Protein phosphorylation in the photosynthetic bacterium Rhodospirillum rubrum

Endogenous protein phosphorylation in cellular fractions from Rhodospirillum rubrum was manifested after exposure to [γ-³²P]ATP. At least six phosphorylated protein bands of 90, 86, 64, 31, 13 and 11 kDa were found in the cell-free extract. Treatment of the 64-kDa band with V₈ protease yielded smaller radioactive bands. Phosphoserine, phosphothreonine and phosphotyrosine were detected after acid hydrolysis of the phosphorylated fractions. Protein phosphorylation in all the fractions was insensitive to cAMP, did not recognize exogenous protein substrates and was rapidly reverted upon elimination of the excess of [γ-³²P]ATP. The chlorophyll-anthena apoprotein from R. rubrum chromatophores overlapped the 13-kDa phosphorylated band during gel filtration by high-pressure liquid chromatography suggesting that it is one of the substrates of the protein kinase(s) of R. rubrum.     

Comparison of extracellular matrix-degrading activities between 64-kDa and 90-kDa gelatinases purified in inhibitor-free forms from human schwannoma cells.

Two kinds of gelatinases (or type IV collagenases), 90-kDa and 64-kDa gelatinases, were purified in a tissue inhibitor of metalloproteinases (TIMP)- or TIMP-2-free form from the serum-free conditioned medium of human schwannoma YST-3 cells, and their activities on extracellular matrix proteins were compared. Sequential chromatographies on a gelatin-Sepharose column, an LCA-agarose column, and a gel filtration column in the presence of 5 M urea yielded 600 micrograms of the 64-kDa enzyme and 45 micrograms of the 90-kDa enzyme from 2.8 liters of the conditioned medium. The purified enzymes showed high gelatinolytic activities without activation by p-aminophenyl mercuric acetate (APMA), indicating that 5 M urea used in the final chromatography not only dissociated the inhibitors from the progelatinases but also activated the proenzymes. The inhibitor-free gelatinases showed a much higher activity than the APMA-activated inhibitor-bound enzymes. The specific activity of the 90-kDa enzyme was nearly 25 times higher than that of the 64-kDa enzyme. The 90-kDa gelatinase hydrolyzed type I collagen as well as native and pepsin-treated type IV collagens at 30 degrees C, while at 37 degrees C it potently hydrolyzed types I, III, and IV collagens but not fibronectin or laminin. The 64-kDa gelatinase showed a similar substrate specificity to that of the 90-kDa enzyme, except that it did not hydrolyze type I collagen and native type IV collagen at 30 degrees C.     

Novel gelatinolytic activities in rat organs.

Novel gelatinolytic activities in both latent and active forms were detected in the normal organs of rat by gelatin zymography. Multiple active bands were detected in the extracts from the skin, jejunum, muscle, and kidney without any activation. These activities were inhibited by 1,10-phenanthroline or leupeptin, nor by E64, suggesting that these activities were derived from metallo-proteinases or serine-proteinases. Some gelatinolytic active bands were newly induced or enhanced by p-aminophenylmercuric acetate. These results suggest that matrix degrading activities due to metallo- and serine-proteinases were constitutively expressed in various rat normal organs.     

Nematicidal activity of Lysobacter capsici YS1215 and the role of gelatinolytic proteins against root-knot nematodes

Lysobacter capsici YS1215 is a soil-borne strain that could inhibit the growth of phytopathogenic fungi, including Phytophthora capsici, Rhizoctonia solani and Fusarium oxysporum, as well as root-knot nematodes. The effect of different concentrations of bacterial culture filtrate (BCF) of L. capsici YS1215 on the mortality of second-stage juveniles (J2) of Meloidogyne incognita was studied using 24-well plates. The J2 mortality increased with increasing concentrations of BCF. YS1215 also produces gelatinases in the culture filtrate. To study its role in nematicidal activities, the partial purification and the characterisation of gelatinolytic proteins were done from the culture medium of the YS1215. The partially purified proteins showed three clear bands with molecular weights estimated using zymography to be 255.7, 232.1 and 146.4 kDa. The optimal pH and temperature for the proteins were 8.0 and 40°C, respectively. The activity of the proteins was inhibited by ethylenediaminetetraacetic acid, FeCl₃ and 1,10-phenanthroline, whereas it was activated by MnCl₂. The proteins may belong to the group of metalloproteases. Moreover, the proteins could hydrolyse skimmed milk, collagen, gelatin and bovine serum albumin (BSA) as substrates, but not casein. The proteins could induce 75% J2 mortality in five days and degrade the J2 bodies. The present study demonstrates the role of the gelatinolytic proteins in the nematicidal potential of L. capsici YS1215.     

