Latency Of Acid Phosphatase Research Articles

Biochemical effects of gentamicin on rat kidney cortex: II. Analytical subfractionation after short-term, high-dose treatment

As a first step in studies on the molecular mechanism(s) underlying gentamicin toxicity, the effect of treating rats with this aminoglycoside antibiotic (100 mg/kg once or twice daily for 3 days) on the analytical subfractionation of the kidney cortex has been examined. DNA was used as a marker for the nuclei, cytochrome oxidase for mitochondria, acid phosphatase for lysosomes, catalase for peroxisomes (with reservations; see the companion paper), NADPH-cytochrome c reductase for the endoplasmic reticulum, p-nitrophenyl-α-mannosidase (at pH 5.5) for the Golgi apparatus, AMPase for the plasma membrane in general and alkaline phosphatase for the brush border, and lactate dehydrogenase for the cytosol. In addition, the presumptive lysosomal hydrolases N-acetyl-β- d-glucosaminidase, p-nitrophenyl-α-mannosidase (at pH 4.5), cathepsin D, and DNase II were monitored. Electron microscopy was also performed on the subfractions obtained. The only significant biochemical changes brought about by gentamicin treatment were that N-acetyl-β- d-glucosaminidase demonstrated both a greater total activity and a larger enrichment in the 104,000 g av pellet, while p-nitrophenyl-α-mannosidase at pH 4.5 demonstrated the same total activity and a greater enrichment in the 104,000 g av pellet. Since myeloid bodies were shown by electron microscopy to sediment primarily with the 500 g av and 10,000 g av pellets, the biochemical changes seen cannot be associated with these morphological structures. These findings suggest that selective changes in a certain subpopulation(s) of lysosomes or in certain lysosomal enzymes may be involved in the early stages of gentamicin toxicity. On the other hand, no lysosomal membrane damage was observed here, since both the latency of acid phosphatase and the recovery of this activity in the soluble cytosol were unchanged. The present investigation may also have relevance for the dosage and duration of gentamicin treatment chosen in clinical situations.

Changes in lysosomal properties of alveolar macrophages of rats treated with chlorphentermine

Rats were treated with chlorphentermine (30 mg/kg) 5 days per week for 4 weeks. Alveolar macrophages washed from the lungs were greatly enlarged and filled with lamellated inclusions. Lysosomal acid hydrolase activity was increased in these cells when compared to macrophages from untreated rats. Chlorphentermine treatment appeared to have marked effects on lysosomal stability. The latency of acid phosphatase was increased. The effects on β- N-acetyl-glucosaminidase were somewhat different, however, with the latency of this enzyme characterized by an increased lysosomal fragility. These findings indicated that in addition to producing morphological changes within alveolar macrophages, chlorphentermine administration leads to lysosomal changes as well.

Effect of fasting on the biochemical properties of perfused rat liver

Livers from rats which had been either fed ad libitum or fasted for 48 hr were perfused with bovine erythrocytes in Krebs-Ringer buffer containing 3% bovine serum albumin, and their function was studied. No difference was noted in the ability of the livers to produce bile. Those from fed rats maintained their initial rate of urea synthesis (1.5 μmol/min) for 60 min, while those from fasted rats maintained this rate for only 20 min. At the end of these periods the livers from both groups of rats synthesized urea at lower rates. Potassium was released into the accellular fraction of the perfusate at a rate of 0.3 μeq/min 60 min after the start of perfusing livers from fed rats and 20 min after the start of perfusing those from fasted rats. Prior to these times, little or no potassium was released. When compared with unperfused controls, perfusion for 3 hr had no effect on the latency and sedimentability of β-glucuronidase or on the sedimentability of acid phosphatase in homogenates of the livers from rats which had been fed ad libitum. There was, however, a small but statistically sigificant decrease in the latency of acid phosphatase. Perfusion of livers from fasted rats resulted in marked and statistically significant falls in the latency and sedimentability of acid phosphatase and in the sedimentability of β-glucuronidase. The fall in the latency of β-glucuronidase was not statistically significant. It was concluded that the livers from rats which had been fasted for 48 hr were less able to tolerate the stress of perfusion in vitro than those from rats which had been fed ad libitum.

Freezing of rat-liver lysosomes to -196 degrees C in the presence and absence of dimethylsulphoxide.

The latency of acid phosphatase and β‐glucuronidase in homogenates of rat liver, prepared in 0.25 M glycerol‐2‐phosphate (pH 7.0), decreased after freezing to –196 °C. The latencies obtained were virtually independent of cooling rate in the range of 2–225 °C/min and the latency of acid phosphatase always suffered a greater decrease than that of β‐glucuronidase.The fall in latency was always less in the presence of 7.5% (1.06 M) dimethylsulphoxide and and the protection afforded to the latency of acid phosphatase was greater than that given to β‐glucuronidase. The sedimentability of both enzymes responded to freezing in a manner similar to that found for latency.The sedimentability of acid phosphatase decreased after exposure to high concentrations of sucrose or glycerol‐2‐phosphate. We suggest that this response, as well as the decrease in latency found after freezing and thawing, results from osmotic pressure gradients which arise when normally non‐penetrating molecules enter the lysosomes under a high concentration gradient.The decrease in sedimentability of acid phosphatase after exposure to high concentrations of glycerol‐2‐phosphate was accompanied by a decrease in the specific total activity of the enzyme and it appears that this enzyme is readily inactivated in the presence of high concentrations of this substrate.

