Properties and Cellular Localization of Chitin Synthetase in Phycomyces blakesleeanus

Abstract

Abstract The response of Phycomyces sporangiophores to various stimuli show up as changes in the elongation rate of the cell wall, a structure largely composed of chitin fibrils. The enzyme chitin synthetase was chosen as the subject of this study for the possible role that regulation of its activity might play in the behavioral outputs. A simple assay for the chitin synthetase has been developed. Membrane preparations from the mycelia of Phycomyces were found to catalyze the synthesis of chitin from UDP-N-acetyl-d-glucosamine. Both Mg2+ and N-acetyl-d-glucosamine stimulate the enzyme activity. The pH optimum for the enzyme activity is about 6.5, and the temperature optimum is about 28°. The Km for UDP-N-acetyl-d-glucosamine is 0.6 mm. The antibiotic polyoxin D competitively inhibits the activity at levels which are comparable to those required for the inhibition of mycelial growth. In the presence of polyoxin D (0.1 mm), germinating spores of Phycomyces develop into protoplast-like structures. At concentrations up to 0.5 mm, ATP stimulates the enzyme activity and at 2 mm, cyclic 3' : 5'-AMP is slightly inhibitory. The enzyme activity was found in the Phycomyces mycelia of different ages as well as in the sporangiophores of each of the five developmental stages. Phycomyces mycelial homogenate was fractionated through a series of differential and isopynic centrifugations. Each fraction was studied for its (a) specific and total chitin synthetase activities; (b) morphology under the electron microscope; and (c) specific and total activities of the marker enzymes 5'-nucleotidase (plasma membrane), glucose 6-phosphatase (endoplasmic reticulum), succinic-2-(p-indophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium-reductase (mitochondria), catalase (peroxisomes) and acid phosphatase (lysosomes). The fraction which exhibited the highest specific activity contained essentially only membraneous structures. The distribution of the specific chitin synthetase activity coincided with that of 5'-nucleotidase activity and did not coincide with that of any of the other marker enzymes. These results suggest that the chitin synthetase is a plasma membrane-bound enzyme. Autoradiography studies showed that along the sporangiophore the growing zone (where cell elongation takes place) has higher chitin synthetase activity than the nongrowing zone.