Histochemical localization of acid and alkaline phosphatases and glucose-6-phosphatase of the hepatopancreas of the crab, Scylla serrata (Forskål)

Simultaneous histochemical localization of non-specific monophosphate esterases, acid and alkaline phosphatases, as well as a specific monophosphate esterase, glucose-6-phosphatase has been made on the hepatopancreas of the marine crab, Scylla serrata (Forskål). Maximum activity of the 3 enzymes was observed in the juvenile and mature absorptive cells. Lining cells of the main hepatopancreatic duct exhibited a moderate activity of the 3 enzymes whereas the embryonic and fibrillar cells and connective tissue of the gland showed negative reactions for the 3 enzymes. The secretory cells showed a positive reaction for these enzymes only at the brush border.Bilateral eyestalk removed evoked a rise in the activity of the 3 enzymes within 2–4 h. The same effect was observed after injection of eyestalk extract to both normal and eyestalkless animals followed by restoration to the normal level after 24 h.The present observations indicate that glucose-6-phosphatase and acid phosphatase may be under the direct influence of eyestalk hormone(s) while alkaline phosphatase activity appears to be related to changes in the substrate. The physiological significance of the various cell types and enzymes is discussed.     

Distribution of lysosomal enzymes in different types of rat liver cells.

Eight lysosomal enzymes were measured in different types of rat liver cells. Hepatocytes were purified by low speed centrifugation of a cell suspension obtained by treating the perfused liver with collagenase. Nonparenchymal cells (NPC) were purified by centrifugation after treating the initial cell suspension with pronase, which selectively destroys the parenchymal cells (PC). Kupffer cells were found to attach selectively to tissue culture dishes after overnight culture of an NPC suspension. The specific activity of lysosomal enzymes was generally higher in NPC than in hepatocytes, but the different enzymes were concentrated to different degrees in the NPC. Specific activity of acid phosphatase was 1.7 times higher in NPC than in hepatocytes. Specific activity of acid DNAase, on the other hand, was 8 times higher in NPC than in hepatocytes. Other enzymes showed intermediate values. Assuming that 30% of the liver cells are nonparenchymal it may be calculated that from 7% (acid phosphatase) to 25% (acid DNAase) of the hepatic lysosomal enzymes are present in the NPC. The pattern of lysosomal enzymes in cultured Kupffer cells was similar to that of the NPC from which the Kupffer cells were derived. Cathepsin D and β-glucuronidase were, however, elevated in Kupffer cells as compared with NPC. The enzyme pattern in Kupffer cells was almost identical with that of rat peritoneal macrophages.     

Studies on the alkaline phosphatase and 5'-nucleotidase of Dictyostelium discoideum.

The alkaline phosphatase and 5′-nucleotidase activities of Dictyostelium discoideum are due to two distinct enzymes. Both enzymes are membrane bound, but over 90% of the 5′-nucleotidase activity is solubilized when the crude membrane fraction of the cell is treated with phospholipase C under conditions that release only 10% of the alkaline phosphatase.Part of the alkaline phosphatase activity can be detected in whole cells, suggesting that some of the enzyme molecules are located on the exterior surface of the plasma membrane. In contrast very low 5′-nucleotidase activity can be detected in whole cells. When membrane preparations, isolated from cells that had been surface labeled with ¹²⁵I, were subjected to sedimentation equilibrium on sucrose density gradients, the majority of the ¹²⁵I-radioactivity cosedimented with the alkaline phosphatase and 5′-nucleotidase activites, suggesting that both enzymes are plasma membrane components.The two enzymes have distinctly different pH optima, but otherwise their properties are remarkably similar. Both enzymes are inhibited by cyanide, sulfhydryl inhibitors and sulfhydryl reagents, although in each case the 5′-nucleotidase is slightly more susceptible. Both enzymes are inhibited by the levamisole analogue, R 8231, but the alkaline phosphatase is inhibited to a somewhat greater extent. Both enzymes are activated by incubation at 50 °C but inactivated by higher temperatures.The two enzymes increase in activity at identical times during differentiation, suggesting that they are under coordinate developmental control.     

