A cDNA coding for glucoamylase P of Hormoconis resinae was cloned using a synthetic oligonucleotide probe coding for a peptide fragment of the purified enzyme and polyclonal anti-glucoamylase antibodies. Nucleotide-sequence analysis revealed an open reading frame of 1848 base pairs coding for a protein of 616 amino-acid residues. Comparison with other fungal glucoamylase amino-acid sequences showed homologies of 37-48%. The glucoamylase cDNA, when introduced into Saccharomyces cerevisiae under the control of the yeast ADC1 promoter, directed the secretion of active glucoamylase P into the growth medium.

Two extracellular glucoamylases (EC 3.2.1.3), glucoamylase P and glucoamylase S, were purified to homogeneity from the culture medium of Hormoconis resinae (ATCC 20495; formerly Cl&sprium resinae) by a new method. Their apparent molecular masses (71 kDa glucoamylase P; 78 kDa glucoamylase S) and catalytic properties agreed well with those previously reported in the literature. Heat inactivation studies suggested that the high debranching (1,6-glycosidic) activity of glucoamylase P preparations (measured with pullulan) may reside in the same protein molecule as its l,&glycosidic activity (measured with soluble starch). Although glucoamylase S had virtually no debranching activity, it cross-reacted with polyclonal antibodies raised against glucoamylase P, and the two enzymes had very similar amino acid compositions. However, peptide mapping and amino-terminal sequencing studies of the peptides showed that the two enzymes have different sequences and must be encoded by different genes. IntroductionEnzymes that degrade poly-and oligosaccharides are commercially important for the food and fermentation industries. Glucoamylases (1,4-glucan glucohydrolases ; EC 3.2.1 .3) release glucose units sequentially from the nonreducing end of polymeric carbohydrates. The ability of glucoamylases to hydrolyse 1,6-glycosidic bonds is called debranching activity and is of great importance in industrial processes requiring complete degradation of starch to glucose.Glucoamylases are produced by a wide variety of micro-organisms (Manjunath et al., 1983). Most of the known glucoamylases have very low activity towards 1,6-glycosidic bonds. The analytical measurement of debranching activity is not straightforward, since the activity of glucoamylases on substrates containing only 1,6-glycosidic bonds is usually quite low. To avoid this difficulty, substrates like pullulan (every third bond 1,6-glycosidic) are often used to estimate 1,6-hydrolysing activity. Many fungal glucoamylases have been reported to exist in multiple forms (Lineback & Bauman, 1970;Tsuboi et al., 1974;Yamasaki et al., 1977;Takahashi et al., 1978Takahashi et al., , 1981. In order to clone the appropriate gene it is essential to know if these multiple forms are coded by different genes, or if they are results of post-translational al. (1984) these two forms of the enzyme are products of two differently spliced mRNA molecules coded by the same gene, whereas Svensson et al. (1986) have pointed out that proteolytic modification also affects the multiplicity of this enzyme.The fungus Hormoconis resinae (ATCC 20495; formerly Cladosporium resinae) has been reported to produce two forms of glucoamylase which exhibit different substrate specificities and have different molecular masses and PI values (McCleary & Anderson, 1980). The smaller glucoamylase P has a very high debranching activity, while the larger glucoamylase S has virtually no debranching activity. In the present work, we examined whether the 1,4-and 1,6-glycosidic activities of glucoamylase P are both functions...

Uteroglobin, a progesterone‐regulated secretory protein in rabbit uterus, was used as a marker protein for studies on progestational activity of various natural and synthetic androgens. All the androgens investigated were able to induce uteroglobin synthesis in rabbit uterus; some of the synthetic androgens were even better inducers than progesterone itself. Our results suggest that androgens elicit their regulatory action on uteroglobin synthesis by way of progestin receptor mechanism, since: (i) There was an intimate correlation between the in vitro binding affinity to progestin receptor and the in vivo potency of the androgens to induce uteroglobin synthesis; (ii) Androgens were able to translocate cytosol progestin receptors to uterine nuclei; (iii) Dose‐response curves for uteroglobin induction were parallel for androgens and progestins, and (iv) Flutamide, a non‐steroidal antiandrogen, did not abolish androgen‐induced synthesis of uteroglobin or androgen‐promoted nuclear translocation of cytosol progestin receptors. Both progesterone and androgens seem to control uteroglobin synthesis through mechanisms involving formation of new mRNA species, since in each case there was an increase in the uterine preuteroglobin‐mRNA activity, as evaluated by a cell‐free in vitro translation assay, which correlated with the amount of uteroglobin secreted into the uterine fluid. Some of the androgens studied (7α, 17α‐dimethyl‐19‐nortestosterone, 17α‐ethyl‐19‐nortestosterone and 11‐methylene‐17α‐methyl‐19‐nortestosterone) enhanced uteroglobin synthesis to the same or greater extent than progesterone. Interestingly, these steroids are also known to be very potent androgens. The progestational actions of androgens may be applicable to human tissues, too, since all the androgens investigated were bound by the human uterine progestin receptor in a fashion identical with the rabbit receptor.

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