Enzyme Preparations

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Microsomal preparations from the sources listed under Subheading 2.1. contain all the firmly associated appropriate glycosyl transferases, sulfotransferases, deacetylase, and epimerases for glycosaminoglycan synthesis plus firmly associated endogenous nascent proteoglycan primers on which the glycosaminoglycan chains can be initiated and/or extended and modified by sulfation and epimerization. Attempts to obtain similarly active preparations from many other tissues or cells have been unsuccessful, apparently due to limited levels of nascent proteoglycan primer, even though the glycosaminoglycan-synthesizing enzymes are present. Soluble enzymes that apparently lack the membrane-spanning region may also be present in supernatant fractions from tissue homogenates, serum, or media of cell cultures. Recombinant enzymes have proven to be valuable in identifying the specificities of some of the transferase reactions (see Chapter Shworak and Rosenberg).

1. Microsomes are prepared from tissues by first suspending 2-5 g of tissue in 4 volumes of 0.25 M sucrose containing protease inhibitors (1 mM phenylmethylsulfonylfluoride, 10 mM EDTA, 1 mM N-ethyl maleimide, 0.5 mg/mL leupeptin and 0.1 mg/mL pepstatin) in case there might be slight protease modifications of the enzymes (see Note 10), and homogenizing 3 to 5 times with a Polytron at the maximum speed for 1 min each. The homogenate is centrifuged at 12,000g (occasionally 10,000g or 20,000g) in a fixed-angle rotor for 20 min, and the supernatant fluid is then centrifuged at 105,000g for 1 h. The pellets are suspended in 0.25 M sucrose and recentrifuged at 105,000g for 1 h to wash the resulting microsomal fraction (see Note 11). Greatest experience has been with microsomal preparations from chick embryo epiphyseal cartilage (see Note 12) and mouse mastocytomas.

2. Similar procedures are used to obtain a microsomal fraction from lesser amounts of cultured cells except that sonication in short bursts (30 s) at maximal power with a microtip is utilized for cell disruption.

3. Fractionation of chick embryo epiphyseal cartilage microsomes to provide Golgi is performed by a sucrose density-gradient procedure that is a minor modification of that utilized by others for Golgi subfractionation with liver and cultured cells (see Note 13). A microsomal pellet from fresh, unfrozen epiphyseal cartilage from 100 seventeen-day-old chick embryos is resuspended by using a Dounce homogenizer with a loose-fitting pestle in 0.5 mL of 0.25 M sucrose. A 0.2 mL portion of the fresh microsomal suspension is then fractionated on a six-step sucrose density gradient consisting of 4.0 mL of 55%, 1.4 mL of 40%, 2.2 mL of 35%, 2.2 mL of 30%, 2.0-mL of 25%, and 1.0 mL of 20% (wt/wt) sucrose containing 10 mM Tris-HCl, pH 8.0. After centrifugation for 40 h at 4°C in a Beckman SW-40 rotor, fractions are collected and used directly or frozen for further use in the assay of various enzyme activities (see Note 14). Protein is estimated by the method of Lowry et al. (40). Although Golgi fractions have not been prepared from other sources for investigation of proteoglycan synthesis, similar fractionations should be applicable.

4. For solubilization of membrane-bound enzymes, microsomal pellets are suspended in 10 mM HEPES buffer, pH 7.2, containing 10 mM MgCl2, 2 mM CaCl2, 1 mM DTT, 20% glycerol, and protease inhibitors. To this suspension an equal volume of the buffer solution containing 2% Triton X-100 is added. The suspension is gently mixed for 60 min and cen-trifuged at 105,000g for 90 min. The supernatant is saved and the pellets are resuspended in the original buffer. An equal volume of the buffer solution containing 2% Triton X-100 and 2.0 M NaCl is added. After incubation for 60 min with gentle mixing, the suspension is centrifuged at 105,000g for 90 min. The supernatant is combined with the first supernatant and used as the solubilized enzyme. By this two-step procedure, 90% or more of the glyco-syl transferase and sulfotransferase activities are solubilized (see Notes 15, and 16). Soluble glycosaminoglycan-synthesizing enzyme activities are found in cell culture medium, serum, and various body fluids, probably as a result of proteolytic cleavage from in vivo or intact cells presumably between the catalytic domain and transmembrane domain of the membrane-bound forms of these enzymes.

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