Toxic effects of ozone on murine L929 fibroblasts. Enzyme inactivation and glutathione depletion.

Exposure of L929 murine fibroblasts to ozone resulted in K+ leakage and inhibition of several enzymes. Most sensitive to ozone exposure were glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase. The activities of another cytosolic enzyme, lactate dehydrogenase, the mitochondrial enzymes glutamate dehydrogenase, succinate dehydrogenase, cytochrome c oxidase and the activity of the lysosomal enzymes acid phosphatase and beta-glucuronidase were, initially, not or only slightly affected. The localization of the lysosomal enzymes did not change during ozone exposure. After prolonged exposure complete deterioration of the cells was observed and all enzyme activities declined. The activity of the enzymes was also monitored during ozone exposure of a sonicated cell suspension and it was shown that all these enzymes are in fact susceptible to ozone. These observations clearly demonstrate that, besides the structure and amino acid composition of an enzyme, the localization in the cell plays an important role in its susceptibility to ozone. The intracellular levels of reduced and oxidized glutathione were affected as well. The ATP content, however, proved to be insensitive to ozone exposure.     

Resolution and purification of three periplasmic phosphatases of Salmonella typhimurium.

A survey of Salmonella typhimurium enzymes possessing phosphatase or phosphodiesterase activity was made using several different growth conditions. These studies revealed the presence of three major enzymes, all of which were subsequently purified: a cyclic 2' ,3'-nucleotide phosphodiesterase (EC 3.1.4.d), an acid hexose phosphatase (EC 3.1.3.2), and a nonspecific acid phosphatase (EC 3.1.3.2). A fourth enzyme hydrolyzed bis-(p-nitrophenyl)phosphate but none of the other substrates tested. No evidence was found for the existence of an alkaline phosphatase (EC 3.1.3.1) or a specific 5'-nucleotidase (EC 3.1.3.5) in S. typhimurium LT2. All three phosphatases could be measured efficiently in intact cells, which suggested a periplasmic location; however, they were not readily released by osmotic shock procedures. The nonspecific acid phosphatase, which was purified to apparent homogeneity, yielded a single polypeptide band on both sodium dodecyl sulfate and acidic urea gel electrophoretic systems.     

Activity and distribution of lysosomal enzymes during collagenous matrix-induced cartilage,bone, and bone marrow development

The appearance of the lysosomal enzymes acid phosphatase, arylsulfatase, and β-glucuronidase was studied during endochondral bone and bone marrow formation induced by implantation of demineralized bone matrix. The activities of acid phosphatase and β-glucuronidase gradually increased from the stage of mesenchymal cell proliferation on Day 3 onward to reach a peak on Day 13, during maximal bone remodeling. However, arysulfatase activity exhibited a sharp increase on Day 9, associated with the onset of cartilage hypertrophy and chondrolysis. The peak of arylsulfatase activity was also attained on Day 13. The activities of all three enzymes declined on Day 15 but acid phosphatase again exhibited an increase during hematopoietic bone marrow differentiation on Days 19–21. Histochemical and ultrastructural studies revealed intense lysosomal enzyme activity in macrophage-like cells on Day 7 and thereafter. During chondrolysis and bone remodeling, these cells were present in a perivascular location. Osteoclasts also exhibited strong reactivity for the lysosomal enzymes. Due to its characteristic temporal appearance during development of endochondral bone, arysulfatase may be used as a marker enzyme for chondrolysis and bone resorption.     

Regulation of two phosphatases and a cyclic phosphodiesterase of Salmonella typhimurium.

