Glucose repression and autolysis ofSaccharomyces cerevisiae cells: alterations in the cytochemical localization of acid phosphatase

Summary Allerations in the localization of acid phosphatase inSaccharomyces cerevisiae during glucose repression and during autolysis have been studied. Cell morphology becomes distinctly changed after only 2 h in the presence of high glucose concentration while after 3 h of glucose repression the majority of the mitochondirial structures resemble promitochondria. Yeast cells repressed for 6 h contain almost completely degraded mitochondrial structures and numerous lipid droplets in the central vacuole and cytoplasm. Destruction of mitochondria is accompanied by the accumulation of acid phosphatase in these organelles and in the cytoplasm whereas its activity in the central vacuole is lowered, most probably because of the leakage of the enzyme into the cytoplasm.No preferential breakdown of mitochondria is observed during autolysis. On the contrary, mitochondria are apparently the last to be degraded. Digestion of cytoplasmic regions and membranous elements occurs intravacuolarly after sequestration by protrusions of the central vacuole which are formed at the initial stages of autolysis. Acid phosphatase is not released from the central vacuole, suggesting indirectly that vacuole enzymes do not migrate into the cytoplasm during autolysis.     

Serotonin catabolism and the formation and fate of 5-hydroxyindole thiazolidine carboxylic acid

Serotonin (5-HT) functions as a neurotransmitter and neuromodulator in both the central and enteric nervous systems of mammals. The dynamic degradation of 5-HT metabolites in 5-HT-containing nervous system tissues is monitored by capillary electrophoresis with wavelength-resolved laser-induced native fluorescence detection in an effort to investigate known and novel 5-HT catabolic pathways. Tissue samples from wild type mice, genetically altered mice, Long Evans rats, and cultured differentiated rat pheochromocytoma PC-12 cells, are analyzed before and after incubation with excess 5-HT. From these experiments, several new compounds are detected. One metabolite, identified as 5-hydroxyindole thiazoladine carboxylic acid (5-HITCA), has been selected for further study. In 5-HT-incubated central and enteric nervous system tissue samples and differentiated PC-12 cells, 5-HITCA forms at levels equivalent to 5-hydroxyindole acetic acid, via a condensation reaction between L-cysteine and 5-hydroxyindole acetaldehyde. In the enteric nervous system, 5-HITCA is detected without the addition of 5-HT. The levels of L-cysteine and homocysteine in rat brain mitochondria are measured between 80 and 140 microm and 1.9 and 3.4 microm, respectively, demonstrating that 5-HITCA can be formed using available, free L-cysteine in these tissues. The lack of significant accumulation of 5-HITCA in the central and enteric nervous systems, along with data showing the degradation of 5-HITCA into 5-hydroxyindole acetaldehyde, suggests that an equilibrium coupled to the enzyme, aldehyde dehydrogenase type 2, prevents the accumulation of 5-HITCA. Even so, the formation of 5-HITCA represents a catabolic pathway of 5-HT that can affect the levels of 5-HT-derived compounds in the body.     

Acid Phosphatase Activity in Mouse Brain Infected with Venezuelan Equine Encephalomyelitis Virus

The mode of development of Venezuelan equine encephalomyelitis virus and the activity of acid phosphatase in the central nervous system of newborn mice were investigated. Precursor particles appeared to be formed in masses of viroplasm, migrating to the membrane of the Golgi cisterns and vacuoles or to the plasma membrane and being transformed into mature viral particles by budding. Mature viral particles were also found in the lumen of the blood vessels and around the myelin sheath of axons. Increased number of Golgi complexes and depletion of polysomes were the main ultrastructural alterations of the nerve cells. Acid phosphatase activity was found to be increased in the Golgi cisterns, vacuoles, and lysosomes of nerve cells. The presence of acid phosphatase activity in the rough endoplasmic reticulum and perinuclear cisterns suggests increased production of the enzyme in the nerve cells infected with Venezuelan equine encephalomyelitis virus.     

