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R C Ruth

Publications and source records attributed to R C Ruth.

At least 19 recordsLinked to original sources
PubMed1989

Is the ATP-dependent protection of lysosomes against osmotic lysis a function of the lysosomal proton pump.

Rat liver lysosomes have been used to characterize further the effects of ATP on lysosomal stability during incubation at 37 degrees C at hypo-osmolarity. As previously reported, when the osmotically-supporting solute is the salt of a strong base (K+), ATP protects against lysis during incubation. However, if the osmotically-supporting solute is the salt of a weak base, e.g. Tris HCl or NH4Cl, ATP actually promotes lysis during incubation. Thus, ATP can exert destabilizing as well as protective effects on lysosomes. The destabilizing effect is eliminated by protonophores. The protective effect in the presence of potassium salts is not eliminated by protonophores. Moreover, when incubation is in the presence of a salt of a weak base, protonophores actually cause an ATP-dependent protective effect to be established. The destabilizing effect occurs at 37 degrees C, but not at 0 degrees C. The Mg++-dependence of the destabilizing effect was found to be similar to that found earlier for the ATP-dependent protective effect, insofar as only 1 mM MgCl2 in the presence of 1mM EDTA is sufficient for nearly maximal stimulation of both effects. The destabilizing effect may result from a H+ ion gradient across the lysosomal membrane which is maintained by the lysosomal ATP-dependent proton pump. The protective effect, on the other hand, does not depend on such a gradient being maintained; on the contrary, protonophores appear to act as enablers of the protective effect. The question that remains to be answered is: does the protective effect derive in some way from the same ATP-driven mechanism which constitutes the proton pump? Some possible answers to this question are considered.

Adenosine Triphosphate↗
PubMed1983

Effects of ATP on lysosomes: protection against hyperosmolar KCl.

After incubation at 37 degrees C in isosmolar (200 mM) KCl, rat liver lysosomes are susceptible to damage caused by brief exposure to hyperosmolar (greater than 200 mM) KCl. Lysosomes that are exposed to hyperosmolar KCl do not undergo significant lysis as long as they are maintained at hyperosmolar conditions; however, they will lyse on being returned to lower osmolarities. If the hyperosmolar KCl-treated lysosomes are intermittently transferred into equally hyperosmolar sucrose, they no longer undergo lysis on subsequent exposure to lower osmolarities; this confirms the reversible nature of the hyperosmolar KCl-induced damage. Thus the hypothesis that the hyperosmolar KCl damage involves an isosmotic permeating of the lysosome by KCl appears reasonable. The increase caused by the hyperosmolar KCl in free activity of beta-N-acetyl-D-glucosaminidase is reduced by about 50% by ATP but not by ATP analogues. ATP protects provided that it is added either before, or simultaneously with, exposure of the lysosomes to hyperosmolar KCl. However, if the ATP is not added until the lysosomes are already in the presence of the hyperosmolar KCl, it does not reverse the damaging effects of the KCl even though actual lysis has not yet occurred at the time of the ATP addition. The protective effect is established very rapidly, because ATP added simultaneously with the addition of the hyperosmolar KCl protects to the same extent as does ATP added any time prior to the KCl addition. The protective effect requires Mg2+ and is not supported by Ca2+. Maximal protection is provided by 5 X 10(-4) M ATP. It is postulated that ATP protects lysosomes by reducing an increase in intralysosomal concentration of KCl, which occurs when incubated lysosomes are exposed to hyperosmolar KCl.

Acetylglucosaminidase↗
PubMed1982

Effects of ATP on lysosomes: inhibition of the loss of latency caused by cooling.

Triton-filled lysosomes from rat liver and a crude lysosomal fraction from livers of rats not injected with Triton have been used to study the ability of ATP to protect lysosomes against the effects of incubation in vitro at 37 degrees C. When incubation was carried out in a sucrose-NH4Cl medium at pH 8.0 or in buffer containing 0.15 M KCl at pH 7.4, ATP protected against the loss of lysosomal enzyme latency that occurred at 37 degrees C and also against the additional loss of latency that occurred when the incubated lysosomes were cooled to 0 degrees C. The inhibition of the effects of cooling was found to result from a decrease in the loss of latency caused specifically by cooling the lysosomes through the temperature range of the membrane phase transition (15-0 degrees C); ATP did not affect the basic rate of loss of latency at 0 degrees C. Protection was minimal when incubation was carried out in 1 mM ethylenediaminetetraacetic acid without added divalent metal ion and was enhanced by the addition of either Mg2+ or Ca2+. Kinetic studies were carried out on the ATP-protective action against the effects of cooling by adding ATP to lysosomes that had been incubated at 37 degrees C and that were then cooled to 0 degrees C at various times after addition of the ATP. Susceptibility to the effects of cooling continued to decrease until the ATP had been present at 37 degrees C for approximately 3 min before cooling. The concentration of ATP required for maximum initial rates of protection was found to be 10(-3) M or possibly lower. The rate of decrease in susceptibility to the effects of cooling was much slower when ATP was added at 22 than at 37 degrees C, suggesting that enzyme action may be involved in the protective mechanism. Possible mechanisms for the ATP-protective action are discussed.

