Phosphotyrosyl-protein phosphatase of TCRC-2 cells

Homogenization of TCRC-2 cells yielded a phosphotyrosyl-protein phosphatase with a specific activity approximately 10-=fold higher in particulate than in soluble fractions. Over 90% of the phosphotyrosyl-protein phosphatase associated with the particles was solubilized with 1.0% Nonidet P-40. Chromatography of the detergent-solubilized particulate fraction on either wheat germ lectin-Sepharose or histone-Sepharose columns separated two major components of phosphatase activity. One peak (eluted with 200 mM NaCl from histone-Sepharose or with N-acetylglucosamine from the lectin column) contained both phosphotyrosyl- and phosphoseryl-protein phosphatase as well as p-nitrophenyl phosphatase activities. The other peak (eluted with 1.0 M NaCl from histone-Sepharose or not bound to the lectin column) contained essentially only phosphoseryl-protein phosphatase activity. Various agents (EDTA, p-nitrophenyl phosphate, fluoride) showed considerable differences in their ability to inhibit the two phosphatase fractions; of these, the most potent and selective inhibitor was orthovanadate. At micromolar concentrations, vanadate inhibited the fraction containing phosphotyrosyl-protein phosphatase and failed to inhibit the fraction containing only phosphoseryl-protein phosphatase activity. These data show that the particulate forms of phosphotyrosyl-protein phosphatase and p-nitrophenyl phosphatase represent the activities of very similar or identical proteins.     

Multiple forms of alkaline phosphatase in untreated and 5-bromo-2'-deoxyuridine-treated choriocarcinoma cells

The alkaline phosphatases present in choriocarcinoma cells, either untreated or treated with 5-bromo-2′-deoxyuridine (BrdUrd), were purified and characterized. Three forms of phosphatase [I, IIa (or IIIa), and IIb (or IIIb)]were isolated from both the untreated and BrdUrd-treated cells. Although BrdUrd induced the synthesis of all three forms of alkaline phosphatase in these cells, the synthesis of forms IIa and IIb was, however, preferentially stimulated. The forms of phosphatase in choriocarcinoma cells resembled each other in their kinetic properties and thermal lability, but differed in their molecular weights and in their electrophoretic mobilities in nondenaturing polyacrylamide gels. All three phosphatases were inactivated by antiserum to term-placental alkaline phosphatase. The alkaline phosphatases from choriocarcinoma cells differed, however, from the enzyme from term placentas in several physicochemical properties. The phosphatases from choriocarcinoma cells had a lower K_m value for p-nitrophenyl phosphate, were more sensitive to inhibition by l-leucine, levamisole, l-p-bromotetramisole, and EDTA, and were more heat-labile. Phosphatase I comigrated with term-placental alkaline phosphatase on nondenaturing polyacrylamide electrophoretic gels, but phosphatases IIa and IIb migrated more slowly. The apparent molecular weights of phosphatase forms I, IIa, and IIb were estimated by gel filtration and polyacrylamide gel electrophoresis to be 115,000, 240,000, and 510,000, respectively. Although three molecular forms of alkaline phosphatase occurred in choriocarcinoma cells, the subunit molecular weight of these phosphatases appeared to be identical to each other and to the subunit of term-placental alkaline phosphatase (63,000 MW). The alkaline phosphatase in choriocarcinoma cells therefore exists in the dimeric, tetrameric, and octameric forms.     

