Stromelysin-1 is a member of a tissue metalloproteinase family whose members are all capable of degrading extracellular matrix components. A truncated form of human fibroblast prostromelysin 1 lacking the C-terminal, hemopexin-like domain has been expressed in Escherichia coli and purified to homogeneity. Treatment of this short form of prostromelysin with (aminophenyl)mercuric acetate resulted in activation and loss of the propeptide in a manner identical with the wild-type, full-length protein. Kinetic comparisons using Nle11-substance P as a substrate showed that the wild-type stromelysin and the truncated form of the enzyme had similar kcat and Km values. Likewise, both enzymes displayed similar Ki values for a hydroxamate-containing peptide inhibitor. Taken together, these results indicate that the C-terminal portion of stromelysin is not required for proper folding of the catalytic domain, maintenance of the enzyme in a latent form, activation with an organomercurial, cleavage of a peptide substrate, or interaction with an inhibitor. Moreover, the active short form of stromelysin displayed a reduction in the C-terminal heterogeneity, a characteristic degradation of the full-length stromelysin, and thereby provides a more suitable protein for future structural studies.

An enriched glucan synthase fraction was obtained from red beet root microsomes by sequential extraction with the detergents 3-1(3-cholamidopropyl)dimethylammoniol-l-propanesulfonate and digitonin. The digitonin suspension was centrifuged on a glycerol gradient, where a glucan synthase peak with a specific activity of 30-to 40-fold over microsomes was recovered. Most protein contaminants were found in the gradient pellet. The glucan synthase-containing fraction was largely free of plasma membrane and tonoplast-derived ATPase activity and was enriched with a protein subunit of 68 kilodaltons.Membrane-bound f,-glucan syntheses are a class of enzymes believed to be important in cell wall biosynthesis. Over the last several years, a great deal of effort has been directed towards understanding the properties and functions of these enzymes. Most work with glucan syntheses has been performed using isolated membrane fractions from various plant and microbial sources (5, 9,16,27,28, 30).To date, glucan syntheses have proven to be extremely difficult to purify. One problem has been stability in detergent (2,12,13,21,26 Membrane Isolation. Microsomes were isolated as previously described (8, 30).Glucan Synthase Assay. Glucan synthase assays were conducted in 100 ul assay mixtures containing 1 mm UDP-['4C] glucose (0.1 mCi per mmol), 5 mM MgCl2, 5 mm cellobiose, and 50 mM Tris-HCl (pH 7.0). The level of protein used varied between 2 and 50 tig, depending on the relative purity of the sample to be assayed. The mixtures were incubated at 30°C for 5 min and the reactions were then terminated by heating at 90 to 100°C for 10 min. Glucose incorporation was measured by the method of Smith and Stone (1975) as follows: Each reaction mixture was spotted on a Whatman GF/A filter disk (2.4 cm diameter), dried, and placed in a culture tube.The tubes were placed on ice and washed successively with 10 ml of 66% (w/v) ethanol containing 0.85 mm EDTA, 10 ml of 66% (w/v) ethanol, and 10 ml of 70% ethanol. Filters were rinsed with acetone, air dried, and counted. Quenching was corrected by counting known amounts of UDP-['4C]glucose that had been spotted on filters.Protein Determinations. Protein concentrations were measured by the Coomassie blue protein assay (7).Extraction of Microsomes with Digitonin. To solutions containing microsomes (0.85-2.0 ml) at protein concentrations of 2.4 to 5.3 mg per ml, an equal volume of a buffer containing 2% (w/v) digitonin, 5 mM MgCl2, and 50 mM Tris-HCl (pH 7.0) was added dropwise with stirring. The Glycerol Density Gradient Centrifugation. Linear gradients were made by combining 1.75 ml each of 12% (w/w) and 60% (w/w) glycerol in 5 mM MgCl2, 0.1 % digitonin, and 50 mM Tris-HCI (pH 7.0) on a gradient maker. In one experiment (Fig. 1), a www.plantphysiol.org on May 10, 2018 -Published by Downloaded from

Enzyme assaysThe effect of hydrogen peroxide and phenolic compounds on the decolorization of betanin and a betaxanthin preparation by horseradish peroxidase (HRP) was examined. Betanin was decolorized at a greater rate than the betaxanthm pigments and both reactions were HzOz-dependent. Betaxanthin was more prone to oxidatic decolorization than beta&. 2,4-Dichlorophenol, resorcinol and o-toluidine stimulated the decolorization of both pigments. Guaiaco1 enhanced the peroxidatic decolorization of both pigments to a small extent, but inhibited the oxidatic breakdown of betaxanthin. Possible implications of these results are discussed.The standard 1 ml assay mixture contained approximately 12 PM betanin (As38 = 0.8-1.1; E = 65,000 L-cm-l-M-') or 20 PM betaxanthin (A476 = 0.5-0.6; E = 25,000 L-cm-'*M-i) in 100 PM citrate-phosphate buffer, pH 3.4, and 0 or 10 PM H202. In assays containing phenolics, final concentrations are given with individual results. Reactions were initiated by the addition of HRP and absorbance losses measured at 25°C using a Gilford 2600 spectrophotometer. Rates of nonenzymic pigment loss were negligible.

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As the first step in determining whether polysaccharide biosynthetic enzymes can be utilized to improve textural properties in cut vegetables, the glucan synthase II of red beet root microsomes and tissue slices was characterized. In microsomes, maximal rates of glucose incorporation using uridine diphosphate glucose (UDPG) were obtained at pH 7.0 with cellobiose and 5 mM Mg2+. Approximately 30% of the substrate was incorporated into products. Products were partially solubilized by treatment with /3(l-*-3)and d(l-*-4)-glucanases. Subcellular localization experiments demonstrated that the enzyme is located predominantly in the plasma membrane, with lesser levels associated with Golgi-derived vesicles. UDPG was readily incorporated into tissue disks, with the bulk of [14C]glucose associated with the 5000g fraction.