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An endogalactosaminidase has been purified 34-fold from the culture filtrate of Streptomyces griseus. This enzyme cleaves GalN-GalN linkages in oligogalactosaminoglycan, a galactosamine-rich oligosaccharide isolated from the culture filtrate of a Neurospora mutant. Since some or all of the GalN-GalN bonds in this molecule link positions 1 and 4, and are in the alpha-configuration, we are probably dealing with an endo-alpha-(1 leads to 4)-galactosaminidase, bu this characterization is only tentative because the few bonds cleaved by the enzyme could have a different structure. The enzyme is inactive towards N-acetyl-oligogalactosaminoglycan and chitosan. The endogalactosaminidase preparations also cleave high molecular weight galactosaminoglycan (obtained from Neurospora) into fragments greater than or equal to 10(4) daltons in molecular weight, and catalyze the release of Neurospora sporelings from the glass surfaces to which they are anchored. Galactosaminoglycan-cleaving and sporeling-releasing activities elute jointly from DEAE-cellulose columns. This observation provides further support for an earlier proposal that the sporelings are anchored to the glass by means of galactosaminoglycan molecules.

The inactivation of Neurospora crassa conidia by galactosaminoglycan isolated from cultures of this organism was followed by measuring colony-forming ability and ability to take up radiolabeled metabolites. When kinetic data on the loss of transport function and on killing were analyzed by use of target theory, it appeared that few “hits” are required for inactivation. However, studies with radio-labeled galactosaminoglycan mucopolysaccharides showed that cells receiving a single lethal hit have approximately 10 5 galactosaminoglycan molecules bound to them.

SUMMARY: Forward mutations at the pyr locus in Neurospora, and back mutations at arg, were scored by using the ‘suppressor method’. This method is efficient and well suited for mutagen screening. Nitrous acid, ethyl-methane sulphonate and ultraviolet radiation were effective as mutagens; 8-ethoxycaffeine yielded inconclusive results. The same pattern of forward mutations at pyr, but different frequencies of back mutations at the arg locus, were obtained with the three agents. In this Neurospora system, nitrous acid appeared to induce mutations which affected simultaneously both DNA strands.

SUMMARY: About 200 forward mutants of Neurospora obtained as suppressors of arg were studied. All fell in the pyr region, and all appeared deficient in aspartic transcarbamylase (ATC) to various degrees: extreme deficiencies in ATC (pyr-N alleles) resulted in pyrimidine-dependence as well as suppression of arg, while partial ATC deficiencies (pyr su-arg lleles) led only to suppression. Five different complementation groups were represented among the pyr-N alleles studied. Evidence is presented which suggests that pyr-N and pyr su-arg lleles affect ATC via different mechanisms.

The conditions required for in vitro complementation between a pair ot mutants deficient in aspartic transcarbamylase were determined. It was observed that complementation in vitro and in vivo produces varieties of aspartic transcarbamylase which are distinguishable from the wild type enzyme and from each other on the basis of thermal stability and other criteria. The significance of these findings with reference to the molecular mechanism of complementation is discussed.

original Fil: Reissig, José L.. Instituto de Investigaciones Bioquímicas Fundación Campomar; Argentina Fil: Strominger, Jack L.. National Institute of Arthritis and Metabolic Diseases. National Institutes of Health. Bethesda. Maryland; Estados Unidos Fil: Leloir, Luis Federico. Instituto de Investigaciones Bioquímicas Fundación Campomar; Argentina Blanco y negro 8 páginas en pdf LFL-PI-O-ART. Artículos científicos Unidad documental simple AR-HYL-2018

A system for the screening of reversions in an arginine-dependent strain of Neurospora has been developed.Reversion resulted from any one of three events:(a) Back mutation of the originalarggene.(b) Forward mutation at thepyrlocus. This has the pleiotropic effect of suppressingargwhile determining a pyrimidine requirement. Interaction with the unmutated nuclei of the same conidium permits growth on minimal medium.(c) Forward mutation at thepyrlocus to alleles of intermediate activity. These alleles suppressarg, but do not create a pyrimidine requirement.

1. 1. Two enzyme system capable of synthesizing carbamyl phosphate in cell-free extracts of Bacillus subtilis were studied after separation from each other by fractionation with (NH 4 2 SO 4 . 2. 2. Carbamyl phosphate synthetase utilizes as substrates ATP ( K m = 4.9 mM), glutamine ( K m = 1.8 mM) and bicarbonate; and has a pH optimum of 7.5. It is inhibited by arginine and pyrimidines, and its formation is repressed by uracil, with a concerted effect of arginine at lower uracil concentrations. A biosynthetic role for this enzyme is thus indicated. 3. 3. Carbamate kinase (ATP:carbamate phosphotransferase, EC 2.7.2.2) uses ATP ( K m = 15 mM) and ammonium carbamate ( K m = 8mM), but not glutamine, as substrates. Its pH optimum is 8.5. It is more stable than carbamyl phosphate synthetase. It is partially inhibited by glutamine and arginine, and induced by arginine. This induction suggests a catabolic role. 4. 4. Single mutational events lead to auxotrophy for arginine and pyrimidines, and marked reductions in the levels of carbamyl phosphate synthetase and carbamate kinase. The double requirement can be most simply understood if a single synthetase feeds into a carbamyl phosphate pool common to the arginine and the pyrimidine pathways. The pleiotropic effect on the two enzyme systems points to additional interrelationships. Perhaps the two enzymes share structural or regulatory elements.

