No abstract

No abstract

No abstract

No abstract

Evidence is presented that ligandin, an intracellular protein involved in the binding of such anions as bilirubin, indocyanine green, and penicillin, is identical to glutathione S-transferase B (EC 2.5.1.18), an enzyme catalyzing the conjugation of glutathione with such electrophiles as 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, iodomethane, ethacrynic acid, and bromosulfophthalein. The proteins, isolated by distinct methods, have the same specificity for substrates and for ligands, react in identical fashion to antibody pioduced against ligandin, bear entirely sinlilar physical characteristics and amino acid composition, and are both induced in response to phenobarbital. Indocyanine green, one of the ligands that is not effective as a substrate, was showh to competitively inhibit the conjugation reaction. It is suggested that specificity is directed toward compounds with electrophilic sites.ligandin is a cytoplasmic protein found in abundance in the liver of rat, man, and other species. This protein is capable of binding noncovalently a large number of compounds, which includes bilirubin, heme, benzyl penicillin, certain steroids, and such dyes as bromosulfophthalein and indocyanine green (1, 2). Phylogenetic, ontogenetic, induction, and competition studies support the hypothesis that ligandin is a major determinant of the net flux of organic anions from plasma into the liver (3-7). Fractionation of the protein on the basis of any one of its binding activities has resulted in apparently identical, highly purified preparations (7-9); Thus, the term, ligandin (2), is synonymous with that of azo-dye carcinogenbinding protein (8), corticosteroid binding I protein (9), and Y protein (7)$, §.The glutathione S-transferases (EC 2.5.1.18) from rat liver (iO-i3) have sevieral physical properties in common with rat liver ligandin (2). When crude liver extracts were subjected to filtration on Sephadex G-75, the fraction containing ligandin also served as a source of enzymatic activity for the conjugation of glutathione (GSH) with 1,2-dichloro-4-nitrobenzene (14). However, subsequent fractionation resulted in removal of alriost all GSH transferase activity with this substrate despite virtually complete recovery of ligandin (t, 15).Since four of the GSH transferases of rat liver (transferases A, B; C, and E) have been purified to homogeneity (11-13), (w/v) and the precipitate was removed by centrifugation after an additional 4 hr at 4°. The superratant fluid was used directly for enzyme assays with 1,2-ehloro-4-nitrobenzene. The precipitate was washed twice with phosphate-buffered saline at pH 7.4, containing 2%o polyethylene glycol. The residue was dissolved in 0.5 M NaOH, and absorbance at 280 nm was determined.

Glucanase (endo-0-1,3-glucan 3-glucanohydrolase, EC 3.2.1.6, laminarinase, callase) and chitinase (poly-0-1,4-[2-acetamido-2-deDxyl-D-glucoside glycanohydrolase, EC 3.2.1.14) were extracted from ethylene-treated bean (Phaseolus vulgaris L. cv. Red Kidney) leaves and purified on hiydroxvapatite and carboxymethyl Sephadex columns. The glucanase prepared was homogeneous as judged by anaIlvtical centrifugation data, electrophoresis, and antibodyantigen reactions. On the basis of gel filtration, antibodyantigen reactions, and amino acid analysis, the molecular weight was estimated to be between 11,300 and 12,300. However, ultracentrifugation gave a higher estimate of 34,000. The glucanase had an isoelectric point near pH 11 and was specific for B-1,3-linkages. The chitinase was only partially purified as judged by electrophoretic behavior.Enzyme systems capable of generating (13) and degrading (8) ,B-1 ,3-glucans have been described. The f-1,3-glucans themselves are widespread and have been associated with callose, leaf and stem hairs, root hairs, cystoliths, pollen mother cells, laticifers, pollen grains, pollen tube walls, wounded parenchyma cells (10), ovules (12), development of microspores from tetrads (15), and cell walls (17). fl-1,3-Glucans are also important components of carbohydrate reserves of cereals, algae, and fungi (5).A number of roles have been proposed for glucanase (fl-1,3-glucanase, laminarinase, callase) in plants. These include degradation of seed glucans (11), control of cell elongation (17, 20) (see however 9, 26), regulation of pollen tube growth (25), cell expansion of yeast (27), fertilization (12, 15), and removal of phloem callose (8).In an earlier paper (1), we described an increase in glucanase in ethylene-treated bean leaves that was associated with protein synthesis de novo and correlated with the removal of callose from the phloem. In order to characterize the enzyme more fully and to evaluate the role of the enzyme in the normal phys- iology of the plant, we have selected a purification procedure that resulted in the preparation of electrophoretically pure glucanase. MATERIALS AND METHODS Preparation of Glucanase. Bean (Phaseolus vulgaris L. cv.Red Kidney) leaves (1,300 g) treated for 3 days with 10 ,ul/liter of ethylene (2) were homogenized with 1,400 ml of water in a Waring Blendor. Except where noted, this and subsequent steps were performed at 5 C. The homogenate was filtered through cheesecloth and centrifuged at 10,000g for 10 min, and the precipitate was discarded (step 1). The supernatant was heated to 60 C for 10 min by placing the flask containing the homogenate in a boiling water bath, stirring rapidly, and monitoring the temperature rise until 60 C was reached. The flask was then removed from the bath, cooled slowly at room temperature, and, after 10 min, placed in an ice bath to lower the temperature to 5 C. The denatured protein was removed by centrifuging at 10,000g for 10 min (step 2).The 60 C supernatant was mixed with diethylaminoethyl cellulose (Cellex-D,...

The initial enzymic step in mercapturic acid formation is catalyzed by glutathione S-transferase.Several species of this enzyme, designated as transferases a, p, y, 6 and E on the basis of increasing isoelectric points, were isolated from human liver. Evidence is presented that each of the purified species is homogeneous with respect to sodium dodecylsulfate -gel electrophoresis. Transferases a, p and E each appear as a single band on gel electrofocusing; transferases y and 6 are present as two and three bands, respectively, with each band catalytically active. Amino acid analysis indicated the five transferases to be either very closely related or identical in this respect.All enzyme species have a molecular weight of about 48 500 and consist of two apparently identical subunits. The spectrum of substrates is the same for each although the enzymes differ slightly in specific activity. As is the case for the rat liver enzymes, each of the human transferases binds bilirubin although this compound is not a substrate.

No abstract