Gelatin SDS-PAGE Research Articles

Advanced Glycation End Products, Oxidative Stress and Metalloproteinases are altered in the Cerebral Microvasculature during Aging

Biological aging is associated with an increased incidence of cerebrovascular disease. Recent findings indicate that oxidative stress promoting age-related changes of cerebral circulation are involved in neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease. The aim of this study was to evaluate the contribution of cerebral microvessels to the oxidative stress during brain aging, by: (i) assessment of precursors for advanced glycation end products (AGE) formation, (ii) activities of antioxidant enzymes, namely superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione disulfide reductase (GR), and (iii) the activities of metalloproteinases (MMPs), MMP-2 and MMP-9, involved in synaptogenesis and memory consolidation. The experiments were performed on two groups of male Wistar rats: 15 young (3-6 months old) and 15 aged (18-24 months old) animals. The cerebral microvessels were isolated by mechanical homogenization, the concentration of protein carbonyls and the activity of antioxidant enzymes were evaluated by spectrophotometry, and gelatin SDS-PAGE zymography was employed to evaluate MMP-2 and MMP-9 activities. The results showed that, by comparison with young rats, aged brain microvessels contain: (i) approximately 106 % increase of protein carbonyls production; (ii) approximately 68% higher GPx activity, unmodified activities of SOD and GR; (iii) approximately 30% diminishment in MMP-2 activity, and the specific occurrence of MMP-9 enzyme. The data suggest that the age-related changes of microvessels could increase the propensity for cerebral diseases and might represent, at least in part, a prerequisite for the deterioration of mental and physical status in the elderly.

Metalloproteases and egg-hatching in Pediculus humanus, the body (clothes) louse of humans (Phthiraptera: Insecta)

To investigate the biochemical components of egg-hatch in the body louse, Pediculus humanus, egg-shell-washings (ESW) were collected during the first 2 h post-hatching and analysed by gelatin SDS-PAGE. These ESW contained proteases with molecular mass in the range of 25-100 kDa; the most abundant proteases were approximately 25 kDa. The 3 main regions of protease activity in the one-dimensional gelatin SDS-PAGE gels resolved to at least 23 distinct regions of protease activity when analysed by two-dimensional gelatin SDS-PAGE, with iso-electric points spread over the entire 3 to 10 pH range. Mechanistic characterization indicated that the ESW contained proteases of the metallo-class, inhibited by both 1,10-phenanthroline and EDTA. Several protease inhibitors were tested for their ability to inhibit louse egg-hatch in vitro. The metalloprotease inhibitor 1,10-phenanthroline and the aminopeptidase inhibitor bestatin significantly inhibited (P<0.05) louse egg-hatch (100% and 58%, respectively). The presence of metalloproteases at the time of egg-hatch and the inhibition of egg-hatch in P. humanus by metalloprotease inhibitors suggests a crucial role for these proteases in the hatching of this medically important parasite.

Alleviation of dormancy in walnut kernels by moist chilling is independent from storage protein mobilization

We studied the effects of moist chilling and warming on storage protein mobilization in walnut (Juglans regia L.) kernels to assess the metabolic inhibition theory, which states that dormant seeds are unable to utilize their own food reserves and that cold conditions allow germination by activating hydrolases involved in reserve mobilization. Stratifying kernels at 5 degrees C for 40 days enhanced their germination. During both cold stratification and warm incubation of kernels, storage protein mobilization occurred in cotyledons rather than axes. Kernel amino acid concentration increased during protein mobilization, with axes of warm-incubated kernels having particularly high amino acid concentrations. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) of the soluble protein fractions from both cold-stratified and warm-incubated cotyledons revealed increased band intensities of putative glutelins (19-22 and 32-35 kDa). A very high molecular weight protease was detected by gelatin SDS-PAGE that was most active at acid to neutral pHs in imbibed, cold stratified and germinated kernels suggesting the protease(s) was synthesized earlier in the mature seeds. Thus, in dormant walnut kernels there is no block to protein mobilization, and imbibition alone is sufficient to initiate proteolysis. Catalase activity was higher in warm-incubated kernels than in cold-stratified kernels, suggesting that seed aging is hastened under warm conditions and that cold stratification in walnut kernels might involve activation of cellular repair mechanisms.