Studies with a pair of Neurospora mutants deficient in aspartate transcarbamylase (carbamoylphosphate: l -aspartate carbamoyltransferase, EC 2.1.3.2) showed the following: 1. 1. Aspartate transcarbamylase activity could be reconstituted by incubation in vitro of ribosomes from one mutant with soluble proteins from the other, or vice versa . This system will be referred to as the ribosomal complementation system. Ribosomes from the two mutants failed to complement each other in vitro . 2. 2. The ribosomal complementation system did not require 2-mercaptoethanol unlike the system for complementation between soluble proteins in vitro . The 2-mercaptoethanol requirement of the soluble system could not be circumvented by the addition of ribosomes from a third mutant, which is transcarbamylase deficient and complementation negative. 3. 3. The aspartate transcarbamylase generated by the ribosomal complementation system remained ribosome-bound after repeated washings. 4. 4. The thermostability of the aspartate transcarbamylase generated by the soluble system depended upon the input ratio of the two mutant extracts, while the thermostability of the product of the ribosomal system was independent of the input ratio and indistinguishable from that of an aspartate transcarbamylase obtained by complementation in vivo . These results are interpreted as tentative indications that complementation is intercistronic. 5. 5. The ribosomal system is suitable for the study of hybrids between wild and mutant enzymes.

Publisher Summary This chapter discusses the mutase activity of phosphoacetylglucosamine from Neurospora . A convenient method for following mutase activity is to incubate the enzyme preparation with N -acetylglucosamine-l-P and catalytic amounts of glucose-l,6-P. The N -acetylglucosamine-6-P formed is measured by a color test for N -acetylamino sugars, which is negative for N -acetylglucosamine-l-P. This assay is not applicable in the presence of phosphatase activity. Phosphatase activity interferes by releasing N -acetylglucosamine. In its presence, it is best to assay mutase activity by following the increment in acid-stable phosphate which results from migration to position 6. The incubation is carried out as in the standard assay, and the reaction is stopped by the addition of 0.3 ml 10% trichloroacetic acid. After centrifugation, an aliquot of the supernatant fluid is used for the determination of acid-labile inorganic phosphate by the method of Fiske and SubbaRow. Phosphate disappearance—that is, stabilization—is calculated by comparison with an unincubated mixture. Only the Neurospora and submaxillary gland activities are associated with enzymes different from phosphoglucomutase.

Neurospora produces a mucopolysaccharide (called MP) which inhibits its growth, causes vacuolation and agglutination of its cells, and precipitates its purified membrane protein. Cultures of a colonial strain display a phase of slow growth; the induction of this phase is traced to the production of MP by the mold. Stationary-phase cultures of wild type also produce MP. MP is a polymer of galactosamine, its amino groups only partially acetylated, probably containing other minor components. MP molecular weight is approximately 10 6 . Complete acetylation abolishes the biological activities of MP. It is suggested that the regulatory effect of MP is mediated by its interaction with the protoplasmic membrane.

Recent work on the regulation of growth and differentiation in metazoan cells has conjured up a fascinating picture of the cell surface as a prime substation in the coding and decoding of regulatory signals. I am referring to studies on contact inhibition and growth control in mammalian cells (Burger, 1971, 1973; Dulbecco, 19714; Sambrook, 1972; Stoker, 1972), on lectin-induced transformation of resting into proliferating lymphocytes (Greaves and Bauminger, 1972; Lindahl-Kiessling, 1972; Rubin et al., 1972), on immune response (Mitchison, 1971), on the action of polypeptide hormones (Rodbell, 1972), on specific cell adhesion as related to morphogenesis (Moscona, 1968; Steinberg, 1964; McGuire, 1972), on fertilization and early development (Tyler, 1965; Metz, 1967; Tomkins and Rodman, 1971), on the action of diphtheria toxin (Pappenheimer et al., 1972), etc. In contrast, the microbial surface seems quite dull. But is it? Evidence to the contrary has been known for a number of years. Thus, in 1965, Kalckar summarized results indicating that the polymers of the external surface of a bacterium (which he named “ektopolymers”) play a decisive role in determining the repertoire of its social activities. Until very recently interest in the “ektobiology” of microorganisms—social or regulatory—has been limited, but a reversal of this neglect is now evident (see, for instance, Tomasz, 1971). Two factors are probably significant in this reversal.