Acid P-nitrophenyl Phosphatase Research Articles

Subcellular localization of human placental acid phosphatases

An electron microscopic study of acid p-nitrophenyl phosphatase activity in placental tissue has revealed two distinct localizations for this enzyme in the syncytiotrophoblast: within lysosome-like bodies and on the microvillous surface. The surface enzyme is distributed in an apparently random patchwork pattern over the chorionic villi. Evidence is presented which suggests that this nonlysosomal form may be phosphatase III, an enzyme which has previously been partially purified and characterized in vitro. Acid phosphatase activity was not observed in the cytotrophoblastic cells of Langhans' layer, and it was not present on microvilli of epithelial cells from uterine glands. These studies were carried out with a modified Gomori-type incubation medium containing a substantially reduced lead ion concentration together with p-nitrophenyl phosphate.

Subcellular Fractionation of the Colourless Alga Polytomella caeca by Differential and Zonal Centrifugation

SUMMARY: Homogenates from Polytomella caeca were fractionated by differential centrifugation. All the mitochondria (located by measurement of cytochrome c oxidase) and most of the peroxisomes (located by measurement of catalase) sedimented at 105 g min. Most of the NADPH-cytochrome c oxidoreductase, IDPase and acid p-nitrophenyl phosphatase were still present in the supernatant. This supernatant also contained a cytochrome b, cytochromes P-450 and P-416. Zonal centrifugation indicated that catalase and cytochrome oxidase were entirely sedimentable at 6 × 106 g min and that NADPH- and antimycin A-insensitive NADH-cytochrome c oxidoreductases were not mitochondrial. Peroxisomes (ρ = 1.25) were separated from mitochondria (ρ = 1.20) by both rate and isopycnic-zonal centrifugation. Two zones of acid p-nitrophenyl phosphatase activity were detected, one at ρ = 1.15 and the other at ρ = 1.21, but no other acid hydrolases were found. Separation, of polysomes and various microsomal enzymes (IDPase, acid p-nitrophenyl phosphatase, NADH- and NADPH-cytochrome c oxidoreductases) was also achieved.

Subcellular fractionation by zonal centrifugation of glucose-repressed anaerobically grown Saccharomyces carlsbergensis.

1. Homogenates were prepared from sphaeroplasts of anaerobically grown, glucoserepressed Saccharomyces carlsbergensis, and the distributions of marker enzymes investigated after zonal centrifugation on sucrose gradients containing 2mm-MgCl(2). 2. These homogenates contained no detectable cytochrome c oxidase, succinate-cytochrome c oxidoreductase, succinate-ferricyanide oxidoreductase, l(+)-lactate-cytochrome c oxidoreductase or catalase. Cytochromes a+a(3) and c were not detected. 3. Zonal centrifugation of whole homogenates indicated complex density distributions of the sedimentable portions of NADH- and NADPH-cytochrome c oxidoreductases, adenosine triphosphatases (ATPases), adenosine pyrophosphatase (ADPase), pyrophosphatase and acid p-nitrophenyl phosphatase. Several different ATPases were distinguished on the basis of their sensitivities to oligomycin and ouabain. 4. Differential centrifugation of whole homogenates at 10(5)g-min left 80-90% of the protein, dithionite-reducible cytochrome b, acid hydrolases and pyrophosphatase in a supernatant (S(1)) together with 65 and 56% of the NADH- and NADPH-cytochrome c oxidoreductases respectively, 25% of the ATPases and 71% of the adenosine monophosphatase. 5. Further analysis of supernatant S(1) revealed the presence of a class of small particles containing NADPH-cytochrome c oxidoreductases and ATPases. 6. At least four different populations of large particles were distinguished. 7. Electron microscopy indicated that one of these corresponded to ;promitochondria' as described by other workers.

Subcellular fractionation by differential and zonal centrifugation of aerobically grown glucose-de-repressed Saccharomyces carlsbergensis.