Partial purification and characterization of phosphotyrosyl-protein phosphatase from Ehrlich ascites tumor cells

We have previously described a phosphotyrosylprotein phosphatase in membrane vesicles from human epidermoid carcinoma A431 cells which is inhibited by micromolar concentration of Zn2+ and is insensitive to ethylenediaminetetraacetic acid (EDTA) and NaF [Brautigan, D. L., Bornstein, P., & Gallis, B. (1981) J. Biol. Chem. 256, 6519-6522]. Here we present the identification and partial purification of a similar enzyme from lysates of Ehrlich ascites tumor cells. the enzyme was purified by using diethylaminoethyl-Sephadex, Zn2+ affinity, and Sephadex G-75 chromatography. During purification, the phosphatase was separated into at least three fractions, all of which exhibited very similar properties and an apparent molecular weight of 40 000 upon gel filtration. The enzyme dephosphorylated phosphotyrosine (P-Tyr)-containing carboxymethylated and succinylated (CM-SC) phosphorylase with an apparent Km of 0.8 microM, as well as P-Tyr containing casein and epidermal growth factor (EGF) receptor kinase, but did not dephosphorylate P-Ser-phosphorylase. The phosphatase was inhibited by Zn2+ at micromolar concentrations (K0.5 with EGF receptor kinase = 5 X 10(-6) M; with CM-SC phosphorylase = 3.3 X 10(-5) M) but not by millimolar concentrations of EDTA and NaF. No inhibition was seen with 1 mM tetramisole, a specific inhibitor of alkaline phosphatases. P-Tyr inhibited the enzyme by 50% at 0.4 X 10(-3) M, while Tyr, Pi, PPi, and p-nitrophenyl phosphate, an excellent substrate for alkaline phosphatases and structurally very similar to P-Tyr, exerted partial inhibition at concentrations above 10(-3) M. The pH optimum was found to be 6.5-7, depending on the substrate used. Very little activity was seen below pH 5 and above pH 8.5. These properties clearly distinguish this enzyme from alkaline phosphatases, as well as the neutral and acidic protein phosphatases so far described, and therefore define it as a new enzyme of the phosphatase family--a phosphotyrosyl-protein phosphatase.     

The p-nitrophenyl phosphatase activity of muscle carbonic anhydrase

Carbonic anhydrase III from rabbit muscle, a newly discovered major isoenzyme of carbonic anhydrase, has been found to be also a p-nitrophenyl phosphatase, an activity which is not associated with carbonic anhydrases I and II. The p-nitrophenyl phosphatase activity has been shown to chromatograph with the CO₂ hydratase activity; both activities are associated with each of its sulfhydryl oxidation subforms; and both activities follow the same pattern of pH stability. This phosphomonoesterase activity of carbonic anhydrase III has an acidic pH optimum (<5.3); its true substrate appears to be the phosphomonoanion with a K_m of 2.8 mm. It is competitively inhibited by the typical acid phosphatase inhibitors phosphate (K_i = 1.22 × 10^?3M), arsenate (K_i = 1.17 × 10^?3M), and molybdate (K_i = 1.34 × 10^?7M), with these inhibitors having no effect on the CO₂ hydratase or the p-nitrophenyl acetate esterase activities of carbonic anhydrase III. The p-nitrophenyl acetate esterase activity of carbonic anhydrase III, on the other hand, has the sigmoidal pH profile with an inflection at neutral pH, typical of carbonic anhydrases for all of their substrates, and is inhibitable by acetazolamide (a highly specific carbonic anhydrase inhibitor) to the same degree as the CO₂ hydratase activity. The acid phosphatase-like activity of carbonic anhydrase III is slightly inhibited by acetazolamide at acidic pH, and inhibited to nearly the same degree at neutral pH. These data are taken to suggest that the phosphatase activity follows a mechanism different from that of the CO₂ hydratase and p-nitrophenyl acetate esterase activities and that there is some overlap of the binding sites.     

Insulin-receptor phosphotyrosyl-protein phosphatases.

