Abstract
Single crystals of the protein lysin (Mr = 16,070) from the spermatozoa of the red abalone (Haliotis rufescens) have been obtained by the vapor diffusion technique, using as precipitants a 32.5% saturated solution of (NH4)2SO4 (incubation at 18 degrees C) or a 5% w/v polyethyleneglycol 8000 solution (incubation at 29 degrees C), both in Bis-Tris-iminodiacetic acid buffers of pH 7.0. The addition to the droplets of EDTA, other carboxylate-containing polyanions, and/or organic solvents improved the size and quality of the crystals and, especially with (NH4)2SO4, addition of EDTA, and/or organic solvents produced a change in crystal habit which resulted in crystals more elongated in the b direction. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with a = 52.3 A, b = 46.0 A, and c = 81.5 A and one molecule per asymmetric unit. The crystals diffract to 2.3 A resolution. The molecular structure of lysin is relevant to the nonenzymatic mechanism by which the protein dissolves a hole in the egg vitelline layer during fertilization.
Highlights
The addition to the droplets of EDTA, other carboxylate-containing polyanions, and/or organic solvents improved the size and quality of the crystals and, especially with
The molecular structure of lysin is relevant to the nonenzymatic mechanism by which the protein dissolves a hole in the egg vitelline layer during fertilization
Sperm of the red abalone Haliotis rufescens have a large acrosome granule containing two major proteins, lysin being one of them. It is a cationic protein which can be obtained in high yield and purity from the acrosome granule and which displays potent activity in dissolving the egg vitelline layer [1]
Summary
Lysin Preparation-Lysin was isolated from sperm of H. rufescens as previously described [1] and stored at -20 “C. Except for a very minor contaminant of M, 26,000, the preparation showed a single major band by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis performed as described [1]. Aliquots of the frozen preparation, containing 0.9 mg of protein per ml in 50 mM Tris-HCl,- 10. A small amount of aggregated protein which formed during concentration was removed by a 5-min centrifugation at 2,000 X g. The supernatant solution containing 8.5-13.5 mg of protein/ml (Bio-Rad protein assay with bovine y-globulin as standard) was divided into aliquots and stored at -70 “C. Reservoirs contained 1 ml of precipitant solution. Droplets were a mixture of 5 ~1 of concentrated protein solution plus 5 ~1 of the reservoir solution plus 2 ~1 of any additive, when used. For PEG experiments, an additional 10 ~1 of 4 M NaCl were added to the reservoir solutions after mixing the droplets, in order to prevent water condensation on the cover glasses which occurs at low PEG concentrations.
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