Cell Debris Fraction Research Articles

Cellular distribution of inorganic mercury and its relation to cytotoxicity in bovine kidney cell cultures.

A bovine kidney cell culture system was used to assess what relationship mercuric chloride (HgCl2) uptake and subcellular distribution had to cytotoxicity. Twenty-four-hour incubations with 0.05-50 micro M HgCl2 elicited a concentration-related, with 1.0 nmol/10(6) cells at the IC50. Measurement of Hg uptake over the 24-h exposure period revealed a multiphasic process. Peak accumulation was attained by 1 h and was followed by extrusion and plateauing of intracellular Hg levels. Least-squares regression analysis of the cytotoxicity and cellular uptake data indicated a potential relationship between the Hg uptake and cytotoxicity (r = 0.91). However, the subcellular distribution of Hg was not concentration-related. Mitochondria and soluble protein fractions accounted for greater than 65% of the cell-associated Hg at all concentrations. The remaining Hg was distributed between the microsomal (6-10%) and nuclear and cell debris (11-22%) fractions at all concentrations tested. Less than 20% of the total cell-associated Hg was bound with metallothionein-like protein.

Subcellular distribution of abnormal proteins in rabbit reticulocytes: Effects of cellular maturation, phenylhydrazine and inhibitors of ATP synthesis

Inhibitors of ATP synthesis (cyanide, dinitrophenol, fluoride) inhibited proteolysis of pulse-labelled abnormal proteins in rabbit reticulocytes and caused an accumulation of the aberrant polypeptides in the cell debris fraction in a manner analogous to phenylhydrazine-induced Heinz bodies. When the reticulocytes were separated into age-groups by sedimentation through discontinuous gradients of bovine serum albumin, the ability of the cells to degrade puromycin peptides decreased with increasing cellular maturity and, in the more mature cells, up to 40% of the labelled abnormal polypeptide remained associated with the cell debris fraction at the end of the chase period. It is suggested that the association of the abnormal polypeptide with the cell debris fraction is a consequence of a maturation-induced loss, or an inhibitor-induced inactivation of the cellular proteolytic activity.

Effect of dimercaptosuccinate on the accumulation and distribution of cadmium in the liver and kidney of the rat

The effect of dimercaptosuccinic acid (DMSA) on the tissue distribution, renal and hepatic subcellular localization of Cd 2+, Zn 2+ and Cu 2+ in Cd 2+-pretreated male rats and on the tissue distribution of Zn 2+ and Cu 2+ in the normal rat was studied. Cd 2+-pretreated rats which received 3 × 1 mg Cd 2+/kg body wt s.c. at 48 hr intervals followed after 7 days by DMSA (50 mg/kg body wt i.p.) daily for 17 days had total hepatic Cd 2+ concentrations 25 per cent lower than Cd 2+-pretreated controls (P < 0.01). DMSA did not influence the concentration or distribution of Cd 2+ in the liver cytosol whereas in the mitochondrial-lysosomal and nuclei + cell debris fractions the Cd 2+ concentration was reduced by 54 and 48 per cent respectively. Total renal and hepatic Cu 2+ concentrations were increased by Cd 2+ treatment and reduced by treatment of the Cd 2+-exposed animal with DMSA. In the liver cytosol Cu 2+, Zn 2+ and Cd 2+ accumulated in the metallothionein fraction and none was mobilized from the cytosol by DMSA. In the kidney cytosol Cu 2+ accumulated in fractions in addition to metallothionein and was eliminated from each of these fractions following treatment with DMSA. It is concluded that the high affinity of metallothionein for these cations prevented their elimination from the cytosol and that the interaction of Cd 2+ and Cu 2+ with DMSA occurred in the particulate fraction and therefore delayed the response to DMSA treatment. It is suggested that long term treatment with DMSA, although ineffective in mobilizing Cd 2+ from the kidney may provide a useful therapeutic measure to reduce the liver burden of Cd 2+ and the high renal Cu 2+ concentrations in the Cd 2+-exposed animal.

Choline acetyltransferase of rat retina a comparison of its properties with the enzyme from brain

1. Kinetic and other properties of choline acetyltransferase (ChAC, E.C.2.3.1.6) of rat retina was studied, and a comparison was made with the enzyme from brain. 2. Subcellular distribution studies of ChAc from both retina and brain revealed that in both tissues the enzyme was concentrated in the fraction containing synaptosomes and mitochondria (P 1). However, considerable activity was also detectable in the cell-debris fraction (P 1) and in the final supernatant (S 2). 3. The enzyme from both sources was easily obtained in a soluble form. The K m for the enzyme was of the order of 10 −5M and its optimum activity was found to be at pH 7.4–7.6. 4. For each of the parameters studied ChAc from retina and brain resembled each other very closely.