The regulation of three Salmonella typhimurium phosphatases in reponse to different nutritional limitations has been studied. Two enzymes, an acid hexose phosphatase (EC 3.1.3.2) and a cyclic phosphodiesterase (EC 3.1.4.d), appear to be regulated by the cyclic adenosine 3' ,5'-monophosphate (AMP) catabolite repression system. Levels of these enzymes increased in cells grown on poor carbon sources but not in cells grown on poor nitrogen or phosphorus sources. Mutants lacking adenyl cyclase did not produce elevated levels of these enzymes in response to carbon limitation unless cyclic AMP was supplied. Mutants lacking the cyclic AMP receptor protein did not produce elevated levels of these enzymes in response to carbon limitation regardless of the presence of cyclic AMP. Since no specific induction of either enzyme could be demonstrated, these enzymes appear to be controlled solely by the cyclic AMP system. Nonspecific acid phsphatase activity (EC 3.1.3.2) increased in response to carbon, nitrogen, phosphorus, or sulfur limitation. The extent of the increase depended on growth rate, with slower growth rates favoring greater increases, and on the type of limitation. Limitation for either carbon or phosphorus resulted in maximum increases, whereas severe limitation of Mg2+ caused only a slight increase. The increase in nonspecific acid phosphatase during carbon limitation was apparently not mediated by the catabolite repression system since mutants lacking adenyl cyclase or the cyclic AMP receptor protein still produced elevated levels of this enzyme during carbon starvation. Nor did the increase during phosphorus limitation appear to be mediated by the alkaline phosphatase regulatory system. A strain of Salmonella bearing a chromosomal mutation, which caused constitutive production of alkaline phosphatase (introduced by an episome from Escherichia coli), did not have constitutive levels of nonspecific acid phosphatase.     

Thymidine as synchronizing agent. 3. Persistence of cell cycle patterns of phosphatase activities and elevation of nuclease activity during inhibition of DNA synthesis

Synchronous cultures of HeLa cells were obtained by selective detachment of cells in mitosis and fluctuations in enzyme activity were followed during the subsequent cell cycle. The enzymes measured were alkaline and acid phosphatases and a nuclease active on denatured DNA at alkaline pH (alkaline DNase). Each of these enzymes showed a different pattern of activity in the cell cycle, but a temporal relationship to the DNA synthetic phase was apparent in each case. Treatment of the cultures at the beginning of the cell cycle with 15 mM thymidine did not alter the subsequent pattern of fluctuations in activity of alkaline phosphatase or of acid phosphatase, although DNA synthesis was fully inhibited by this treatment. This indicates that the pattern of activity of some enzymes is not linked to DNA replication. On the other hand, the pattern of fluctuations in the activity of alkaline DNase was abolished by thymidine treatment, and elevation of the activity of this enzyme was observed. These results suggest complex and variable relationships between phases of the cell cycle and enzyme activity, and show that inhibition of DNA synthesis is not a suitable procedure for induction of culture synchrony if enzyme activities are to be studied.     

A light- and electron-microscopic study of enzymes in the embryonic shell-forming tissue of the freshwater snail, Biomphalaria glabrata

Abstract. The mode of formation of the molluscan exoskeleton is still poorly understood, but studies on adult snails indicate that enzymes involved in vertebrate bone formation also participate in mollusc shell formation. The enzymes peroxidase, alkaline phosphatase, and acid phosphatase are expressed in a constant pattern and help to identify the different zones of the adult shell-forming tissue. The present study evaluates whether the expression of these enzymes is also a tool for the identification of the developing zones of the embryonic shell-forming tissue. Thus, we analyzed the temporal and spatial activity of the above-mentioned enzymes and of tartrate-resistant acid phosphatase in the shell forming tissues in Biomphalaria glabrata. Embryos of different age groups and adults were studied; alkaline phosphatase activity was seen in very young embryos in the shell field invagination prior to the secretion of any shell material, while peroxidase activity was present from the start of the periostracum production. Acid phosphatase, found in considerable amounts in yolk granules and albumen cells, appeared in the embryonic shell-forming tissue in relatively few Golgi stacks. Tartrate-resistant phosphatase was not present in embryos, but was found in adults in the same zone of the mantle edge as acid phosphatase. Using the enzymes as cell markers, the differentiation of the embryonic shell-forming tissue to the different zones of the adult mantle edge could clearly be followed.     

