Alkaline ribonuclease and phosphodiesterase activity in rat liver plasma membranes

The ribonuclease and phosphodiesterase activities of rat liver plasma membranes, purified from the crude nuclear fraction by centrifugation in an A-XII zonal rotor and flotation, were examined and compared. The plasma membrane is responsible for between 65 and 90% of the phosphodiesterase activity of the cell and between 25 and 30% of the particulate ribonuclease activity measured at pH8.7 in the presence of 7.5mm-MgCl(2). Both enzymes were most active between pH8.5 and 8.9. Close to the pH optimum, both enzymes were more active in Tris buffer than in Bicine or glycine buffer. Both plasma-membrane phosphodiesterase and ribonuclease were strongly activated by Mg(2+), there being at least a 12-fold difference between the activity in the presence of Mg(2+) and of EDTA. There is, however, a difference in the response of the enzymes to Mg(2+) and EDTA in that the phosphodiesterase is fully activated by 1.0mm-MgCl(2) and fully inhibited by 1.0mm-EDTA, whereas the ribonuclease requires 7.5mm-MgCl(2) for full activation and 5mm-EDTA for full inhibition. Density-gradient centrifugation has indicated that on solubilization in Triton X-100 most of the ribonuclease activity is released into a small fragment of the same size as that containing the phosphodiesterase activity. The relationship between the two activities is discussed in view of these results.     

Estrogen-induced changes in the maltase activity of rat uterus

The effect of estrogen administration on uterine maltase activities at pH 4.3, 5.6 and 6.2 have been studied in the ovarleotomized immature rat. Estradiol (in total doses of 0.5, 1.0 and 2.0 /ug) for 3 consecutive days resulted in increased uterine maltase levels at pH 4.3 and pH 5.6, though the activity at pH 6.3 remained unchanged. A single injection of 2/ug estradiol resulted in an increase in the enzyme activities at pH 4.3 and pH 5.6; this was detectable in 36 hours and reached peak levels 48 hours after the hormone injection. Actinomycin and cycloheximide blocked the estradiol-induced increase in the enzyme activities. Pretreatment with clomiphene also neutralized estradiol-induced increases in the enzyme activities.     

Purification of a calcium dependent ribonuclease from Xenopus laevis.

We have purified a Ca2+ dependent ribonuclease from the oocytes of Xenopus leavis. Two properties of this ribonuclease set it apart from other known nucleases. First, Ca2+ was required for ribonuclease activity, and Mg2+ would not substitute. Second, the enzyme specifically degraded RNA and digestion of double or single stranded DNA was not observed. Ca2+ dependent ribonuclease activity of the purified 36-kDa protein was directly observed after renaturation of the protein following electrophoresis in an SDS-Laemmli gel. In addition, the enzyme was shown to have endoribonuclease activity at numerous sites. The Ca2+ dependence suggests that the ribonuclease activity may be modulated by changes in the level of intracellular Ca2+ and thereby provide a direct link to signal transduction systems.     

Change of nuclear ribonuclease H activity following deoxyribonucleic acid synthesis in liver of protein-deficient rat

The activities of RNA polymerase I and Mg²⁺ - dependent ribonuclease H (RNase H) become doubled in the liver nuclei of rats that are fed for several days a diet that lacks amino acids or protein. Both Mn²⁺- and Mg²⁺- dependent RNases H activities increased two- to three-fold in the liver nuclei of rats in which hepatic DNA replication has been induced by a dietal manipulation.     

Human pancreatic ribonuclease presents higher endonucleolytic activity than ribonuclease A

Analyzing the pattern of oligonucleotide formation induced by HP-RNase cleavage shows that the enzyme does not act randomly and follows a more endonucleolytic pattern when compared to RNase A. The enzyme prefers the binding and cleavage of longer substrate molecules, especially when the phosphodiester bond that is broken is 8-11 nucleotides away from at least one of the ends of the substrate molecule. This more endonucleolytic pattern is more appropriate for an enzyme with a regulatory role. Deleting two positive charges on the N-terminus (Arg4 and Lys6) modifies this pattern of external/internal phosphodiester bond cleavage preference, and produces a more exonucleolytic enzyme. These residues may reinforce the strength of a non-catalytic secondary phosphate binding (p₂) or, alternatively, constitute a new non-catalytic phosphate binding subsite (p₃).     

Ultrafiltration membrane with ribonuclease activity

Summary RNase was immobilized by entrapment in polymer matrix during the formation of the ultrafiltration membrane. The resulting membrane can hydrolyze RNA with simultaneous separation of reaction products (nucleotides) and RNA. The RNase activity has not decreased after one month of operation.     

Multicompartmental distribution of ribonuclease II in mouse liver

Thirty percent of RNase II (EC 3.1.27.5) is present in the cytosol of mouse liver where it exists in an inactive complex with a protein inhibitor. The remaining 70% of RNase II is active, soluble enzyme unassociated with inhibitor and is distributed in a ratio of 1.3 to 1 between the lumen of reticular elements and the interior of heavy particles. Although heavy particle RNase II resembles acid hydrolases in centrifugal behavior, in other tests including density shift experiments the resemblance is incomplete. In experiments employing lysis induced by L-amino acid methyl esters, RNase II activity is much more latent than the activity of the lysosomal marker, acid RNase. It is postulated that the heavy particle component of RNase II is contained in a secretory vesicle rather than in classic lysosomes.     

Estrogen-induced membrane alterations and growth associated with proteinase activity in endometrial cells

Endometrial cells isolated from uteri of ovariectomized rats were treated in vitro with 1 X 10(-9) M estradiol-17 beta (E2beta) to analyze early changes in membrane properties during hormone-induced growth. After 30-min exposure to E2beta at 22 degrees C, cells exhibited an enhanced capacity to bind erythrocytes (hemadsorption) in the presence of concanavalin A (Con A) to 237% of the level in paired controls. Fluorescence microscopy revealted that approximately 25% of cells exposed to E2beta, but not estradiol-17 alpha (E2alpha), showed a redistribution into polar clusters of Con A-binding sites that were dispersed in random patches at the external surfaces of control cells. These hormore-induced membrane alterations were abolished by prior treatment of cells with inhibitors of thiol proteinase activity of the cathepsin B1 (CB1) type, such as leupeptin and iodoacetate. Leupeptin at 4.5 X 10(-7) M also reduced the affinity of [3H]E2beta binding to intact cells but did not influence specific binding of the hormone to macromolecular components of cytosol. A pronounced increase in the availability of endogenous CB1, But not of alkaline phosphatase, succinate, or lactate dehydrogenase, in the extracellular media was elicited within 30 min after E2beta treatment. In cells cultured in chemically defined medium for up to 48 h, E2beta, but not E2alpha, enhanced cell proliferation and stimulated [3H]thymidine incorporation into macromolecular form. These E2beta-induced effects were abolished by prior treatment of cells with liposome-entrapped leupeptin at a final concentration of 7 X 10(-8) M. The net rate of intercellular adhesion among endometrial cells was also enhanced by E2beta. This hormonal response was diminished by prior exposure to leupeptin. Fractionation of cells by selection for adhesiveness due to E2beta exposure for 30 min yielded a subpopulation of rapidly dividing cells which surpassed their less adhesive counterparts in cathepsin secretion and in Con A-mediated hemadsorption. These results indicate that leupeptin-sensitive proteinase activity may contribute to membrane and growth modifications elicited by E2beta treatment in endometrial cells.