Acetylglucosaminidase↗
PubMed1980

Mechanisms of loss of latency of lysosomal enzymes. Effects of incubation on the properties of lysosomal membranes.

1. The effects of sucrose and KCl on the loss of latency of lysosomal enzymes caused by incubation at 37 degrees C, pH 7.4, were examined by using Triton-filled lysosomes from rat liver and two fractions from livers of rats not injected with Triton. 2. After incubation, the percentage free activity of lysosomal enzymes was measured before and after cooling to 0 degrees C in order to determine the amount of latency lost at 37 degrees C without cooling and the additional amount lost on cooling the incubated lysosomes to 0 degrees C. 3. The latency that is lost without cooling is first decreased and then increased by increasing the osmotic strength of the incubation medium with KCl, or with sucrose in the presence of KCl. However, if the osmotic strength is increased with sucrose alone, loss of latency is decreased up to 0.25M-sucrose, but is increased only slightly at higher sucrose concentrations. Apparently the lysosome is permeated by hyperosmolar KCl but not by sucrose during incubation. 4. If the osmotic strength of the assay medium is increased with KCl, the loss of latency caused by incubation for 60 min in hyperosmolar KCl is repressed. Thus it appears that a KCl-permeated lysosome can be obtained which is relatively stable until exposure to lower osmolarities. 5. The loss of latency caused by cooling incubated lysosomes to 0 degrees C is largely eliminated if the osmotic strength of the medium in which the lysosomes are cooled is raised sufficiently with either sucrose or KCl. 6. Osmotic-fragility curves were obtained after incubation for 1 and 60 min at iso-osmoticity (0.2M-KCl or 0.25 M-sucrose). Although little loss of latency occurs at iso-osmoticity, lysosomes incubated for 60 min display greatly increased fragility on exposure to hypo-osmolar KCl, hypo-osmolar sucrose or hyperosmolar KCl. 7. It is suggested that permeability to KCl at 37 degrees C and the increase in fragility on exposure to hypo-osmolar conditions are both consequences of injury, probably from enzymic action, sustained by the lysosomal membrane during incubation at 37 degrees C.

Animals↗
PubMed1980

Lysosomal changes in an animal model of myocardial ischemia. Treatment with methylprednisolone.

In this work we have employed a new extraction buffer for isolation of cardiac lysosomes from control and ischemia canine tissue. Compared to previous techniques, this buffer (0.6 M KCl, 0.25 M sucrose) enabled a 300% increase in the yield of particulate cardiac lysosomes and allowed acceptable levels of specific activity to be maintained. It also permitted great enrichment of a membrane-bound enzyme localized to a microsomal fraction, rotenone-insensitive NADH-cytochrome c reductase (RINCR). Ischemia was produced by ligation of the left anterior descending coronary artery for 2 hr, and myocardial blood flow (MBF) was measured using 85Sr labeled microspheres (15 microns). Enzymatic changes were measured in endocardial tissue of the ischemic left ventricular wall for comparison with control nonischemic samples. N-Acetyl-beta-glucosaminidase (NAG) was the most reliable lysosomal marker enzyme; this was depleted significantly (P < 0.001) in particulate lysosomal fractions; significant increases in the supernatant (P < 0.001) were found in areas of ischemia that were less than 25% of the control MBF. Lysosomal latency also diminished significantly during ischemia. Loss of total activity of RINCR in the microsomal fraction was highly significant (P < 0.001) in the areas of profound ischemia. The above data were compared with those from animals in which methylprednisolone (30 mg/kg) was administered 30 min prior to ligation of the coronary artery. Between the two groups (untreated versus methylprednisolone-treated animals) no significant differences could be found in total losses of NAG and RINCR or the rate at which these enzymatic changes ocurred. Lysosomal latency studies also revealed lack of significant changes between both groups. Thus, no significant "protective" effects of methylprednisolone could be found after 2 hr of ischemia.

Acetylglucosaminidase↗
PubMed1978

The temperature-dependence of the loss of latency of lysosomal enzymes.