Intramitochondrial proteolytic activity of rat liver]

Highly purified mitochondria and lysosomes are isolated from rat liver homogenate. pH optimum of proteolytic activity with respect to proteins of own structures and to mitochondrial structural protein is investigated. The purification of mitochondria from lysosomes is found to be accompanied by the change of proteolytic activity pH optimum from 5.0 to 6.0 in coarse and purified mitochondria respectively. Comparative study of structural protein hydrolysis products with enzyme preparations from purified mitochondria and lysosomes has revealed differences in the spectrum of the reaction products. The data obtained suggest a presence of a proteolytic enzyme in rat liver mitochondria.     

Ultracytochemical characterization of non-specific acid phosphatase activities in Saccharomyces cerevisiae.

Glutaraldehyde prefixation causes a considerable inactivation of the acid phosphatase of yeast protoplasts in dependence on the duration of aldehyde influence. Lead ions necessary for ultracytochemical demonstration effect a still stronger inhibition of enzymatic activity. Prefixation, however, protects the enzyme from further inhibition by lead. At pH 4.4 in intact cells acid phosphatase activities are mainly localized in the periplasmic space and in vesicles fused with the plasma membrane. The cell wall and cytoplasm usually remain free of reaction products. On the cell surface activities are found in form of globular lead deposits. At pH 5.2 and 6.3 the periplasmic activity appears decreased compared to that at lower pH values and the intracellular activity is increased. The plasma membrane of protoplasts is completely free of precipitates. The intracellular activity sites of protoplasts (cisternae of endoplasmic reticulum and/or Golgi-like system, small vesicles, central vacuole, nuclear envelope) are the same as for intact cells. The occurrence of at least two forms of acid phosphatase in S. cerevisiae id deduced.     

小麦受精过程中酸性磷酸酶的超微细胞化学定位

小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ ）受精前成熟胚囊,除胚囊中央细胞的合点端细胞质中有酸性磷酸酶外,其余部位均未发现酸性磷酸酶。受精时期,以下部位存在酸性磷酸酶活性：卵细胞的细胞核内一部分染色质和细胞质中大部分线粒体;精、卵核融合时两核的核周腔内;退化助细胞合点端细胞质和一些液泡内;进入雌性细胞中的两个精核;胚囊各成员细胞的细胞壁及胚囊周围珠心细胞的细胞壁。二细胞原胚中未见有酸性磷酸酶。早期胚乳游离核染色质上有酸性磷酸酶。小麦受精过程酸性磷酸酶的分布特点可能与卵细胞生理状态的变化和细胞质中线粒体的改组、助细胞的退化、精核的生理状态以及精核与卵核的核膜融合等有关。     

Ultrastructure and cytochemical localization of acid phosphatase of laticifers in Euphorbia kansui Liou

Acid phosphatase (AcPase) activities are involved in the degeneration process of cytoplasm in plants. In this study, acid phosphatase was detected by the method of lead nitrate and cytochemical electron microscopy during the development of nonarticulated laticifers in Euphorbia kansui Liou. The most important feature in the differentiation of the laticifers in E. kansui is that the development of small vacuoles arises from endoplasmic reticulum (ER). The mature laticifers possess a thin layer of electron-dense peripheral cytoplasm in which the organelle cannot be distinguished and a large central vacuole filled with latex particles. AcPase cytochemistry studies show AcPase reaction products congregated into heaps are distributed along the tonoplast of central vacuole and around the mitochondria and plastids. Some small vacuoles which develop at later developmental stages of laticifers contain AcPase reaction products. As a result, the central vacuole is formed by cellular autophagy and fusion of small vacuoles which apparently arises from ER.     

Solubilization and partial characterization of a phosphoprotein phosphatase from human myelin.

The phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) solubilized from human central nervous system myelin has been shown to possess a comparatively high degree of specificity towards myelin basic protein, a constituent of the membrane and most likely its natural substrate, rather than the mixed histones. The enzyme has a pH optimum of 7.5. Hydrolysis of both the substrates is stimulated by dithiothreitol and is almost completely dependent upon the presence of divalent metal ions. The maximum rate of dephosphorylation of basic protein is attained in the presence of 125 micrometer Mn2+ whereas a much higher concentration of Mg2+ (50--100 mM) is required for the optimal dephosphorylation of histones. The dephosphorylation of basic protein was also stimulated by Triton X-100 (0.15%, v/v) and was shown to result from a 3-fold increase in the V of the reaction catalyzed by the phosphatase. The apparent Km values for basic protein and histones were unaffected by the presence of Triton X-100 and were found to be approx. 1 and approx. 160 micrometer, respectively. Under optimal conditions of assay, the phosphatase cleaved approx. 32 and approx. 0.7 nmol of orthophosphate.min-1.mg-1 of protein from basic protein and histones, respectively.     

A cytochemical study of the localization of acid phosphatase in Saccharomyces cerevisiae at different growth phases.

Synopsis The localization of acid phosphatase in the yeastSaccharomyces cerevisiae at different growth phases has been studied. It was shown to be crucial for authentic location of acid phosphatase that the cytochemical reaction be performed on whole cells. Dimethylsulphoxide was used to alleviate the effects of fixation of the yeast cells with glutaraldehyde; the sulphoxide did not affect the distribution of acid phosphatase in the cells. It has been established that in exponentially-growing cells acid phosphatase is localized mostly in small vacuolar compartments. In mature cells, the bulk of acid phosphatase is found in the central vacuole, although a significant amount of the enzyme is detectable in the plasma membrane and the adjacent vesicles.     

Enzyme histochemical characteristics of human and kitten odontoclasts and kitten osteoclasts: a comparative study using whole cells

Synopsis Methods for the histochemical demonstration of enzymes in whole cell preparations of odontoclasts and osteoclasts are described. Enzyme histochemical characteristics of human and kitten odontoclasts from resorbing primary teeth and of osteoclasts from kitten femur metaphyses were determined and compared. The enzyme profiles, times for the appearance of detectable reaction product, intensity of the reactions and localization of the reaction products were similar in all three types of giant cell. These findings suggest that odontoclasts have enzyme properties and metabolic functions similar to those of osteoclasts. Species differences appear to be minor, although the NADP-dependent enzymes are less active in human than in kitten odontoclasts. Both odontoclasts and osteoclasts are rich in enzymes concerned with energy production and possess considerable activity of enzymes usually associated with catabolic functions. Metabolic pathways are well developed in respect of the utilization of succinic, malic, glutamic, lactic and isocitric acids, -hydroxybutyric acid and glucose-6-phosphate, and they also possess phosphatases, non-specific esterases and leucine naphthylamidase. The distribution of enzyme reaction products for the individual enzymes demonstrated is consistent with the presence in these cells of large numbers of mitochondria and lysosome-like organelles. Considerable phosphatase activity is demonstrable in both odontoclasts and osteoclasts at both neutral and acid pH.     

Photographic microdensitometry for evaluation of acid phosphatase activity at the electron microscope level

Summary The dry mass of reaction products in ultrathin sections was determined using electron micrographs of polystyrene spheres of known weight deposited on Formvar membranes and evaluating the negatives photometrically. This method was applied to the quantification of the final reaction product of the acid phosphatase reaction in a model system in which enzyme was incorporated in gelatin. The enzyme activity was demonstrated by the lead precipitation method and quantified by direct microphotometry at the light microscope level. Models were then embedded and sectioned for electron microscopy. Microphotometric values afforded by the electron negatives were in linear correlation with incubation times and enzyme concentration. Section thickness and its possible variations due to deformation or contamination under the electron beam were also evaluated. Measurements of lysosomal acid phosphatase activity in rat kidney sections served to illustrate the application of the technique.     

