Acid Phosphatase Isozymes Research Articles

Occurrence and expression of acid phosphatase of Hymenoscyphus ericae (Read) Korf & Kernan, in isolation or associated with plant roots

The activity of acid phosphatase produced in pure culture by the endomycorrhizal fungus Hymenoscyphus ericae (Read) Korf & Kernan (H. ericae LPA 2) was inhibited by high phosphorus levels, alkaline pH, fluoride, molybdate and mannosidase, and activated by concanavalin A. Over 80% of the enzyme activity was due to two wall-bound acid phosphatase isozymes with the characteristics of mannose-rich glycoproteins. Antiserum was raised against the major, low-molecular-weight wall isozyme and its activity tested by immunoblotting and ELISA. The antiserum cross reacted 100% with exocellular (excreted) and 28% with cytoplasmic cellular fractions of H. ericae (LPA 2) cultures, and showed high reactivity with other strains of H. ericae but not with fungal isolates from Erica hispidula L. or E. mauritanica L. Ultrastructural localization of acid phosphatase by cytoenzymology and indirect immunogold labelling confirmed its association with the fungal wall in pure culture and showed that the influence of a high phosphorus level, fluoride and molybdate is through inactivation of the enzyme. Intense acid phosphatase activity, sensitive to the latter inhibitors, was also present on external hyphae growing over a host or non-host root but it was weak or absent from intracellular hyphae where these developed within a host root. Indirect immunolabelling confirmed that this acid phosphatase was of fungal origin and that the specific inhibitory effect of host cells is due to inactivation of the enzyme rather than repression of its synthesis. Possible implications of fungal acid phosphatase in ericoid endomycorrhizal infection processes are discussed together with mechanisms that may be regulating the enzyme activity.

Intervarietal and interspecific chimera formation by in vitro graft-culture method in Brassica

An efficient chimera formation method by tissue culture combined with grafting was studied in Brassica. Cabbage cultivars "Ruby-ball" and "YR-ranpou" (Brassica oleracea) were used for intervarietal chimera formation. Seven-day-old seedlings leaving one of two cotyledons were approach-grafted and cultured in vitro (AGSC method). Chimeric shoots were obtained by the subculture of directly growing chimeric leaves (DGCL) from grafted part and cross-cut section of the united part after the graft culture. These were rooted and grown to complete chimera. An approach-grafted culture method was also available for interspecific chimera formation between "Komatsuna" (B. campestris) and "Ruby-ball" (B. oleracea), resulting in 20% formation per culture. Interspecific chimeras were identified as complex and peripheral-sectorial type by microscopic observation and the electrophoretic analysis of acid phosphatase isozyme pattern. The AGSC method was more effective than the usual Winkler's graft method and a simple mixed culture of heterogeneous cells or tissues.

Characterization of cationic acid phosphatase isozyme from rat liver mitochondria.

Acid phosphatase isozyme was highly purified from rat liver mitochondrial fraction. The enzyme showed an isoelectric point value of above 9.5 on isoelectric focusing, and the apparent molecular weight was estimated to be 32000 by Sephadex G-100 gel filtration or 16000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme catalyzed the hydrolysis of adenosine 5'-triphosphate, adenosine 5'-diphosphate, thiamine pyrophosphate, inorganic pyrophosphate, and phosphoprotein such as casein and phosvitin, but not of several phosphomonoesters, except for p-nitrophenyl phosphate and o-phosphotyrosine. The enzyme was not inhibited by L-(+)-tartrate, and was significantly activated by Fe2+ and reducing agents such as ascorbic acid, L-cysteine,and dithiothreitol. The enzyme was found to be distributed in various rat tissues including liver, spleen, kidney, small intestine, lung, stomach, brain and heart, but not in skeletal muscle.

Survey of isozyme polymorphism for clonal identification inMusa.: I. Esterase, acid phosphatase and catalase

The isozymes of esterase, acid phosphatase and catalase were investigated electrophoretically in 44 Indian Musa cultivars belonging to the AB, AAB, ABB and AAA genomic groups. A high degree of poly...

Partial sequence of acid phosphatase-1(1) gene (Aps-1(1)) linked to nematode resistance gene (Mi) of tomato.

