Histochemical Localization of Enzymes in the Cucurbitaceae Acid Phosphatases: Acid Phosphatases

Histochemical localization of acid phosphatases was successfullycarried out on the steins of Cucumis, Cucwbita and Colocynthis—threeof the most important genera of the Cucurbitaceae whose speciesare cultivated for their fruits in the tropics. The localizationsshowed generally widespread activity and similarity in distributionfor this enzyme in the tissues of these plants. For each plantacid phosphatase activity decreased with age. The possible significanceof these localizations has been discussed in the light of thedistributional evidence and the roles usually ascribed to thisenzyme. Conventional histochemical techniques for the localizationof acid phosphatase have also been evaluated.     

Lipoteichoic Acid Localization in Mesosomal Vesicles of Staphylococcus aureus

Mesosomal vesicles and plasma membranes of Staphylococcus aureus ATCC 6538P have been prepared and examined for the presence of lipoteichoic acid. Lipids were first removed by treatment with pyridine-acetic acid-butanol (22:31:100, vol/vol/vol) and chloroform-methanol (2:1, vol/vol). Subsequently, lipoteichoic acid was removed with 40% phenol in water. The lipoteichoic acid from mesosomal vesicles was characterized by (i) equimolar glycerol and phosphate, (ii) alanine upon hydrolysis (2 N NH₄OH, 18 h, 22 C), and (iii) fatty acids, diglycerol triphosphate, glycerol monophosphate, and glycerol diphosphate upon alkaline hydrolysis (1 N NaOH, 3h, 100 C). The plasma membranes contained no lipoteichoic acid. The presence in mesosomal vesicles of 18% of the dry weight as lipoteichoic acid and its absence from plasma membranes provide the first major chemical differences between these organelles. A study of the lipoteichoic acid content in various fractions of the cell showed that the mesosomal vesicles were the major and probably the sole site for the localization of the lipoteichoic acid in these organisms. A new method for the preparation of mesosomes in increased yields is reported. A theory for the control of cell division involving lipoteichoic acid and the mesosome is proposed.     

Alkaline Phosphatases

Our knowledge of the structure and function of alkaline phosphatases has increased greatly in recent years. The crystal structure of the human placental isozyme has enabled us to probe salient features of the mammalian enzymes that differ from those of the bacterial enzymes. The availability of knockout mice deficient in each of the murine alkaline phosphatase isozymes has also given deep insights into their in vivo role. This has been particularly true for probing the biological role of bone alkaline phosphatase during skeletal mineralization. Due to space constraints this mini-review focuses exclusively on structural and functional features of mammalian alkaline phosphatases as identified by crystallography and probed by site-directed mutagenesis and kinetic analysis. An emphasis is also placed on the substrate specificity of alkaline phosphatases, their catalytic properties as phosphohydrolases as well as phosphodiesterases and their structural and functional relatedness to a large superfamily of enzymes that includes nucleotide pyrophosphatase/phosphodiesterase.     

Histochemical Localization of Alkaline Phosphatases and {beta}-Glucosidases in Cucurbitaceae

The distribution of alkaline phosphatases and ß-glucosidasesin the tissues of the stems of Colocynthls citrullus, Cucumissativus and Cucurbita pepo was studied. There was similarityin the pattern of distribution for these two groups of enzymesand that of acid phosphatases. The activities of these enzymesvaried not only from plant to plant but also from tissue totissue. However both enzymes showed increasing activity withplant age and localization was correspondingly more restricted.The significance of these localizations is discussed.     

Phytochrome Control of Acid and Alkaline Phosphatases in Etiolated Shoot Apices of Sorghum bicolor

A short pulse of red light or continuous far-red light enhancedthe activities of acid and alkaline phosphatases over the valuesof the dark controls in 5-day-old etiolated seedlings of Sorghumbicolor. For 30 min after the red light pulse 100% of the red/far-redphotoreversibilities was maintained for the acid and 80% forthe alkaline phosphatases. Thereafter, the "photoreversibilityescape reaction" was fast, being completed within 180 min. Cycloheximideas well as 6-methyl purine markedly inhibited red light enhancementof the activities of the phosphatases, but chloramphenicol,lincomiycin and rifamycin SV were ineffective. In spite of photoregulationof both the phosphatases at the time of de novo synthesis, itappears that control of the acid and alkaline phosphatases maybe affected by two independent initial actions of phytochrome. ¹ Present address: Biologisches Institut II, University of Freiburg,FRG. (Received August 4, 1984; Accepted April 3, 1985)     

Association of Matrix Acid and Alkaline Phosphatases with Mineralization of Cartilage and Endochondral Bone