The Ultracytochemical Localzation of Acid Phosphatase Activity in Wheat During Fertilization

No acid phosphatase activity was observed in the mature embryo sac of wheat (Triticum aestivum) except the chalazal cytoplasm Of the central cell before fertilization. During fertilization, acid phosphataseactivity was observed in the following loci: part of chromatin of the egg nucleus and most of the mitochondria in the egg cytoplasm; the perinuclear spaces of the egg and sperm nuclei at the fusion of the egg and sperm nuclei; the chalazal cytoplasm and some vacuoles of the degenerated synergid; two sperm nuclei within the cytoplasm of female cells; the cell wall of each cell of the embryo sac and that of the nucellar cells surrounding the embryo sac. No acid phosphatase was observed in the two-celled proembryo. Dense enzyme reaction product was localized in the chromatin of the free nuclei at early stage of the endosperm. The characteristic of acid phosphatase distribution during fertilization may be associated with the physiological change of the egg Cell, the reorganization of mitochondria in the egg cell cytoplasm, the degeneration of one of the two synergids, the physiological state of the sperm nuclei and the nuclear membrane fusion of the egg and sperm nuclei.     

Immobilization of BSA, enzymes and cells of Bacillus stearothermophilus onto cellulose, polygalacturonic acid and starch based graft copolymers containing maleic anhydride

Poly(maleic anhydride styrene) graft copolymers of cellulose, pectin polygalacturonic acid salt, calcium polygalacturonate, and starch were prepared and used to immobilize proteins. The cellulose grafts coupled quite appreciable quantities of acid phosphatase, glucose oxidase, and trypsin. However, the general retention of activity was somewhat disappointing. Further investigation with acid phosphatase showed that the amount of enzyme immobilized increased as the amount of anhydride in the graft copolymer increased but no such relationship existed for the enzymic activity. The cellulose graft copolymers were hydrolyzed and it appeared that the carboxyl group aided adsorption of the enzyme. Attempts to couple acid phosphatase using CMC through the free carboxyl groups, created by hydrolysis, gave only a small increase in the extent of protein coupling. However, the unhydrolyzed system gave a useful degree of immobilization of cells of Bacillus stearothermophilus, as did a poly(maleic anhydride/styrene)-cocellulose system. Attempts to improve the activity by using grafts based on other polysaccharide supports met with mixed success. Pectin products were soluble. Polygalacturonic acid products were partially soluble and extremely high levels of enzymic activity were obtained. This was probably due in part to the hydrophilic nature of the system, which also encouraged absorption of the enzyme. Attempts were made to reduce the solubility by using the calcium pectinate salt. Immobilization of acid phosphatase and trypsin resulted in inceased protein coupling but relatively poor activities were attained. A starch based system gave similar results. Calcium polygalacturonate was used to prepare an insoluble graft copolymeric system containing acrylonitrile-comaleic anhydride. The resulting gels gave excellent coupling with acid phosphatase which had a very good retention of activity.     

A Simple Screening Procedure for Evaluating Central Nervous System Tissue Sections Showing Structural and Cytochemical Alterations of the Blood-Brain Barrier

A simple method for rapidly screening and evaluating many areas of central nervous system tissue before and after flat embedding in Beem capsules is described. This method uses light microscopy to select regions surrounding needle track injuries of brain tissue for subsequent fine structural and enzyme cytochemical analysis of the blood-brain barrier. The mouse cerebral cortex was sectioned with a tissue chopper at 40-50 μm and reacted with diaminobenzidine to demonstrate the presence of exogenous horseradish peroxidase near an injured central nervous system site. Following the enzyme reaction, both osmicated and unosmicated tissue slices were processed for routine electron microscopy, infiltrated with unpolymerized resin, and evaluated on glass slides by light microscopy prior to flat embedding and polymerization. Numerous tissue specimens can be screened in this way for maximum information per tissue slice, and extra tissue samples can be polymerized on the glass slides and conveniently stored for future sectioning.     