Effect of triparanol (mer 29) on rat liver lysosomes: cytochemical and biochemical study.

Rats treated with triparanol (MER-29) develop numerous membranous inclusions-“myeloid” bodies in the cytoplasm of liver cells. The “myeloid” bodies did not show cytochemically demonstrable acid phosphatase. Instead diffuse activity was observed throughout the cytoplasm. Biochemically, acid phosphatase was found in the liver lysosomal fraction obtained from triparanol treated rats. This fraction, however, did not show the structure-linked latency of acid phosphatase which is characteristic of normal lysosomes. It is suggested that “myeloid” bodies are lysosomes with altered membranes.

Effect of increasing concentrations of salt on the lysosomes of rat liver and Tetrrahymena pyriformis

When cells are cooled below their freezing point there will be an increase in the concentration of solutes surrounding the subcellular organelles and, for that reason, we have studied the effect of different concentrations of a model solute, KCl, on the lysosomes of rat liver and Tetrahymena pyriformis. The latency of acid phosphatase and β-glucuronidase in homogenates of rat liver fell progressively after exposure to increasing concentrations of KCl and, although the sedimentable acid phosphatase also declined, the decrease was less marked. After exposure of homogenates of T. pyriformis to increasing concentrations of KCl, there was no decrease in the sedimentability of acid phosphatase although a progressive decline in the latency of the enzyme was demonstrated. The rat liver lysosomal membrane was shown to be only slighly permeable to KCl although the lysosomes of T. pyriformis were much more permeable to this salt. It seems possible that the disruptive effect of high concentrations of KCl is due to transiet osmotic pressure gradients which may arise when the concentration is reduced by dilution.

The effect of dimethylsulphoxide on the permeability of the lysosomal membrane

The latency of acid phosphatase in sucrose homogenates of rat liver and Tetrahymena pyriformis was found to decrease progressively on exposure to concentrations of dimethylsulphoxide between 5 and 25% (v/v). A progressive decrease in latency was also found in homogenates of rat liver prepared with potassium gluconate although, in this instance, the extent of the decrease was much less than with homogenates prepared in sucrose. With homogenates of rat liver prepared in ß-glycerophosphate, dimethylsulphoxide in concentrations up to 10% had little effect on the latency of acid phosphatase, although it decreased on exposure to higher concentrations.

The effect of dimethylsulphoxide on the permeability of the lysosomal membrane

The latency of acid phosphatase in sucrose homogenates of rat liver and Tetrahymena pyriformis was found to decrease progressively on exposure to concentrations of dimethylsulphoxide between 5 and 25% (v/v). A progressive decrease in latency was also found in homogenates of rat liver prepared with potassium gluconate although, in this instance, the extent of the decrease was much less than with homogenates prepared in sucrose. With homogenates of rat liver prepared in β-glycerophosphate, dimethylsulphoxide in concentrations up to 10% had little effect on the latency of acid phosphatase, although it decreased on exposure to higher concentrations.

Variation in the latency of acid phosphatase and ribonuclease throughout the growth cycle of Tetrahymena pyriformis.

AbstractThe latency of both acid phosphatase and ribonuclease was studied throughout the growth cycle of the ciliate protozoan Tetrahymena pyriformis and was found to be remarkably low in cells in the logarithmic phase of growth. The latency increased progressively throughout the log phase until it reached a maximum just after the cells had entered the stationary phase.The specific activity of acid phosphatase remained constant throughout the whole of the growth cycle while that of ribonuclease decreased as the cells began to leave log phase.The possibility is discussed that all rapidly dividing cells have a high proportion of their acid hydrolases outside the lysosomes and that these free hydrolases may have a function in cell division.

Effects of ovariectomy and hypervitaminosis A on lysosomes of the rat conceptus.

AbstractThis report is concerned with some biochemical effects of ovariectomy and hypervitaminosis A on cell organelles, with particular emphasis on the lysosomes, of embryos, yolk sacs, and placental labyrinths of 13‐day pregnant rats. The two parameters considered were lysosomal stability and the distribution of enzymes in fractions obtained by differential centrifugation. Lysosomal stability was determined by measuring (1) the amount of “free” enzyme activity, i.e., the amount of enzyme in the cytoplasmic extracts of the three tissues measured in the presence of isotonic sucrose and (2) the amount of “nonsedimentable” enzyme. Excess vitamin A produced no change in the latency of acid phosphatase or β‐glucuronidase in any of the tissues when compared with controls. Following ovariectomy there was marked labilization of both enzymes in embryos, increased labilization of β‐glucuronidase in placentas, and little change in yolk sacs.The distributions of the lysosomal enzymes in control fractions were not the same in the three tissues, indicating differences in size, shape, and/or density of the lysosomes. There was no change in the distribution of reference enzymes after hypervitaminosis A. Ovariectomy produced increased solubilization of β‐glucuronidase in the three tissues and a lesser increase in soluble acid phosphatase in embryos, indicating lack of uniformity in the lysosomes.

Lysosomes in human placenta.

SINCE their discovery1, lysosomes have been described in many tissues. Here I report the presence of lysosomes characterized by staining with acridine orange2,3 and assays of the concentration and latency of acid phosphatase in the “lysosomal” pellet, in human term placenta.