Identification of collagen binding domain residues that govern catalytic activities of matrix metalloproteinase-2 (MMP-2)

An innovative approach to enhance the selectivity of matrix metalloproteinase (MMP) inhibitors comprises targeting these inhibitors to catalytically required substrate binding sites (exosites) that are located outside the catalytic cleft. In MMP-2, positioning of collagen substrate molecules occurs via a unique fibronectin-like domain (CBD) that contains three distinct modular collagen binding sites. To characterize the contributions of these exosites to gelatinolysis by MMP-2, seven MMP-2 variants were generated with single, or concurrent double and triple alanine substitutions in the three fibronectin type II modules of the CBD. Circular dichroism spectroscopy verified that recombinant MMP-2 wild-type (WT) and variants had the same fold. Moreover, the MMP-2 WT and variants had the same activity on a short FRET peptide substrate that is hydrolyzed independently of CBD binding. Among single-point variants, substitution in the module 3 binding site had greatest impact on the affinity of MMP-2 for gelatin. Simultaneous substitutions in two or three CBD modules further reduced gelatin binding. The rates of gelatinolysis of MMP-2 variants were reduced by 20–40% following single-point substitutions, by 60–75% after double-point modifications, and by > 90% for triple-point variants. Intriguingly, the three CBD modules contributed differentially to cleavage of dissociated α-1(I) and α-2(I) collagen chains. Importantly, kinetic analyses (k_cat/K_m) revealed that catalysis of a triple-helical FRET peptide substrate by MMP-2 relied primarily on the module 3 binding site. Thus, we have identified three collagen binding site residues that are essential for gelatinolysis and constitute promising targets for selective inhibition of MMP-2.     

The isolation, characterization, and molecular cloning of a 75-kDa gelatinase B-like enzyme, a member of the matrix metalloproteinase (MMP) family. An avian enzyme that is MMP-9-like in its cell expression pattern but diverges from mammalian gelatinase B in sequence and biochemical properties

We have isolated a novel 75-kDa gelatinase from a chicken macrophage cell line, HD11. Biochemical and immunological characterization of the purified enzyme demonstrated that it is distinct from the chicken 72-kDa gelatinase A (MMP-2). The enzyme is capable of specific gelatin binding and rapid gelatin cleavage. Incubation with an organomercurial compound (p-aminophenylmercuric acetate) induces proteolytic processing and activation of this enzyme, and the resultant gelatinolytic activity is sensitive to both zinc chelators and tissue inhibitors of metalloproteinases. A full-length cDNA for the enzyme has been cloned, and sequence analysis demonstrated that the enzyme possesses the characteristic multidomain structure of an MMP gelatinase including a cysteine switch prodomain, three fibronectin type II repeats, a catalytic zinc binding region, and a hemopexin-like domain. The 75-kDa gelatinase is produced by phorbol ester-treated chicken bone marrow cells, monocytes, and polymorphonuclear leukocytes, cell types that charac- teristically produce the 92-kDa mammalian gelatinase B (MMP-9). The absence of a 90-110-kDa gelatinase in these cell types indicates that the 75-kDa gelatinase is likely the avian counterpart of gelatinase B. However, the protein is only 59% identical to human gelatinase B, whereas all previously cloned chicken MMP homologues are 75-90% identical to their human counterparts. In addition, the new 75-kDa chicken gelatinase lacks the type V collagen domain that is found in all mammalian gelatinase Bs. Furthermore, the secreted enzyme appears structurally distinct from known gelatinase Bs and the activated enzyme can cleave fibronectin, which is not a substrate for mammalian gelatinase B. Thus the results of this study indicate that a second MMP gelatinase exists in chickens, and although it is MMP-9/gelatinase B-like in its overall domain structure and expression pattern, it appears to be biochemically divergent from mammalian gelatinase B.     