Calmodulin-dependent protein phosphatase has been proposed to be an important phosphotyrosyl-protein phosphatase. The ability of the enzyme to attack autophosphorylated insulin receptor was examined and compared with the known ability of the enzyme to act on autophosphorylated epidermal-growth-factor (EGF) receptor. Purified calmodulin-dependent protein phosphatase was shown to catalyse the complete dephosphorylation of phosphotyrosyl-(insulin receptor). When compared at similar concentrations, 32P-labelled EGF receptor was dephosphorylated at greater than 3 times the rate of 32P-labelled insulin receptor; both dephosphorylations exhibited similar dependence on metal ions and calmodulin. Native phosphotyrosyl-protein phosphatases in cell extracts were also characterized. With rat liver, heart or brain, most (75%) of the native phosphatase activity against both 32P-labelled insulin and EGF receptors was recovered in the particulate fraction of the cell, with only 25% in the soluble fraction. This subcellular distribution contrasts with results of previous studies using artificial substrates, which found most of the phosphotyrosyl-protein phosphatase activity in the soluble fraction of the cell. Properties of particulate and soluble phosphatase activity against 32P-labelled insulin and EGF receptors are reported. The contribution of calmodulin-dependent protein phosphatase activity to phosphotyrosyl-protein phosphatase activity in cell fractions was determined by utilizing the unique metal-ion dependence of calmodulin-dependent protein phosphatase. Whereas Ni2+ (1 mM) markedly activated the calmodulin-dependent protein phosphatase, it was found to inhibit potently both particulate and soluble phosphotyrosyl-protein phosphatase activity. In fractions from rat liver, brain and heart, total phosphotyrosyl-protein phosphatase activity against both 32P-labelled receptors was inhibited by 99.5 +/- 6% (mean +/- S.E.M., 30 observations) by Ni2+. Results of Ni2+ inhibition studies were confirmed by other methods. It is concluded that in cell extracts phosphotyrosyl-protein phosphatases other than calmodulin-dependent protein phosphatase are the major phosphotyrosyl-(insulin receptor) and -(EGF receptor) phosphatases.     

Phosphotyrosyl-protein phosphatase. Specific inhibition by Zn

Epidermal growth factor stimulates a cyclic AMP-independent protein kinase associated with membrane vesicles derived from human epidermoid carcinoma cells (Carpenter, G., King, L., Jr., and Cohen, S. (1979) J. Biol. Chem. 254,. 4884-4891). The kinase specifically phosphorylates tyrosyl residues in a Mr = 150,000 membrane protein (Ushiro, H., and Cohen, S. (1980) J. Biol. Chem. 255, 8363-8365). We show that the reverse reaction, catalyzed by a phosphotyrosyl-protein phosphatase associated with the membrane, is inhibited by Zn2+. Dephosphorylation of phosphotyrosyl residues in the Mr = 150,000 protein is completely inhibited by Zn2+ at concentrations as low as 10 microM, whereas other divalent cations have no substantial effect. Inhibition of the phosphatase was reversed by EDTA and the activity in membrane preparations was increased by EDTA or fluoride, agents commonly thought to be phosphatase inhibitors. Acid hydrolysis of the membrane proteins followed by analysis of phosphoamino acids by two-dimensional electrophoresis revealed that the phosphatase hydrolyzed phosphotyrosyl in preference to phosphoseryl residues. The specific inhibition of this phosphatase activity by low concentrations of Zn2+ may be indicative of the physiological importance of Zn2+ in the regulation of cellular phosphotyrosyl-protein levels.     

Enzymatic characteristics of an ApaH-like phosphatase,PrpA, and a diadenosine tetraphosphate hydrolase,ApaH, from Myxococcus xanthus

We characterized the activities of the Myxococcus xanthus ApaH-like phosphatases PrpA and ApaH, which share homologies with both phosphoprotein phosphatases and diadenosine tetraphosphate (Ap₄A) hydrolases. PrpA exhibited a phosphatase activity towards p-nitrophenyl phosphate (pNPP), tyrosine phosphopeptide and tyrosine-phosphorylated protein, and a weak hydrolase activity towards Ap_nA and ATP. In the presence of Mn²⁺, PrpA hydrolyzed Ap₄A into AMP and ATP, whereas in the presence of Co²⁺ PrpA hydrolyzed Ap₄A into two molecules of ADP. ApaH exhibited high phosphatase activity towards pNPP, and hydrolase activity towards Ap_nA and ATP. Mn²⁺ was required for ApaH-mediated pNPP dephosphorylation and ATP hydrolysis, whereas Co²⁺ was required for Ap_nA hydrolysis. Thus, PrpA and ApaH may function mainly as a tyrosine protein phosphatase and an Ap_nA hydrolase, respectively.     