Phospholipase activity in rat liver mitochondria studied by the use of endogenous substrates

The hydrolysis of endogenous phosphatidyl ethanolamine and lecithin in rat liver mitochondria has been studied by using mitochondria from rats injected with ethanolamine-1,2-(14)C or choline-1,2-(14)C. A phospholipase A-like enzyme has been demonstrated, which catalyzes the hydrolysis of one fatty acid ester linkage in phosphatidyl ethanolamine and lecithin. Phosphatidyl ethanolamine is hydrolyzed in preference to lecithin and the main reaction products are free fatty acids and lysophosphatidyl ethanolamine. The further breakdown of lysophospholipids appears to be limited in mitochondria, which indicates that lysophospholipase activity is mainly located extramitochondrially. The enzymic system is greatly stimulated by calcium ions, and also slightly by magnesium ions, while EDTA inhibits it almost completely. These findings are discussed in relation to previous observations on the effect of calcium and of EDTA on the functions of mitochondria. The possible function of the mitochondrial phospholipase for the formation of phospholipids with special fatty acids at the alpha- and -position is discussed.     

N-glycolylneuraminic acid conjugates: implications of their absence in mammalian biochemistry

N-glycolylneuraminic acid (Neu5Gc) is one of the two most common forms of sialic acids present in glycoproteins and glycolipids of mammalian tissues. It is synthesized from the most ubiquitous sialic acid, N-acetylneuraminic acid (Neu5Ac) in a hydroxylation reaction catalysed by the enzyme Neu5Ac hydroxylase. Though Neu5Gc conjugates are prevalent in many tissues of mammals, they are absent in glycolipids and only trace amounts are present in glycoproteins of the brain and central nervous system. In humans Neu5Ac is the main sialic acid as Neu5Ac hydroxylase is inactive due to mutation of its gene. The importance of sialic acids in biochemical phenomena and the distinct roles played by specific forms of these amino sugars is adequately reflected in functional studies of selectin and sialoadhesin families of adhesion molecules. The absence of Neu5Gc, therefore, in tissues of humans and brain of mammals has raised interest, especially with regard to its impact on biochemical differences evident between humans and other mammals. It is suggested that though Neu5Gc conjugates are important in cellular interactions, their presence in brain and the central nervous system is deleterious to the latter's normal functions. Their interaction with other cellular components to form supramolecular associations is indicated that may have a bearing on major biochemical differences, a few of which are presently evident between humans and other mammals.     

Lysosomal origin of the chloragosomes in the chloragogenous tissue of the earthworm Eisenia foetida: cytochemical demonstration of acid phosphatase activity

Summary The cytochemical localization of the lysosomal marker enzyme acid phosphatase was studied in the chloragogenous tissue of earthworms. The Gomori lead technique and the cerium capture technique were utilized. Both techniques demonstrated the chloragosomal location of this enzyme. Only a small proportion of chloragosomes presented reactivity, which suggests that these organelles are distinctly heterogeneous. The reaction product was localized in the periphery of chloragosomes, suggesting a membrane-bound compartmentalization of acid phosphatase. In addition, degenerating mitochondria and membrane whorls were observed in some chloragosomes, indicating the possibility that these organelles perform autophagosomal functions.     

An ultrastructural study of acid phosphatase localization in Phaseolus vulgaris xylem by the use of an azo-dye method.

The localization of acid phosphatase during xylem development has been examined in the bean, Phaseolus vulgaris. The azo dye, the final reaction product, is initially prominent in the dictyosomes, vesicles apparently participating in secondary wall formation, and in the middle lamella of the young vessel element. Final reaction particles are also present in mitochondria, chloroplasts, and certain vacuoles and are sparsely scattered in the cytoplasm. At a later stage of vessel differentiation, the azo dye is concentrated in the disintegrating cytoplasm and along the fibrils of the partially hydrolysed primary wall and middle lamella. In the mature vessel element, the azo dye is still present along the disintegrated primary wall at the side of the vessel and covers the secondary wall. In the parenchyma cell adjacent to the vessel element, acid phosphatase localization is found in the dictyosomes, endoplasmic reticulum, mitochondria, small vacuoles, and the middle lamella. The controls from all stages of vessel element development were free of azo dye particles. The concentration of acid phosphatase along the secondary walls of the mature vessels and in the middle lamella between other cells indicates that this enzyme has other functions besides autolysis of the cytoplasm and primary cell wall. Acid phosphatase may participate in the formation of the secondary wall and may also have a role in the secretion and transport of sugars.     

