scholarly journals Intralysosomal hydrolysis of glycyl-l-phenylalanine 2-naphthylamide

1984 ◽  
Vol 219 (3) ◽  
pp. 965-970 ◽  
Author(s):  
M Jadot ◽  
C Colmant ◽  
S Wattiaux-De Coninck ◽  
R Wattiaux
Keyword(s):  
Membrane Permeability ◽  
High Activity ◽  
Incubation Medium ◽  
Total Activity ◽  
Mitochondrial Fraction ◽  
Cathepsin C ◽  
Permeability Studies ◽  
Triton X ◽  
Hydrolysis Of ◽  
Free Activity

Glycyl-L-phenylalanine 2-naphthylamide (Gly-L-Phe-2-NNap), a cathepsin C substrate, induces an increase of the free and unsedimentable activities of this enzyme when incubated with a total mitochondrial fraction of rat liver. 1 mM-ZnSO4 considerably inhibits the cathepsin C total activity, measured with Gly-L-Phe-2-NNap as the substrate, in the presence of Triton X-100. The inhibition is markedly less pronounced when the free activity is determined; a high activity remains that depends on the integrity of the lysosomes; it decreases as the free activity of N-acetylglucosaminidase increases when lysosomes are subjected to treatments able to disrupt their membrane. Cathepsin C activity is reduced when thioethylamine hydrochloride is omitted from the incubation medium. Under these conditions at 37 degrees C, the free activity equals the total activity, although the lysosomes are intact, as indicated by the low free activity of N-acetylglucosaminidase. 1 mM-ZnSO4 strikingly inhibits the total activity, whereas more than 80% of the free activity remains. These observations are presented as evidence that Gly-L-Phe-2-NNap can possibly cause a disruption of the lysosomes as a result of its hydrolysis inside these organelles. In the presence of ZnSO4, intralysosomal hydrolysis becomes apparent, owing to a preferential inhibition by Zn2+ of extralysosomal hydrolysis; in the absence of thioethylamine hydrochloride, it is measurable because the disruption of lysosomes by Gly-L-Phe-2-NNap is delayed as a result of a slow-down of the reaction. The usefulness of Gly-L-Phe-2-NNap and related dipeptidyl naphthylamides in lysosomal-membrane-permeability studies is emphasized.

Lysosomal Triglyceride Lipase from the Lateral Line Tissue of Rainbow Trout (Salmo gairdneri)

1971 ◽  
Vol 28 (7) ◽  
pp. 1015-1018 ◽  
Author(s):  
E. Bilinski ◽  
R. E. E. Jonas ◽  
Y. C. Lau
Keyword(s):  
Rainbow Trout ◽  
Lateral Line ◽  
Lysosomal Enzymes ◽  
Lipolytic Activity ◽  
Mitochondrial Fraction ◽  
Maximum Activity ◽  
Salmo Gairdneri ◽  
Acid Lipase ◽  
Triton X ◽  
Hydrolysis Of

An acid lipase active toward tripalmitin and having the characteristics of lysosomal enzymes was shown to occur in the red lateral line tissue of rainbow trout (Salmo gairdneri). The enzyme showed maximum activity at pH 4–4.5. Triton X-100 (0.2–2.0%) strongly stimulated the activity of the acid lipase, but it inhibited markedly the lipolytic activity above pH 7. NaF (20 mM) and Na-p-chloromercuriphenyl-sulfonate (1 mM) partially inhibited the acid lipase. Fractionation of the total homogenate by differential centrifugation in 0.25 M sucrose showed that the acid lipase was present at highest concentration in the light mitochondrial fraction. Palmitic acid and dipalmitin were the two main products of hydrolysis of tripalmitin.

scholarly journals Structural equivalents of latency for lysosome hydrolases

1975 ◽  
Vol 146 (1) ◽  
pp. 97-108 ◽  
Author(s):  
F M Baccino ◽  
M F Zuretti
Keyword(s):  
Substrate Concentration ◽  
Osmotic Shock ◽  
Total Activity ◽  
Hydrolytic Activity ◽  
Linear Increase ◽  
Permeability Barrier ◽  
Significant Stimulation ◽  
Triton X ◽  
Free Activity

