Studies on the intracellular status and localization of mouse pancreas ribonucleases
Creators: name: Morrill, Gene A.
Publisher: name: University of Utah
Year: 2012
Subjects: Purine Ribonuclease
Homogeneity

description: 1. The evidence presented in this thesis indicates that mouse pancreas ribonuclease and beef pancreas ribonuclease have quite similar catalytic properties. Both enzymes cause a partial breakdown of RNA and split only secondary phosphate esters of pyrimidine-3'-phosphates.  Further, both enzymes have similar pH optima and general stability to storage and handling.  2. Mouse pancreas ribonuclease can be chromatographed on columns of the carboxylic ion exchange resin IRC-50 (SE-64).  When 0.2 M sodium phosphate buffer, pH 6.47, extracts of mouse pancreas are prepared and chromatographed, the enzymatically active material was eluted in two broad zones. However, when 0.25 N. sulfuric acid extracts of mouse pancreas are chromatographed, two sharp, distinct peaks of ribonuclease activity can be recognized, indicating that an alteration of the protein molecule occurred during the acid treatment. pH 7.5:5.0 ribonuclease activity ratios for the individual fractions indicate an enzymatic as well as chromatographic heterogeneity both within a given peak as well as between the peaks from a sulfuric acid extract of mouse pancreas. These chromatographic elution components have been further studied by limited rechromatography and under conditions of pH gradient elution.  3.  The intracellular distribution of the ribonucleases of mouse pancreas was studied by differential centrifugation of pancreas homo-genates prepared in 0.25 M sucrose. Approximately 70 per cent of the ribonuclease activity in the tissue was associated with three (of one nuclear and nine cytoplasmic) isolated cell fractions. The highest level of ribonuclease activity was found in the cytoplasmic f" microsomal fraction (35 per cent), with lesser amounts being found in the nuclear dense granule fraction (20 per cent) and the cell "soluble" or supernatant fraction (15 per cent).  4.  The effect of treating the nuclear and cytoplasmic pellets isolated from mouse pancreas homogenates with 0o25 N. sulfuric acid by the method of Hirs et al. was studied.  Large decreases in the activity of the nuclear fraction as well as moderate increases in the "cytoplasmic f" microsomal fraction were observed, and the significance of these changes is discussed.  5« The cell supernatant contained a significant level of ribonuclease activity when the tissue was homogenized in sucrose, and this level could be increased four or five fold by treatment with a strong acid or by column chromatography. This inactive of "latent" ribonuclease may be bound to a protein inhibitor which is removed by an acid-inactivation or by chromatography. The relationships of these findings to other studies on cellular enzyme inhibitors are discussed.  6.  Chromatographic elution studies were carried out on both the 0.2 M sodium phosphate buffer extracts and the 0.25 N. sulfuric acid extracts of four ribonuclease "rich" cell fractions.  Differential analyses of the elution patterns from these cell fractions indicate, based on stability or lability to acid treatment and intracellular localization, that at least six distinct forms of the enzyme may be present in the 0o2 M sodium phosphate buffer extracts of mouse pancreas. Further, two of the six ribonucleases have specific intracellular locations, and only one of the six enzymes appears to be present in each of the fractions investigated.  7.  Mouse pancreas has been incubated in vitro with a parasympathomimetic drug (pilocarpine) and with the hormone pancreozymin. Secretion (active extrusion) of both amylase and ribonuclease and an increase in respiratory rate occurred during incubation. Large differences in both the stimulated and non-stimulated extrusion of enzyme were found in different strains of mice. The means of killing the animals and pre-treatment of the tissue also affected secretion.  Chromatographic analyses of the ribonuclease "actively extruded" into the incubation medium by maximal in. vitro stimulation with pilocarpine or pancreozymin suggests the following hypothesis:  The enzyme associated with the nuclear fraction is contained in dense secretory granules which are selectively released on in vitro pilocarpine stimulation. In contrast, a less dense secretory granule, present in the so-called "principle zymogen fraction" of the cell, is selectively released by in vitro pancreozymin stimulation. In addition, the appearance of very large amounts of ninhydrin positive material (normally present almost exclusively in the soluble portion of the cell) in the incubation medium further suggests an involvement of the cellular cytoplasm in the secretory mechanism.  9. The cytochemical implications of the intracellular status and function of mouse pancreatic ribonuclease and the relationships of these findings to previous studies on cellular ribonucleases are discussed.