1. Homogenates were prepared from sphaeroplasts of aerobically grown glucose-de-repressed Saccharomyces carlsbergensis and the distributions of marker enzymes were investigated after differential centrifugation. Cytochrome c oxidase and cytochrome c were sedimented almost completely at 10(5)g-min, and this fraction also contained 37% of the catalase, 27% of the acid p-nitrophenyl phosphatase, 53 and 54% respectively of the NADH- and NADPH-cytochrome c oxidoreductases. 2. Zonal centrifugation indicated complex density distributions of the sedimentable portions of these enzymes and of adenosine triphosphatases and suggested the presence of two mitochondrial populations, as well as a bimodal distribution of peroxisomes and heterogeneity of the acid p-nitrophenyl phosphatase-containing particles. 3. Several different adenosine triphosphatases were distinguished in a post-mitochondrial supernatant that contained no mitochondrial fragments; these enzymes varied in their sensitivities to oligomycin and ouabain and their distributions were different from those of pyrophosphatase, adenosine phosphatase and adenosine pyrophosphatase. 4. The distribution of NADPH-cytochrome c oxidoreductase demonstrated that it cannot be used in S. carlsbergensis as a specific marker enzyme for the microsomal fraction. Glucose 6-phosphatase, inosine pyrophosphatase, cytochrome P-450 and five other enzymes frequently assigned to microsomal fractions of mammalian origin were not detected in yeast under these growth conditions.

The microsomal fraction from Tetrahymena pyriformis strain ST: characterization and subfractionation.

SUMMARY Post-mitochondrial supernatants isolated from Tetrahymena pyriformis contained high specific activities of antimycin A-insensitive NADH- and NADPH- cytochrome c oxidoreductases, acid and alkaline p-nitrophenylphosphatases, DNase and ATPase; many of the enzymes associated with mammalian microsomes were not present at detectable levels. The only haem detected was protohaem; cytochromes b 5, P 450 and P 422 were present in microsomal fractions, and ethyl morphine and aniline HC1 produced distinct changes in difference spectra. Post-mitochondrial supernatants were subfractionated by density gradient centrifugation. The distributions of NADH- and NADPH-cytochrome c oxidoreductases were different, and several distinct particles containing the latter enzyme system were distinguished. The distributions of acid p-nitrophenylphosphatase, acid DNase and ATPase were all different and indicative of several functionally distinct membrane systems. Alkaline p-nitrophenylphosphatase was non-sedimentable.

FURTHER BIOCHEMICAL AND MORPHOLOGICAL STUDIES OF GRANULE FRACTIONS FROM RABBIT HETEROPHIL LEUKOCYTES

Fractionation of rabbit heterophil leukocyte homogenates by isopycnic centrifugation as well as by zonal sedimentation has helped to characterize further the particulate components of these cells. Four classes have been identified: (A) Large (0.5-0.8 microm) and dense (1.26) azurophil or primary granules, containing all the myeloperoxidase, one-third of the lysozyme, and a major proportion of the lysosomal acid hydrolase activities of the cells. (B) Smaller (0.25-0.40 microm) and less dense (1.23) specific or secondary granules, containing 90% of the alkaline phosphatase and the remainder of the lysozyme activities, but very little if any acid hydrolases. (C) Particles of low density (1.20), containing the remainder of the lysosomal acid hydrolases. This fraction was heterogeneous, but showed abundant small rod- or dumbbell-shaped particles of moderate electron opacity, surrounded by a single membrane (tertiary granules?). The possible origin of these lysosomes from contaminating macrophages could not be ruled out but appeared unlikely. (D) Slowly sedimenting material of very low density (1.14), made up of large, empty vesicular membrane structures, and containing 10% of the alkaline phosphatase, and all of a thiol-dependent acid p-nitrophenyl phosphatase, an enzyme clearly different from the lysosomal acid phosphatase.

Intracellular Distribution of p-Nitrophenyl-Phosphatase in Plants

Less than 10% of the acid p-nitrophenylphosphatase activity of plant extracts was sedimentable under a variety of conditions including zonal centrifugation. The sedimentable fraction did not show the latency properties characteristic of animal lysosomes. We conclude that, at least operationally, plant acid phosphatase is a soluble enzyme and that it is not suitable as a lysosomal marker. The plant tissues employed were pea cotyledons, cauliflower florets, turnip storage organs, onion embryos, and leaves of red oak and soybean.