Amino acid requirements and proteolytic activity of Streptococcus sanguis.

The growth response of Streptococcus sanguis groups 1:A and 1:B in a complete chemically defined medium was not influenced by the oxygen concentration of the growth atmosphere. All of the cultures required cysteine and arginine; tyrosine and branched-chain amino acids were frequently required. Proteolysis of casein, mucin, and the anionic proteins of germfree rat saliva by S. sanguis was demonstrated. Hydrolytic activity toward casein was found in the soluble contents of the cells and in the cellular debris after disruption of the cells, with the soluble fractions exhibiting greater proteolytic activity toward casein. The soluble fractions from S. sanguis did not hydrolyze mucin, but this substrate was hydrolyzed by the cell debris fraction. When the amino acid requirements and proteolytic activity of S. sanguis and S. mutans were compared, these two oral streptococcal species exhibited distinct and characteristic differences.

Amino Acid Requirements and Proteolytic Activity of Streptococcus sanguis

The growth response of Streptococcus sanguis groups 1:A and 1:B in a complete chemically defined medium was not influenced by the oxygen concentration of the growth atmosphere. All of the cultures required cysteine and arginine; tyrosine and branched-chain amino acids were frequently required. Proteolysis of casein, mucin, and the anionic proteins of germfree rat saliva by S. sanguis was demonstrated. Hydrolytic activity toward casein was found in the soluble contents of the cells and in the cellular debris after disruption of the cells, with the soluble fractions exhibiting greater proteolytic activity toward casein. The soluble fractions from S. sanguis did not hydrolyze mucin, but this substrate was hydrolyzed by the cell debris fraction. When the amino acid requirements and proteolytic activity of S. sanguis and S. mutans were compared, these two oral streptococcal species exhibited distinct and characteristic differences.

Cell-bound glucosyltransferase activity of Streptococcus sanguis strain 804

Cell-bound glucosyltransferase (GT) activity of washed cells of Streptococcus sanguis, strain 804, represents about 12 per cent of the total GT activity of glucose-grown cultures of this organism. During culture, cell-associated GT activity could be detected prior to its being found extracellularly. Sonication of the washed cells harvested at the stationary phase indicated that predominant proportions of GT and β-fructofuranosidase (FH) activities were associated with the cell debris fraction. Extracellular fibrillar material, demonstrated by electron microscopic examination, was synthesized by washed cells incubated with sucrose and chemical analysis indicated glucan formation. Incubation with sucrose also promoted the osmotic diffusion of proteins from the intracellular phase. Sodium fluoride inhibited cell metabolism without effecting the formation of glucan or the release of proteins into the supernatant fluid. Maltose in equimolar concentration with sucrose considerably reduced the synthesis of insoluble glucans. Washed cells incubated with sucrose did not release any GT.

Insulin metabolizing enzyme activities in human placental tissue.

The nature of insulin-degrading activity in the human placenta has been examined. Two soluble enzyme activities, I and II, have been demonstrated, in addition to an activity in the cell debris fraction. Activity I is sulfhydryl dependent and can be partially resolved into two components on C-100 Sephadex chromatography. The minor component chromatographs with activity II in the void volume. The major portion is of smaller molecular weight, as evidenced by its greater elution volume. Activity I has a pH optimum of 7.2 to 7.4 and a broad range of substrate specificity. These properties, along with the demonstration of glutathione-insulin transhydrogenase activity in placental supernatant, raise the possibility that a substantial part of activity I is due to a glutathione-insulin transhydrogenase. Activity II is not sulfhydryl dependent but appears to be stimulated by sulfhydryl and inhibited by p-chloromercuribenzoate. It has a pH optimum of 6.3 to 6.6, shows greater specificity for insulin than does activity I and is less readily inactivated at 60° C. (t1/2, I = 25 min. II = 120 min.). It shows the same elution volume on G-150 Sephadex as rat muscle protease. The characteristics of activity II suggest that it is a sulfhydryl protease similar to that observed in rat skeletal muscle.

Isolation and characterization of poly A polymerase from cell debris of E. coli.

A poly A polymerase was found in cell debris fraction of E. coli when extracted with a neutral detergent. The enzyme protein was purified and characterized. As one of its biological roles, this enzyme was identified as a component of DNA dependent RNA polymerase.

Evidence for the absorption of immunoreactive intrinsic factor into the intestinal epithelial cell during vitamin B 12 absorption.