1. Structure-linked latency, a trait for most lysosome hydrolase activities, is customarily ascribed to the permeability-barrier function performed by the particle-limiting membrane, which shields enzyme sites from externally added substrates. 2. The influence of various substrate concentrations on the reaction rate has been measured for both free (non-latent) and total (completely unmasked by Triton X-100) hydrolase activities in rat liver cell-free preparations. The substrates were: β-glycerophosphate, phenolphthalein mono-beta-glucuronide. p-nitrophenyl N-acetyl-beta-D-glucosaminide and p-nitrophenyl β-D-galactopyranoside. The ratio (free activity/total activity) X 100 is called fractional free activity at any given substrate concentration. 3. The fractional free activity of β-glucuronidase and β-N-acetylglucosaminidase were clearly independent of substrate concentration, over the range examined, in both homogenates and lysosome-rich fractions. The fractional free activity of acid phosphatase appeared to be either unaffected (homogenate) or even depressed (lysosome-rich fraction) by increasing the β-glycerophosphate concentration. The fractional free activity of β-galactosidase consistently showed a non-linear increase with increasing substrate concentration in both homogenates and lysosome-rich fractions. 4. Procedures such as treatment with digitonin, hypo-osmotic shock and acid autolysis, although effective in causing varying degrees of resolution of the latency of lysosome hydrolase activities, were unable to modify appreciably the pattern of dependence or independence of their fractional free activities on substrate concentration, as compared with that exhibited by control preparations. Ouabain did not affect the free β-N-acetylglucosaminidase activity of liver homogenates at all. 5. Preincubation of control preparations with β-glycerophosphate or p-nitrophenyl α-galactoside did not result in any significant stimulation of the free hydrolytic activity toward these substrates. 6. The results consistently support the view that the membrane of “intact” lysosomes is virtually impermeable to all the substrates tested, except for p-nitrophenyl α-galactoside, for which the evidence is contradictory. Moreover the progressive unmasking of the hydrolase activities produced by these procedures in vitro reflects the increasing proportion of enzyme sites that are fully accessible to their substrates rather than a graded increase in the permeability of the lysosomal membrane.

Creatine kinase isoenzymes in cerebrospinal fluid in a case of brain damage.

1976 ◽  
Vol 22 (8) ◽  
pp. 1405-1407 ◽  
Author(s):  
P M Bayer ◽  
F Gabl ◽  
G Granditsch ◽  
K Widhalm ◽  
H Zyman ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Creatine Kinase ◽  
High Activity ◽  
Brain Damage ◽  
Acute Phase ◽  
Clinical Importance ◽  
Total Activity ◽  
Spinal Fluid ◽  
Consumption Coagulopathy ◽  
Creatine Kinase Isoenzymes

Abstract We present a case of a 11/2-year-old boy with toxic enteritis, consecutive consumption coagulopathy, and sever brain damage. During the acute phase we found high activity of the BB isoenzyme of creatine kinase in cerebrospinal fluid, but not in the serum. Isoenzyme MM could also be found in the spinal fluid (37.9% of the total activity). We conclude that analysis for creatine kinase isoenzymes in spinal fluid is of clinical importance.

scholarly journals The roles of phospholipase D and a GTP-binding protein in guanosine 5′-[γ-thio]triphosphate-stimulated hydrolysis of phosphatidylcholine in rat liver plasma membranes

1990 ◽  
Vol 272 (3) ◽  
pp. 749-753 ◽  
Author(s):  
K M Hurst ◽  
B P Hughes ◽  
G J Barritt
Keyword(s):  
Rat Liver ◽  
Phospholipase D ◽  
Plasma Membranes ◽  
Regulatory Protein ◽  
Gtp Binding ◽  
Liver Plasma Membranes ◽  
Low Concentrations ◽  
Rat Liver Plasma Membranes ◽  
Triton X ◽  
Hydrolysis Of

1. Guanosine 5′-[gamma-thio]triphosphate (GTP[S]) stimulated by 50% the rate of release of [3H]choline and [3H]phosphorylcholine in rat liver plasma membranes labelled with [3H]choline. About 70% of the radioactivity released in the presence of GTP[S] was [3H]choline and 30% was [3H]phosphorylcholine. 2. The hydrolysis of phosphorylcholine to choline and the conversion of choline to phosphorylcholine did not contribute to the formation of [3H]choline and [3H]phosphorylcholine respectively. 3. The release of [3H]choline from membranes was inhibited by low concentrations of SDS or Triton X-100. Considerably higher concentrations of the detergents were required to inhibit the release of [3H]phosphorylcholine. 4. Guanosine 5′-[beta gamma-imido]triphosphate and guanosine 5′-[alpha beta-methylene]triphosphate, but not adenosine 5′-[gamma-thio]-triphosphate, stimulated [3H]choline release to the same extent as did GTP[S]. The GTP[S]-stimulated [3H]choline release was inhibited by guanosine 5′-[beta-thio]diphosphate, GDP and GTP but not by GMP. 5. It is concluded that, in rat liver plasma membranes, (a) GTP[S]-stimulated hydrolysis of phosphatidylcholine is catalysed predominantly by phospholipase D with some contribution from phospholipase C, and (b) the stimulation of phosphatidylcholine hydrolysis by GTP[s] occurs via a GTP-binding regulatory protein.

scholarly journals Sporadic Emergence of Klebsiella pneumoniae Strains Resistant to Cefepime and Cefpirome in Greek Hospitals

1998 ◽  
Vol 36 (1) ◽  
pp. 266-268 ◽  
Author(s):  
L. S. Tzouvelekis ◽  
E. Tzelepi ◽  
E. Prinarakis ◽  
M. Gazouli ◽  
A. Katrahoura ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Membrane Permeability ◽  
Outer Membrane ◽  
Outer Membrane Permeability ◽  
Greek Hospitals ◽  
Hydrolysis Of

The sporadic emergence of Klebsiella pneumoniae strains resistant to cefepime and cefpirome was observed in Greek hospitals during 1996. Examination of six epidemiologically distinct strains and clones selected in vitro provided indications that resistance is due to the cooperation of decreased outer membrane permeability and hydrolysis of the cephalosporins by SHV-5 β-lactamase, which was produced in large amounts.

