Abstract:
An in vitro investigation of some important factors controlling the
activity of chitin synthase in cell-free extracts of two Mortierella
species has been carried out. Mixed membrane fractions from mycelial
homogenates of Mortierella candelabrum and Mortierella pusilla were found
to catalyse the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine
into an insoluble product characterized as chitin by its insolubility in
weak acid and alkali, and the release of glucosamine and diacetylchitobiose
on hydrolysis with a strong acid and chitinase, respectively. Apparent Km
values for UDP-GlcNAc were 1.8 mM and 2.0 mM for M. pusilla and
~ candelabrum, respectively. Polyoxin D was found to be a very potent
competitive inhibitor with values of the constant of inhibition, Ki' for both
species about three orders of magnitude lower than theKm for UDP-GlcNAc.
A divalent cation, Mg+2 , Mn+2 or Co+2 , was required for activity.
N-acetylglucosamine, the monomer of chitin, stimulated the activity of the
enzyme. The crude enzyme preparation of ~ candelabrum, unlike that of
~ pusilla, showed an absolute requirement for both Mg+2 and N-acetylglucosamine.
Large differences in response to exogenous proteases were
noted in the ratio of active to inactive chitin synthase of the two species.
A fifteen fold or greater increase was obtained after treatment with acid
protease (from Aspergillussaitoi) as compared to a two- to four-fold
activation of the M. pusilla membrane preparation treated similarly. During storage at 4°C over 48 hours, an endogenous activation of chitin
synthase of ~ pus ilIa was achieved, comparable to that obtained by
exogenous protease treatment. The high speed supernatant of both species
inhibited the chitin synthase activity of the mixed membrane fractions.
The inhibitor of ~ pus ilIa was effective against the pre-activated enzyme
whereas that of M. candelabrum inhibited the activated enzyme. Several
possibilities are discussed as to the role of the different factors
regulating the enzyme activity. The suggestion is made from the properties
of chitin synthase in the two species that in vivo a delicate balance exists
between the activation and inactivation of the enzyme which is responsible
for the pattern of wall growth of each fungus.
