Cytochemical Analysis of Enzyme Activity during Regeneration in Blepharisma intermedium Bhandary

SYNOPSIS. Transection and repair in Blepharisma intermedium modify the distribution of several enzymes. The number of active phagosomes is reduced and there are increased staining reactions for acid phosphatase, the nucleases and type-B esterases in the ground cytoplasm. 5'-Nucleotidase activity is localized in the cytoplasmic matrix. Except for ribonuclease, the enzymes may have a perimacronuclear deposition at some period during regeneration. Acid phosphatase may be involved in development of the oral membranelles. Adenosine triphosphatase and lactic dehydrogenase have a moderate staining intensity whereas malic and succinic dehydrogenases have a strong reaction in the cytoplasm; in addition, these enzymes may be localized in the kinetics involved in the neoformation of the oral concrescence. Nuclear reorganization is attended by an increased frequency of peripheral dehydrogenase activity in the late stages of regeneration. These observations are compared with catalytic systems known to function in protozoan metabolism and in vertebrate regeneration.     

The subcellular localization of the changes in ribonuclease and phosphatase activities of the mouse kidney produced by castration and testosterone

Male mice were castrated at 2 months of age and a pellet of testosterone propionate was implanted subcutaneously ten days later. After 14 days, normal, castrated and androgen treated mice were autopsied simultaneously. The kidneys of each group were homogenized, pooled and fractionated by centrifugation into the various subcellular components. The alkaline ribonuclease and phosphatase activities were localized in the microsomal fraction and changed in inverse relationship to the previously observed changes in the rate of protein biosynthesis and the concentration of microsomal and polysomal RNA induced by castration and androgen administration. Esterase activity also was localized in the microsomal fraction and its specific activity decreased after castration and was restored to normal by testosterone. The activities of acid ribonuclease, acid phosphatase and the several marker enzymes (glucose-6-phosphatase, nucleotidase, succinic dehydrogenase and glucose-6-phosphate dehydrogenase) changed in direct proportion to the changes in weight of the kidney after castration and androgen stimulation.     

Molybdenum nutrition of isolates of four Aspergillus species

The molybdenum requirement for growth and conidial formation by Aspergillus flavus, A. terreus, and A. sulphureus was found to be 0.2 ppb, which was one-fifth that of an A. niger isolate. Molybdenum deficiency depressed growth, conidial formation, dry weight, soluble protein, and the specific activities of nitrate reductase, succinic dehydrogenase, and aconitase in all the isolates of Aspergillus studied, but the specific activities of catalase and peroxidase were depressed only in isolates of A. niger, A. terreus, and A. flavus. Also, molybdenum deficiency stimulated the specific activities of acid phosphatase and ribonuclease in the A. flavus isolate, although the specific activities of these enzymes decreased in other isolates. Eighteen hours after the addition of molybdenum (5 ppb) to molybdenum-deficient (0.02 ppb) cultures of A. niger, the specific activities of catalase, peroxidase and succinic dehydrogenase were restored in the absence of cycloheximide, while the specific activity of nitrate reductase was recovered even in the presence of the inhibitor. There was no effect on the specific activities of aconitase and acid phosphatase following the addition of molybdenum to molybdenum-deficient cultures of A. niger.     

The localization of enzymes in the cotyledons of Pisum arvense L. during germination

Summary Enzyme activity is not uniformly distributed through the cotyledon of Pisum arvense. Initially the peripheral region, certain scattered cells of the storage tissue and the procambium show a high level of activity of succinic dehydrogenase, cytochrome oxidase, acid phosphatase and esterase. Activity of acid phosphatase declines sharply after the first day of germination; activity of the other enzymes declines after about three days. In the storage tissue, where activity is lower initially, it declines after about five days and is correlated with the disappearance of the reserves. The pattern of alkaline phosphatase activity is similar except that activity is lower in the procambium but increases in the sieve-elements during differentiation of the phloem. 5-nucleotidase and glucose-6-phosphatase activity is low throughout the cotyledon but it also increases to a significant level in the sieve-elements. Activity of starch synthesizing enzymes is high in the parenchymatous bundle sheath, where they may be involved in the pathway from lipids to soluble carbohydrates.     

