Optimization of Saliva Collection and Immunochromatographic Detection of Salivary Pepsin for Point-of-Care Testing of Laryngopharyngeal Reflux.

Salivary pepsin is a promising marker for the non-invasive diagnosis of laryngopharyngeal reflux (LPR). For reliable results regarding pepsin in saliva, it is critical to standardize the collection, storage, and pre-processing methods. In this study, we optimized the saliva collection protocols, including storage conditions, i.e., solution, temperature, and time, and the pre-processing filter for pepsin. Moreover, we prepared a simple immunochromatographic strip for the rapid detection of pepsin and evaluated its sensing performance. As a result, we selected a polypropylene (PP) filter as the pre-processing filter for salivary pepsin in low resource settings, such as those where point of care testing (POCT) is conducted. This filter showed a similar efficiency to the centrifuge (standard method). Finally, we detected the pepsin using gold nanoparticles conjugated with monoclonal pepsin antibody. Under optimized conditions, the lower limit of detection for pepsin test strips was determined as 0.01 µg/mL. Furthermore, we successfully detected the salivary pepsin in real saliva samples of LPR patients, which were pre-processed by the PP filter. Therefore, we expect that our saliva collection protocol and pepsin immunochromatographic strip can be utilized as useful tools for a non-invasive diagnosis/screening of LPR in POCT.

Purification and identification of antioxidant peptides from walnut (Juglans regia L.) protein hydrolysates.

Walnut proteins were hydrolyzed separately using three different proteases to obtain antioxidant peptides. The antioxidant activities of the hydrolysates were measured using 1,1-diphenyl-2-picryl hydrazyl (DPPH) assay. Among hydrolysates, pepsin hydrolysate obtained by 3h exhibited the highest antioxidant activities, which could also quench the hydroxyl radical, chelate ferrous ion, exhibit reducing power and inhibit the lipid peroxidation. Then, 3-h pepsin hydrolysates were purified sequentially by ultrafiltration, gel filtration and RP-HPLC. The sequence of the peptide with the highest antioxidative activity was identified to be Ala-Asp-Ala-Phe (423.23 Da) using RP-HPLC-ESI-MS, which was identified for the first time from walnut protein hydrolysates. Last, the inhibition of the peptide on lipid peroxidation was similar with that of reduced glutathione (GSH). These results indicate that the protein hydrolysates and/or its isolated peptides may be effectively used as food additives.

Imobilização de pepsina em membranas liofilizadas de quitosana e O-carboximetilquitosana / Pepsin immobilization into lyophilized chitosan and O-carboxymethilchitosan membranes

Enzimas são proteínas utilizadas em processos tecnológicos diversos. Estas enzimas dependendo do tipo e grau de pureza são geralmente caras. Comumente as enzimas exigem controle contínuo do processo no que se refere à temperatura, pH, agitação, entre outros, e após o uso são descartadas, o que torna o custo do processo mais elevado. Em decorrência disto, a imobilização de enzimas em suportes insolúveis e inertes, vem sendo proposta com resultados promissores de manutenção e até mesmo aumento da atividade enzimática, resistência mecânica, térmica e de pH, bem como por apresentar maior facilidade de remoção da enzima do sistema e possibilitar sua reutilização. Por causa disto, diferentes tipos de suportes vêem sendo estudados, dentre estes, os materiais poliméricos, tem recebido atenção especial. A quitosana é um polímero natural, biocompatível, biodegradável e atóxico. É obtida de fontes renováveis provenientes do descarte de cascas de crustáceos da indústria de alimentos, o que constitui um fator ambiental importante atualmente. Neste trabalho a enzima pepsina foi imobilizada em membranas liofilizadas de quitosana e O-carboximetilquitosana reticuladas ou não com glutaraldeído. A pepsina imobilizada na membrana de quitosana reticulada com glutaraldeído manteve sua atividade enzimática e o suporte apresentou propriedades físico-químicas de resistência a solubilização em pH ácido, o qual é necessário para atividade da pepsina. O processo de liofilização preservou a estrutura do suporte e não comprometeu a atividade enzimática. Demonstrando que o processo de liofilização é viável para secagem e incorporação de enzimas