MT1-MMP Controls Tumor-induced Angiogenesis through the Release of Semaphorin 4D

The semaphorins are a family of proteins originally identified as regulators of axon growth that recently have been implicated in blood vessel development. The plexins are high affinity receptors for the semaphorins and are responsible for initiation of signaling upon ligation. Emerging evidence indicates that many human cancers overexpress Semaphorin 4D, which promotes neovascularization upon stimulating its receptor, Plexin-B1, on endothelial cells. However, to exert its pro-angiogenic functions, Semaphorin 4D must be processed and released from its membrane bound form to act in a paracrine manner on endothelial cells. Here we show that Semaphorin 4D is a novel target for the membrane-tethered collagenase membrane type 1-matrix metalloproteinase. We demonstrate that this metalloproteinase, which is not expressed in normal or immortal but non-tumorigenic epithelial cell lines, was present in several head and neck squamous cell carcinoma cell lines and was required for processing and release of Semaphorin 4D into its soluble form from these cells, thereby inducing endothelial cell chemotaxis in vitro and blood vessel growth in vivo. These results suggest that the proteolytic cleavage of Semaphorin 4D may provide a novel molecular mechanism by which membrane type 1-matrix metalloproteinase controls tumor-induced angiogenesis.

Effects of crocetin on the matrix metalloproteinases in cardiac hypertrophy induced by norepinephrine in rats

The effects of crocetin on the cardiac hypertrophy induced by long-term treatment with norepinephrine (NE) in rats have been investigated. The activities of matrix metalloproteinases (MMP-2 and MMP-9) have been assayed by gelatin SDS-PAGE zymography. The expressions of MMP-2 and MMP-9 were detected by RT-PCR. ATPase activity and hydroxyproline contents were measured with a commercial kit. The results show that crocetin blocked the development of left ventricular hypertrophy induced by NE, decreased the level of collagen in myocardium, enhanced both the Na+–K+ ATPase activity in cardiac tissue and the Ca2+–Mg2+ ATPase activity in mitochondria and inhibited significantly the activity of MMP-2 and the expressions of MMP-2 and MMP-9. These results suggest that crocetin may prevent cardiac hypertrophy induced by NE in rats.

Protease activity of Blastocystis hominis

Parasite-derived proteases are important for the parasite life cycle and the pathogenesis of the disease they produce. Proteases of intestinal protozoan parasite Blastocystis hominis were studied for the first time with azocasein assays and gelatin SDS-PAGE analysis. Parasitic lysates were found to have high protease activity and nine protease bands of low (20-33 kDa) and high (44-75 kDa) molecular weights were reported. Proteases were found to be pH-dependent and highest proteolytic activity was observed at neutral pH. Inhibition studies showed that B. hominis isolate B, like many other protozoan parasites, contains mainly cysteine proteases.

Use of inhibitors to identify essential cysteine proteinases of Trichomonas vaginalis.

Designing cysteine proteinase inhibitors as antitrichomonal drugs requires knowledge of which cysteine proteinases are essential to the parasite. In an attempt to obtain such information, the effects of a number of cysteine proteinase inhibitors on trichomonad growth in vitro and proteinase activity were investigated. The broad specificity inhibitor trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (known as E-64) had little effect on growth of Trichomonas vaginalis (27% inhibition at 280 microM, none at 28 microM) even though the addition of 2.8 microM E-64 to growth medium resulted in inhibition of all but two (apparent molecular masses: 35 k and 49 k) of the parasite's proteinases detected by gelatin SDS-PAGE. This shows that many of the parasite's cysteine proteinases are not essential for growth in axenic culture. In contrast, a peptidyl acyloxymethyl ketone, N-benzoyloxycarbonyl-Phe-Ala-CH2OCO-(2,6,-(CF3)2)Ph, at 16 microM killed T. vaginalis and severely inhibited growth of Tritrichomonas foetus. Exposure of Trichomonas vaginalis to 16 microM of this compound for 1 h resulted in both the 35 kDa and 49 kDa proteinases being inhibited, whereas some other proteinases were unaffected. Similar distinctions between the inhibitor sensitivity of the parasite's cysteine proteinases were apparent when a biotinylated peptidyl diazomethyl ketone was used to detect active proteinases. These data suggest that the growth inhibitory effects of the peptidyl acyloxymethyl ketone are through inhibition of cysteine proteinases that are not affected when the parasites are grown in the presence of E-64. At least one of these enzymes, which include the 35 kDa and 49 kDa cysteine proteinases, must be essential and so a suitable target for chemotherapeutic attack.

Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania) amazonensis promastigotes

Here we report the subcellular localization of an intracellular serine protease of 68 kDa in axenic promastigotes of Leishmania (Leishmania) amazonensis, using subcellular fractionation, enzymatic assays, immunoblotting, and immunocytochemistry. All fractions were evaluated by transmission electron microscopy and the serine protease activity was measured during the cell fractionation procedure using alpha-N-r-tosyl-L-arginine methyl ester (L-TAME) as substrate, phenylmethylsulphone fluoride (PMSF) and L-1-tosylamino-2-phenylethylchloromethylketone (TPCK) as specific inhibitors. The enzymatic activity was detected mainly in a membranous vesicular fraction (6.5-fold enrichment relative to the whole homogenate), but also in a crude plasma membrane fraction (2.0-fold). Analysis by SDS-PAGE gelatin under reducing conditions demonstrated that the major proteolytic activity was found in a 68 kDa protein in all fractions studied. A protein with identical molecular weight was also recognized in immunoblots by a polyclonal antibody against serine protease (anti-SP), with higher immunoreactivity in the vesicular fraction. Electron microscopic immunolocalization using the same polyclonal antibody showed the enzyme present at the cell surface, as well as in cytoplasmic membranous compartments of the parasite. Our findings indicate that the internal location of this serine protease in L. amazonensis is mainly restricted to the membranes of intracellular compartments resembling endocytic/exocytic elements.

Gnathostoma binucleatum: Excretion–secretion antigen analysis obtained from advanced third-stage larvae in in vitro culture

The paper describes an introductory characterization of antigenic stimulation of excretion–secretion products (ESP) of Gnathostoma binucleatum advanced third-stage larvae cultured in vitro and proteinases present in this products. Excretory and secretory proteins were obtained after 10 larvae were maintained in 5% CO 2 RPMI medium. The supernatant was collected each week for two months. The proteins were dialyzed, concentrated, and separated in 10% SDS–PAGE gels under reducing conditions and transferred to nitrocellulose paper for immunoblot analyses. G. binucleatum immunized mice serum was used to determine protein antigenicity. Four proteins of 40, 80, 120, and 208 kDa persisted for two months and three proteins, 80, 120, and 208 kDa were recognized for antibodies of mice. In SDS–PAGE gelatin substrate gels ESP resolved as two proteins with molecular weight of 80 and 208 kDa that were sensitive to a metalloproteinase inhibitor, and thus it may be inferred that they might be used for diagnosis of gnathostomiasis.

Lysophosphatidic acid enhances epithelial ovarian carcinoma invasion through the increased expression of interleukin-8

Objective. We previously reported that lysophosphatidic acid (LPA) stimulates cellular invasion of ovarian cancer (OVCA) cells by enhancing membrane-type-1-matrix metalloproteinase (MT1-MMP)-mediated activation of MMP2. Here, we investigate a second mechanism in which LPA enhances cellular invasion through the increased expression of IL-8, independent of the expression or activation of MMP2. Methods. Epithelial ovarian carcinoma cells (DOV 13) were exposed to LPA (80 μM) and IL-8 (100 ng/ml) for 24 h. IL-8 expression was quantified by enzyme-linked immunosorbent assay (ELISA). Cellular invasion (Matrigel invasion), migration (colloidal gold), and urinary-type plasminogen activator (uPA) activity (colorimetric assay) were quantified. Conditioned medium was also assayed for MMP activation and expression by SDS–PAGE gelatin zymography, ELISA, and Western blotting. In addition, IL-8 neutralizing antibody and MMP inhibitors were employed. Results. Our results found LPA to increase IL-8 expression threefold. IL-8 did not affect cellular migration, MMP2 activation, or uPA expression. However, exposure to various concentrations of IL-8 increased cellular invasiveness. Using an IL-8 blocking antibody and various MMP inhibitors, we determined that the increase in invasion was IL-8-dependent, while independent of the activation of MMP2 or MMP9. We further determined IL-8 exposure increased the expression of matrilysin (MMP7). Cells exposed to LPA and IL-8 resulted in a synergistic effect on cellular invasion. Adding the IL-8 blocking antibody, slightly decreased cellular invasion, indicating LPA in part, increases cellular invasion through the increased expression of IL-8. Conclusion. We have identified a separate mechanism of enhanced cellular invasion, which is independent of MMP2 activation and involves the increased expression of IL-8 and subsequent increased expression of MMP7.

