A 13-glucanase (Z-glucanase) from Zymolyase was freed from a protease (Z-protease) by affinity chromatography on a2-macroglobulin-Sepharose columns and used to solubilize proteins from isolated cell walls of Saccharomyces cerevisiae. The cell wall proteins were labeled with 12'I and subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis and autoradiography. The bulk of the labeled material had very low mobility. Its mannoprotein nature was demonstrated by precipitation with specific antibodies and by conversion to a band with an average molecular weight of 94,000 after incubation with endo-1-Nacetylglucosaminidase. The intact mannoproteins were hydrolyzed by Z-protease, but were resistant to the enzyme when the carbohydrate was first removed by endo-oN -acetylglucosaminidase. In intact cells, lysis of cell walls by Z-glucanase required a previous incubation with Z-protease, which led to solubilization of most of the 125I-labeled proteins. Other proteases that did not attack the cell wall mannoproteins were unable to substitute for Z-protease. The specific effect of Z-protease is consistent with the notion that mannoproteins form a surface layer of the cell wall that penetrates the wall to some depth and shields glucans from attack by, Zglucanase. Mannoproteins, however, do not appear to cover the inner face of the cell wall, because isolated cell walls, in contrast to intact cells, were completely solubilized by Z-glucanase in the absence of protease. The function of mannoproteins in determining cell wall porosity was highlighted by the finding that horseradish peroxidase (Mr, 40,000) causes lysis of cells that had been treated with Z-protease. Depletion of mannoproteins by Z-protease also resulted in the disappearance of a darkly stained surface layer of the cell wall, as observed by electron microscopy. Other agents that facilitate cell lysis by Z-glucanase, such as 2-mercaptoethanol, digitonin, and high concentration of salts, caused little or no solubilization of mannoprotein. We assume that they perturb and loosen the structure of the mannoprotein network, thereby increasing its porosity. The implications of our results for the construction of the yeast cell wall and the anchoring of mannoprotein to the cell are discussed.

Nocardiosis is a well-described infection in immunocompromised patients, and has been rarely documented in patients with AIDS. Nocardia asteroides is the most frequently isolated etiologic agent. Rare cases are due to N. brasiliensis and N. otitidiscaviarum. This work describes the first case of nocardiosis in Italy caused by N. otitidiscaviarum in an AIDS patient. A 31 year-old intravenous drug abuser with a diagnosis of full-blown AIDS, presented with high fever and lymphadenitis with a fistula draining copious purulent discharge. Broad-spectrum antibiotic therapy was initiated, but the patient did not shown any improvement. Direct examination of the pus revealed numerous gram positive rods. When cultured N. otitidiscavarium was isolated and identified by morphological and biochemical tests.

Nocardia asteroids and Nocardia brasiliensis whole-cell extracts were used as antigens to generate monoclonal antibodies (MAbs). Six stable hybrid cell lines secreting anti-Nocardia spp. MAbs were obtained. These were characterized by enzyme-linked immunosorbent assay, Western blot (immunoblot), and immunofluorescence assay. Although all the MAbs exhibited different degrees of cross-reactivity with N. asteroids and N. brasiliensis antigens as well as with culture-filtrate antigens from Mycobacteria spp., they have the potential for use as reagents in the purification of Nocardia antigens.

Like many other bacteria, Nocardia sp. possess acid phosphatase activity. In N. brasiliensis, a human and animal pathogen, this activity was resolved into two enzyme forms by native gel electrophoresis. One (isozyme I) was partially purified and characterized. It exhibited an estimated molecular weight on SDS-PAGE of 50 kDa, a pH optimum of 5.2, and a Km value of 1.25 mM for p-nitrophenylphosphate. The N. brasiliensis enzyme was stable at 4 degrees C for at least 24 h, but readily inactivated at 60 degrees C. Ammonium molybdate, sodium fluoride and L-(+)-tartrate were found to be potent inhibitors of the enzyme. Although its function is presently unknown, by analogy to other bacterial systems it could be envisioned to play an important role in the physiology and pathogenicity of the microorganism.

Nocardia brasiliensis possess proteolytic activities that can be readily detected in a variety of media. In a modified formulation of a growth imedium originally used for Streptonrces aureofaciens, N. brasiliensis was found to secrete proteolytic enzymes, one of which was capable of hydrolyzing casein. This enzyme was purified to homogeneity from cell-free culture filtrates of N. brasiliensis. The purification procedure included ion-exchange chromatogrtphy on carboxymethyl-Sepharose, gel filtration on Sephadex G-100, and affinity chromatography, using a hemoglobin-Sepharose resin. The molecular weight of the N. brasiliensis protease was found to be 25,000 by gel filtration and 35,000 by sodium dodecyl sulfate-discontinuous gel electrophoresis. The enzyme is inhibited by o-phenanthroline and 8-hydroxyquinoline-5-sulfonic acid but is not affected by EDTA. Average values for its kinetic parameters were 0.288 jxmol of hemoglobin solubilized per min per mg of enzyme for Vmax and 0.76 mM for K, using hemoglobin as the substrate.