Enzymes are proteins used in a wide variety of biotechnological processes. Commonly, enzymes require stringent conditions, such as a particular pH, temperature, stirring, etc. In chemical and biochemical reactions, purified enzymes can be rather costly and additionally, must be discarded after each use, which is still less economical. As a result of this, enzyme immobilization on insoluble and inert supports has been studied as a manner to overcome these problems and optimize enzymes use. Promising results of greater immobilized enzyme activity and stability over a broader range of pH and temperature have been reported. As well, immobilized enzymes can be easily removed from the system and reused. Various materials have been employed as enzymes supports, among then, the polymers have received special attention. Chitosan is a natural polymer that presents biocompatibility, biodegradability and nontoxicity. Chitosan is obtained from crustacean shell wastes discarded by the food industry, and recover this material constitutes an important environmental factor nowadays. In this work the enzyme pepsin was immobilized on freezedried chitosan and O-Carboxymethylchitosan membranes crosslinked or not with glutaraldehyde. Pepsin immobilized on chitosan membrane crosslinked with glutaraldehyde maintained its enzymatic activity and the polymer support provided physicochemical properties such resistance to dissolution in acid pH. Acid pH is required for pepsin activity. The freeze-drying process preserved the support structure and did not compromise the enzymatic activity. Demonstrating that, freeze drying process, is viable for drying and enzymes incorporation

Peptide inhibitor modified magnetic particles for pepsin separation.

Synthetic heptapeptide containing D-amino acid residues (Val-D-Leu-Pro-Phe-Phe-Val-D-Leu) was coupled to glyoxal-activated magnetic agarose particles via the free peptide amino group. The peptide-modified magnetic particles were used for the separation of pepsins. Porcine pepsin A and human pepsin A were adsorbed to the magnetic peptide-modified affinity carrier, while the rat pepsin C and human pepsin C did not interact with the immobilized ligand. Conditions of pepsin adsorption to peptide-modified magnetic particles, as well as elution buffers were optimized. Porcine pepsin A did not interact with the immobilized peptide in the presence of pepsin inhibitor pepstatin A, indicating that the enzyme binding site is involved in the studied interaction. The elaborated method represents a rapid and simple technique not only for the separation of pepsins but also, in combination with MS, for the enzyme detection and determination.

Evaluación de la actividad de extractos marinos sobre Plasmodium falciparum in vitro y aspártico proteasas / Evaluation of the marine extracts action on Plasmodium falciparum in vitro and on aspartic proteases

Introducción: la búsqueda de nuevas drogas o alternativas terapéuticas para el tratamiento de la malaria es una alta prioridad en la lucha por el control de esta enfermedad. En la actualidad, varios estudios se concentran en la evaluación de inhibidores de proteasas de tipo aspártico presentes en la vacuola digestiva de Plasmodium falciparum, las cuales son parte de las enzimas que participan en la degradación de la hemoglobina. Los escasos reportes en la literatura sobre la purificación de inhibidores de proteasas aspárticas a partir de organismos marinos sugieren que constituyen una fuente de este tipo de moléculas prácticamente inexplorada. Métodos: las especies de invertebrados marinos Phallusia nigra, Bugula sp., Lyssodendoryx isodictyalis, Ascidia sydneiensis, Microscosmus goanus, Holothuria mexicana, Lytechinus variegatus y Echinaster sp. fueron colectadas en la localidad de Puerto Esperanza, Pinar del Río, en abril de 2006 y se prepararon extractos etanólicos. Se realizó la evaluación antimalárica in vitro contra Plasmodium falciparum de estos extractos con valores descriptivos de eficacia comparables a los utilizados internacionalmente. Los resultados se relacionaron con los hallazgos de los ensayos de inhibición de la actividad enzimática de pepsina como modelo de proteasa aspártica y con el perfil químico de metabolitos secundarios en estos extractos. Resultados: se encontró una buena reproducibilidad de la actividad antimalárica de los extractos de P. nigra, M. goanus y L. isodictyalis con concentraciones inhibitorias medias menores que 50 µg/mL. El extracto de M. goanus mostró la posible presencia de un inhibidor de pepsina. El perfil químico obtenido para las ascidias se corresponde con los principales compuestos reportados para las familias Pyuridae y Ascidiidae. La actividad antimalárica, así como la actividad inhibidora de pepsina, pudiera ser atribuida a algunos de los grupos de metabolitos secundarios detectados...