A rapid micro-assay method for gelatinolytic activity using tritium-labeled heat-denatured polymeric collagen as a substrate and its application to the detection of enzymes involved in collagen metabolism

A rapid micro-assay method for gelatinolytic activitiy has been developed using 3H-labeled heat-denatured polymeric collagen (gelatin) as a substrate to investigate enzymes involved in the post-collagenase catabolism of collagen. The method is based on the incubation of gelatin with enzyme followed by determination of the enzyme digestion products soluble in 67% dioxane. It is sensitive enough to detect microgram levels of gelatin fragments, and can be employed over wide ranges of pH and ionic strength. By applying the method to an embryonic chick skin culture system, three gelatinolytic enzyme fractions which showed high, limited and no caseinolytic activities were demonstrated to be separable by gel chromatography.     

Expression of soluble and functional full-length human matrix metalloproteinase-2 in Escherichia coli

Characterization of the matrix metalloproteinase-2 (MMP-2) substrates and understanding of its function remain difficult because up to date preparations containing minor amounts of other eukaryotic proteins that are co-purified with MMP-2 are still used. In this work, the expression of a soluble and functional full-length recombinant human MMP-2 (rhMMP-2) in the cytoplasm of Escherichia coli is reported, and the purification of this metalloproteinase is described. Culture of this bacterium at 18 °C culminated in maintenance of the soluble and functional rhMMP-2 in the soluble fraction of the E. coli lysate and its purification by affinity with gelatin-sepharose yielded approximately 0.12 mg/L of medium. Western Blotting and zymographic analysis revealed that the most abundant form was the 72-kDa MMP-2, but some gelatinolytic bands corresponding to proteins with lower molecular weight were also detected. The obtained rhMMP-2 was demonstrated to be functional in a gelatinolytic fluorimetric assay, suggesting that the purified rhMMP-2 was correctly folded. The method described here involves fewer steps, is less expensive, and is less prone to contamination with other proteinases and MMP inhibitors as compared to expression of rhMMP-2 in eukaryotic tissue culture. This protocol will facilitate the use of the full-length rhMMP-2 expressed in bacteria and will certainly help researchers to acquire new knowledge about the substrates and biological activities of this important proteinase.     

Matrix metalloproteinase‐2 and ‐9 activities in human keloids,hypertrophic and atrophic scars: a pilot study

Proteolytic degradation of extracellular matrix is one of the principal features of cutaneous wound healing but little is known about the activities of gelatinases; matrix metalloproteinase‐2 (MMP‐2) and matrix metalloproteinase‐9 (MMP‐9) on abnormal scar formation. The aim of this study is to determine collagen levels and the gelatinase activities in tissue from hypertrophic scars, atrophic scars, keloids and donor skin in 36 patients and 14 donors. Gelatinase levels (proenzyme + active enzyme) were determined by ELISA and their activities by gelatin zymography. MMP‐9 activity was undetectable in gelatin zymography analysis. Pro‐MMP‐2 levels (median) were highest in normal skin group 53.58 (36.40–75.11) OD µg^?1 protein, while active MMP‐2 levels were highest in keloid group 52.53 (42.47–61.51) OD µg^?1 protein. The active/pro ratio was the highest in keloid group 0.97 followed by hypertrophic scar, normal skin and atrophic scar groups 0.69 > 0.54 > 0.48, respectively. According to results of our study, the two‐phase theory of the duration of hypertrophic scar and keloid formation can be supported by the data of tissue collagen and gelatinase analysis. This study is the first to relate scar formation relationship in regard to gelatinase activation ratio in a keloid, hypertrophic and atrophic scar patient group which is chosen appropriate in age and sex. Copyright © 2009 John Wiley & Sons, Ltd.     