Untersuchungen über den Einfluss von Aflatoxin B1 auf die Morphologie und die cytochemisch fassbare Aktivität einiger Enzyme von Mucor hiemalis (Mucorales)

Zusammenfassung Mit Hilfe cytochemischer Methoden wurde der Einfluß verschiedener Konzentrationen von Aflatoxin B₁ (100 ppm, 10 ppm, 0 ppm) auf die Enzyme Succinat-Dehydrogenase, L (+)-Lactat: NAD-Oxydoreduktase, Alkohol-Dehydrogenase, L-Isocitrat: NAD-Oxydoreduktase, Malat dehydrierendes Enzym (NAD als Akzeptor), NADPH₂-Cytochrom c-Reduktase, Cytochromoxydase und saure Phosphatase des PilzesMucor hiemalis untersucht. Succinat-Dehydrogenase, Alkohol-Dehydrogenase, L-Isocitrat: NAD-Oxydoreduktase und saure Phosphatase wurden bereits durch 10 ppm des Mycotoxins in ihren Aktivitäten vermindert. Atypische Sporenkeimung und verringerter Durchmesser der Hyphen waren die äußeren Kennzeichen der Aflatoxin B₁-Intoxikation. Die Ähnlichkeit des Eingriffs von Aflatoxin B₁ in den Stoffwechsel der Leber junger Enten und in denjenigen von Zellen vonMucor hiemalis wird diskutiert.

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Investigations of the influence of aflatoxin B₁ on the morphology and the cytochemically detectable activities of some enzymes ofMucor hiemalis (Mucorales). Using cytochemical methods the influence of various concentrations of aflatoxin B₁ (100 ppm 10 ppm, 0 ppm) on the enzymes succinic dehydrogenase, L(+)-lactate: NAD oxidoreductase, alcohol dehydrogenase, L-isocitrate: NAD oxidoreductase, malate dehydrogenating enzyme (NAD as acceptor), NADPH₂: cytochrome reductase, cytochrome oxidase and acid phosphatase of the fungusMucor hiemalis was investigated. The activity of succinic dehydrogenase, alcohol dehydrogenase, L-isocitrate: NAD oxidoreductase and acid phosphatase was reduced by the addition of only 10 ppm of the mycotoxin. The morphological symptoms of aflatoxin B₁ intoxication were atypical germination of the spores and reduced diameter of the hyphae. The similarity between the interference of aflatoxin B₁ with metabolism in the liver of ducklings and that of the cells ofMucor hiemalis is the subject of discussion.

     

Lysosomes in Tetrahymena pyriformis. I. Some properties and lysosomal localization of acid hydrolases

In homogenates of Tetrahymena pyriformis, five hydrolases — phosphatase, ribonuclease, deoxyribonuclease, proteinase, amylase — with acid pH optima were found. Over 75% of their activity is sedimentable with a centrifugal force of 250,000 g. min. Only 17% of the acid phosphatase and ribonuclease is active when assayed in the presence of 0.25 M sucrose at 0°. Exposure to a lowered osmotic pressure, freezing and thawing, and incubation at temperatures over 0° result in activation of the latent phosphatase and ribonuclease. After isopycnic centrifugation in a sucrose density gradient the hydrolases show a broad distribution which differs greatly from those of enzymes associated with mitochondria (succinate dehydrogenase) or with peroxisomes (catalase). The results are interpreted as evidence that the five acid hydrolases studied are localized in lysosomes which represent a distinct population of subcellular particles in Tetrahymena.     

Age changes in the mitochondria and succinoxidase system of the Leydig cell

Summary The succinic dehydrogenase and cytochrome oxidase content of the Leydig cell were studied by histochemical means in the mouse during the first ten weeks of postnatal life; ultrastructural details of Leydig mitochondria were examined at the same time.Both enzymes appear at the end of the third week; mitochondria are present at all ages and do not change with advancing maturity. All three constituents increase with age, the increase is most marked during the 4th–6th weeks of life, and all increase at a compound rate of about 12% per day. The three components are closely related, though some succinic dehydrogenase may have an extra-mitochondrial locus; their increase is thought to be merely an expression of Leydig tissue growth and does not represent a true rise in cellular or mitochondrial enzyme concentrations. Acknowledgment. The author is grateful for the research facilities provided in the Anatomy Department of the University of Glasgow.     

