I. Studies on the Motility and Biochemistry of Cilia. II. Chitinase Activity during Drosophila Development

Biology

Winicur, Sandra ; Mitchell, Herschel K.

University:California Institute of Technology

Department:Biology

关键词: Biology;   

Others  :  https://thesis.library.caltech.edu/10499/1/Winicur_S_1971.pdf

美国|英语

来源: Caltech THESIS

【 摘 要 】

Part I

Cilia isolated from Tetrahymena after inducing ciliary shedding by addition of calcium chloride to cells suspended in an ethanolsolution have been shown capable of motility in the presence of ATP. The potential for motility was restored by treating the isolated cilia with glycerol, ethylene glycol or digitonin-sucrose solutions. Up to 80% of these cilia were motile upon addition of ATP or ADP. These cilia showed an optimum reactivation temperature of 16° C.

The cilia are most motile in concentrated solution. Addition of xylose and dextrose, both of which are present in high concen­tration in native cilia, cause a sharp increas in percent motility.

The ATPase activity was also studied under different conditions of motility.

Regeneration of cilia by Tetrahymena deciliated by pH 5 treat­ment was observed. This regeneration took about 7 hours.

Part II

Before both larval molts in Drosophila melanogaster, the chitinin the cuticle is digested to a significant degree by the molting fluid. A spurt of chitinase activity appears just before each molt, drops sharply after the first molt and begins to rise again just about thetime that chitin degradation becomes visible in electron micrographs. The level of enzyme activity per mg of soluble protein or per mg of wet weight reached just before the second molt is about twice that before the first, and this declines gradually after the molt until puparium formation.

The activity measured per individual larva however starts a slow rise after hatching with a slight peak at the second molt and continues to rise to a point mid way between the second molt and puparium for­mation.This indicates that some of the measured chitinase activitymay be due to the activity of another enzyme, either a different chitinase, a lysozyme, or possibly a chitin synthetase, which would be most active during the third instar.

Data existsupporting the presence of more than one enzyme with chitinase activity. Two fractions can be separated by ammonium sulfate precipitation and three peaks can be separated on a DEAE-Sephadex column.

The enzyme activity is stable, with no loosely bound cofactor and a single isoelectric point about 3.8. Chitinase activity was measured by a viscometric assay on a substrate of chitosan, a partially deacety­lated solubilized product of chitin.