Effect of external CO2 concentrations on protein synthesis in the green algae Scenedesmus obliquus (Turp.) Kütz and Chlorella vulgaris (Kosikov)

Unicellular algae grown under low-CO₂ conditions (0.03% CO₂) have developed a means of concentrating CO₂ at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase. Cells with the CO₂-concentrating mechanism (CCM) acquire the ability to accumulate inorganic carbon to a level higher than that obtained by simple diffusion. To identify proteins which are involved in the organization of the CCM, cells of Scenedesumus obliquus and Chlorella vulgaris grown in high CO₂ (5% CO₂ in air) were transferred to low-CO₂ (0.03%) conditions in the presence of ³⁵SO _inf4 ^sup2? and, thereafter, polypeptides labeled with ³⁵S were detected. Under low-CO₂ conditions the inducton of 36-, 39-, 94- and 110- to 116kDa polypeptides were particularly observed in S. obliquus and 16-, 19-, 27-, 36-, 38- and 45-kDa polypeptides were induced in C. vulgaris. Western blots with antibodies raised against 37-kDa subunits of the periplasmic carbonic anhydrase (CA) of Chlamydomonas reinhardtii showed immunoreactive bands with the 39-kDa polypeptide in the whole-cell homogenates from S. obliquus and with 36 and 38-kDa polypeptides in both high- and low-CO₂grown cells of C. vulgaris. Anti-pea-chloroplast CA antibodies cross-reacted with a single polypeptide of 30 kDa in the whole-cell homogenates but not with thylakoid membranes. The CA activity was associated with soluble and membrane-bound fractions, except thylakoid membranes.     

Comparative analysis of the kinetic parameters and thermal stability of two matrix metalloproteinases expressed in the developing sea urchin embryo

The extracellular matrix is now recognized as a dynamic structure which influences cellular properties. Many matrix metalloproteinase activities have been identified and characterized in vertebrates and constitute important agents in controlling the composition of the extracellular matrix. We have begun a study of matrix metalloproteinase activities in the developing sea urchin embryo. Using sea urchin peristome collagen or gelatin as physiological substrates we have determined the kinetic parameters, K_m and V_max, for an 87 kDa gelatinase activity expressed in late stage sea urchin embryos. We also determined the kinetic parameters K_m, V_max and k_cat, for a 41 kDa species, expressed in the early sea urchin embryo, which possesses both collagenase and gelatinase activities. All values determined were similar to those reported in the literature for vertebrate collagenases and gelatinases and K_m values in the micromolar range suggest that both species possess physiologically relevant activities. Both activities have previously been shown to require Ca²⁺ for activity. Using an assay for quantitating the cleavage of gelatin into trichloroacetic acid soluble peptides we report here markedly different effects of Ca²⁺ on the thermal denaturation profiles of the gelatinases. This latter finding may be indicative of different modes of action for this activating cation. Collectively, these results demonstrate both similarities and differences between vertebrate and invertebrate sea urchin gelatinases.     

A versatile assay for gelatinases using succinylated gelatin.

A spectrophotometric assay using succinylated gelatin as substrate is described for measuring the catalytic activity of gelatinases. The assay is based on measurement of primary amines exposed as a result of hydrolysis of the substrate by gelatinases. Comparison of hydrolysis by matrix metalloproteinase (MMP) 1, 2, 3, 7, 9 indicated that succinylated gelatin was primarily digested by MMP-2 and -9. The assay is rapid (<60 min), specific, suitable for measuring gelatinolytic activity of enzymes and high volume screening of MMP-2 and -9 inhibitors. Sensitivity of the assay is comparable to that of gelatin zymography, under similar experimental conditions. Thus, the assay combines ease and rapidity of assays based on synthetic peptide substrates with specificity of the gelatin zymography technique.     