A Serine Protease from a Detergent-soluble Extract of Leishmania (Leishmania) amazonensis

Proteases mediate important crucial functions in parasitic diseases, and their characterization contributes to the understanding of host-parasite interaction. A serine protease was purified about 43-fold with a total recovery of 60% from a detergent-soluble extract of promastigotes of Leishmania amazonensis. The purification procedures included aprotinin-agarose affinity chromatography and gel filtration high performance liquid chromatography. The molecular mass of active enzyme was 110 kDa by native gel filtration HPLC and by SDS-PAGE gelatin under non-reducing conditions. Under conditions of reduction using SDS-PAGE gelatin analyses the activity of enzyme was observed in two proteins of 60 and 45 kDa, suggesting that the enzyme may be considered as a dimer. The Leishmania protease was not glycosylated, and its isoelectric point (pI) was around 4.8. The maximal protease activity was at pH 7.0 and 28 degrees C, using a-N-o-tosyl-L-arginyl-methyl ester (L-TAME) as substrate. Assays of thermal stability indicated that this enzyme was totally denatured after pre-treatment at 42 degrees C for 12 min and preserved only 20% of its activity after pre-treatment at 37 degrees C for 24 h, in the absence of substrate. Hemoglobin, bovine serum albumin (BSA), ovalbumin and gelatin were hydrolyzed by Leishmania protease. Inhibition studies indicated that the enzyme belonged to a serine protease class because of a significant impediment by serine protease inhibitors such as benzamidine, aprotinin, and antipain. The activity of the present serine protease is negatively modulated by calcium and zinc and positively modulated by manganese ions. This is the first study that reports the purification of a protease from a detergent-soluble extract of Leishmania species.

Candida guilliermondii isolated from HIV-infected human secretes a 50 kDa serine proteinase that cleaves a broad spectrum of proteinaceous substrates

Non- albicans Candida species cause 35–65% of all candidemias in the general population, especially in immunosuppressed individuals. Here, we describe a case of a 19-year-old HIV-infected man with pneumonia due to a yeast-like organism. This clinical yeast isolate was identified as Candida guilliermondii through mycological tests. C. guilliermondii was cultivated in brain heart infusion medium for 48 h at 37 °C. After sequential centrifugation and concentration steps, the free-cell culture supernatant was obtained and extracellular proteolytic activity was assayed firstly using gelatin-SDS–PAGE. A 50 kDa proteolytic enzyme was detected with activity at physiological pH. This activity was completely blocked by 10 mM phenylmethylsulphonyl fluoride (PMSF), a serine proteinase inhibitor, suggesting that this extracellular proteinase belongs to the serine proteinase class. E-64, a strong cysteine proteinase inhibitor, and pepstatin A, a specific aspartic proteolytic inhibitor, did not interfere with the 50 kDa proteinase. Conversely, a zinc-metalloproteinase inhibitor (1,10-phenanthroline) restrained the proteinase activity released by C. guilliermondii by approximately 50%. Proteinases are a well-known class of enzymes that participate in a vast context of yeast–host interactions. In an effort to establish a functional implication for this extracellular serine-type enzyme, we investigated its capacity to hydrolyze some serum proteins and extracellular matrix components. We demonstrated that the 50 kDa exocellular serine proteinase cleaved human serum albumin, non-immune human immunoglobulin G, human fibronectin and human placental laminin, generating low molecular mass polypeptides. Collectively, these results showed for the first time the ability of an extracellular proteolytic enzyme other than aspartic-type proteinases in destroying a broad spectrum of relevant host proteins by a clinical species of non- albicans Candida.

Tissue distribution, inhibition and activation of gelatinolytic activities in Atlantic cod ( Gadus morhua)

Gelatinolytic activities in fish tissues with properties like matrix metalloproteinases (MMPs) have been paid little attention. However, they have been proposed to participate in post mortem degradation during storage and the disintegration of pericellular connective tissue during spawning. In this paper the distribution of gelatinolytic activities in liver, heart, muscle, gill, and male and female gonad of Atlantic cod ( Gadus morhua) was studied by using gelatin SDS-PAGE, proteinase inhibitors, gelatin and lentil lectin Sepharose affinity chromatography. The amount of gelatin degrading enzymes varied from tissue to tissue. Most of the gelatin binding enzymes were found to be matrix metalloproteinases by adding galardin, a broad range MMP inhibitor, to the incubation buffer. A 72 kDa form of cod gelatin degrading enzyme had properties similar to human proMMP-2, as it could be activated by p-aminophenylmercuric acetate and trypsin. Like the human MMP-2 it did not bind to lentil lectin. An 83 kDa cod gelatin degrading enzyme had properties similar to the 92 kDa progelatinase B (proMMP-9). These properties were also similar to that of the 72 kDa form, except that the 83 kDa cod gelatinase was bound to lentil lectin, showing that it is a glycoprotein like MMP-9.