Incorporation of urease into liposomes.

1. Plasma membranes were isolated from ascites hepatoma AH-130 and rat livers with or without partial hepatectomy or bile duct ligation. Reciprocal manifestations of two marker enzymes for plasma membranes were observed in these membrane preparations; alkaline phosphatase activity was found much higher in the hepatoma membrane than in any preparations of the liver membranes, whereas 5'-nucleotidase activity was much lower in the former than in the latter. 2. Effects of lectins and anti-plasma membrane antiserum on these two marker enzymes were examined. The results showed that about 50% of apparent activity of 5'-nucleotidase found in the hepatoma membrane was exhibited by alkaline phosphatase. 3. Localizations of alkaline phosphatase and 5'-nucleotidase in polyacrylamide gels after electrophoresis were demonstrated using 5'-AMP and 5-Br, 4-Cl-indoxylphosphate as substrate. There was a difference in electrophoretic mobility between the alkaline phosphatase of the hepatoma and that of the liver.     

The surface membrane of the small intestinal epithelial cell. I. Localization of adenyl cyclase.

The subcellular distribution of adenyl cyclase was investigated in small intestinal epithelial cells. Enterocytes were isolated, disrupted and the resulting membranes fractionated by differential and sucrose gradient centrifugation. Separation of luminal (brush border) and contra-luminal (basolateral) plasma membrane was achieved on a discontinuous sucrose gradient. The activity of adenyl cyclase was followed during fractionation in relation to other enzymes, notably those considered as markers for luminal and contraluminal plasma membrane. The luminal membrane was identified by the membrane-bound enzymes sucrase and alkaline phosphatase and the basolateral region by (Na+ + K+)-ATPase. Enrichment of the former two enzymes in purified luminal plasma membrane was 8-fold over cells and that of (Na+ + K+)-ATPase in purified bisolateral plasma membranes was 13-fold. F--activated adenyl cyclase co-purified with (Na+ + K+)-ATPase, suggesting a common localization on the plasma membrane. The distribution of K+-stimulated phosphatase and 5'-nucleotidase also followed (Na+ + K+)-ATPase during fractionation.     

Enzymes involved in the metabolism of flavin nucleotides in Streptomyces olivaceus

The work was aimed at studying enzymes involved in the metabolism of flavin nucleotides, namely, riboflavin kinase (EC 2.7.1.26) and FAD pyrophosphorylase (EC 2.7.7.2), as well as flavin mononucleotide hydrolysis by acid phosphatase (EC 3.1.3.2) and alkaline phosphatase (EC 3.1.3.1) in Streptomyces olivaceus actively producing vitamin B12. No correlation could be established between changes in the activity of the above enzymes during the culture growth and the qualitative composition of flavins. The enzyme activity was assayed using, as an enzyme preparation, both intact cells and a cell-free extract obtained by disintegrating the mycelium with different techniques. The screening effect of phosphatases exerted when the activity of riboflavin kinase was assayed could be partly eliminated by adding sodium fluoride to the incubation medium. The localisation of the above enzymes in the cytoplasm is discussed.     

Subcellular distribution of marker enzymes in cells of a minute fungus, Fusidium sp. 100-3

An electron microscope cytochemical technique was used to determine the subcellular distribution of marker enzymes in Fusidium sp. 100-3 cells. Nucleoside diphosphatase was found in the nuclear envelope and intracytoplasmic membrane segment. Thiamine pyrophosphatase was found to be associated with the mesosomes. Cytochrome c (oxidase) activity was found only in the mitochondrial cristae. Strong alkaline phosphatase activity was present in the vacuole; in addition, the enzyme activity was discretely dispersed throughout the cytoplasm without any association with any membrane material. The overall characteristics of the cell ultrastructure and subcellular enzyme distribution of Fusidium sp. 100-3 cells compare fairly well with those of a fungal cell. But there are considerable differences from the characteristics of higher eucaryotic cells. Detailed data on the marker enzymes distribution in a variety of fungal cells are not available. Therefore, it is not possible to conclude whether the marker enzyme distribution of Fusidium sp. 100-3 cells is unique or is typical of any fungal organism. Detailed studies of cell ultrastructure of and marker enzyme distribution in minute fungal cells and their comparison to the ultrastructure of and marker enzyme distribution in other fungal organisms may be helpful in understanding the phylogenetic and ontogenic development of subcellular organelles.     