Purification and characterization of a fibrino(geno)lytic protease from cultured natural isolate of a cyanobacterium, Anabaena fertilissima

An isolate of the cyanobacterium Anabaena from paddy fields was cultured and identified as Anabaena fertilissima based on morphometric features and 16S rRNA gene sequence matching. Cell extracts prepared using bead beater hydrolyzed casein. The caseinolytic protease with native molecular mass of 49 kDa was purified using ammonium sulfate fractionation, hydrophobic, affinity and ion-exchange chromatography, and gel filtration. Upon sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the purified protease was resolved in 17-kDa homologue of microcompartment protein and 27-kDa fragment of unknown protein. The enzyme in native state was digested with gelatin and fibrin in substrate gels producing bands corresponding to ca. 49 kDa. Moreover, a plasmin-specific substrate d-Val-Leu-Lys p-nitroanilide was also hydrolyzed with apparent K _m?=?0.18 mM and V _max?=?4.9?×?10^?7?M?s^?1; while Ca²⁺ stimulated, phenylmethanesulfonyl fluoride, leupeptin, and chelators completely abolished the amidolytic activity. The enzyme exhibited pH and temperature stability over a wide range. Upon incubation with fibrinogen, the Aα- and Bβ-chains preferentially cleaved, though the products thus resolved on SDS-PAGE moved at masses different from those of thrombin- and plasmin hydrolysates, and unlike thrombin, cross-linking of fibrinopeptides was not observed. In the plate assays, fibrinolysis was revealed at comparable strengths to that of plasmin, and the dissolute so obtained upon SDS-PAGE lacked bands corresponding to γ-dimer. Consequently, the degraded D-Dimer peptides appeared. The cyanobacterial protease displayed several unique properties not found in microbial and snake venom fibrinolytic enzymes.     

Low-CO2-inducible protein synthesis in the green alga Dunaliella tertiolecta

In the green marine alga Dunaliella tertiolecta, a CO₂-concentrating mechanism is induced when the cells are grown under low-CO₂ conditions (0.03% CO₂). To identify proteins induced under low-CO₂ conditions the cells were labelled with ³⁵SO₄ ^2–, and seven polypeptides with molecular weights of 45, 47, 49, 55, 60, 68 and 100 kDa were detected. The induction of these polypeptides was observed when cells grown in high CO₂ (5% CO₂ in air) were switched to low CO₂, but only while the cultures were growing in light. Immunoblot analysis of total cell protein against pea chloroplastic carbonic anhydrase polyclonal antibodies showed immunoreactive 30-kDa bands in both high- and low-CO₂-grown cells and an aditional 49-kDa band exclusively in low-CO₂-grown cells. The 30-kDa protein was shown to be located in the chloroplast. Western blot analysis of the plasmamembrane fraction against corn plasma-membrane AT-Pase polyclonal antibodies showed 60-kDa bands in both high- and low-CO₂ cell types as well as an immunoreactive 100-kDa band occurring only in low-CO₂-grown cells. These results suggest that there are two distinct forms of both carbonic anhydrase and plasma-membrane ATPase, and that one form of each of them can be regulated by the CO₂ concentration.Abbreviations CA carbonic anhydrase - DIC dissolved inorganic carbon (CO₂+ HCO₃ ^–) - CCM CO₂-concentrating mechanism - low CO₂ air containing 0.03% CO₂ - high CO₂ air supplemented with 5% CO₂ (v/v) We thank Prof. John Coleman for providing antibodies raised against pea chloroplast CA, Dr. James V. Moroney for providing antibodies raised against the 37-kDa periplasmic carbonic anhydrase of CO₂ Chlamydomonas reinhardtii, and Prof. Leonard T. Robert for a gift of corn plasma-membrane 100-kDa ATPase antibodies. We thank Dr. Jeanine Olsen (University of Groningen, the Netherlands) for style comments. This work was supported by the Institute Tecnológico de Canarias (Spain).     