HISTOCHEMICAL LOCALIZATION OF ENZYME ACTIVITIES IN ROOT MERISTEM CELLS

Avers , Charlotte J. (Douglass Coll., Rutgers—The State University, New Brunswick, N. J.) Histochemical localization of enzyme activities in root meristem cells. Amer. Jour. Bot. 48(2): 137–143. Illus. 1961,—Particle counts were made in epidermal cells of the root meristem of 2 grasses after exposure of living seedlings to various substrates involved in dehydrogenation reactions. Hydrolytic enzyme activities also were recorded for 1 of these species. The mean number of particles stained with Janus green B was about 90 for each species, but significantly lower counts were obtained in all the dehydrogenase tests. With lactate, pyruvate, glutamate, citrate, and isocitrate as substrates, Phleum cells showed about 25% of the Janus green count, while Panicum cells were about 33% active. These substrates are known to be oxidized by DPN-linked enzymes. The succinic dehydrogenase counts were about 50% of the Janus green total, and 60–70% particle activity was recorded with hexose-phosphate substrates which are probably oxidized in TPN-mediated reactions. In Phleum, particulate activity occurred in the adenosine triphosphatase and aryl sulfatase tests, but a non-particulate distribution characterized 5-nucleotidase activity. The particle counts in the ATPase tests were not significantly different from the Janus green counts, but the 85% particle activity in the aryl sulfatase tests was significantly lower than the Janus green results. These intracellular distributions were compared with those obtained by various authors using biochemical and histochemical techniques and were found to be in close agreement. It was suggested that the evidence indicated intracellular differentiation of at least one kind of cellular organelle, which in all probability was mitochondria.     

Effect of hypoxia on phosphoesterases,oxidative and glycolytic enzymes in the rabbit common carotid artery

Summary An enzyme-histochemical study was performed on the rabbit common carotid artery at periods ranging from one to seventeen days following double-ligation and injections of human , human pre- serum lipoproteins and a physiologic saline into the lumen. The alterations in enzyme activities compared to the contralateral carotid (control) were studied for DPN diaphorase, succinic acid dehydrogenase (SDH), lactic acid dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G-6-pH DH), ATPase, AMPase, acid and alkaline phosphatase. At earlier time intervals there was a general reduction in oxidative enzyme and ATPase activities concomitant with a general increase in LDH and acid phosphatase activities. At later times oxidative enzyme and ATPase activities returned to their control levels and actually increased in the thickened intima. LDH and acid phosphatase activities remained above the control levels, especially in the thickened intima. Focal areas, presumably necrotic, demonstrated complete loss of activity for all enzymes studied. AMPase activity did not differ from the controls throughout this study, while G-6-pH DH and alkaline phosphatase activities were found only sparsely in the adventitia. The same general pattern of alteration in enzyme activity was found regardless of the substance injected into the ligated artery. Arguments for the use of this experimental model for studies on the pathogenesis of atherosclerosis are given.     

Effect of enterotoxigenic Escherichia coli heat stable toxin on intestinal lysosomal enzymes in the suckling rat

A preliminary study on 9 suckling Wistar rats, which received E. coli stable toxin, and on 12 sham-operated controls showed that acid phosphatase, the marker enzyme for lysosome, was significantly increased in the infected group whereas alkaline phosphatase, glucose 6-phosphatase, succinic dehydrogenase, and proteinase, the marker enzymes for brush border, microsome, mitochondria, and the soluble fraction, respectively, remained unaffected. The results suggest that lysosome, the subcellular organelle responsible for intracellular digestion could be modified by E. coli stable toxin. In another set of experiments, where 7 infected suckling rats and 7 sham-operated controls were used, the maximal activities of lysosomal enzymes (released by Triton X-100) were found to be increased in the infected group confirming the results obtained in the preliminary experiment. The values of the ratio between maximal and basal activity (an expression of the degree of retention of enzymes to lysosome) of acid phosphatase and cathepsin D were also significantly increased, indicating that lysosomal membrane may also be stabilized during the infection. The increased activities of lysosomal enzymes and the increased lysosomal membrane stability suggest that intracellular digestion by lysosome could be increased during E. coli stable toxin infection.     