Background: the search for new drugs or therapeutic alternatives for malaria treatment is a high priority in the struggle against this disease. At present, several studies are focused on the evaluation of aspartic protease inhibitors present in the digestive vacuole of Plasmodium falciparum, which are part of the enzymes involved in hemoglobin degradation. The few reports in literature on the purification of aspartic proteases inhibitors from marine organisms suggest that they are a practically unexplored source of this type of molecules. Methods: marine invertebrate species Phallusia nigra, Bugula sp., Lyssodendoryx isodictyalis, Ascidia sydneiensis, Microscosmus goanus, Holothuria mexicana, Lytechinus variegatus y Echinaster sp.were detected in Puerto Esperanza area, Pinar del Rio province, on April 2006 and then ethanol extracts were prepared. In vitro antimalarial evaluation of these extracts against Plasmodium falciparum, with descriptive efficacy values being comparable with those used worldwide. The results were associated to the findings of aspartic protease model-like pepsin enzymatic action inhibition tests and to the chemical profile of secondary metabolites in these extracts. RESULTS: good reproducibility of antimalarial action of P. nigra, M. goanus y L. isodictyalis extracts was found, being the average inhibitory concentrations lower than 50 µg/mL. M. goanus extract showed a possible pepsin inhibitor. The chemical profile for ascidians corresponded to the main compounds reported in Pyuridae y Ascidiidae families. The antimalarial activity as well as the pepsin inhibitory activity might be attributed to some of the detected secondary metabolites. Conclusions: the breaking-up of these extracts is recommended in order to isolate the chemical compounds involved in the studied biological activities.

Purificacion y actividad de la pepsina de pollo / Purification and assay of chicken pepsin

The proteolitic enzyme pepsin (EC 3.4.23.1) was purified from chicken stomach by a modification of the method of Bohak (1970): after homogenazing the raw material, the zymogen was extracted with NaCl and NaHCO3, activated with 3N HCl and precipitated with NaCl (28 per cent final concentration). The precipitate was lyophilised; fractions of it were suspended in 0.02N HCl. The solution was filtered through a column (2.6 x 80 cm) of Sephadex G-100 at an elution rate of 8-10 ml x cm-2 x h-1. Two protein peaks were obtained, the first one corresponding to the pepsin (2.0 mg/ml in pooled fractions). The milk clotting activity of the enzyme was determined on skimmed milk as a substrate (Berridge, 1955). Its proteolitic activity on the artificial substrate N-acetyl-L-phenylalanyl-L-3, 5-diiodo tyrosin (APD) also was determined (Rick-Fritsch, 1974). Mean clotting activity value was 5.52 UC, higher (P < 0.01) than that of the reference chymosin (0.64 UC). The activity with APD was unsatisfactory, due to very high absorbance values of the blanks. It is concluded, that the purification steps followed in this trial are simple and rapid, conferring a strong stimulus to using chicken pepsin as a clotting agent for the industrial production of pasteurized white cheese.

Isolation and characterization of a high molecular weight type IV collagenase isolated from human carcinoma tissue.

A proform of high molecular weight type IV collagenase was isolated and purified 1230-fold from human metastatic carcinoma tissue. Like matrix metalloproteinases (MMPs), the enzyme was activated by trypsin and degraded type IV collagen and gelatin at a neutral pH, the activity was inhibited by EDTA and o-phenanthroline. However, the molecular weight was much higher than MMPs which degraded type IV collagen, gelatinase A (MMP-2; 72 kDa gelatinase/type IV collagenase) (EC 3.4.24.24), gelatinase B (MMP-9; 92 kDa gelatinase/type IV collagenase) (EC 3.4.24.35), stromelysin-1 (MMP-3; 57 kDa) (EC 3.4.24.17) and stromelysin-2 (MMP-10; 57 kDa) (EC 3.4.24.22). The other significant difference from MMPs was that the enzyme was not activated by 4-aminophenylmercuric acetate nor inhibited by TIMP. Taking together these results, this high molecular weight type IV collagenase might be a newly found enzyme different from MMPs or might have the same configuration as MMPs already reported.

Identification and characterization of a novel matrix-degrading protease from hormone-dependent human breast cancer cells.

