Isolation and characterization of ß-glucosidases from Aspergillus nidulans mutant USDB 1183.

Two extracellular ß-glucosidases (cellobiase, EC 3.2.1.21), I and II, from Aspergillus nidulans USDB 1183 were purified to homogeneity with molecular weights of 240,000 and 78,000, respectively. Both hydrolysed laminaribiose, ß-gentiobiose, cellobiose, p-nitrophenyl-ß-L-glucoside, phenyl-ß-L-glucoside, o-nitrophenyl-ß-L-glucoside, salicin and methyl-ß-L-glucoside but not α-linked disaccharides. Both were competitively inhibited by glucose and non-competitively (mixed) inhibited by glucono-1,5-lactone. ß-Glucosidase I was more susceptible to inhibition by Ag(+) and less inhibited by Fe(2+) and Fe(3+) than ß-glucosidase II.

Protoplast fusion of beta-glucosidase-producing Aspergillus niger strains.

Protoplast fusion, induced by polyethylene glycol and Ca2+, was carried out between two auxotrophic strains of Aspergillus niger. The fusion frequency ranged from 6.2 x 10(-2) - 9.1 x 10(-2). After induced haploidization of a diploid, various segregants showing combinations of the parental genetic markers were isolated. Unlike diploids, haploid segregants exhibited greater variations in their morphology and beta-glucosidase activities. One segregant showed a 2.5-fold increase in beta-glucosidase activity over those of the parents. Thus, this method appears promising for creating new recombinant strains of A. niger with improved beta-glucosidase activities.

Fatty acid modulation of antiestrogen action and antiestrogen-binding protein in cultured lymphoid cells.

Nonsteroidal antiestrogens reversibly and specifically inhibited the proliferation of two estrogen receptor-negative lymphoid cell lines (EL4 and Raji) in a dose-dependent manner. [3H]Thymidine incorporation of concanavalin A-stimulated primary splenocytes was also inhibited by 10(-6) M clomiphene (1-[4-(2-diethylaminoethoxy)phenyl]-1,2-diphenyl-2-chloroethylene). The antiproliferative effect could be prevented by the simultaneous presence in the growth medium of 10(-5) M linoleic acid or 10(-5) M arachidonic acid but not by 10(-6) M estradiol. Both lymphoid cell lines had high affinity antiestrogen-binding sites whose affinity could be altered by conditions of growth. Growth of EL4 cells in RPMI 1640 medium supplemented with charcoal-pretreated 5% fetal calf serum (charcoal-stripped medium) resulted in significantly higher affinity (Kd 0.54 nM +/- 0.11 nM; n = 6) than growth in medium supplemented with untreated serum (complete medium) (Kd = 1.68 nM +/- 0.48 nM; n = 6) (p less than 0.001). This change in affinity was partly due to removal of fatty acids from the growth medium by charcoal pretreatment, since addition of 10(-5) M linoleic acid or 10(-5) M gamma-linolenic to charcoal-stripped medium decreased the affinity of the antiestrogen-binding protein. In contrast, growth in 10(-5) M stearic acid or 10(-5) M oleic acid did not significantly alter the affinity of the antiestrogen-binding protein, whereas 10(-5) M palmitic acid significantly increased its affinity. The same fatty acids were also tested for their intrinsic effects on EL4 cell proliferation. Oleic, linoleic and gamma-linolenic acids were growth stimulatory while stearic and palmitic acids were not. Thus linoleic and gamma-linolenic acids whose presence in the growth medium was associated with decreased affinity of [3H]tamoxifen (1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenylbut-1(Z)-ene) binding to the intracellular antiestrogen-binding protein were also growth stimulatory. Unsaturated fatty acids have previously been shown to inhibit binding of [3H]tamoxifen to the antiestrogen-binding protein in a cell-free system. The present observations demonstrate that unsaturated fatty acids also modify the affinity of the antiestrogen-binding protein in intact cells.