Role of DL α-lipoic acid in gentamicin induced nephrotoxicity

The effect of DL -lipoic acid on the nephrotoxic potential of gentamicin was examined. Intraperitoneal injection of gentamicin (100 mg/kg/day) to rats resulted in decreased activity of the glycolytic enzymes-hexokinase, phosphoglucoisomerase, aldolase and lactate dehydrogenase. The two gluconeogenic enzymes—glucose-6-phosphatase and fructose-1, 6-diphosphatase, the transmembrane enzymes namely the Na⁺, K⁺-ATPase, Ca²⁺-ATPase, Mg²⁺-ATPase and the brushborder enzyme alkaline phosphatase, also showed decreased activities. This decrease in the activities of ATPases and alkaline phosphatase suggests basolateral and brush border membrane damage. Decreased activity of the TCA cycle enzymes isocitrate dehydrogenase (ICDH), succinate dehydrogenase (SDH) and malate dehydrogenase (MDH), suggests a loss in mitochondrial integrity. These biochemical disturbances were effectively counteracted by lipoic acid administration. Lipoic acid administration by gastric intubation at two different concentrations (10 mg and 25 mg/kg/day) brought about an increase in the activity of the glycolytic enzymes, ATPases and the TCA cycle enzymes. The gluconeogenic enzymes however showed a further decrease in their activities at both the concentrations of lipoic acid administered. These observations shed light on the nephroprotective action of lipoic acid against experimental aminoglycoside toxicity and the protection afforded at 25 mg/kg/day of lipoic acid was noted to be higher than that at 10 mg level.     

Metabolism of the anaerobic formation of succinic acid bySaccharomyces cerevisiae

1. Succinic acid is formed in amounts of 0.2–1.7 g/l by fermenting yeasts of the genusSaccharomyces during the exponential growth phase. No differences were observed between the various species, respiratory deficient mutants and wild type strains.
2. At low glucose concentrations the formation of succinic acid depended on the amount of sugar fermented. However, the nitrogen source was found to be of greater importance than the carbon source.
3. Of all nitrogen sources, glutamate yielded the highest amounts of succinic acid. Glutamate led to an oxidative and aspartate to a reductive formation of succinic acid.
4. A reductive formation of succinic acid by the citric acid cycle enzymes was observed with malate. This was partially inhibited by malonate. No evidence was obtained that the glyoxylate cycle is involved in succinic acid formation by yeasts.
5. Anaerobically grown cells ofSaccharomyces cerevisiae contained α-ketoglutarate dehydrogenase. Its activity was found in the 175000 x g sediment after fractionated centrifugation. The specific activity increased 6-fold after growth on glutamate as compared with cells grown on ammonium sulfate.
6. The specific activities of malate dehydrogenase, fumarase, succinate dehydrogenase, succinylcoenzymeA synthetase, α-ketoglutarate dehydrogenase and glutamate dehydrogenase (nicotinamide adenine dinucleotide dependent) were determined in yeast cells grown on glutamate or ammonium sulfate. Similar results were obtained with a wild type strain and a respiratory deficient mutant. The latter did not contain succinate dehydrogenase.
7. In fermenting yeasts succinic acid is mainly formed from glutamate by oxidation.

     

STUDIES ON THE MECHANISM OF HYDROGEN TRANSPORT IN ANIMAL TISSUES : VII. INHIBITION BY RIBONUCLEASE

1. The effect of ribonuclease on various enzyme systems was studied as one approach to the problem of whether or not these enzymes are contained in macromolecules of ribonucleoprotein nature in protoplasm. 2. Ribonuclease inhibited CoI-cytochrome c reductase, succinic dehydrogenase, and cytochrome oxidase, all of which require cytochrome c in order to function. Ribonuclease did not act on cytochrome c. 3. Ribonuclease did not inhibit urease, xanthine oxidase, catalase, alkaline phosphatase, or adenosine triphosphatase under the conditions employed. 4. It was suggested that ribonuclease acted sterically by preventing contact between cytochrome c and its activating centers. 5. It was suggested that the enzymes inhibited may be contained in a ribonucleoprotein of macromolecular dimensions but that the enzymes not inhibited are not necessarily excluded from such a complex by the data presented. 6. Further evidence against the Szent-Györgyi theory of hydrogen transport was presented and discussed.     