A novel matrix-degrading enzyme was identified from human breast cancer cells. This enzyme appears as major gelatinase in hormone-dependent breast cancer cell lines and has as an apparent molecular mass of 80 kDa on gelatin zymography. The 80-kDa enzyme has a unique metal ion specificity. In addition to calcium ions, the gelatinolytic activity can be supported by manganese and/or magnesium. Unlike 92- and 72-kDa gelatinases and other known members of the metalloproteinase family, the 80-kDa protease is not activated by p-aminophenylmercuric acetate and its gelatinolytic activity is not inhibited by tissue inhibitor of metalloproteinase 2. It is active over the pH range 7.5-9.5 with an optimum at pH 8.5. The enzyme degrades gelatin and type IV collagen. The proteolytic activity of the enzyme is inhibited by EDTA and leupeptin. These unique features clearly distinguish the 80-kDa protease from the known 92-and 72-kDa gelatinases. The expression of 80-kDa enzyme can be detected in hormone-dependent human breast cancer cell lines in vitro and in tumors grown from these cells in athymic nude mice.

Human neutrophil gelatinase is a collagenase type IV.

Secreted gelatinase from human neutrophils was purified as a 94 kDa polypeptide. Gelatinolytic and type IV collagenolytic activities of the purified protein were measured and compared. Immunoglobulins purified from antisera raised against gelatinase inhibited both the gelatinase and type IV collagenase activities. There was no cross-reaction in the inhibition with type I collagenase while the three metalloproteases were similarly inhibited by recombinant tissue inhibitor of metalloproteases. Purified gelatinase degraded denatured type I and native type IV collagens; there was no proteolysis of native type I collagen.

Expression of human recombinant 72 kDa gelatinase and tissue inhibitor of metalloproteinase-2 (TIMP-2): characterization of complex and free enzyme.

The human 72 kDa gelatinase/type IV collagenase is a metalloproteinase that is thought to play a role in metastasis and angiogenesis. The 72 kDa progelatinase can be isolated from conditioned media as a complex with the tissue inhibitor of metalloproteinase-2 (TIMP-2). To investigate 72 kDa gelatinase-TIMP-2 interactions and to compare the activity of the complex versus that of the free enzyme, we have expressed and purified human 72 kDa progelatinase and TIMP-2 as single proteins in a recombinant vaccinia virus mammalian cell expression system. The recombinant 72 kDa progelatinase was able to bind TIMP-2, and it digested gelatin and collagen type IV after activation by p-aminophenylmercuric acid (APMA). The specific activity of the recombinant free enzyme was 20-fold higher than the activity of an APMA-treated stoichiometric complex of recombinant 72 kDa progelatinase and TIMP-2. Also, TIMP-2 caused an 86% inhibition of activity when added to the activated enzyme at a 1:1 molar ratio. Activation of the free recombinant 72 kDa progelatinase yielded the 62 kDa species and two fragments of 46 and 35 kDa that cross-reacted with monoclonal antibodies to the 72 kDa proenzyme. TIMP-2 inhibited the conversion of the recombinant proenzyme to the 62 kDa species and the appearance of the 45 and 35 kDa bands. These results suggest that TIMP-2 is not only a potent inhibitor of the activated enzyme but also prevents the generation of low-molecular-mass species and full enzymic activity from the zymogen.

Subcellular localization and release of human neutrophil gelatinase, confirming the existence of separate gelatinase-containing granules.

An e.l.i.s.a. was developed using specific polyclonal rabbit antibodies against human neutrophil gelatinase. This assay, in contrast to the functional assay, is independent of activation of gelatinase, and is specific for the detection of gelatinase in both its reduced and unreduced forms. Using this assay, we were able to demonstrate a difference between the subcellular localization of gelatinase on the one hand, and the subcellular localization of vitamin B-12-binding protein, lactoferrin and cytochrome b558 on the other hand. The latter three co-localized in fractions of slightly higher density than gelatinase on a two-layer Percoll density gradient. Furthermore, the release of gelatinase exceeded the release of vitamin B-12-binding protein as well as lactoferrin by a factor of 3-6 following stimulation with formylmethionyl-leucyl-phenylalanine, leukotriene B4 and other soluble stimuli. Thus, although gelatinase has previously been found to co-localize with lactoferrin on immuno-electron microscopy, we confirm the existence of gelatinase-rich and lactoferrin- and vitamin B-12-binding-protein-poor granules, that are lighter and mobilized more easily than specific granules. These gelatinase-containing granules are not the store of cytochrome b558.