The activities of acid and alkaline phosphatases were localized by enzyme histochemistry in the chondroepiphyses of 5 week old rabbits. Using paraformaldehyde-lysine-periodate as fixative, the activity of acid phosphatase was particularly well preserved and could be demonstrated not only in osteoclasts, but also in chondrocytes as well as in the cartilage and early endochondral matrices. The acid phosphatase in the chondrocytes and the matrix was tartrate-resistant, but inhibited by 2mM sodium fluoride, whereas for osteoclasts 50–100mM sodium fluoride were required for inhibition. Simultaneous localisation of both acid and alkaline phosphatase activities was possible in tissue that had been fixed in 85% ethanol and processed immediately. In the growth plates of the secondary ossification centre and the physis, there was a sequential localisation of the two phosphatases associated with chondrocyte maturation. The matrix surrounding immature epiphyseal chondrocytes or resting/proliferating growth plate chondrocytes contained weak acid phosphatase activity. Maturing chondrocytes were positive for alkaline phosphatase which spread to the matrix in the pre-mineralising zone, in a pattern that was consistent with the known location of matrix vesicles. The region of strong alkaline phosphatase activity was the precise region where acid phosphatase activity was reduced. With the onset of cartilage calcification, alkaline phosphatase activity disappeared, but strong acid phosphatase activity was found in close association with the early mineral deposition. Acid phosphatase activity was also present in the matrix of the endochondral bone, but was only found in early spicules which had recently mineralised. The results suggest that alkaline phosphatase activity is required in preparation of mineralization, whereas acid phosphatase activity might have a contributory role during the early progression of mineral formation.     

Metallophosphoryl and Apophosphoryl Alkaline Phosphatases.

The noncovalent phosphate (E-P) and covalent phosphory (E-P) complexes of Zn(II), Cd(II), and apoalkaline phosphatases of Escherichia coli have been studied by stopped flow kinetic methods and 32P-labeling techniques. With 2,4-dinitrophenylphosphate as substrate, preincubation of the Zn(II) enzyme with Pi at pH 8 slows the pre-steady state burst rate, but does not affect the burst magnitude of 1 mol of ROH per enzyme dimer. Preincubation of the enzyme with Pi at pH 5.5 reduces the burst magnitude by one-half, as well as reducing the burst rate. Reduction of the burst magnitude as a function of the pH of the preincubation with Pi follows the same function as that previously established for the formation of E-P. Hence, ROP phosphorylates the enzyme by displacing phosphate from E-P during a pre-steady state reaction, while E-P turns over at the steady state velocity.     

Histochemical Localization of Phosphatases in Mycoplasma gallisepticum

Histochemical studies of adenosine triphosphatase and acid phosphatase activity were performed on Mycoplasma gallisepticum. The adenosine triphosphatase activity appears to be localized in the bleb and infrableb regions exclusively and is associated with the cell membrane; acid phosphatase activity is localized in the infrableb region and does not appear to be membrane-associated. These findings are consistent with data from biochemical studies of Mycoplasma cell fractions but, unlike them, reveal that adenosine triphosphatase activity is restricted to a particular part of the cell membrane.     

Localization of Phosphatases in Trypanosoma rhodesiense1

ABSTRACT. Phosphatase activity in Trypanosoma rhodesiense has been examined histochemically by light and electron microscopy and by enzymatic assay in homogenate fractions. Using a method with lead as capture ion, acid phosphatase was found in lysosome-like vesicles and in the flagellar pocket. No alkaline adenosine triphosphatase (ATPase) was detectable by this method. Direct assay of p-nitrophenylphosphatase activity in homogenate fractions showed that acid phosphatase activity was strongly membrane-bound, but that activity at pH 9 was minimal in both soluble and particulate fractions. “Endogenous” ATPase activity was localized specifically and reproducibly in the mitochondrial membranes and under the plasma membrane of the flagellum. This nonenzymic reaction product could not be eradicated by glycerol extraction or glucose depletion. Unlike the membrane staining, which was manifest only after lead treatment, heat-resistant electron-dense material was found in the matrix of lysosomal vesicles in trypanosomes fixed in glutaraldehyde only and not subjected to further treatment with heavy metal reagents. X-ray emission analysis showed the presence of calcium and phosphorus, indicating that the matrix might have a phosphate storage function.     