Independent Intramolecular Mediators of Folding, Activity, and Inhibition for the Plasmodium falciparum Cysteine Protease Falcipain-2

The Plasmodium falciparum cysteine protease falcipain-2 is a trophozoite hemoglobinase and potential antimalarial drug target. Unlike other studied papain family proteases, falcipain-2 does not require its prodomain for folding to active enzyme. Rather, folding is mediated by an amino-terminal extension of the mature protease. As in related enzymes, the prodomain is a potent inhibitor of falcipain-2. We now report further functional evaluation of the domains of falcipain-2 and related plasmodial proteases. The minimum requirement for folding of falcipain-2 and four related plasmodial cysteine proteases was inclusion of a 14-15-residue amino-terminal folding domain, beginning with a conserved Tyr. Chimeras of the falcipain-2 catalytic domain with extensions from six other plasmodial proteases folded normally and had kinetic parameters (k(cat)/K(m) 124,000-195,000 M(-1) s(-1)) similar to those of recombinant falcipain-2 (k(cat)/K(m) 120,000 M(-1) s(-1)), indicating that the folding domain is functionally conserved across the falcipain-2 subfamily. Correct folding also occurred when the catalytic domain was refolded with a separate prodomain-folding domain construct but not with an isolated folding domain peptide. Thus, the prodomain mediated interaction between the other two domains when they were not covalently bound. The prodomain-catalytic domain interaction was independent of the active site, because it was blocked by free inactive catalytic domain but not by the active site-binding peptide leupeptin. The folded catalytic domain retained activity after purification from the prodomain-folding domain construct (k(cat)/K(m) 168,000 M(-1) s(-1)), indicating that the folding domain is not required for activity once folding has been achieved. Activity was lost after nonreducing gelatin SDS-PAGE but not native gelatin PAGE, indicating that correct disulfide bonds are insufficient to direct appropriate folding. Our results identify unique features of the falcipain-2 subfamily with independent mediation of activity, folding, and inhibition.

Enzymes with gelatinolytic activity can be found in Tityus bahiensis and Tityus serrulatus venoms

Enzymes with gelatinolytic activity were detected in Tityus bahiensis and Tityus serrulatus venom. Their activity was optimal at pH 8.0 in SDS–PAGE–gelatin. They were inhibited by PMSF but not by iodoacetamide, pepstatin or phenantrolin in the assay conditions used. This suggests that these enzymes are serine proteases. The presence of metal ions did not affect the proteolytic activity of these enzymes. Several possible functions may be envisaged for these enzymes: in tissue permeabilization, pancreatitis and toxin processing.

Proteolytic activity of cultured Pseudoperkinsus tapetis extracellular products

Several pathogenic protozoan release proteases are necessary for host invasion and initiation of infection. We have identified proteolytic activities in extracellular proteins secreted by the clam parasite Pseudoperkinsus tapetis (Mesomycetozoa) in vitro. The protein concentration of the P. tapetis extracellular products (ECP) increased only during the first week of culture. The appearance of new proteins of 10 and 157 kDa at the second week sample and of 12 kDa at the third week sample was shown by SDS-PAGE. The protease activity rapidly increased in the first 3 weeks of culture, and five clear bands of 23, 29, 60, 67 and 96 kDa with proteolytic activity were detected in the ECP on gelatin SDS-PAGE. Using inhibitors, the proteases were identified as members of the Ca 2+ dependent, serine protease family. Their optimum pH was higher than pH 9.4. The protease activity of the P. tapetis ECP was different than that described for Perkinsus marinus, an oyster pathogen very similar morphologically to the clam parasite and member of the genus in which P. tapetis had been initially included.