Activation of TIMP-2/progelatinase A complex by stromelysin.

Progelatinase A was purified as a complex with TIMP-2 from the conditioned medium of a human glioblastoma cell line. The TIMP-2/progelatinase complex was resistant to the activation by p-aminophenylmercuric acetic acid (APMA), and showed less than 10% of the activity of the TIMP-2-free active enzyme. When the complex was incubated with stromelysin in the presence of APMA, the 64-kDa progelatinase was effectively converted to the 57-kDa mature enzyme, increasing its gelatinolytic activity about 8-fold. These results suggest that stromelysin is a natural activator of TIMP-2-bound progelatinase A.

Sequential degradation of interstitial collagen by metalloproteinases extracted from tumors of murine ascites hepatomas.

Gelatinolytic and collagenolytic proteinases were separately isolated by different extraction methods from the mouse ascites hepatoma MH134, and from rat ascites hepatoma AH109A. The activities of two proteinases in each extract showed no significant differences, but after trypsin activation the activities of proteinases from the highly metastatic MH134 were significantly increased compared to the enzyme activities in AH109A, which has low metastatic potential. The total activities of collagenase and gelatinase were increased 7.2- and 5.1-fold; their specific activities were increased 5.2- and 4.8-fold, respectively. Gelatinase and collagenase from MH134 were characterized on gelatin zymography. The gelatinase had a molecular weight of 99 and activation by 4-aminophenylmercuric acetate (APMA) or trypsin resulted in its conversion to 79 or 79-95 kD, respectively. The collagenase revealed a major gelatinolytic band at 89 kD, which was converted to 85 and 70 kD by APMA-activation, and a minor gelatinolytic band at 60 kD. These proteinases could degrade native type I collagen to small fragments in a cooperative manner. Trypsin inhibitor, which affects the trypsin activation of latent gelatinase, was extracted together with gelatinase. The inhibitory activity of the enzyme from AH109A showed a 4.1-fold higher specific activity and 3.7-fold greater total activity than that from MH134. The proteinase(s) capable of activating the gelatinase was also extracted from MH134.

The purification of tissue inhibitor of metalloproteinases-2 from its 72 kDa progelatinase complex. Demonstration of the biochemical similarities of tissue inhibitor of metalloproteinases-2 and tissue inhibitor of metalloproteinases-1.

Human gingival fibroblasts in culture were shown to secrete a 72 kDa progelatinase, of which a proportion in the medium was found to be complexed with tissue inhibitor of metalloproteinases-2 (TIMP-2). A purification procedure was devised to purify free enzyme and inhibitor. We also describe the purification of both 95 kDa progelatinase bound to TIMP-1 and free 95 kDa progelatinase from the medium of U937 cells. A polyclonal antiserum to TIMP-2 was prepared and it was shown that TIMP-1 and TIMP-2 are antigenically distinct. The ability to form stable complexes and the relative inhibitory activities of TIMP-1 and TIMP-2 towards 95 kDa and 72 kDa gelatinases, collagenase, stromelysins 1 and 2 and punctuated metalloproteinase were determined; only minor differences were found. Complex-formation between TIMP-2 and 72 kDa progelatinase was demonstrated not to reduce the metalloproteinase-inhibitory activity of TIMP-2, a finding that led to the characterization of high-molecular-mass TIMP activity. Competition experiments between progelatinases and active gelatinases for TIMPs indicated that the affinity of TIMPs for progelatinases is weaker than that for active gelatinases. In a study of the effects of TIMP-1 and TIMP-2 on progelatinase self-cleavage we found that both TIMP-1 and TIMP-2 inhibit the conversion of 95 kDa and 72 kDa progelatinases and prostromelysin into lower-molecular-mass forms. TIMP capable of complexing with progelatinase was shown to be no more efficient an inhibitor of gelatinase self-cleavage than TIMP not able to complex with progelatinase.

Preferential inhibition of 72- and 92-kDa gelatinases by tissue inhibitor of metalloproteinases-2.

