhibitory, suggesting that mussel MMPs require divalent cations for their proteolytic activity; in fact, the presence of exogenously added divalent ions significantly protected the MMPs from inhibition. No inhibition was detected with serine or cysteine proteinase inhibitors. The specific vertebrate inhibitors as well as the classical vertebrate activator of MMPs were without effect, whereas sulphydryl reducing agents had a strong inhibitory effect. Mussel MMPs showed an exponential curve of thermal-dependent decay that was not protected by the presence of metal ions. Overall the results indicate both similarities and differences between invertebrate and vertebrate gelatinases, providing information for understanding the biological role of these ancient proteinases.     

Degradation of Host Heme Proteins by Lysine- and Arginine-Specific Cysteine Proteinases (Gingipains) of Porphyromonas gingivalis

Porphyromonas gingivalis can use hemoglobin bound to haptoglobin and heme complexed to hemopexin as heme sources; however, the mechanism by which hemin is released from these proteins has not been defined. In the present study, using a variety of analytical methods, we demonstrate that lysine-specific cysteine proteinase of P. gingivalis (gingipain K, Kgp) can efficiently cleave hemoglobin, hemopexin, haptoglobin, and transferrin. Degradation of hemopexin and transferrin in human serum by Kgp was also detected; however, we did not observe extensive degradation of hemoglobin in serum by Kgp. Likewise the beta-chain of haptoglobin was partially protected from degradation by Kgp in a haptoglobin-hemoglobin complex. Arginine-specific gingipains (gingipains R) were also found to degrade hemopexin and transferrin in serum; however, this was observed only at relatively high concentrations of these enzymes. Growth of P. gingivalis strain A7436 in a minimal media with normal human serum as a source of heme correlated not only with the ability of the organism to degrade hemoglobin, haptoglobin, hemopexin, and transferrin but also with an increase in gingipain K and gingipain R activity. The ability of gingipain K to cleave hemoglobin, haptoglobin, and hemopexin may provide P. gingivalis with a usable source of heme for growth and may contribute to the proliferation of P. gingivalis within periodontal pockets in which erythrocytes are abundant.     

Recombinant expression and purification of an enzymatically active cysteine proteinase of the protozoan parasite Entamoeba histolytica.

Cysteine proteinases and in particular cysteine proteinase 5 (EhCP5) of Entamoeba histolytica are considered important for ameba pathogenicity. To study EhCP5 in more detail a protocol was elaborated to produce considerable amounts of the enzyme in its active form. The protein was expressed in Escherichia coli as a histidine-tagged pro-enzyme and purified to homogeneity under denaturing conditions in the presence of guanidine-HCl using nickel affinity chromatography. Renaturation was performed by 100-fold dilution in a buffer containing reduced and oxidized thiols, which led to soluble but enzymatically inactive pro-enzyme. Further processing and activation was achieved in the presence of 10 mM DTT and 0.04% SDS at 37 degrees C. Recombinant enzyme (rEhCP5) was indistinguishable from native EhCP5 purified from E. histolytica lysates. Both runs in SDS-PAGE under reducing and nonreducing conditions at positions corresponding to 27 and 29 kDa, respectively, had the same pH optima and displayed similar specific activity against azocasein. Moreover, both enzymes were active against a broad spectrum of biological and synthetic substrates such as mucin, fibrinogen, collagen, human hemoglobin, bovine serum albumin, gelatin, human IgG, Z-Arg-Arg-pNA, and Z-Ala-Arg-Arg-pNA, but not against Z-Phe-Arg-pNA. The identity of rEhCP5 as a cysteine proteinase was confirmed by inhibition with specific cysteine proteinase inhibitors. In contrast, various compounds known to specifically inhibit aspartic, metallo, or serine proteinases had no effect on rEhCP5 activity.     

Supernatant proteolytic activities of High-Five insect cells grown in serum-free culture

The proteolytic activity of High-Five insect cell culture supernatants was analysed using substrate gel electrophoresis (zymography). During growth in serum-free media, High-Five cells constitutively expressed and secreted proteases that were active on casein gel but not on gelatin or bovine serum albumin gels. Two main protease bands were visible at about 41–42 kDa and 32–33 kDa. By addition of various protease inhibitors in the incubation buffer, the proteases were identified as metalloproteases as complete and specific inhibition of the proteolytic activities was only obtained by 1,10-phenanthroline.     

The purification and characterization of a glomerular-basement-membrane-degrading neutral proteinase from rat mesangial cells.