A partial amino acid sequence of acid phosphatase-1(1) (apase-1(1)), one of acid phosphatase isozymes of tomato, was identified. This information enabled us to synthesize degenerated primer pools of oligonucleotides for polymerase chain reactions (PCR) using cDNA for poly(A)+ RNA of tomato leaves as a template. As a result, a 135-bp, then a 467-bp PCR product were obtained. Nucleotide sequencing of these two PCR products gave a total of 522-bp sequence that was identified as a part of the Asp-1(1) gene judging from the amino acid sequence deduced from it. Using the 135-bp PCR product as a probe, we detected the restriction fragment length polymorphism (RFLP) in two different lines of tomato by genomic Southern blot analysis. We also did pulsed-field gel electrophoresis (PFGE) and Southern blot analysis to search for suitable fragments to clone into a YAC vector. As a result, a single band with a size that could be cloned into a YAC vector was detected when the genomic DNA was digested with some kinds of restriction enzymes.

Phosphate-starvation response in plant cells: de novo synthesis and degradation of acid phosphatases.

Induction of phosphatase activity is an important component of the plant cell response to phosphate deficiency. Suspension cell cultures of Brassica nigra contain two major inducible acid phosphatase (APase) isozymes; vacuolar phosphoenolpyruvate (PEP) APase and cell wall nonspecific APase. Polyclonal antibodies raised against purified PEP-APase crossreacted specifically with both isozymes. Furthermore, anti-(PEP-APase) IgG detected proteins from a wide range of higher plants, suggesting that the major plant APase isozymes have diverged from a common ancestral form. Quantification on immunoblots indicated that in B. nigra suspension cells experiencing transition from Pi sufficiency to deficiency or vice versa, the amount of total antigenic APase protein correlated closely with total enzyme activity. This was also shown in intact plant roots. Therefore, the activity was governed by the synthesis and degradation of APases. Increases in the amounts of both major APase isozymes occurred simultaneously following Pi deprivation of B. nigra suspension cells, suggesting the involvement of a common regulatory mechanism.

Embryo Culture of Lycopersicon esculentum × L. peruvianum Hybrid Genotypes Possessing Heat-stable Resistance to Meloidogyne incognita

Genotypes of Lycopersicon peruvianum (L.) Mill. and L. peruvianum var. glandulosum (Rick), selected from accessions that possess resistance to Meloidogyne incognita [(Kofoid and White) Chitwood] at high soil temperature (30C), were used as male parents in crosses with L. esculentum (Mill.) susceptible cultivars UC82, Lukullus, Tropic, and male-sterile line ms-31, respectively. The incongruity barrier between the two plant species was overcome by embryo callus and embryo cloning techniques. Hybridity of the F, progeny obtained from each cross was confirmed by differences in leaf and flower morphology, plant growth habits, and by acid phosphatase isozyme phenotypes using polyacrylamide gel electrophoresis. In greenhouse inoculation experiments, F1 plants were highly resistant to M. incognita in soil at 25 and 30C. These results confirmed the successful transfer and expression of heat-stable resistance to M. incognita from L. peruvianum to hybrids with L. esculentum as a preliminary step to introgressing additional root-knot nematode resistance into tomato.

Inheritance, linkage relationship and chromosomal localization of the glutamate oxaloacetate transaminase, acid phosphatase and diaphorase isozyme genes in Secale cereale L.

Genetic analysis of the inheritance of glutamate oxaloacetate transaminase (GOT), acid phosphatase (ACP) and diaphorase (DIA) in leaf tissue of rye revealed the involvement of four Got, four Acp and three Dia loci. Linkage analysis led to the arrangement of these and other previously described isozyme loci into four linkage groups located on chromosomes 3Rq, 4Rq, 6R, and 7Rq, respectively. Implications of these findings for possible translocation differences between chromosomes of S. cereale and S. montanum are discussed. A χ (2) component analysis which makes use of the entire potential of the information provided by codominantly inherited traits such as isozymes is described.

Regulation of acid and alkaline phosphatases of Cladosporium cucumerinum by inorganic phosphate

Cladosporium cucumerinum produced multiple isozymes of extracellular acid phosphatase, in response to phosphate concentration in the medium and related to growth phase. Production of the largest, a 174 kDa molecular form, was not affected by culture conditions and this isozyme could be a constitutive enzyme. Another form, with a mol. wt of 59 kDa, was subject to phosphate repression and was secreted to the culture medium in the early phase of fungal growth. Two minor forms, which exhibited mol. wt of 389 and 562 kDa, were detected in culture filtrates at the end of exponential growth. A single 174 kDa molecular form of acid phosphatase was detected in mycelial extracts. Furthermore, two forms of alkaline phosphatase were determined in culture filtrates and mycelial extracts in response to the phosphate level in the medium. A repressible alkaline phosphatase, with a mol. wt of 174 kDa and a form which was promoted by phosphate, with a mol. wt of 98 kDa were also found.