Transformed human fibroblasts secrete two structurally and functionally related inhibitors of matrix metalloproteinases, tissue inhibitor of metalloproteinases (TIMP) 1 and 2. In assays measuring the relative inhibitory capability of TIMP-1 and TIMP-2 against autoactivated 72-kDa gelatinase, which consists of two major active peptides and several inactive fragments, TIMP-2 was more effective than TIMP-1. The isolated 42.5-kDa active fragment that formed as a result of the autoactivation of 72-kDa gelatinase showed the greatest preference for TIMP-2; at half-maximal inhibition, TIMP-2 was greater than 10-fold more effective than TIMP-1. TIMP-2 was also greater than 2-fold more effective than TIMP-1 at inhibiting 72-kDa gelatinase-TIMP-2 complexes activated with 4-aminophenylmercuric acetate, and greater than 7-fold more effective than TIMP-1 at inhibiting 92-kDa gelatinase activated with 4-aminophenylmercuric acetate. Furthermore, these active gelatinases preferentially bound 125I-TIMP-2 when incubated with equal amounts of radiolabeled TIMP-1 and TIMP-2. The ratios of 125I-TIMP-2/125I-TIMP-1 binding to 92-kDa gelatinase, autoactivated 72-kDa gelatinase, and 42.5-kDa fragment were 4.4, 10, and 33, respectively. On the other hand, interstitial collagenase was inhibited by TIMP-1 greater than 2-fold more effectively than TIMP-2 in assays measuring cleavage of loose collagen fibrils.

Regulation of the autoactivation of human 72-kDa progelatinase by tissue inhibitor of metalloproteinases-2.

To study the activation of human 72-kDa gelatinase, and its relation to tissue inhibitor of metalloproteinases 2 (TIMP-2), we purified human 72-kDa progelatinase both as a complex with TIMP-2 and as a free proteinase. Activation of progelatinase-TIMP-2 complexes with 4-aminophenylmercuric acetate yielded gelatinolytically active enzyme migrating at 62 kDa. TIMP-2 remained bound to the active enzyme. Removal of TIMP-2 from progelatinase by reverse-phase high performance liquid chromatography in the presence of trifluoroacetic acid, followed by complete dialysis in neutral pH buffer, resulted in multiple fragments. These fragments were formed as a result of the cleavage of 72-kDa progelatinase at several locations. Cleavage at the amino terminus was restricted to the removal of the propeptide, except in the case of degradation leading to inactive fragments. Two active species autocatalytically evolved upon removal of TIMP-2 from progelatinase. The 62 kDa-activated gelatinase lacked the amino-terminal propeptide, which is known to be removed upon treatment with 4-aminophenylmercuric acetate. In addition, an active 42.5-kDa fragment lacking both the propeptide and a portion of the carboxyl terminus was formed. This low-molecular-weight active form of 72-kDa progelatinase retained its ability to bind and degrade gelatin. Self-activation and degradation of 72-kDa progelatinase can be prevented by agents that inhibit metalloproteinases, including 1,10-phenanthroline. Evidence presented here suggests that TIMP-2 binds to a stabilization site that is independent of the active site. This stabilization site does not bind TIMP-1 (TIMP). Occupation of this site by TIMP-2 prevents autocatalytic activation and degradation but does not prevent gelatinolysis by the enzyme-inhibitor complex.

Natural human monocyte gelatinase and its inhibitor.

Gelatinases produced by stimulated peripheral blood monocytes were detected by substrate zymography and were compared with those derived from tumor cells. Stimulated monocytes were found to produce an 85 kDa gelatinase which co-migrated upon electrophoretic separation and cross-reacted in immunoprecipitation experiments with a phorbol ester inducible metalloprotease from human tumor cells. The intact natural gelatinase (85 kDa), a high molecular weight and complexed gelatinase as well as a proteolytic fragment (25 kDa) were purified by substrate- and antibody-affinity chromatography techniques. Aminoterminal sequence analysis showed that natural monocyte gelatinase occurs as a truncated form of tumor cell gelatinase/type IV collagenase. Furthermore, peripheral blood monocytes were found to also produce a tissue inhibitor of metalloproteases (TIMP). TIMP was co-purified with gelatinase on gelatin sepharose and identified by microsequencing. The balanced and regulated production of gelatinase and TIMP might be important in monocyte migration and tissue remodeling.

The complex between a tissue inhibitor of metalloproteinases (TIMP-2) and 72-kDa progelatinase is a metalloproteinase inhibitor.

