Protein kinases of the chick oviduct: a study of the cytoplasmic and nuclear enzymes.

Subcellular fractionation of oviduct tissue from estrogen-treated chicks indicated that the bulk of the protein kinase activity of this tissue is located in the cytoplasmic and nuclear fractions, DEAE-cellulose chromatography of cytosol revealed a major peak of cAMP stimulatable activity eluting at 0.2 M KCl. This peak was further characterized and found to exhibit properties consistent with cytoplasmic cAMP dependent protein kinases isolated from other tissues; it had a Km for ATP of 2 X 10(-5) M, preferred basic proteins such as histones, as substrate, and had a M of 165 000. Addition of 10(-6) M cAMP caused the holoenzyme to dissociate into cAMP binding regulatory subunit and a protein kinase catalytic subunit. Extraction of purified oviduct nuclei with 0.3 M KCl released greater than 80% of the kinase activity in this fraction. Upon elution from phospho-cellulose, the nuclear extract was resolved into two equal peaks of kinase activity (designated I and II). Peak I had a sedimentation coefficient of 3S and a Km for ATP of 13 muM. while peak II had a sedimentation coefficient of 6S and a Km for ATP of 9 muM. Both enzymes preferred alpha-casein as a substrate over phosvitin or whole histone, although they exhibited different salt-activity profiles. The cytoplasmic and nuclear enzymes were well separated on phospho-cellulose and this resin was used to quantitate the amount of cAMP dependent histone kinase activity in the nucleus and the amount of casein kinase activity in the cytosol. Protein kinase activity in nuclei from estrogen-stimulated chicks was found to be 40% greater than hormone-withdrawn animals. This increase in activity was not due to translocation of the cytoplasmic protein kinase in response to hormone, but to an increase in nuclear (casein) kinase activity. During the course of this work, we observed small but significant amounts of cAMP binding activity very tightly bound to the nuclear fraction. Solubilization of the binding activity by sonication in high salt allowed comparison studies to be performed which indicated that the nuclear binding protein is identical with the cytoplasmic cAMP binding regulatory subunit. The possible role of the nuclear binding activity is discussed.     

Pollination and nucellus inAbelmoschus esculentus

Pollination induced dissolution of nucellar cells at the micropylar end forming a passage for pollen tube entry even when the pollen tubes are in the stylar region.     

Non-equivalence of YEPD and synthetic complete media in yeast reversion studies

In yeast reversion studies, assay of the total number of cells is made by plating irradiated cells on agar plates containing yeast extract, peptone and dextrose (YEPD) medium. The number of revertants are scored by plating cells on synthetic complete (SC) medium deficient in the particular nutrient for which the reversion is tested. In this procedure equivalence for cell survival between the YEPD and the SC media is always assumed. However it is shown in this paper that this assumption is valid only up to dose levels where cell killing is not significant. At high doses, survivals on the two media differ significantly from each other for both high and low LET radiations. This difference influences the slope of the reversion frequency curve at high doses. Since the reversion frequency is expressed with reference to the number of survivors after a given radiation dose, it is essential to see that the same chance of survival is offered to the reverted and unreverted cells.Even though reversion is reported to vary linearly with dose, it is found that this linearity is restricted only to dose levels where cell killing is not significant. At higher doses, the reversion frequency varies in a very complex manner with dose for both high and low LET radiations. The complexity depends further on the reference medium chosen.     

Self-association of diisopropylphosphoryl-alpha-chymotrypsin: the involvement of active site of the enzyme in its self-association behaviour

The self-association of diisopropylphosphoryl(DIP)-alpha-chymotrypsin is studied in order to find out whether the active site of the enzyme is involved in its self-association behaviour or not. Sedimentation coefficient as well as the weight-average (Archibald) molecular weight data are obtained as a function of concentration using an analytical ultracentrifugation technique. The analysis indicated that the experimental data fits the model of indefinite self-association. The comparison of the data with earlier data on alpha-chymotrypsin revealed that after the modification at the active site, the association constant for the self-association is reduced by about 47%, and the system deviated from ideality. Results showed further that Ser-195, at the active site, appears to be involved in the self-association behaviour of alpha-chymotrypsin; however, the participation of other groups at the active site is also implicated.