The distribution of protease activities on liver cell fractions

Abstract

PRELIMINARY NOTES 451tively) are high, and nearly equally so, during the initial intervals. The RNA phosphorus would appear to move as follows: Sp --+ DS --+ RNP. The evidence on the manner in which ribonucleic acid participates in protein synthesis is still fragmentary. If such a participation requires the rapid renewal of the nucleic acid, our observations would suggest that the ribonucleic acid of the RNP particles of the microsomes is not directly involved in the formation of microsomal protein, though the protein precursors, activated in the Sp fraction, may be assembled in the microsomes to form the final polymer. It is perhaps significant that the cytoplasmic supernatant fraction, in which the enzymic activation of amino acids s has been shown to occur ~,7, also contained the ribonucleic acid exhibiting the highest initial uptake of asp.We shall submit more detailed information on these studies in due course. This work, which benefited from the technical assistance of Miss EDITH A. LAWRENCE, has been supported by research grants from the U.S. Public Health Service, the National Science Foundation and the Rockefeller Foundation. The distribution of protease activities on liver cell fractionsUsing hemoglobin as a substrate, DE DUVE and co-workersZ, 2 found the highest cathepsin activity in the light mitochondria fraction of liver homogenates. Earlier MAVER AND GRECO 3 had found that the total mitochondria fraction contains the highest percentage of the total protease activity of liver homogenates not only when hemoglobin, but also when benzoyl-l-argininamide was used as a substrate.As cathepsin is the collective name for the tissue proteinases and other proteases, e.g. dipeptidases and a carboxypeptidase, also occur in the tissues we thought it would be interesting to study the protease activities of the liver cell fractions with synthetic substrates, specific for various proteases. 1:5 rat liver homogenates were prepared in o.25 M sucrose. The homogenate was layered on 0.35 M sucrose and centrifuged for 5-5 rain at 9oo × g in order to sediment the nuclear fraction. The supernatant containing the other subcellular components was centrifuged for 12 min at 18,ooo x g. The total mitochondria sedimented in this way could be separated into the light and the heavy fraction by resuspending them in o.25 M sucrose, centrifuging for 8 rain at ii,ooo × g, and resuspending the slightly pink light mitochondria layer (easily distinguishable from the dark yellow heavy mitochondria layer) by causing a slow rotatory movement in the supernatant by means of a small pestle fitted into the centrifuge tube. The suspension obtained in this way was then transferred to another centrifuge tube and the light mitochondria spun down by centrifuging for 12 min at i8,ooo × g. The whole procedure was carried out at about 2 ° C.The final supernatant obtained by sedimenting the total mitochondria was not fractionated further; thus it still contained the microsomes. In order to avoid damage as much as possible the sedimented fractions were n...

Intestinal Dipeptidases. IV. Studies on the Release and Subcellular Distribution of Intestinal Dipeptidases of the Mucosa Cells of the Pig

“…The results are presented in Table 11. They are also in agreement with previous observations on homogenate of other tissues (Rademaker andSoons 1957, Hanson andBlech 1959). These findings also confirmed the results reported recently by Robinson (1963) in his study of the subcellular fractions of rat intestinal mucosa homogenate, where the dipeptidases were assayed with the ninhydrin method.…”

“…These findings also confirmed the results reported recently by Robinson (1963) in his study of the subcellular fractions of rat intestinal mucosa homogenate, where the dipeptidases were assayed with the ninhydrin method. They are also in agreement with previous observations on homogenate of other tissues (Rademaker andSoons 1957, Hanson andBlech 1959). Variations of the homogenizing procedure both in respect to time and force gave no difference in the distribution of the activities.…”

The Absorption of Glycine and Glycine Oligopeptides by the Rat

“…This is in keeping with the observations that many tissues including liver (Hanson & Blech, 1959;Rademaker & Soons, 1957), kidney (Hanson & Blech, 1959;Robinson et al, 1953;Capobianco & Vescia, 1967), skeletal muscle (Schwartz, 1953;Smith, 1948;Pina, Hamabata & Laguna, 1962), uterus (Smith, 1948) and brain (Uzman, Rumley & van -de Noort, 1961), contain significant amounts of glycylglycine peptide hydrolase.…”

Cell Fractionation of Anterior Pituitary Glands from Beef and Pig

“…The proteinase may be a definite constituent of both the cell "sap" and the microsomes, or it may be associated with the lighter components of the endoplasmic reticulum, some of which may not have sedimented during centrifugation. Rademaker and Soons (20) studied the intracellular distribution of peptidases and proteinases in liver and found that certain enzymes were located only in the supernatant, others were absent from the supernatant, and still others were found in both particulate components and supernatant. In the present study acid proteinase is assumed to be localized only in particulate elements 0ysosomes?).…”