The Molecular Weight and Other Biophysical Properties of Bromegrass Mosaic Virus

Data are presented which show that bromegrass mosaic virus has a particularly low molecular weight and nucleic acid content. A molecular weight of 4.6 × 10⁶ was calculated from the sedimentation coefficient, S°_20,w = 86.2S, the diffusion coefficient, D_20,w = 1.55 × 10^-7 cm²/sec., and an assumed partial specific volume, [UNK] = 0.708 ml/gm. The virus has a ribonucleic acid content of 1.0 × 10⁶ atomic mass units. Electrophoresis experiments showed that the virus is stable in 0.10 ionic strength buffers in the pH range 3-6. Breakdown of the virus was observed outside this pH range. Some characteristics of the breakdown products are described.     

Gelatin degradation assay reveals MMP-9 inhibitors and function of O-glycosylated domain

AIM: To establish a novel, sensitive and high-throughput gelatinolytic assay to define new inhibitors and compare domain deletion mutants of gelatinase B/matrix metalloproteinase (MMP)-9. METHODS: Fluorogenic Dye-quenched (DQ)TM-gelatin was used as a substrate and biochemical parameters (substrate and enzyme concentrations, DMSO solvent concentrations) were optimized to establish a highthroughput assay system. Various small-sized libraries (ChemDiv, InterBioScreen and ChemBridge) of hetero-cyclic, drug-like substances were tested and compared with prototypic inhibitors. RESULTS: First, we designed a test system with gelatin as a natural substrate. Second, the assay was validated by selecting a novel pyrimidine-2,4,6-trione (barbitu- rate) inhibitor. Third, and in line with present structural data on collagenolysis, it was found that deletion of the O-glycosylated region significantly decreased gelatinolytic activity (kcat/kM ± 40% less than full-length MMP-9). CONCLUSION: The DQTM-gelatin assay is useful in high-throughput drug screening and exosite targeting. We demonstrate that flexibility between the catalytic and hemopexin domain is functionally critical for gelatinolysis.     

Role of the conserved histidine and aspartic acid residues in activity and stabilization of human gelatinase B: An example of matrix metalloproteinases

Gelatinase B (MMP-9), a member of the matrix metalloproteinase family, is a zinc- and calcium-dependent endopeptidase that is known to play a role in tumor cell invasion and in destruction of cartilage in arthritis. It contains a conserved sequence⁴⁰⁰His-(X)₃-His-(X)₂₈-Asp-Asp-(X)₂-⁴³⁶Gly, the function of which is under investigation. The conserved Asp-432 and Asp-433 residues were individually replaced with Gly; these substitutions reduced the gelatinolytic activity of the enzyme to 23% and 0%, respectively. Replacing Asp-433 with Glu, however, decreased the gelatinolytic activity of the enzyme by 93% and proteolytic activity of the enzyme for the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH₂ substrate by 79%. The wild-type and D432G and D433E mutant enzymes had similarK _m values for the synthetic substrate and similarK _i values for the competitive inhibitor, GM6001. Thek _cat/K _m values for D432G and D433E mutant enzymes, however, were reduced by a factor of 4 and their K _a ^Ca values were increased by four- and sixfold, respectively. The significance of His-400 in the activity of the enzyme was assessed by replacing this residue with Ala and Phe. Both H400A and H400F mutants were inactive toward gelatin substrate. These data demonstrate that Asp-432, Asp-433, and His-400 residues are important for the activity of gelatinase B. His-400 may act as a zinc-binding ligand similar to the His-197 in interstitial collagenase (MMP-7) and Asp-432 and Asp-433 residues are probably involved in stabilization of the active site of the enzyme. The His-400 and Asp-433 residues are conserved in all members of the MMP family. Therefore, our results are relevant to this group as a whole.Abbreviations MMP Matrix metalloproteinase - TIMP tissue inhibitor of metalloproteinase - IPTG isopropyl-D-thiogalactoside - APMA 4-aminophenyl-mercuric acetate - PCR polymerase chain reaction - Dpa 3(2,4-di-nitrophenyl) diaminopropionic acid - Mca 7-methoxycoumarin acetic acid     

Purification of an insect derived recombinant human ADAMTS-1 reveals novel gelatin (type I collagen) degrading activities