Purification and characterization of an acid phosphatase that displays phosphotyrosyl-protein phosphatase activity from bovine cortical bone matrix

An acid phosphatase activity that displayed phosphotyrosyl-protein phosphatase has been purified from bovine cortical bone matrix to apparent homogeneity. The overall yield of the enzyme activity was greater than 25%, and overall purification was approximately 2000-fold with a specific activity of 8.15 mumol of p-nitrophenyl phosphate hydrolyzed per min/mg of protein at pH 5.5 and 37 degrees C. The purified enzyme was judged to be purified based on its appearance as a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (silver staining technique). The enzyme could be classified as a band 5-type tartrate-resistant acid phosphatase isoenzyme. The apparent molecular weight of this enzyme activity was determined to be 34,600 by gel filtration and 32,500 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of reducing agent, indicating that the active enzyme is a single polypeptide chain. Kinetic evaluations revealed that the acid phosphatase activity appeared to catalyze its reaction by a pseudo Uni Bi hydrolytic two-step transfer reaction mechanism and was competitively inhibited by transition state analogs of Pi. The enzyme activity was also sensitive to reducing agents and several divalent metal ions. Substrate specificity evaluation showed that this purified bovine skeletal acid phosphatase was capable of hydrolyzing nucleotide tri- and diphosphates, phosphotyrosine, and phosphotyrosyl histones, but not nucleotide monophosphates, phosphoserine, phosphothreonine, phosphoseryl histones, or low molecular weight phosphoryl esters. Further examination of the phosphotyrosyl-protein phosphatase activity indicated that the optimal pH at a fixed substrate concentration (50 nM phosphohistones) for this activity was 7.0. Kinetic analysis of the phosphotyrosyl-protein phosphatase activity indicated that the purified enzyme had an apparent Vmax of approximately 60 nmol of [32P]phosphate hydrolyzed from [32P]phosphotyrosyl histones per min/mg of protein at pH 7.0 and an apparent Km for phosphotyrosyl proteins of approximately 450 nM phosphate group. In summary, the results of these studies represent the first purification of a skeletal acid phosphatase to apparent homogeneity. Our observation that this purified bovine bone matrix acid phosphatase was able to dephosphorylate phosphotyrosyl proteins at neutral pH is consistent with our suggestion that this enzyme may function as a phosphotyrosyl-protein phosphatase in vivo.     

Phosphoprotein phosphatase in the central nervous system of Manduca sexta.

The existence and some enzymological properties of phosphoprotein phosphatase (EC 3.1.3.16) have been established in the larval central nervous system of the tobacco hornworm, Manduca sexta (Lepidoptera: Sphingidae). A simple, sensitive and reproducible assay employing 32-P-labeled protamine as a phosphoprotein substrate was employed to measure phosphatase activity in both soluble and particulate fractions of the insect nerve cord. The specific activity of soluble phosphatase in the Manduca sexta central nervous system is of the same order of magnitude as that in mammalian brain. Nerve cord phosphoprotamine phosphatase activity may be stimulated by a variety of monovalent salts, the optimal concentration of NaCl or KCl being 0.2 molar. Activity does not appear to be dependent on bivalent metals and is stimulated by EDTA. A reduced sulfhydryl group is obligatory for maximum activity. Phosphatase could be greatly inhibited by sodium fluoride, ATP and GTP. Cyclic AMP and cyclic GMP are without effect on enzyme activity. Although most of the phosphatase activity in the insect nerve cord appears to be of cytosolic origin, much latent activity can be unmasked by incubating membranous fractions with Triton X-100. In contrast to soluble phosphatase, the detergent-solubilized activity is moderately stimulated by Mn-2+.?