1. When Triton-filled lysosomes from rat liver are incubated for up to 50min at 37 degrees C, pH7.4, in 0.25m-sucrose, no loss of latency of N-acetyl-beta-glucosaminidase or p-nitrophenyl phosphatase occurs unless the incubated lysosomes are cooled to approx. 15 degrees C. 2. It is suggested that a phase change takes place in the incubated lysosomal membranes on cooling; it starts at approx. 15 degrees C and probably is not complete at 0 degrees C. 3. Incubation of the lysosomes causes an increased potential for loss of latency of the lysosomal enzymes. This potential is not fully expressed at elevated temperature (e.g. 37 degrees C), but is expressed on cooling. 4. The increase at elevated temperature in potential for loss of latency exhibits biphasic kinetics, with an initial rapid phase followed by a slower phase, which is linear with respect to time. The extra loss of latency resulting from the rapid phase in proportional to the temperature of the incubation. 5. Arrhenius plots of the increase is potential for loss of latency during the slow phase for N-acetyl-beta-glucosaminidase and p-nitrophenyl phosphatase exhibit marked deviations from linearity beginning at approx. 15 degrees C. This suggests that the increase in potential for loss of latency is affected by a phase change that occurs around this temperature. 6. Activation energies for the increase in potential for loss of latency at and above 22 degrees C are 53.1+/-5.4kJ/mol (12.7+/-1.3kcal/mol) for N-acetyl-beta-glucosaminidase and 45.2+/-7.5kJ/mol (10.8+/-1.8kcal/mol) for p-nitrophenyl phosphatase. It is postulated that these energies reflect enzymic action, the products of which cause loss of latency to occur on cooling.

4-Nitrophenylphosphatase↗
PubMed1975

Structural and functional characteristics of membrane fractions from cardiomyopathic and dystrophic muscle.

Subcellular fractions from ventricular muscle of the cardiomyopathic Syrian hamster and breast muscle from the dystrophic chicken were prepared by zonal centrifugation in sucrose gradients. This tenchnique permitted the analysis of a wide spectrum of subcellular particles prior to sedimentation, thus avoiding loss of activity through particle clumping. Substantial differences in the distribution of beta-glucuronidase and p-nitrophenyl-phosphatase in zonal fractions from myopathic and control tissues provided evidence for contamination of fractions containing fragmented sarcoplasmic reticulum by lysosomes. This contamination may contribute signifantly to the altered lipid composition and Ca2+ transport function of the myopathic preparations.

Adenosine Triphosphatases↗
PubMed1975

Lysosomes of cardiac and skeletal muscle: resolution by zonal centrifugation.

The application of zonal centrifugation to the analysis of homogenates of cardiac and skeletal muscle permits selection of fractions that are enriched in markers for lysosomes, sarcolemma, sarcoplasmic reticulum, and mitochondria. The method of disruption of normal and pathological tissue alters significantly the distribution of total protein and peaks of enzymatic activity on the gradient. Total activities of cathepsin, N-acetyl-beta-glucosaminidase, beta-glucuronidase, and para-nitrophenylphosphatase are distributed at different concentrations of sucrose on the gradient. Beta-Glucuronidase appears to "mark" the sarcoplasmic reticulum, as well as lysosomes, of skeletal muscle, para-Nitrophenylphosphatase, a common marker of acid phosphatase of lysosomes, is enriched in those fractions of cardiac muscle containing the highest specific activity of ouabain-inhibited Na-K-ATPase. Thus, these two enzymes appear to have a localization in at least two separate organelles. On the other hand, these results may indicate the isolation of several "populations" of lysosomes that are associated constantly with distribution peaks of other organelles. In any event, attempts to correlate changes in structure of organelles of normal and pathological specimens of tissue with functional impairment, e.g., Ca2+ uptake, activity of Na-K-ATPase, etc., must include consideration of dual localization of enzymatic markers or cross contamination by populations of other organelles.

4-Nitrophenylphosphatase↗
PubMed1975

Phospholipase A and acid lipase activity during release of lysosomal hydrolases.

Hydrolysis of cardiac and hepatic lysosomal phospholipids by endogenous phospholipase A occurs during incubation at 37 degrees C at pH 5.0. Lysophospholipids and free fatty acids accumulate in association with release of hydrolases from the lysosomes into the supernatant. Acid-active neutral lipid lipases contribute to the release of free fatty acids. Albumin inhibits the production of these surface-active lipids as well as the release of hydrolases. The soluble phospholipase A is inhibited by albumin, soluble protein (cytoplasmic), heparin, and protamine sulfate. Thus, hydrolysis of lysosomal lipids, catalyzed by endogenous phospholipases, as well as acid-active neutral lipid lipases, may contribute significantly to the increased permeability, swelling, and subsequent lysis of lysosomes. Stabilization of the lysosomal membrane is associated with integrity of the structural lipids of the membrane.

Animals↗