Lactobacilli as effectors of host functions: no influence on the activities of enzymes in enterocytes of mice.

Preparations of lactobacilli are often used as dietary supplements to improve the growth and efficiency in utilizing food of animals of commercial value. We tested in an experimental model whether the effects of lactobacilli on growth of and food utilization by animals may be due to alteration of the activities of absorptive enzymes in the small bowel. Germfree mice housed in isolators under tightly controlled conditions were monoassociated with one of four strains of indigenous Lactobacillus spp. From 1 to 5 weeks later, the activity of alkaline phosphatase was assayed in homogenates of segments of the upper small intestines of the associated animals and of matched germfree controls. The specific activity of the enzyme was the same in the mice in the two groups. In other experiments, epithelial cells were isolated from the upper small intestines of mice associated with eight Lactobacillus strains (octa-associated) and from those of matched germfree mice and assayed for alkaline phosphatase, phosphodiesterase, and thymidine kinase activities. The epithelial cells were harvested sequentially from the tips of the villi toward the crypts of Lieberkühn of the intestines. In all preparations, mice of both types yielded an equivalent mass (wet weight) of cells. The protein content of the cells reflected the mass. The activities of the microvillous membrane enzymes alkaline phosphatase and phosphodiesterase and the cytosol enzyme thymidine kinase were the same whether or not the animals contained the bacteria. Therefore, any effects on animal growth and food utilization observed when lactobacilli are used as dietary supplements may not be due to a direct alteration by the bacteria of the absorptive enzymes of the host animal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Directed Evolution of Metabolic Pathways in Microbial Populations. I. Modification of the Acid Phosphatase Ph Optimum in S. CEREVISIAE

An experimental system for directing the evolution of enzymes and metabolic pathways in microbial populations is proposed and an initial test of its power is provided.-The test involved an attempt to genetically enhance certain functional properties of the enzyme acid phosphatase in S. cerevisiae by constructing an environment in which the functional changes desired would be "adaptive". Naturally occurring mutations in a population of 10(9) cells were automatically and continuously screened, over 1,000 generations, for their effect on the efficiency (K(m)) and activity of acid phosphatase at pH 6, and for their effect on the efficiency of orthophosphate metabolism.-The first adaptation observed, M1, was due to a single mutational event that effected a 30% increase in the efficiency of orthophosphate metabolism. The second, M2, effected an adaptive shift in the pH optimum of acid phosphatase and an increase in its activity over a wide range of pH values (an increment of 60% at pH 6). M2 was shown to result from a single mutational event in the region of the acid phosphatase structural gene. The third, M3, effected cell clumping, an adaptation to the culture apparatus that had no effect on phosphate metabolism.-The power of this system for directing the evolution of enzymes and of metabolic pathways is discussed in terms of the kinetic properties of the experimental system and in terms of the results obtained.     

Acid phosphatase and adenosine triphosphatase activities in the cell wall of baker's yeast.

In order to establish whether a specific adenosine triphosphatase is present in yeast cell wall, hydrolysis rates for p-nitrophenylphosphate (acid phosphatase activity) and for ATP (ATPase activity) were compared under various conditions. Rate determinations were made with both, intact cells and with preparations containing secreted enzymes from protoplasts. Acid phosphatase and ATPase activities had the same pH profile and were susceptible in the same way to the repression by orthophosphate and to the inhibition by 2-deoxyglucose. The Lineweaver-Burk plot shows biphasic kinetic behaviour for the hydrolysis of either p-nitrophenylphosphate or ATP. This suggests the existence of two enzymes with different affinities for the substrates, or one enzyme with at least two active sites. The two activities differ in thermostability and only one activity could be completely abolished by heat treatment. The thermostable enzyme activity had K-m values of 0.475 mM for p-nitrophenylphosphate, and 0.040 mM for ATP. ATP behaved as a partially competitive inhibitor of p-nitrophenylphosphate hydrolysis. Substrate competition studies showed that only a non-specific acid phosphatase is responsible for the hydrolysis of ATP.     