Multidrug salt forms of norfloxacin with non-steroidal anti-inflammatory drugs: solubility and membrane permeability studies

CrystEngComm ◽  
2018 ◽  
Vol 20 (41) ◽  
pp. 6420-6429 ◽  
Author(s):  
Biswajit Bhattacharya ◽  
Amit Mondal ◽  
Saundray Raj Soni ◽  
Susobhan Das ◽  
Surojit Bhunia ◽  
...  
Keyword(s):  
Membrane Permeability ◽  
Mefenamic Acid ◽  
Dissolution Properties ◽  
Inflammatory Drugs ◽  
Anti Inflammatory ◽  
Permeability Studies ◽  
Salt Forms

Dissolution properties and membrane permeability studies were conducted for four newly prepared multidrug salts of norfloxacin with four NSAIDs, diclofenac, diflunisal, mefenamic acid and indomethacin.

Secretion of tetraethylammonium by proximal tubules of rabbit kidneys

1983 ◽  
Vol 245 (2) ◽  
pp. F238-F246 ◽  
Author(s):  
C. Schali ◽  
L. Schild ◽  
J. Overney ◽  
F. Roch-Ramel
Keyword(s):  
Membrane Permeability ◽  
Cellular Uptake ◽  
Incubation Medium ◽  
Proximal Tubules ◽  
Tissue Water ◽  
Secretory Rate ◽  
Luminal Membrane ◽  
Secretory Transport ◽  
Energy Dependent

The secretory transport of tetraethylammonium (TEA) was investigated in perfused and nonperfused isolated S1, S2, and S3 segments of proximal tubules from rabbit kidneys. In the perfused tubules the transepithelial net secretory flux and in nonperfused tubules the TEA cellular uptake were saturable (Km = 67 microM, Vmax = 2,480 fmol X min-1 X mm-1 in perfused S2 segments), energy dependent, and inhibited by mepiperphenidol. The net secretory flux of TEA (J b leads to j TEA) at a bath TEA concentration of 40 microM differed for the three segments and decreased in the order S1 greater than S2 greater than S3. The concentration of TEA in the perfusate leaving the tubule was approximately twice as great and the intracellular TEA concentration approximately 40 times as great as that in the bath. In nonperfused segments (40 microM TEA in the incubation medium) the TEA tissue water-to-medium ratio reached 100. In the three segments the ability to accumulate TEA across the peritubular membrane, thus, was similar, but the transepithelial secretory flux differed significantly. The differences in secretory rate between the three segments presumably result from differences in the luminal membrane permeability.

scholarly journals Phosphomonoesterase hydrolysis of polyphosphoinositides in rat kidney: Properties and subcellular localization of the enzyme system

1975 ◽  
Vol 150 (3) ◽  
pp. 537-551 ◽  
Author(s):  
P H Cooper ◽  
J N Hawthorne
Keyword(s):  
Phosphatase Activity ◽  
Metal Ion ◽  
Rat Kidney ◽  
Gradient Centrifugation ◽  
Sodium Deoxycholate ◽  
Subcellular Fractionation ◽  
Kidney Cortex ◽  
Ph Optimum ◽  
Triton X ◽  
Hydrolysis Of

Tthe properties of diphosphoinositide and triphosphoinositide phosphatases from rat kidney homogenate were studied in an assay system in which non-specific phosphatase activity was eliminated. The enzymes were not completely metal-ion dependent and were activated by Mg2+. The detergent sodium deoxycholate, Triton X-100 and Cutscum inhibited the reaction; cetyltrimethylammonium bromide only activated when added with the subtrates and in the presence Mg2+. Both enzymes had a pH optimum of 7.5. Ca2+ and Li+ both activated triphosphoinositide phosphatase, but Ca2+ inhibited and L+ had little effect on diphosphoinositide phosphatase. Cyclic AMP had no effect on either enzyme. The enzymes were three times more active in kidney cortex than in the medulla. On subcellular fractionation of kidney-cortex homogenates by differential and density-gradient centrifugation, the distribution of the enzymes resembled that of thiamin pyrophosphatase (assayed in the absence of ATP), suggesting localization in the Golgi complex. However, the distribution differed from that of the liver Golgimarker galactosyltransferase. Activities of both diphosphoinositide and triphosphoinositide phosphatases and thiamin pyrophosphatase were low in purified brush-border fragments. Further experiments indicate that at least part of the phosphatase activity is soluble.

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