ADAMTS-1 (A Disintegrin And Metalloprotease with ThromboSpondin repeats) is a member of a family of secreted proteolytic enzymes with a complex modular structure. These enzymes are characterised by an N-terminal metalloproteinase domain, a disintegrin-like domain and a carboxyl terminal region containing variable numbers of a repeat sequence with homology to thrombospondin-1. The expression of the gene for ADAMTS-1 has been associated with inflammation, ovulation, angiogenesis, cellular proliferation and bone formation. ADAMTS-1 can proteolytically process large proteoglycans indicating a potential role in extracellular matrix turnover. In this study, we have tested ADAMTS-1 activity in gelatin zymogram assays. Since previous data demonstrate that ADAMTS-1 is a matrix metalloproteinase (MMP) substrate and is highly unstable in conditioned medium from eukaryotic cell types, we created an insect cell line expressing human ADAMTS-1. We isolated an epitope tagged full-length recombinant ADAMTS-1 from serum free insect cell conditioned medium. The purified protein had aggrecanase activity and appears as two major bands on the silver stained SDS-PAGE corresponding well to a pro-domain on form of 115 kDa and a pro-domain off form of 90 kDa. Using denatured type I collagen in zymographic analysis we demonstrate that ADAMTS-1 has a previously unreported gelatinolytic activity. Also, we notice that processing of its C-terminal region by an apparently autocatalytic process reveals a 27 kDa species with gelatinolytic activity. Furthermore, we show that MMP2 but not MMP13 remove ADAMTS-1 specific gelatin zymopraphic zones.     

Sulfation of (1→3)-β-d-glucan from the fruiting bodies of Russula virescens and antitumor activities of the modifiers

A water-insoluble (1→3)-β-d-glucan isolated from the fresh fruiting bodies of Russula virescens was sulfated using sulfur trioxide-pyridine complex as reagent in dimethyl sulfoxide. Depending on the reaction conditions, the products showed different degrees of sulfation (DS) ranging from 0.17 to 1.17 and different weight average molecular weights (M_ws) ranging from 2.5 × 10⁴ to 1.2 × 10⁵ Da. Moreover, the antitumor activities of the five sulfated derivatives against Sarcoma 180 tumor cell were tested both in vitro and in vivo. The results indicated that the native (1→3)-β-d-glucan did not show antitumor activity, while the sulfated derivatives exhibited enhanced antitumor activities. This study demonstrated that DS and M_w could influence the antitumor activities of the sulfated derivatives.     

Determination of matrix metalloproteinase activity using biotinylated gelatin

Matrix metalloproteinases (MMPs) and, specifically, MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are strongly associated with malignant progression and matrix remodeling. These enzymes are a subject of intensive studies involving screening of comprehensive chemical libraries of synthetic inhibitors. There is no simple method available for measurement of activity of gelatinases and related MMPs. Here, we report a simple, inexpensive, and highly sensitive assay for MMP activity. The assay performed in a 96-well microtiter plate format employs biotin-labeled gelatin (denatured collagen type I) as a substrate. Following the substrate cleavage, only the proteolytic fragments bearing biotin moieties are captured by streptavidin coated on the plastic surface and the captured fragments with at least two biotin molecules should be revealed by streptavidin conjugated with horseradish peroxidase. The frequency of lysine residues is low in collagen type I relative to the MMP cleavage sequences (PXGX). Accordingly, the majority of the cleavage products must be devoid of biotin or possess only one biotin group. Both of these types of fragments cannot be recognized by the horseradish peroxidase-streptavidin conjugate. Therefore, higher gelatinolytic activity is associated with lower signal in the assay. This 2-h assay allows identification of gelatinolytic activity of MMP-2 in concentrations as low as 0.16 ng/ml. The sensitivity of this ELISA-like assay is comparable to that of gelatin zymography, a method widely used to detect gelatinases. However, in contrast to zymography, the assay directly measures the enzymatic activity of MMP samples. The gelatinolytic activity assay permits efficient analyses and screening of the MMP inhibitor panels and allows quantitation of gelatinolytic activity of various MMPs in solution as well as on cell surfaces.