LOCAL REDUCTION OF SPINDLE FIBER BIREFRINGENCE IN LIVING NEPHROTOMA SUTURALIS (LOEW) SPERMATOCYTES INDUCED BY ULTRAVIOLET MICROBEAM IRRADIATION

Irradiation of the mitotic spindle in living Nephrotoma suturalis (Loew) spermatocytes with an ultraviolet microbeam of controlled dose produced a localized area of reduced birefringence in the spindle fibers. The birefringence was reduced only at the site irradiated, and only on the spindle fibers irradiated. Areas of reduced birefringence, whether produced during metaphase or during anaphase, immediately began to move toward the pole in the direction of the chromosomal fiber, even though the associated chromosomes did not necessarily move poleward. Both the poleward and the chromosomal sides of the area of reduced birefringence on each chromosomal fiber moved poleward with about the same, constant, velocity. On the average, the areas of reduced birefringence moved poleward with about the same velocities as did the chromosomes during anaphase. The area of reduced birefringence was interpreted as a region in which most, though not necessarily all, of the previously oriented material was disoriented by the irradiation. The poleward movement of the areas of reduced birefringence indicates that the spindle fibers are not static, nonchangeable structures. The poleward movement possibly represents the manner in which the birefringent spindle fibers normally become organized. All the experiments reported were on primary spermatocytes which completed the second meiotic division subsequent to the experimentation. Since both the irradiated and the control cells completed the two meiotic divisions, the movement and irradiation effects studied in the first division were nondegenerative.     

THE ASSOCIATION OF ACETYLCHOLINESTERASE AND MEMBRANE IN SUBCELLULAR FRACTIONS OF THE ELECTRIC TISSUE OF ELECTROPHORUS

Subcellular fractions of the electric tissue of the main organ of the eel Electrophorus electricus were prepared in sucrose media by differential centrifugation and differential discontinuous gradient centrifugation. The distributions of acetylcholinesterase, cytochrome oxidase, DNA, and protein were determined. The appearance of the fractions was determined by phase contrast microscopy and by electron microscopy. A fraction prepared by differectial centrifugation at 30,000 g for 20 minutes in 0.89 M sucrose contained 63 per cent of the total acetylcholinesterase activity at 4 times the specific activity of that of the tissue homogenate. A subfraction prepared by centrifugation in a discontinuous density gradient showed a peak of total and relative specific acetylcholinesterase activity of 35 per cent and 1.9, respectively. The average over-all purification was 7 times. The acetylcholinesterase peak was below the cytochrome oxidase peak and above the DNA peak in the density gradient. The presence of acetylcholinesterase in the fractions was correlated with the presence of large fragments of the cell membrane; however, the presence of other tissue components was noted. The acetylcholinesterase associated with membrane was found to be activated by incubation with sodium deoxycholate. The possible use of the peak fraction containing membranes rich in acetylcholinesterase for the investigation of other components of the acetylcholine system and of other properties of the membrane is discussed.     

Egg Membrane Lytic Activity of Sperm Extract and its Significance in Relation to Sperm Entry in Hydroides hexagonus (Annelida)

Previous electron microscope studies indicated that the individual spermatozoön of Hydroides hexagonus forms a hole in the vitelline membrane by means of lysis. Other observations established that the hole is real, being visible in living material during sperm entry. During the present investigation sea water extracts from frozen-thawed sperm were tested for lytic effect on the membrane. In normal living eggs the membrane appears as a single thick envelope, but in electron micrographs of sections it is seen to consist of a narrow outer border layer, a wide principal or middle layer, and a narrow inner border layer. After immersion in sperm extract the outer border layer elevates but does not dissolve, the middle layer liquefies and disappears, and the inner border layer seems not to change. This is interpreted as lysis of the middle layer. The extract exerted the same effect on fertilized and unfertilized eggs. In electron micrographs the sections treated with extract greatly resemble that part of the membrane which has been penetrated by the individual spermatozoön. It is concluded that the individual spermatozoön, too, exerts a lytic effect. Together, the present and two earlier studies are considered clearly to demonstrate that in Hydroides the individual spermatozoön does indeed make an entry hole in the egg membrane by applying lytic material to that part of the membrane in its own vicinity.     

Immunoenzymological Evidence Suggesting a Change in Conformation of Adenylic Acid Deaminase and Creatine Kinase during Substrate Combination

The kinetics of inhibition of 5′-adenylic acid deaminase and creatine-ATP transphosphorylase by their respective antibodies are studied and rate constants of combination are ascertained. It is shown that the single substrate 5′-adenylic acid (AMP) of deaminase “protects” the enzyme against antibody inhibition. However, phosphate, a competitive inhibitor of the highly specific deaminase, enhances combination with antibody. With creatine kinase, however, addition of either of the substrates, alone or in combination with the required magnesium, each of which separately bind to the enzyme, does not prevent inhibition of the enzyme by its antibody. However, the “working” enzyme combined with all substrates is “protected” against antibody inhibition.