Matrix sequestration of matrix metalloproteinases may be important for the facilitation of remodelling events and the migration of cells through the extracellular matrix. Using an ELISA technique we studied the ability of pro and active forms of gelatinases A and B (GLA and GLB) to bind to matrix components and the contribution made by the different enzyme domains. Pro and active forms of GLA and GLB bound to type-I and type-IV collagens, gelatin and laminin films. Binding to collagens occurred exclusively via the N-terminal portion of the molecule in both of the gelatinases; deletion of the fibronectin-like domain in GLA abolished binding. Fibronectin was shown to compete with GLA, confirming that binding occurs through this domain. GLA and GLB competed for binding to collagen type I, whereas collagenase and stromelysin bound to different sites and could be co-localized with the gelatinases. We conclude that gelatinases have different binding specificities from those previously documented for stromelysin and collagenase, which bind through their C-terminal domains to collagen fibrils.
An endoglucanase (1,4-beta-D-glucan glucanohydrolase, EC 22.214.171.124) from the thermophilic anaerobe Clostridium thermocellum was purified to apparent homogeneity without the use of denaturants. No carbohydrate is associated with the endoglucanase. A molecular mass of 76,000 Da was determined by SDS/PAGE. The optimal pH is 7.0 and the enzyme is isoelectric at pH 5.05. The enzyme has a temperature optimum of 70 degrees C and retains approx. 50% of its activity after 48 h at 60 degrees C. Hydrolysis of CM-cellulose takes place with a rapid decrease in viscosity but a slow liberation of reducing sugars, indicating an endoglucanase type of activity. The endoglucanase shows little ability to hydrolyse highly ordered cellulose. Cellobiose inhibits whereas Mg2+ and Ca2+ stimulate the activity. The enzyme is completely inactivated by 1 mM-Hg2+ and is inhibited by a thiol-blocking reagent.
Full instructions are given for the construction of a micro-respirometer. It is based on a membrane-covered platinum electrode of 10 micron diameter that consumes little oxygen. The reaction chamber, made entirely of glass, can be varied in volume between 20 and 100 microliter by adjusting the sealer assembly. Although specifically designed to study the properties of small samples of mitochondria from tiny organisms, it can be used equally well either for oxygen dissolved in aqueous solutions or dry gas mixtures containing oxygen. Measurements can be made with about one-hundredth the amount of material required for commercially available macro-respirometers, and when connected via an impedance-matching device to a pen recorder it gives a stable output of 100-200 mV in air.
1. Whole lettuce plants were incubated with (1) [1- 14 C]acetate, (2) fluoroacetate followed by [1- 14 C]acetate, (3) fluoro[1- 14 C]acetate, (4) fluoro[2- 14 C]acetate or (5) S -carboxy[ 14 C]methylglutathione. 2. Fluoroacetate did not affect the expiration of 14 CO 2 from [1- 14 C]acetate and only a small amount of 14 CO 2 was produced from either fluoro[1- 14 C]-acetate or fluoro[2- 14 C]acetate in 43h. 3. Fluoroacetate at 50mg/kg wet wt. doubled the plant citrate concentration after 43h incubation, and depending on the age and size of the plant 50–100% of the compound was metabolized. 4. With both fluoro[1- 14 C]acetate and fluoro[2- 14 C]acetate all the radioactivity except that in the CO 2 was found in the water-soluble acid fraction. About 2% was in fluorocitrate and the remainder, apart from unchanged fluoroacetate, was in a number of compounds devoid of fluorine but containing nitrogen and sulphur. These were peptide-like and could be separated by chromatography on an amino acid analyser. 5. Identical compounds were obtained from the spontaneous reaction between iodo[2- 14 C]acetate and glutathione, the major product being S -carboxymethylglutathione. 6. S -Carboxymethylcysteine was also isolated and its mass spectrum compared with a commercial sample. 7. Reaction rates of all the monohaloacetates with glutathione were studied at pH7 at 25°C. No reaction was observed with fluoroacetate. 8. The metabolism of fluoroacetate by lettuce is discussed in relation to that of aliphatic and aromatic halogen compounds, including fluoroacetate, by mammalian liver and to the metabolism of fluoroacetate by different plants reported by other workers.