Histochemical studies on reserve substances and enzymes in female gametophyte of Zea mays.

Cytochemical changes during the early development of maize caryopsis are reported. Changes in the localization of different reserve substances (e.g. polysaccharides, proteins, nucleic acids and lipids) and enzymes (acid phosphatase, esterase, lipase, phosphorylase, succinate dehydrogenase, cytochrome oxidase and peroxidase) have been studied in unfertilized and fertilized ovules. Before pollination very feeble enzyme activity (acid phosphatase, succinate dehydrogenase, cytochrome oxidase and peroxidase) was observed. Reserve substances were present in low amounts before pollination. Pollination stimulated the accumulation of several substances and enzymes in the tip of the nucellus, micropylar zone. Just prior to, during and after fertilization, the cells in the micropylar zone had strong reaction for several enzymes indicating temporary enhancement of metabolic activity in the micropylar zone. The role of antipodals in the storage of reserve food products and nutrition of embryo and early stages of endosperm development is discussed. The pattern of enzymatic changes within the embryo sac reflected the biochemical changes operative during quiescent and active stages. The nucellus of Zea mays contains many enzymes required for hydrolysis of reserved food substances. A role of acid phosphatase in autolysis of nucellar cells, after fertilization is suggested. Post-fertilization increase in the activity of enzymes and accumulation of reserve materials is interpreted as reflecting a presumed increase in the metabolic rate relative to growth and differentiation.     

Transformed SV3T3 cells have a reduced lysosomal compartment and lower levels of enzyme activity than 3T3 cells.

The secreted and intracellular activities of a number of lysosomal hydrolases were higher in 3T3 cells than in SV40-transformed cells. The number of lysosomes and their total volume were also much larger in 3T3 cells and the surface area of their lysosomal membranes was almost twice that of SV3T3 cells. These differences alone were not sufficiently large, however, to account for the disparity seen in activity of some enzymes. Gel electrophoresis showed that a number of protein components present in lysosomal membranes purified from 3T3 cells were absent from SV3T3 membrane preparations. The absence of these components may be correlated with the reduced enzyme activity of SV3T3 cells particularly with respect to beta-glucosidase and acid phosphatase, both of which are normally found associated with lysosomal membranes.     

Regulation of thiamine biosynthesis in Saccharomyces cerevisiae.

A pho6 mutant of Saccharomyces cerevisiae, lacking a regulatory gene for the synthesis of periplasmic thiamine-repressible acid phosphatase activity, was found to be auxotrophic for thiamine. The activities of four enzymes involved in the synthesis of thiamine monophosphate were hardly detectable in the crude extract from the pho6 mutant. On the other hand, the activities of these enzymes and thiamine-repressible acid phosphatase in a wild-type strain of S. cerevisiae, H42, decreased with the increase in the concentration of thiamine in yeast cells. These results suggest that thiamine synthesis in S. cerevisiae is subject to a positive regulatory gene, PHO6, whereas it is controlled negatively by the intracellular thiamine level.     

The enzyme cytochemistry of the intracellular organelles in the rat choroid plexus epithelial cell

Electron microscopic cytochemical studies on the rat choroid plexus epithelium have revealed enzymatic sites for the activities of acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase on different organelles. Only the activity of acid phosphatase has been previously described. Acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase were respectively situated mainly in the lysosomes, in the endoplasmic reticulum an nuclear envelope, and in the Golgi complex. These three enzymes can thus be considered as marker enzymes for their respective organelles in the choroid plexus epithelial cells as well as in other tissue cells. The possible function of these enzymes in the choroid plexus epithelial cells is also briefly discussed.