Differential expression of proteolytic enzymes in endosymbiont-harboring Crithidia species

Crithidia oncopelti, Crithidia deanei and Crithidia desouzai are flagellates of the Trypanosomatidae family that present bacterium-like endosymbionts in their cytoplasm. Gelatin–SDS–PAGE analysis was used to characterize cell-associated and extracellular proteinases in these organisms. Our survey indicates that the proteolytic profiles of C. deanei and C. desouzai are identical; that C. oncopelti displays a distinct zymogram; and that species naturally lacking endosymbionts have a more complex extracellular proteolytic activity, which illustrates the heterogeneity of this genus. This is the first report on the presence of cysteine proteinases in the culture supernatant of monoxenic trypanosomatids, and by the use of wild and aposymbiotic strains from C. deanei we also demonstrated that the prokaryote endosymbiont somehow alters quantitatively the expression of extracellular proteinases in this trypanosomatid.

Characterization of proteinase activity in stratified Douglas-fir seeds.

We investigated the effect of stratification on the proteinase activity involved in mobilization of the major soluble approximately 45 kDa storage protein during germination of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seeds. Complete hydrolysis of the approximately 45 kDa protein was observed approximately 7 days after exposure of stratified seeds to germination conditions. Coincident with the onset of mobilization, proteinase activity was detected primarily in microsomal extracts from stratified seeds. Microsomal-associated proteinase activity was most active at pH 8.0 and had a molecular mass > 175 kDa as determined by gelatin SDS-PAGE gels. In vitro digestion of soluble protein extracts indicated that, following stratification, there was a significant increase in proteinase activity and hydrolysis of the approximately 45 kDa storage protein. Whether this increase was a result of activation of preexisting proteinase(s) or de novo synthesis remains unknown. In vitro digestion of soluble protein extracts in the presence of various proteinase inhibitors showed that digestion of the approximately 45 kDa storage protein is mediated primarily by a metalloproteinase and to a lesser degree by a serine proteinase. The accumulation of approximately 25 kDa protein products following in vitro digestion suggests that mobilization of the approximately 45 kDa soluble storage protein is mediated by a multi-step process involving the action of different classes of proteinases.

Localisation of gelatinase activity in epidermal hoof lamellae by in situ zymography.

In situ gelatin zymography is a technique, which utilises a gelatin-based emulsion overlay to detect and, more importantly, localise the gelatinase activity in underlying tissue. Gelatinase A [matrix metalloproteinase-2 (MMP-2)] and gelatinase B [matrix metalloproteinase-9 (MMP-9)] are present in equine hoof homogenates and supernatants from cultured hoof explants by SDS-PAGE gelatin zymography, and it has been assumed that the enzymes are derived solely from matrix and epithelia and not from other sources such as leucocytes. Using in situ zymography, gelatinases are shown to be localised within the equine epidermal hoof lamellae and, more specifically, are apparently produced by epidermal basal and/or parabasal cells. The pattern of expression correlates with that expected based on the progression of pathological changes observed during the onset of laminitis, thus providing further evidence that laminitis pathology probably arises as a result of inadequate local MMP regulation.

Matrix Metalloproteinases Produced by Rat IL-2-Activated NK Cells

We have previously documented that adoptively transferred IL-2-activated NK (A-NK) cells can accumulate within cancer metastases. Electron microscopic studies of pulmonary metastases have revealed that adoptively transferred A-NK cells that accumulate within metastases bind to endothelial cells and are able to traverse basement membranes. We have now extended these morphologic studies. We report that rat A-NK cells produce two matrix metalloproteinases: MMP-2 and MMP-9, as determined by SDS-PAGE gelatin zymography. These activities are inhibited following incubation with BB-94 (batimastat), a specific inhibitor of matrix metalloproteinases but not with 3,4-dichloroisocoumarin, an inhibitor of neutral serine proteases. The identity of MMP-2 was confirmed by Western blots using a polyclonal Ab against human MMP-2, whereas reverse transcriptase-PCR analysis of mRNA extracts of A-NK cells has confirmed the presence of MMP-9. In addition, we report for the first time that A-NK cells can migrate through a model basement membrane-like extracellular matrix. Moreover, the ability of A-NK cells to migrate through this model basement membrane was partially inhibited by BB-94; however, BB-94 has no effect on A-NK cell-mediated cytotoxicity, suggesting that matrix metalloproteinases do not contribute to cytolytic function of A-NK cells. In sum, our studies show that A-NK cells employ BB-94-inhibitable matrix metalloproteinases to degrade extracellular matrices. This suggests that matrix metalloproteinases may play a role in the accumulation of A-NK cells within cancer metastases.