Studies on the pathophysiology of acute renal failure. II. A histochemical study of the proximal tubule of the rat following administration of mercuric chloride.

Acute renal failure was induced in male rats by the subcutaneous injectioon of 4 mg HgC12 per kg body weight. Enzyme activities of the proximal tubule were studied histochemically at six time intervals from 15 min to 24 h. The enzyme studied were alkaline phosphatase, 5'-nucleotidase, acid phosphatase, alpha-glycerophosphate dehydrogenase (NAD-independent), malic dehydrogenase, succinic dehydrogenase, latic dehydrogenase, glucose-6-phosphate dehydrogenase and glucose-6-phosphatase. Decreases in activity were observed for alkaline phosphatase and 5'-nucleotidase after 15 min. Acid phosphatase was decreased after 30 min. These three enzymes returned to control levels after 3 h, but malic dehydrogenase and alpha-glycerophosphate dehydrogenase were decreased at this time interval. Succinic dehydrogenase was first decreased after 6 h. The earliest morphological changes detectable by light microscopy were observed in pars recta tubules in the medullary rays after 6 h, a time when all enzymes studied showed widespread decreased activity throughout the proximal tubule. After 24 h, the pars convoluta appeared morphologically normal but the pars recta was necrotic and exhibited calcification, whereas enzyme activity was decreased (absent in some cases) in both pars convoluta and pars recta. These results support the hypothesis that Hg++, when given in a sublethal dose, is associated with early histochemical changes in the brush border of the proximal tubule, which may be related to early changes in sodium reabsorption and to the subsequent development of acute renal failure. The observation that changes in plasma membrane-associated enzymes occur early and prior to alterations in enzymes of mitochondria and the endoplasmic reticulum suggests that Hg++ interacts initially with the plasma membrane.     

The screening of the enzyme and isoenzyme patterns in seeds ofAllium cepa L.

The screening of enzyme patterns in seeds ofAllium cepa cv. Všetatská revealed the presence of the following enzymes: alcohol dehydrogenase, lactate dehyd ogenase, NAD+- and NADP+-glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase NAD+- and NADP+-malate dehydrogenase, NADH₂- and NADPH₂-tetrazolium reductase catalase, Superoxide dismutase, acid and alkaline phosphatase, L-leucine aminopeptidase, glutamate dehydrogenase, non-specific esterase, and cholinesterase. Altogether 17 enzymes were detected in onion seeds, nine of which had more than three isoenzymes, NAD+-malate dehydrogenase had 8, and non-specific esterase 9 isoenzymes. The demonstration of cholinesterase and Superoxide dismutase activities is remarkable.     

Lysosomal Physiology in Tetrahymena. II. Effect of Culture Age and Temperature on the Extracellular Release of 3 Acid Hydrolases*

Tetrahymena cultures were grown from a single inoculum and collected on 3 successive days corresponding to the log, transitional, and early stationary phases of growth. Cells were washed and incubated for 5 hr in a dilute salt solution. Intra- and extracellular activities of acid phosphatase, α-glucosidase, and ribonuclease were assayed, and extracellular activities corrected for proteolytic degradation. A marked increase in the cellular content of acid phosphatase and significant decreases in α-glucosidase and ribonuclease occurred with advancing culture age. The intracellular changes in enzyme activities during incubation were roughly similar for cells of all ages. Protein content did not change appreciably during incubation. Extracellular A₂₅₅ release, monitored as an indication of the loss of RNA breakdown products, was at a minimum during incubation of transition cells. Significant quantities of all 3 acid hydrolases were released from cells of all ages except for ribonuclease from transition cells. The release of acid phosphatase and α-glucosidase was approximately proportional to the initial cellular content of these enzymes for cells of different ages and in log cells the effect of temperature on the rates of release was described by the Arrhenius equation. Release of ribonuclease, however, was not proportional to its intracellular content nor did it vary with temperature according to the Arrhenius equation. The results suggest that acid phosphatase and α-glucosidase are released via a first-order process.     