Biochemical differences between carrot inbreds differing in plant regeneration potential

Cultures derived from domestic carrot (Daucus carota L.) inbreds were found to vary with respect to regeneration potential as measured by the production of somatic embryos in suspension cultures. A number of biochemical parameters previously reported to distinguish embryogenic from non-embryogenic cultures of other species were measured in these carrot cell lines. Ethylene production was found to be inversely related to regeneration potential. The cell line producing the greatest number of somatic embryos exhibited the lowest rate of ethylene biosynthesis, even when grown on 2, 4-D-containing maintenance medium. A specific isozyme of acid phosphatase was associated with embryogenic calli. Proteins visualized by SDS-PAGE did not discriminate between embryo-forming and proliferating calli in all inbreds.

Regeneration potentiality and isozymic variations during morphogenesis of barley callus

Morphogenic callus cultures were obtained from 7–10 days old immature embryo explants on Murashige and Skoog and Gamborg’s medium supplemented with 2,4-D. In the initial stages of culture the frequency of shoot formation varied from 28% to 65%. After 5 to 6 months of subculturing, the frequency of shoot formation was reduced to 14%. In the initial stages of culture, growth hormones do not seem to be very important for regeneration. Cultures from young and old non-differentiating calli, and calli with shoot and/or root formation at different intervals were analysed for isozymes of esterase, peroxidase and acid phosphatase for studying the morphogenic capacity. With the development of shoot/root, changes in isozymes takes place but no specific isozyme(s) could be related to the process of induction of morphogenesis.

Growth and nutrient allocation in Phaseolus vulgaris L. colonized with endomycorrhizae or Rhizobium

Two cultivars of Phaseolus vulgaris L., one responsive to colonization with microsymbionts (Mexico 309) and one less-responsive (Rio Tibagi) were grown in Leonard jars containing sand/vermiculite under greenhouse conditions. Bean plants were either left non-inoculated (controls) or were inoculated with the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus etunicatum or a strain of Rhizobium leguminosarum bv. phaseoli (UMR-1899). Plants from the Mexico 309 cultivar maintained a higher growth rate, supported proportionately more nodules and mycorrhizae, and assimilated relatively more N or P when colonized by Rhizobium or Glomus, respectively, than did plants of the Rio Tibagi cultivar. Estimated specific nodule activity for Mexico 309 beans was more than twice that of Rio Tibagi, whereas the specific phosphorus uptake rate (SPUR) was 35% greater in the non-inoculated roots of Rio Tibagi compared to Mexico 309. Colonization by G. etunicatum more than doubled the SPUR for each cultivar compared to control roots. New acid phosphatase isozymes appeared in VAM-colonized roots of both cultivars compared to controls. Acid and alkaline phosphatase activities were significantly higher in G. etunicatum-colonized Mexico 309 roots, but not in Rio Tibagi mycorrhizae, compared to uninfected roots. Polyphosphate hydrolase activity was elevated in mycorrhizae of both cultivars compared to control roots. These results indicate that the dependence of a host on a specific endophyte increases when there are limitations to the supply of a nutrient that the endophyte can provide. The greater the increase in absorption or utilization capacity following colonization by the microsymbiont, the greater the dependence by the host. More importantly, identification of enzymatic activities that influence these plant-microbe associations opens the possibility that the specific genes that code for these enzymes could be targeted for future manipulation.

Human red cell acid phosphatase: purification and properties of the A, B and C isozymes

Human red cell acid phosphatase isozymes encoded by three alleles (ACP1*A, ACPI*B and ACP1*C), each of which generates two isozymes, (f) and (s), were purified to homogeneity. The molecular mass of the six isozymes (Af, As, Bf, Bs, Cf and Cs) was estimated to be 17-18 kDa, the mass of the f isozymes probably being slightly higher than that of the s isozymes. It was indicated that the isozymes react with p-nitrophenyl phosphate in the mono anionic state, and that a group with a pKa value of about 6, which may be histidine, is of importance for the catalytic function of the s isozymes. Significant differences between the f and s isozymes were observed with respect to specific activity. Km (p-nitrophenyl phosphate), Ki (p-aminobenzylphosphonic acid), amino acid composition, stability in the presence of urea, thermal stability, retention time in size-exclusion chromatography of the native isozymes and migration in sodium dodecyl sulphate polyacrylamide gel electrophoresis, In contrast, identical or similar properties were observed for the three genetically different f isozymes, and the same was the case for the three s isozymes. It is suggested that the f and s isozymes serve different functions in the cell.