Biochemical Changes Associated With Brassica juncea Seed Development. IV. Acid and Alkaline Phosphatases

Changes in acid and alkaline phosphatase activities in cytoplasmic and wall-bound fractions of developing mustard (Brassica juncea) seed were studied. Growth was measured by seed dry weight and water content. Seed dry weight data were fitted to a cubic polynomial equation. Seed water content and dry matter accumulation was significantly correlated. Cytoplasmic acid and alkaline phosphatase activities were substantially less in the cytoplasmic fraction than the wall-bound fraction. Wall-bound acid phosphatase activity was low initially, but high levels were maintained after day 25, indicating a relationship with dry matter accumulation. The results suggest that acid phosphatase plays an important role during mustard seed development. Received February 19, 1998; accepted May 6, 1999     

Transfection of Staphylococcus aureus with Bacteriophage Deoxyribonucleic Acid

Staphylococcus aureus cells of strain 8325 (N) are competent for phage deoxyribonucleic acid (DNA) when harvested in the early exponential growth phase. Phenotypic expression of the competence requires divalent cations, and calcium ions are most effective. Treatment of phage DNA with deoxyribonuclease completely destroys infectivity and heat-denaturated DNA is not infectious. The highest frequency of transfection is around 10(4) plaque-forming units per mug of DNA.     

Localization of a determinant mediating partial macrolide resistance in Staphylococcus aureus

Four out of more than 8,200 Staphylococcus aureus strains isolated in Japan between 1961 and 1980 were constitutively resistant to a variety of macrolide antibiotics except tylosin and rokitamycin, but susceptible to lincosamide and streptogramin type B antibiotics (PM). The data obtained by agarose gel electrophoresis, CsCl-ethidium bromide density gradient analysis, diagnosis with ATP-dependent deoxyribonuclease, and a test transducing into a rec- mutant with phage 80L2 propagated on PM-resistant S. aureus all suggested that the determinant for the PM-resistance is located in chromosome.     

Surface-Bound Nuclease of Staphylococcus aureus: Localization of the Enzyme

The cellular localization of staphylococcus nuclease, previously known as an exoenzyme, was investigated, and the following results were obtained. (i) When Staphylococcus aureus cells were converted to protoplasts by cell wall lytic enzyme L-11 (a bacteriolytic enzyme purified from Flavobacterium sp. which specifically hydrolyzes amide and peptide linkages of murein layers), over 80% of the cell-bound nuclease was released into the surrounding sucrose medium. (ii) The cell-bound nuclease was associated with the cell-wall membrane fraction of mechanically disrupted cells. (iii) The nuclease activity of cell-wall membrane fractions from cells during early and late stages of protoplast formation were compared. Less activity was found in the late stage. These results suggest that nuclease may be located at or near the surface of the cells. The distribution of cell-bound nuclease in the cell-wall membrane fraction varied with the growth conditions of S. aureus. The activity of alkaline phosphatase, another surface enzyme, was also investigated. Less of this enzyme than nuclease was released when the cells were converted to protoplasts.     

Enzymhistochemical Localization of Alkaline Phosphatases in Germinal Cysts of the Testis of the Frog Rana temporaria (Anura)

Alkaline phosphatases have been demonstrated enzymhistochemically in the testis of the common frog Rana temporaria, caught in the month of January. The follicle cells in the cyst walls and the lamina propria of the seminiferous tubules showed strong enzymatic activity; also a weak activity was observed in the peripheral region of some of the germinal cells lying within the cysts. The possible interaction of alkaline phosphatases in the transport processes across the cyst walls has been discussed.     

Elution of Loosely Bound Acid Phosphatase from Staphylococcus aureus

Strains of Staphylococcus aureus from the International-Blair and the Seto-Wilson series of phage propagating strains were examined for acid phosphatase activity. This enzyme was found to occur in varying amounts in three different fractions: free (6 to 60%), loosely bound (25 to 82%), and firmly bound (0 to 46%). Propagating strain 3A, because of its high activity, was chosen for further study. The rate of enzyme production paralleled cell growth in Trypticase Soy Broth, but followed a biphasic pattern in a semisynthetic casein acid-hydrolysate medium with glyceryl phosphate. Maximal elution of acid phosphatase in the loosely bound fraction, presumably from the surface of cells, occurred in the alkaline pH range. From log-phase cells, elution was maximally effected with buffered 1.0 M KCl (pH 7.5), but stationary-phase cells required twice the concentration of KCl.     

The Deceptive Acid Phosphatases

Determination of the prostatic acid phosphatase is, theoretically, a specific test for carcinoma of the prostate, but the present laboratory techniques have produced too many false positives and false negatives to be dependable. There may be inhibitors or enzymes that interfere with these tests.Until more exact enzymes are discovered, the present acid phosphatases should not be depended upon as a criterion for the type of surgical operation in carcinoma of the prostate, nor, without biopsy, should they be taken as an indication of prostatic malignant disease.