A neutral proteinase, capable of degrading gelatin, has been found in both an active and a latent form in the medium from the culture of rat mesangial cells. The latent form had an Mr of 80,000-100,000 and could be activated with either 4-aminophenylmercuric acetate or prolonged incubation at neutral pH. The active form of the enzyme was extensively purified. The estimated Mr of the purified enzyme on gel filtration was approximately 200,000, indicating that the active enzyme formed aggregates. However, analysis by SDS/polyacrylamide-gel electrophoresis under reducing conditions showed two protein bands, with Mr 68,000 and 66,000. Both proteins were found to contain proteolytic activity when run on SDS/substrate gels. The enzyme was inhibited by EDTA and 1,10-phenanthroline, but not by inhibitors for cysteine, serine or aspartic proteinases. The enzyme did not digest fibronectin, bovine serum albumin, proteoglycan or interstitial collagen. The enzyme degraded pepsin-solubilized placental type V collagen at 31 degrees C, whereas similarly solubilized type IV collagen was only degraded at higher temperatures. In addition, the neutral proteinase degraded native soluble type IV collagen. It also had activity on insoluble type IV collagen of glomerular basement membrane. The above properties suggest that the mesangial neutral proteinase belongs to the gelatinase group of metalloproteinases and that it may play a role in the normal turnover of extracellular glomerular matrix.     

Porphyrin-Mediated Binding to Hemoglobin by the HA2 Domain of Cysteine Proteinases (Gingipains) and Hemagglutinins from the Periodontal Pathogen Porphyromonas gingivalis

Heme binding and uptake are considered fundamental to the growth and virulence of the gram-negative periodontal pathogen Porphyromonas gingivalis. We therefore examined the potential role of the dominant P. gingivalis cysteine proteinases (gingipains) in the acquisition of heme from the environment. A recombinant hemoglobin-binding domain that is conserved between two predominant gingipains (domain HA2) demonstrated tight binding to hemin (Kd = 16 nM), and binding was inhibited by iron-free protoporphyrin IX (Ki = 2.5 microM). Hemoglobin binding to the gingipains and the recombinant HA2 (rHA2) domain (Kd = 2.1 nM) was also inhibited by protoporphyrin IX (Ki = 10 microM), demonstrating an essential interaction between the HA2 domain and the heme moiety in hemoglobin binding. Binding of rHA2 with either hemin, protoporphyrin IX, or hematoporphyrin was abolished by establishing covalent linkage of the protoporphyrin propionic acid side chains to fixed amines, demonstrating specific and directed binding of rHA2 to these protoporphyrins. A monoclonal antibody which recognizes a peptide epitope within the HA2 domain was employed to demonstrate that HA2-associated hemoglobin-binding activity was expressed and released by P. gingivalis cells in a batch culture, in parallel with proteinase activity. Cysteine proteinases from P. gingivalis appear to be multidomain proteins with functions for hemagglutination, erythrocyte lysis, proteolysis, and heme binding, as demonstrated here. Detailed understanding of the biochemical pathways for heme acquisition in P. gingivalis may allow precise targeting of this critical metabolic aspect for periodontal disease prevention.     

Degradation of connective tissue proteins by serine proteases from Streptococcus pneumoniae.

The proteolytic activity of pneumococcal culture supernatants was investigated. Phenylmethylsulfonyl fluoride and diisopropylfluorophosphate inhibited the proteolytic activity by 94% indicating that the enzymes are serine proteases. Zymogram analysis with inhibitors utilizing a non-denaturing gelatin substrate gel revealed two classes of serine proteases; one sensitive to calcium chelators and one resistant. Enzymes from the culture supernatant cleaved fibronectin, fibrinogen, elastin, and laminin; whereas bovine albumin, and the human immunoglobulins, IgG, IgM, and IgA, were not cleaved. These results indicate that pneumococci produce previously unrecognized serine proteases that degrade several tissue and blood proteins.     

Intracellular distribution of neutral proteinases and inhibitors in pig leucocytes. Isolation of two inhibitors of neutral proteinases.

Granule and post-granular-supernatant fractions were obtained from pig leucocyte cells by differential centrifugation in 0.34 M sucrose. Granule extract possesses proteinase activity at acid and at neutral pH. Three groups of neutral and a group of acid proteinases were isolated from granule extracts by chromatography on DEAE-cellulose. In the first group are present elastase-like and plasminogen-activator proteinases, that are inhibited by diisopropylphosphorofluoridate, alpha1-antitrypsin, intracellular leucocyte inhibitor and partly with p-aminomethylbenzoic acid and Trasylol. The second group of neutral proteinases is unstable under the conditions of isolation used the third group of neutral proteinases comprises collagenases that are inhibited by ethylenediamine tetraacetic acid disodium salt, alpha1-antitrypsin and leucocyte inhibitor. The acid proteinases are inhibited only with pepstatin, up to 90%. In the post-granular supernatant was found the acid proteinase activity towards hemoglobin and casein, and non-stable neutral proteolytic activity towards bovine serum albumin and serum gamma globulin. In the post-granular supernatant also the inhibitors of neutral proteinases were found. By gel filtration on Sephadex G-100 and ion-exchange chromatography on CM-cellulose two inhibitors of neutral proteinases were isolated. The majority of the inhibitor capacity (about 80%) of post-granular supernatant was eluted together with ovalbumin (Mr 43000) and the remainder with cytochrome c (12300). These inhibitors inhibit the granule neutral proteinases, acting on all substrates used, but do not inhibit granule acid proteinase. Inhibition effects of post-granular-supernatant inhibitors on trypsin and chymotrypsin were obtained only when bovine serum albumin was used as substrate. Inhibitors of post-granular supernatant are stable at pH 6-8, but unstable in the pH rnage 2-5 and are thermolabile.