Activities of sulfhydryl-related and phenylpropanoid-synthesizing enzymes during the germination ofArabidopsis thaliana seeds

Seeds ofArabidopsis thaliana were sowed in a Murashige and Skoog’s medium and incubated in a tissue culture chamber at 26.2°C. They appeared to germinate at 60–72 hours after sowing. We measured the time-course activities of sulfhydryl-related enzymes such as thioredoxin, thioltransferase (glutaredoxin), thioredoxin reductase, and glutathione reductase. The activities of two enzymes, phenylalanine ammonia-lyase and tyrosine ammonia-lyase, which are involved in phenylpropanoid synthesis, were also measured and compared. Phenylalanine ammonia-lyase activity increased from 48 hours after sowing, whereas tyrosine ammonia-lyase activity increased transiently at the early stage and decreased slightly afterwards. Thioredoxin activity gradually decreased during the germination process. However, thioltransferase activity increased drastically from 60 hours after sowing. Thioredoxin reductase and glutathione reducatase increased rapidly from 36 hours after sowing. Thioredoxin activity was found to be relatively high in the dormant seeds.     

Age-dependent changes in the activities of ATPase and some pyridine nucleotide-linked enzymes in the chick testis.

The ontogeny of some of the enzymes connected with carbohydrate metabolism in the testis were studied in the White-Rock chicks. In the first place testicular growth in these chicks relate to their overall growth as measured by their body weights. ATPase and NAD+-dependent succinic dehydrogenase activities decreased both with advancing age and increasing testicular weight. However, these enzymes showed maximum activities at 17 and 28 weeks respectively. NAD+-linked isocitric dehydrogenase activity continually increased with increasing testicular weight and age. It is suggested that during spermatogenesis the activities of these enzymes are controlled by different developmental mechanisms.     

Development and differentiation of fetal rat sensory ganglia and spinal cord segments in vitro

Summary Fetal spinal ganglia and spinal cord segments with adhering spinal ganglia were explanted on collagen-coated coverslips. They were investigated with enzyme histochemical methods for the existence of hydrolases and dehydrogenases up to 54 days of cultivation.Alkaline phosphatase was located in arachnoid cells and in mantle cells (satellite cells). Butyrylcholinesterase and alpha-glycerophosphate-menadione-tetrazolium reductase were found in mantle cells. Acetylcholinesterase and indoxylesterase were active in the whole neuron; acid phosphatase and alpha-naphthylacetate esterase were restricted to the perikarya.During the process of cell differentiation in vitro alkaline phosphatase decreased in mantle cells. Acid phosphatase became diminished distinctly in spinal ganglion cells. The other neuronal enzymatic activities remained unaltered during the whole period of cultivation. Proliferated Schwann cells were conspicuous by their activity for butyrylcholinesterase. In newly formed myelin sheaths arylsulphatase was active. Lactate dehydrogenase was contained in the perineurium which had developed. Cultures of long duration could contain cytological formations which were reminiscent of sensory end-organs with respect to their enzyme patterns.The enzyme activities of nervous tissues in vitro in their approximation to the situation in situ are discussed.A preliminary report was presented at the 17th Meeting of the Association of German Neuropathologists and Neuroanatomists, in Freiburg, September 1972.The skilled technical assistance of Miss Johanna Sixel and Miss Charlotte Beyer is gratefully acknowledged.     

Effects of Cadmium on Respiration Rate and Activities of Several Enzymes in Soybean Seedlings

Soybean (Glycine max L. ev. Columbus) seedlings grown in culture solution were treated with cadmium as CdSO₄. Final concentrations of cadmium (Cd²⁺) in the solution were 0, 0.45, 0.90, and 1.35 μM. Soybean leaves, analyzed 10 days after Cd²⁺ was added to the culture solution, showed increased respiration rate and activities of malate dehydrogenase, acid phosphatase, ribonuclease, deoxyribonuclease, and peroxidase but decreased activity of carbonic anhydrase. Increased activity of hydrolytic enzymes and peroxidase reflects a general senescence response while the carbonic anhydrase decrease is consistent with an antagonism between cadmium and endogenous zinc. Chlorosis, epinasty, abscission of leaves, and decreased growth rate occurred in seedlings treated with 1.35 μM Cd²⁺.