Differential Androgen Modulation of Acid Phosphatase Isozymes in Primary Cultures of Rat Ventral Prostate Epithelial and Stromal Cells*

The influence of androgen on prostate differentiated cell function was investigated using primary cultures of rat ventral prostate epithelial and stromal cells developed from sexually immature animals (21 days of age). As a biochemical marker of androgen action, total acid phosphatase activity, which comprises both the secretory and lysosomal isoforms, was measured. Testosterone increased total acid phosphatase activity approximately 2-fold in epithelial cell cultures. This increase occurred only after the cessation of cell proliferation (i.e. upon reaching a confluent monolayer). In contrast, stromal cells showed no significant change in total acid phosphatase activity in response to androgen. Polyacrylamide gel isoelectric focusing of total acid phosphatase activity from epithelial and stromal cell extracts revealed that secretory acid phosphatase activity was localized exclusively in the epithelial cells while lysosomal acid phosphatase activity was present in both cell types. Furthermore, the androgen-induced increases in epithelial total acid phosphatase activity were found to result from increases in the secretory isoform.

Iσozymic identification of individual ectornycorrhizas synthesized between Scots pine (Pinus sylvestris L.) and isolates of two species of Suillus

SUMMARYMorphologically similar ectornycorrhizas between Scots pine (Pinus sylvestris L.) seedlings and different geographic isolates of Suillus variegatus (Fr.) O. Kuntze and Suillus bovinus (Fr.) O. Kuntze were synthesized under semi‐aseptic conditions in a sand and peat growth mixture containing ureaformaldehyde as the organic nitrogen source. Fourteen weeks after inoculation significant increases in shoot height were recorded in most of the infected seedlings and in two inoculated treatments where no mycorrhizal infection was detected. Based on the assumption that 50% of the dry mass of these ectomycorrhizas was of fungal origin, the soluble protein content of the synthesized ectomycorrhizas was found to be between 10‐ and 15‐fold greater than in equivalent amounts of the non‐symbiotic tissues (short roofs and axenically grown mycelium of S. variegatus and S. bovinus). Extracts from individual ectornycorrhizas exhibited considerable esterase (EST) and peptidase (PEP) isozyme activities following PAGE and it was confirmed that diagnostic species‐specific fungal isozymes, reproducibly detected in these extracts, were present in extracts of excised sheath tissue. Acid phosphatase (ACP) isozyme activities were also detected in the ectomycorrhizas but were restricted to a single isozyme known to be common to both fungal species. In addition to the identification of taxonomically useful diagnostic fungal isozymes, the lack of detectable host PEP or ACP isozymes in these individual ectomycorrhizas, which also exhibited induced isolate‐specific EST isozymes, provides further information on the biochemical relationships in the ectomycorrhizas.

Iǹtraspecific variation in two species of Suillus from Scots pine (Pinus sylvestris L.) forests based on somatic incompatibility and isozyme analyses

SUMMARYIsolates of Suillus variegatus (Swartz ex Fr.) O. Kuntze and Suillus bovinus (Fr.) O. Kuntze cultured from basidiocarps collected in different geographically located Scots pine (Pinus sylvestris L.) forests were subjected to an analysis of axenic growth rates, somatic incompatibility and electrophoretic isozyme expression. Intraspecific variation in the radial growth rates and at certain putative enzyme loci was detected in these fungal populations which, after tests for somatic incompatibility, were identified as being composed mainly of different clones. Following a numerical analysis of the isozyme data of seven enzyme systems, considerable interspecific dissimilarity (11% similarity) and intraspecific similarity (> 65% similarity) were detected in these fungi. Isozymes of esterase, peptidase and acid phosphatase were conserved within species but differed between these species enabling their unambiguous identification. Isolates of S. variegatus were found in two main clusters that represented the two forest locations from which they were collected. A single putative locus of glutamate oxaloacetate transaminase was found to display isozyroe polymorphism directly related to the forest location of the isolates. No habitat‐related isozyme activities could be detected in the isolates of 5. bovinus which, unlike those of S. variegatus, exhibited isozyme expression that was strongly dependent upon the medium. Isolates of S. variegatus and S. botinus identified as originating from the same vegetative clone by somatic incompatibility testing were found to exhibit 100 and 93‐97% similarity in their respective isozyme activities. These studies indicate that isozyme analyses in conjunction with somatic incompatibility testing will provide powerful tools for the genetic analysis of ectomycorrhizal communities.