Human rheumatoid synovial cells in culture secrete both 72-kDa progelatinase and a complex consisting of 72-kDa progelatinase and a 24-kDa inhibitor of metalloproteinases, TIMP-2. In addition, the culture medium contains TIMP-1, the classical inhibitor of metalloproteinases, with a molecular mass of 30 kDa. TIMP-1 does not form a complex with free 72-kDa progelatinase. Free progelatinase and progelatinase complexed with TIMP-2 can be activated with the organomercury compound p-aminophenylmercury acetate. The activated complex shows less than 10% the enzyme activity of activated free gelatinase. The progelatinase-TIMP-2 complex could be shown to be an inhibitor for other metalloproteinases, such as gelatinase and collagenase secreted by human rheumatoid synovia fibroblasts, as well as for the corresponding enzymes from human neutrophils.

Purification and identification of 91-kDa neutrophil gelatinase. Release by the activating peptide interleukin-8.

Human neutrophils were found to release a 91-kDa gelatinase that is serologically related to tumor-derived gelatinolytic enzymes, as evidenced by immunoprecipitation. In order to identify the neutrophil gelatinase, the activity in conditioned medium from human neutrophil suspensions was purified by affinity chromatography on a gelatin substrate. The 91-kDa active enzyme was further separated from other stainable protein bands by classical SDS PAGE and blotting to a solid support. Amino-terminal sequence analysis of blotted proteins showed that the 91-kDa enzyme is a truncated form of tumor-derived 92-kDa gelatinase (type IV collagenase), lacking eight residues at the NH2-terminus. Sequence analysis of enzymatically inactive cleavage products of this neutrophil gelatinase demonstrated that the gelatin-binding part of the molecule is restricted to the amino-terminal third. Exocytosis of gelatinase-containing granules from neutrophils occurred spontaneously within 6 h after neutrophil plating. When the cells were triggered with the phorbol ester phorbol 12-myristate 13-acetate, a strong secretagogue, rapid gelatinase release was observed. When granulocytes were stimulated with the neutrophil-activating peptide interleukin-8, maximal exocytosis occurred within 1 h. The almost immediate release of neutrophil gelatinase after stimulation of the cells with a chemotactic factor might play a key role in remodeling of the extracellular matrix during granulocyte movement in response to chemotactic stimuli.

Application of N-carboxyalkyl peptides to the inhibition and affinity purification of the porcine matrix metalloproteinases collagenase, gelatinase, and stromelysin.

Several N-carboxyalkyl peptides were synthesized and tested as inhibitors of pig synovial collagenase, 72-kDa gelatinase and stromelysin (matrix metalloproteinases MMP-1, MMP-2, and MMP-3). The most potent of the series, CH3CH2CH2(R,S)CH(COOH)-NH-Leu-Phe-Ala-NH2, competitively inhibited cleavage of dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the Gly-Leu bond by MMP-1 and MMP-2 (KI = 30 and 40 microM, respectively). A similar inhibitory potency was found for MMP-1 with soluble Type I collagen and MMP-3 with substance P as substrate. The inhibitor was coupled to EAH-Sepharose 4B through a C-terminal amide. In the presence of 2 M NaCl at pH 7.2, this matrix bound MMP-1, MMP-2, and MMP-3 from concentrated culture medium of pig synovial membranes. The enzymes coeluted at pH 4.1 and subsequently were resolved by chromatography on DEAE-Sephacel and heparin-Sepharose. Purified MMP-1 catalyzed the o-phenanthroline-sensitive cleavage of collagen into TCA and TCB fragments as well as slower hydrolysis of the alpha 2 chain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of MMP-1 indicated a predominant polypeptide of approximately 44 kDa and minor species of approximately 24 and 21 kDa. The 44-kDa species and one of the smaller polypeptides reacted with an antiserum to residues 195-207 of human fibroblast MMP-1, indicating that porcine MMP-1 contains a similar sequence and that the smaller components were probably derived from MMP-1. Neither MMP-2 nor MMP-3 reacted with this antiserum. Purified porcine MMP-2 degraded gelatin but not collagen and exhibited an apparent Mr of approximately 71 kDa. Additional smaller polypeptides were present, one of which may correspond to tissue inhibitor of metalloproteinases. MMP-3 showed doublets of approximately 47/46 and 26/25 kDa and cleaved substance P at the Gly6-Phe7 bond. This procedure provides a rapid means of obtaining all three MMPs from one source in approximately 15% yield each.