Acid phosphatase-11, a tightly linked molecular marker for root-knot nematode resistance in tomato: from protein to gene,using PCR and degenerate primers containing deoxyinosine

With a view to cloning the root-knot nematode resistance gene Mi in tomato by chromosome walking, we have developed a molecular probe for the tightly linked acid phosphatase-1 (Aps-1) locus. The acid phosphatase-1 allozyme (APS-1¹), encoded by the Aps-1 ¹ allele originating from Lycopersicon peruvianum, was purified to apparent homogeneity from tomato roots and suspension cells. Microsequencing of CNBr and tryptic peptides generated from APS-1¹ provided a partial amino acid sequence, which accounted for approximately 23% of the protein and revealed two stretches of homology with soybean proteins KSH3 and VSP27, comprising 22 matches within 26 amino acid residues. The partial amino acid sequence information enabled us to isolate a 2.4 kb genomic Aps-1 ¹ sequence by means of the polymerase chain reaction (PCR), primed by degenerate pools of oligodeoxyribonucleotides, synthesized on the basis of the amino acid sequences. Synthesis of the 2.4 kb PCR product was specific for genomic templates carrying the L. peruvianum Aps-1 ¹ allele. Crucial to the priming specificity and the synthesis of the 2.4 kb genomic sequence was the use of degenerate primer pools in which the number of different primer species was limited by incorporating deoxyinosine phosphate residues at three and four base ambiguities. In using cDNA as a template, a 490 bp sequence was obtained, indicating a high proportion of intron sequences in the 2.4 kb genomic Aps-1 ¹ sequence. The Aps-1 ¹ origin of the PCR product was confirmed by RFLP (restriction fragment length polymorphism) analysis, using both a chromosome 6 substitution line and a pair of nearly isogenic lines, differing for a small chromosomal region around the Aps-1/Mi loci.     

Alkaline phosphatase activities of anAnabaena sp. from deep-water rice

Anabaena sp. grew with mono- and di-ester phosphate compounds as sources of phosphate, indicating the presence of phosphomonoesterase (PMEase) and phosphodiesterase (PDEase) activities. Cell-bound PMEase and PDEase activities were detected during growth in 0.5 and 10 mg PO₄l^–1 only when the cellular phosphate concentration fell to 0.46% of cell protein and the activities increased as cellular phosphate content decreased. The K_m values for these enzymes were 0.3mm forp-nitrophenyl phosphate and 0.2mm for bis-p-nitrophenyl phosphate, respectively. Only PMEase activity was found extracellularly. The pH optima for PMEase and PDEase were 10.2 and 10.4, respectively, and the temperature optima at pH 10.2 were 37°C and 40°C, respectively. Ca²⁺ increased the enzyme activities while Zn²⁺ caused marked inhibition. The inorganic phosphate repressed the cellular PMEase activity after a lag of 4 h.     

Identification of the adipocyte acid phosphatase as a PAO-sensitive tyrosyl phosphatase.

We have partially purified an 18-kDa cytoplasmic protein from 3T3-L1 cells, which dephosphorylates pNPP and the phosphorylated adipocyte lipid binding protein (ALBP), and have identified it by virtue of kinetic and immunological criteria as an acid phosphatase (EC 3.1.3.2). The cytoplasmic acid phosphatase was inactivated by phenylarsine oxide (PAO) (Kinact = 10 microM), and the inactivation could be reversed by the dithiol, 2,3-dimercaptopropanol (Kreact = 23 microM), but not the monothiol, 2-mercaptoethanol. Cloning of the human adipocyte acid phosphatase revealed that two isoforms exist, termed HAAP alpha and HAAP beta (human adipocyte acid phosphatase), which are distinguished by a 34-amino acid isoform-specific domain. Sequence analysis shows HAAP alpha and HAAP beta share 74% and 90% identity with the bovine liver acid phosphatase, respectively, and 99% identity with both isoenzymes of the human red cell acid phosphatase but no sequence similarity to the protein tyrosine phosphatases (EC 3.1.3.48). HAAP beta has been cloned into Escherichia coli, expressed, and purified as a glutathione S-transferase fusion protein. Recombinant HAAP beta was shown to dephosphorylate pNPP and phosphoALBP and to be inactivated by PAO and inhibited by vanadate (Ki = 17 microM). These results describe the adipocyte acid phosphatase as a cytoplasmic enzyme containing conformationally vicinal cysteine residues with properties that suggest it may dephosphorylate tyrosyl phosphorylated cellular proteins.     

Isolation of brush-border membranes from rat and rabbit colonocytes: Is alkaline phosphatase a marker enzyme?

Summary A method for the isolation of brush-border membranes of large intestinal epithelial cells was developed, which is based on the purification of intact brush-border caps by Percoll^® density-gradient centrifugation followed by separation of the vesiculated brush-border membranes on sucrose gradients. The procedure has two major advantages in comparison to known methods: 1) its first step does not depend on the determination of marker enzymes and 2) the method is applicable to rats as well as rabbits without major modifications. Due to the lack of an accepted marker for the colonic brush-border membrane the validity of the isolation procedure was tested by its application to the small intestine. Rat small intestinal brush-border membranes were enriched 21-fold when compared to the homogenate. The method was used to evaluate alkaline phosphatase as a marker enzyme for the colonic brush-border membrane. The results suggest that alkaline phosphatase is not exclusively localized in the brush-border membrane since this enzyme was also associated with membranes having different physical properties.     