A specific alkaline phosphatase from Saccharomyces cerevisiae with protein phosphatase activity

In this paper, specific PHO13 alkaline phosphatase from Saccharomyces cerevisiae was demonstrated to possess phosphoprotein phosphatase activity on the phosphoseryl proteins histone II-A and casein. The enzyme is a monomeric protein with molecular mass of 60 kDa and hydrolyzes p-nitrophenyl phosphate with maximal activity at pH 8.2 with strong dependence on Mg²⁺ ions and an apparent K_m of 3.6×10⁻⁵ M. No other substrates tested except phosphorylated histone II-A and casein were hydrolyzed at any significant rate. These data suggest that the physiological role of the p-nitrophenyl phosphate-specific phosphatase may involve participation in reversible protein phosphorylation.     

Liver glycogen synthase phosphatase and phosphorylase phosphatase activities in vitro following glucose and glucagon administration

Correlation of the changes in phosphorylase a concentration with the synthase phosphatase velocity in a glycogen particle preparation in the presence of EDTA revealed that the initial synthase phosphatase rate was greatest in extracts from glucose-treated rats and least in extracts from glucagon-treated rats. In all cases the velocity increased with time and with a decrease in phosphorylase a. However, a threshold release of phosphatase activity when phosphorylase a reached a critical level was not observed. The data are compatible with either an independent regulation of synthase phosphatase by glucose and glucagon or regulation of the activity by phosphorylase over a range of phosphorylase a concentrations.     

Co-purification of type I alkaline phosphatase and type I phosphoprotein phosphatase from various animal tissues

A purification procedure, which included ethanol treatment as a step for dissociating the large molecular forms of type I phosphoprotein phosphatase, was employed for the studies of the alkaline phosphatase and phosphoprotein phosphatase activities in bovine brain, heart, spleen, kidney, and uterus, rabbit skeletal muscle and liver, and lobster tail muscle. The results indicate that the major phosphoprotein phosphatase (phosphorylase a as a substrate) and alkaline phosphatase (p-nitrophenyl phosphate as a substrate; Mg²⁺ and dithiothreitol as activators) activities in the extracts of all tissues studied were copurified as an entity of M_r = 35,000. The purified enzymes from different tissues exhibit similar physical and catalytic properties with respect to either the phosphoprotein phosphatase or the alkaline phosphatase activity. The present findings indicate that (a) the M_r = 35,000 species, which represents a catalytic entity of the large molecular forms of type I phosphoprotein phosphatase, is widespread in animal tissues, indicating that it is a multifunctional phosphatase; (b) the association of type I alkaline phosphatase activity with type I phosphoprotein phosphatase is a general phenomenon.     

Action of organophosphorus compounds on the p-nitrophenyl phosphatase of membranes from rabbit and guinea pig polymorphonuclear leucocytes

Organophosphorus compounds such as DFP and its non-phosphorylating analogue, triisopropyl phosphate, stimulate the p-nitrophenyl phosphatase activity of membranes from rabbit and guinea pig leucocytes. Triethyl phosphate and trimethyl phosphate have little effect and the stimulation by triisopropyl phosphate can be distinguished from that of the non-ionic detergent lubrol. It is suggested that the effect on the membrane leading to p-nitrophenyl phosphatase stimulation can be the basis of the inhibition of locomotion and the enhancement of leucocidin action in the leucocyte. The hypothesis that Organophosphorus compounds act on cells exclusively as inhibitors of esterases is criticised. The p-nitrophenyl phosphatases of rabbit and guinea pig leucocyte membranes differ in some respects and it is suggested that a study of the enzymes in related cells may reveal further species differences.     