Biochemical classification of date palm male cultivars

Six male cultivars of Phoenix dactylifera L., Adi, Khukri, Wardi, Ghannami Akhdhar, Ghannami Ahmar and Sumaismi were analysed for esterase (EST), glutamate oxaloace- tate transaminase (GOT), leucine amino-peptidase (LAP), peroxidase (PRX) and acid phosphatase (ACP) isozymes using polyacrylamide gel electrophoresis (PAGE). There were no differences in the isozyme banding pattern between individual plants of the same cultivar. However clear differences were observed between the cultivars tested. Such differences included the number of isozymes, the relative mobility value and the intensity of the bands. The use of isozyme system as a genetic marker for date palm biochemical classification is discussed.

Heterogeneity of lysosomes in human fibroblasts.

Lysosomes are defined traditionally with the marker enzyme acid phosphatase. We showed recently that lysosomes from human fibroblasts can be separated into a light and dense fraction as well as prelysosomal population. We now provide evidence that although acid phosphatase is enriched in all three fractions, the marker enzyme in the prelysosomal compartment is qualitatively distinct from that of the lysosomes. Ultrastructural analysis showed that the acid phosphatase in the prelysosomal vesicles deposited an extremely electron-dense reaction product, entirely obliterating the lumen of the vesicle, in contrast to that of the light and dense lysosomes which deposited a fine and diffuse product scattered throughout the luminal space. Biochemical analysis showed that only 51% of the acid phosphatase in the prelysosomes was inhibited by tartrate, while 80% of that in the lysosomes was tartrate-inhibitable. Immunoprecipitation with antibodies specific for various isozymes of acid phosphatase showed that 39% of the acid phosphatase in the prelysosomes was of the 'lysosomal' type whereas over 50% of the acid phosphatase in the lysosomes was of this type. These results showed that acid phosphatase in the prelysosomes of human cultured fibroblasts can be distinguished from that of the lysosomes cytochemically, biochemically, and immunologically and that lysosomes, as marked by acid phosphatase, are a heterogeneous organelle.

Regeneration responses of callus from different expiants and changes in isozymes during morphogenesis in wheat

Immature embryo and root meristem expiants of wheat were cultured on modified medium of Murashige and Skoog and Gamborg’s medium supplemented with 2,4-dichlorophenoxy acetic acid. Morphogeriic callus cultures were obtained from both the expiants. The frequency of shoot formation varied from 22% to 48% from callus obtained from embryos while only root formation could be induced from root meristem expiants. Cultures from young and old non-differentiating calli, and calli with shoot and/or root formation at different intervals were analysed for isozymes of esterase, peroxidase and acid phosphatase for studying the morphogenic capacity. With the development of shoot and/or root from callus, some conspicuous isozymes appeared which indicates the involvement of these isozymes in root/shoot development rather than in the induction of morphogenesis in callus. Basic isozyme pattern of each enzyme for the callus was retained in all the callus stages.

Comparison of the property of a novel isozyme E (formerly null mutant, 0) with other isozymes of hemolymph acid phosphatase of the silkworm

1. 1. A novel acid phosphatase isozyme E (formerly null mutant 0) was partially purified by ammonium sulfate fractionation, DEAE-Sephacel and Sephacryl S-200 column chromatography, and its properties were compared with those of other isozymes of the silkworm hemolymph. 2. 2. The isozyme E was extremely heat labile and showed lower pH-stability than those of others. 3. 3. Three isozymes hydrolyzed p-nitrophenyl phosphate, α-naphthyl phosphate and glucose-1 phosphate strongly. The isozyme E showed about 50% hydrolyzing activity for α-naphthyl phosphate as compared to those of A and B. 4. 4. Activities of three isozymes were inhibited by tartaric acid, sodium fluoride, ammonium molybdate and potassium diphosphate. Inhibitory effects of Cu 2+ and Hg 2+ were most remarkable against E isozyme.