NAD+ Glycohydrolase of the Plasma Membrane Prepared from Glial and Neuronal Cells

NAD+ glycohydrolase (EC 3.2.2.5) activity was detected in the plasma membrane prepared from the primary culture of rat astrocytes. The enzyme has a broad optimum pH range. From the kinetic analysis, a Michaelis constant of 91.2 microM and a maximum velocity of 0.785 mumol/min/mg protein were obtained. ADPribose exhibited a competitive inhibition with respect to NAD. The inhibition by nicotinamide was shown to be of a non-competitive type. ATP and GTP were found to be competitive inhibitors. NAD+ glycohydrolase activity was not detected in the plasma membrane prepared from the primary culture of neuronal cells of chick embryos.     

Effect of Early Iron Deficiency in Rat on the γ-Aminobutyric Acid Shunt in Brain

Early iron deficiency in rat does not affect the weight or the protein, DNA, and RNA content but results in a slight reduction in gamma-aminobutyric acid (GABA) (13%, p less than 0.01) and glutamic acid (20%, p less than 0.001) content of the brain. The activities of the two GABA shunt enzymes, glutamate dehydrogenase and GABA-transaminase, and of the NAD+-linked isocitrate dehydrogenase (ICDH) were inhibited whereas the glutamic acid decarboxylase, mitochondrial NADP+-linked ICDH, and succinic dehydrogenase activities remained unaltered in brain. On rehabilitation with the iron-supplemented diet for 1 week, these decreased enzyme activities in brain attained the corresponding control values. However, the hepatic nonheme iron content increased to about 80% of the control, after rehabilitation for 2 weeks. A prolonged iron deficiency resulting in decreased levels of glutamate and GABA may lead to endocrinological, neurological, and behavioral alterations.     

Surface antigens of Biomphalaria glabrata (Gastropoda) hemocytes: functional heterogeneity in cell subpopulations recognized by a monoclonal antibody

A hemocyte surface membrane marker (BGH₁) has been identified using hemocyte-specific monoclonal antibodies (mABs) generated by somatic cell fusion methods. The BGH₁ epitope was expressed on a subpopulation of circulating, glass-adherent blood cells from two strains of the snail, Biomphalaria glabrata. Approximately 40% of the circulating hemocytes from the PR albino (M-line) B. glabrata strain were BGH₁^?, compared to a prevalence of 10% BGH₁⁺ cells in the 10-R2 snail strain. When hemocytes were firmly attached and spread on a glass surface, BGH₁⁺ cells were morphologically distinguishable from BGH₁^? cells by their ovoid shape and the presence of short, thin filopodial projections along the ectoplasmic border. In contrast, BGH₁^? hemocytes were more pleomorphic and possessed long, spike-like filopodia. Moreover, the BGH₁ epitope was trypsin-resistant and retained its antigenic reactivity with probe mABs following fixation with paraformaldehyde or paraformaldehyde/MeOH. Fixation with glutaraldehyde, however, significantly reduced mAB binding to the BGH₁ surface epitope. There was no apparent age-dependent expression of the BGH₁ determinant since circulating hemocyte populations in very young (1–2 mm) to adult (10–12 mm) snails were composed of both BGH₁⁺ and BGH₁^? subpopulations. Quantitative shifts in the prevalence of epitope-bearing hemocytes between the smallest snail size class (1–2 mm) and the larger snails (3–4 and 10–12 mm) are believed to be due to a differential production and/or release of BGH₁^? hemocytes within the blood circulation rather than a gradual age-related change in the expression of surface antigens on individual cells. Experiments designed to assess the in vitro phagocytic capability and lysosomal acid phosphatase (APase) activity of mAB-reactive hemocytes revealed that BGH₁⁺ cells, when compared to those lacking the surface marker, were significantly reduced in both their phagocytic and APase-producing activities. Since the PR albino strain of B. glabrata possesses a higher proportion of BGH₁^? hemocytes and a lower total concentration of circulating cells than do snails of the 10-R2 strain, PR albino snails are thus potentially reduced in their natural capacity to mount cellular reactions against foreign materials.     

Pathophysiologic alterations in Biomphalaria glabrata infected with Angiostrongylus costaricensis

Hemolymph glucose, alkaline phosphatase, lactic dehydrogenase, and creatine phosphokinase in Biomphalaria glabrata infected with Angiostrongylus costaricensis were significantly higher on day 27 postinfection (PI) than in uninfected snails. Hemolymph total calcium from infected snails was less on days 6, 12, and 27 PI than that from controls. Total hemolymph protein was similar for controls and infected animals during the entire study. Throughout the study the mean number of amoebocytes/mm³ hemolymph from infected snails was significantly less than that for controls. Mean total wet weights of digestive gland and foot muscle from infected and uninfected snails was similar throughout the study. Mean μg glycogen/mg wet weight of digestive gland from infected snails was significantly greater on days 24, 27, and 28 PI than that from controls. Mean μg glycogen/mg wet weight of foot muscle from infected snails was significantly reduced between days 12 and 28 PI from that of uninfected snails. It is suggested that hemolymph glucose and digestive gland glycogen in infected snails are augmented by glycogen breakdown in the foot muscle of parasitized animals. Elevations in hemolymph enzymes are due to tissue destruction by larvae emerging from the foot muscle of infected snails. Parasite-induced derangements in shell metabolism underlie observed changes in hemolymph calcium in infected snails.     

Cyclic AMP stimulates dephosphorylation of specific proteins in intact S49 mouse lymphoma cells

Two-dimensional gel electrophoresis of proteins labeled with ³²P₁ in S49 mouse lymphoma cells revealed five phosphoproteins that were rapidly and reversibly dephosphorylated in response to elevation of cyclic AMP (cAMP). Under basal conditions, labeling of at least two of these proteins was limited by slow turnover of protein-bound phosphate. The rapid cAMP-mediated dephosphorylation of these species was attributable, therefore, to stimulation of a specific phosphoprotein phosphatase.