Localization of alkaline phosphatase inBacillus intermedius cells

Alkaline phosphatase, an enzyme secreted byBacillus intermedius S3-19 cells to the medium, was also detected in the cell wall, membrane, and cytoplasm. The relative content of alkaline phosphatase in these cell compartments depended on the culture age and cultivation medium. The vegetative growth ofB. intermedius on 0.3% lactate was characterized by increased activity of extracellular and membrane-bound phosphatases. The increase in lactate concentration to 3% did not affect the activity of membrane-bound phosphatase but led to a decrease in the activity of the extracellular enzyme. Na₂HPO₄ at a concentration of 0.01 % diminished the activity of membrane-bound and extracellular phosphatases. CoCl₂ at a concentration of 0.1 mM released membrane-bound phosphatase into the medium. By the onset of sporulation, phosphatase was predominantly localized in the medium and in the cell wall. As is evident from zymograms, the multiple molecular forms of phosphatase varied depending on its cellular localization and growth phase.     

Isolation and characterization of an inhibitor-sensitive and a polycation-stimulated protein phosphatase from rat liver nuclei

Two protein phosphatases were isolated from rat liver nuclei. The enzymes, solubilized from crude chromatin by 1 M NaCl, were resolved by column chromatography on Sephadex G-150, DEAE-Sepharose and heparin-Sepharose. The phosphorylase phosphatase activity of one of the enzymes (inhibitor-sensitive phosphatase) was inhibited by heat-stable phosphatase inhibitor proteins and also by histone H1. This phosphatase had a molecular weight of approx. 35 000 both before and after 4 M urea treatment. Its activity was specific for the β-subunit of phosphorylase kinase. Pretreatment with 0.1 mM ATP inhibited the enzyme only about 10%, and it did not require divalent cations for activity. On the basis of these properties, this nuclear enzyme was identified as the catalytic subunit of phosphatase 1. The other phosphatase (polycation-stimulated phosphatase) was insensitive to inhibition by inhibitor 1, and it was stimulated 10-fold by low concentrations of histone H1 (A_0.5 = 0.6 μM). This enzyme had a molecular weight of approx. 70 000 which was reduced to approx. 35 000 after treatment with 4 M urea. It dephosphorylated both the α- and β-subunits of phosphorylase kinase. The enzyme was inhibited more than 90% by preincubation with 0.1 mM ATP and did not require divalent cations for activity. On the basis of these properties, this nuclear enzyme was identified as phosphatase 2A.     

An insect farnesyl phosphatase homologous to the N-terminal domain of soluble epoxide hydrolase

In insects, farnesyl pyrophosphate (FPP) is converted to juvenile hormone (JH) via a conserved pathway consisting of isoprenoid-derived metabolites. The first step of this pathway is presumed to be hydrolysis of FPP to farnesol in the ring gland. Based on alignment of putative phosphatases from Drosophila melanogaster with the phosphatase domain of soluble epoxide hydrolase, Phos2680 and Phos15739 with conserved phosphatase motifs were identified, cloned and purified. Both D. melanogaster phosphatases hydrolyzed para-nitrophenyl phosphate, however, Phos15739 also hydrolyzed FPP with a K_cat/K_m of 2.1 × 10⁵ M⁻¹ s⁻¹. RT-PCR analysis revealed that Phos15739 was expressed in the ring gland and its expression was correlated with JHIII titer during development of D. melanogaster. N-acetyl-S-geranylgeranyl-l-cysteine was found to be a potent inhibitor of Phos15739 with an IC₅₀ value of 4.4 μM. Thus, our data identify Phos15739 as a FPP phosphatase that likely catalyzes the hydrolysis of FPP to farnesol in D. melanogaster.     