Abstract To investigate the distribution and nature of vitamin B 12 in the subcellular fractions of the ileal epithelial cells during the process of absorption, 57 CoB 12 bound to human intrinsic factor (IF) was incubated in isolated ileal loops of anesthetized guinea pigs and periodically for 120 minutes the animals were killed and the homogenate of the mucosa separated by differential centrifugation into (1) cell debris fraction containing large fragments of partially disrupted surface structures, nuclei, and other cell constituents, including some mitochondria, (2) mitochondrial fraction, (3) microsomes, and (4) cytosol. The particulate composition of each fraction was confirmed using electron microscopy. Radioactivity accumulated continuously during the observation period in the cell debris and mitochondria-rich fractions. Lesser amounts were found with the microsomes and in the cytosol. The nature of the 57 CoB 17 in the cell debris and mitochondrial fractions was further studied by sonicating each fraction to solubilize the radioactivity and then removing the insoluble particulate by centrifugation at 10,000 g. Eighty to 94 per cent and 57 to 78 per cent of the solubilized radioactivity in the supernates of the cell debris and mitochondrial fractions, respectively, could not be extracted by albumin-coated charcoal, indicating that a major fraction of the 57 CoB 12 was bound to a macromolecule. When these supernates were then incubated with antihuman IF antibody, 71 to 84 per cent and 54 to 69 per cent of the radioactivity in the cell debris and mitochondrial fractions, respectively, precipitated with the immunoglobins at 15 per cent Na 2 SO 4 indicating that a major fraction of the absorbed 57 CoB 12 was bound to a large molecule with the immunologic identity of IF. Although significantly less radioactivity in the supernates of these sonicated fractions precipitated at 15 per cent Na 2 SO 4 in the absence of anti-IF antibody, it was still significantly more than precipitated when IF- 57 CoB 12 in gastric juice was subjected to this salt concentration. This finding suggests that either a small fraction of the absorbed immunoreactive IF-B 12 complex has been altered in some way, or that some of the absorbed 57 CoB 12 becomes bound to a different protein which is insoluble at 15 per cent Na 2 SO 4 .

Protein synthesis in neuronal perikarya isolated from cerebral cortex of the immature rat.

Abstract— Homogenates of neuronal perikarya isolated from the cerebral cortex of the 8‐day‐old rat were incubated with [3H]leucine, and the characteristics of the protein synthetic process were studied. Incorporation of leucine into protein was linear up to 90 min, proceeded optimally at pH 7.6 and was stimulated by K+ and NH4+, unaffected by Li+ and inhibited by Na+. Puromycin, cycloheximide, RNAse, sulphhydryl blocking agents and phospholipase A exerted a pronounced inhibition, whereas chloramphenicol and phospholipase C had no effect. About 42 per cent of the total radioactive protein formed in the optimally fortified in uitro system was recovered in non‐sedimentable form. Incorporation into the subcellular fractions of the neuronal perikarya increased steadily with increasing time of incubation. The microsomal fraction acquired the highest specific radioactivity (d.p.m./mg of protein), followed by the mitochondrial and the nuclear + cell debris fractions. The high‐speed soluble fraction exhibited the lowest specific radioactivity.Although the addition of L‐methionine to a suitably fortified incubation medium inhibited neuronal protein synthesis by about 80 per cent, the addition of D‐methionhe, α‐methyl‐DL‐methionine or L‐tryptophan was relatively ineffective by comparison.

Metabolic disposition of [ 131I]iodoinsulin within the rat diaphragm

1. 1. The rate of uptake and release of physiological levels of biologically active [ 131I]iodoinsulin from subcellular components of isolated intact rat diaphragm has been studied. The 100 000 × g supernatant solution contained the largest amount of radioactivity, of which more than 70% was degraded insulin. Of the particulate fractions the cell debris sedimenting at 700 × g incorporated 5 times the radioactivity of the mitochondria and 25 times that of the microsomes. 2. 2. The incorporation of radioactivtiy from [ 131I]iodoinsulin into only one fraction of those studied was significantly suppressed by the addition of unlabeled insulin. This was the fraction of the 100 000 × g supernatant precipitable with 60% saturated (NH 4) 2SO 4. Sephadex G-50 gel filtration of the latter fraction demonstrated components of higher molecular weight than insulin, suggesting the presence of an insulin-specific binding component in the fraction. 3. 3. Two insulin-degrading enzymes were found in the subfractioned diaphragm. one in the 100 000 × g supernatant solution and a second in the cell debris fraction. The supernatant enzyme was 7–8 times more active than the debris enzyme at physiological concentrations of insulin. Both enzymes were active after dialysis in the absence of added GSH. These characteristics and the evidence given in (1) above suggest that the enzymes are distinct from GSH-insulin transhydrogenase and that the degradation of insulin is a accomplished by proteolysis.