ISSN:
0148-7280
Keywords:
acetylcholinesterase
;
sea urchin spermatozoa
;
enzyme purification
;
Life and Medical Sciences
;
Cell & Developmental Biology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
Notes:
Flagella contain the bulk of spermatozoan acetylcholinesterase. Brief sonication of sea urchin sperm suspended in Tris-buffered (pH 8.0), Ca, Mg-free artificial sea water (F-ASW) containing 10 mM ethylene diaminetetracetic acid, (EDTA) doubled the specific activity over that of the intact spermatozoa. Lipids were removed from the solubilized supernatant of the tail membrane fraction by ether extraction. Hydrolysis of acetylthiocholine in the presence of dithiobisnitrobenzoic acid (DTNB) was monitored spectrophotometrically at 412 nm by the Ellman procedure. The enzyme was purified by affinity chromatography on a Sepharose cyanogen bromide gel to which the cholinesterase inhibitor trimethyl (para-aminophenyl) ammonium chloride was coupled. The enzyme was eluted from the column with a discontinous NaCl gradient (0.1-0.5 M). The active fraction recovered at 0.35 M NaCl contained 0.007% of the initial total sperm cell protein with a 500-fold increase in specific activity.Twenty-four hr centrifugation on a 5-20% sucrose density gradient at 50,000g in a Beckman L5-75 centrifuge yielded peaks at 14.7 S and 9.1 S. In the presence of 1% Triton X-100, three peaks appeared: 23.3 S, 13.7 S, and 9.1 S. These sedimentation coefficients resemble those of the electroplax acetylcholinesterase (AChE) forms A8 and A4.Eserine completely inhibited the activity of the purified enzyme, which exhibits a substrate optimum at 4 mM acetylcholine. The activity is depressed by 75% at 10 mM ACh and by 90% at 25 mM. The Km was 2.1 × 10-4 M. In the sperm cell the enzyme that terminates the action of intracellularly synthesized ACh may be involved in controlling ionophoric channels that regulate transmembrane transport of calcium.
Additional Material:
4 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/mrd.1120140405