Alkaline phosphatase in Amoeba proteus

In free-living Amoeba proteus (strain B), 3 phosphatase were found after disc-electrophoresis of 10 microg of protein in PAGE and using 1-naphthyl phosphate as a substrate a pH 9.0. These phosphatases differed in their electrophoretic mobilities - "slow" (1-3 bands), "middle" (one band) and "fast" (one band). In addition to 1-naphthyl phosphate, "slow" phosphatases were able to hydrolyse 2-naphthyl phosphate and p-nitrophenyl phosphate. They were slightly activated by Mg2+, completely inhibited by 3 chelators (EDTA, EGTA and 1,10-phenanthroline), L-cysteine, sodium dodecyl sulfate and Fe2+, Zn2+ and Mn2+ (50 mM), considerably inactivated by orthovanadate, molybdate, phosphatase inhibitor cocktail 1, p-nitrophenyl phosphate, Na2HPO4, DL-dithiothreitol and urea and partly inhibited by H2O2, DL-phenylalanine, 2-mercaptoethanol, phosphatase inhibitor cocktail 2 and Ca2+. Imidazole, L-(+)-tartrate, okadaic acid, NaF and sulfhydryl reagents -p-(hydroxy-mercuri)benzoate and N-ethylmaleimide - had no influence on the activity of "slow" phosphatases. "Middle" and "fast" phosphatases, in contrast to "slow" ones, were not inactivated by 3 chelators. The "middle" phosphatase differed from the "fast" one by smaller resistance to urea, Ca2+, Mn2+, phosphates and H2O2 and greater resistance to dithiothreitol and L-(+)-tartrate. In addition, the "fast" phosphatase was inhibited by L-cysteine but the "middle" one was activated by it. Of 5 tested ions (Mg2+, Cu2+, Mn2+, Ca2+ and Zn2+), only Zn2+ reactivated "slow" phosphatases after their inactivation by EDTA treatment. The reactivation of apoenzyme was only partial (about 35 %). Thus, among phosphatases found in amoebae at pH 9.0, only "slow" ones are Zn-metalloenzymes and may be considered as alkaline phosphatases (EC 3.1.3.1). It still remains uncertain, to which particular phosphatase class "middle" and "fast" phosphatases (pH 9.0) may belong.     

Purification and characterization of Kurloff cell sialoglycoproteins with acid phosphatase activity

The majora2–6 sialoglycoproteins in detergent-extracts of Kurloff cells were purified by anion-exchange andSambucus nigra agglutinin-affinity chromatographies. The similar ultrastructural localisations of (1)S. nigra agglutinin-gold conjugates and (2) acid phosphatase activities on the Kurloff body and particularly on its myelin figures indicated that the majora2-6 sialoglycoproteins of the Kurloff cell had acid phosphatase activity. Two-dimensional electrophoresis showed that these tartrate-sensitive phosphatases corresponded to 2 acidic (pI 3.4–3.7) polypeptides of 36 and 34 kDa. Hydrolysis with peptide-N-glycosidases F gave a 33 kDa apoprotein rich in alanine, glutamic acid, tyrosine and lysin. A lectin-affinity study demonstrated that they contained hybrid type bisected and fucosylatedN-linked oligosaccharides. Cytotoxic properties were previously attributed to Kurloff cells and other studies suggested that not only acid phosphatases but alsoa2-6-linked sialic acid residues themselves may participate in natural killer activity.     

Induction of acid phosphatase in cotton seedlings: Enzyme purification,subunit structure and kinetic properties

About a 16-fold rise in acid phosphatase (EC 3.1.3.2) activity was observed during the early stages of germination of cotton embryos. Administration of cyclobeximide to the germinating embryos significantly blocked the enhancement of acid phosphatase activity. This indicated that translational activity was essential for the induction of enzyme activity. Conclusive proof for the de novo synthesis of the enzyme was obtained by showing the incorporation of ³⁵S from ³⁵SO²⁻₄ into the cysteine residues of the purified acid phosphatase. The enzyme was purified (1046-fold) to electrophoretic homogeneity by ammonium sulphate fractionation, CM-Sephadex C-50 and affinity chromatography on concanavalin A-Agarose. PAGE gave two isozyme bands. The M, of the phosphatase was 200 k as determined by molecular sieving on Sephadex G-200. SDS-PAGE of acid phosphatase revealed a single band of M 55 k. Thus the native enzyme is a tetramer of four identical subunits. The K_m of the enzyme with p-nitrophenyl phosphate was 0.5 mM. Optimal enzyme activity was observed at pH 5.0, using p-nitrophenyl phosphate as substrate. The enzyme activity remained linear for 105 min at 37° and was proportional to the concentration of protein within the range 0.6–2.4 μg.     

Purification and properties of Acid phosphatase from plump and shriveled seeds of triticale

A major triticale (X Triticosecale Wittmack) endosperm acid phosphatase (EC 3.1.2.2) (APase) from sib-lines producing plump and shriveled seed was purified 140- and 230-fold to a specific activity of 94 and 153 micromoles per minute per milligram protein respectively, by ammonium sulfate fractionation, ion-exchange chromatography, chromatofocusing, affinity column chromatography, and gel filtration. The purified enzyme from both materials is a monomeric glycoprotein with an apparent molecular weight of 45,700 ± 500 containing 12% carbohydrate and an apparent isoelectric point of pH 5.9. It hydrolyzes tri- and di-phosphate of nucleosides as well as phosphate esters and exhibits characteristics of ATP-hydrolase and phosphatase. About 2-fold more of the APase was isolated from shriveled seeds, and the purified enzyme exhibited 3- and 5-fold higher V_max for p-nitrophenyl phosphate and ATP, respectively, than that of plump seed. The I₅₀ for Pi concentration was 5.5-fold higher in APase of shriveled seed than the plump one. These varied quantitative and kinetic properties substantiate the role of APase in lines with shriveled seeds being reduction of starch accumulation by depleting substrates and energy supply in the cytosol.     

Boophilus microplus: characterization of larval phosphomonoesterases and isolation of subcellular fractions with high phosphatase activity.

Phosphomonoesterase activity was determined for a 115,000g pellet and soluble fractions resulting from a subcellular fractioning of a homogenate of larval Boophilus microplus. Both fractions showed maximum phosphatase activity at pH 5.5 and 10. Acid phosphatase (EC 3.1.3.2) activity was found to be greatest in the soluble fraction. When the reaction rate was plotted against homogenate concentration, the soluble acid phosphatase deviated from the linear relationship. For both fractions different thermostability patterns were obtained, inactlvation beginning for the alkaline phosphatase (EC 3.1.3.1) at 45–55 C. When the effect of substrate concentration on activity was studied, deviations from the typical hyperbolic behavior were observed. Homogenization of larvae with 5 mm EDTA buffer failed to yield a low-speed pellet with high alkaline phosphatase activity, as it is expected if absorptive structures sediment. Moreover, total alkaline phosphatase activity recovered by this method is significantly lower than activity recovered when homogenization is carried out without EDTA. Alternately, homogenization with 10 mM Tris buffer and 0.25 M sucrose gave 27,000g and 115,000g fractions with high phosphatase activity when fractioned by centrifugation. Alkaline treatment of the 115,000g fraction with 10 mM Tris buffer, pH 7.8, failed to separate endoplasmic reticulum contaminants without loss of phosphatase activity. When the 115,000g fraction was centrifuged in a sucrose density gradient, two activity peaks, coincident for both acid and alkaline phosphatases, were obtained. Antigenic analysis showed the existence of similar antigenic determinants in both peaks “immunologically” presented in different ways.     

Isolation and expression of a maize type 1 protein phosphatase

The dephosphorylation of phosphoproteins by protein phosphatases represents an important mechanism for regulating specific cellular processes in eukaryotic cells. The aim of the present study was to examine the structural and biochemical characteristics of a specific class of protein Ser/Thr phosphatases (type 1 protein phosphatases) which have received very little attention in higher plants. A cDNA clone (ZmPP1) was isolated from a maize (Zea mays L.) cDNA library. The deduced amino acid sequence is 80% identical with a 292-amino acid core region of rabbit and yeast type 1 protein phosphatase catalytic subunit. Southern blot analysis indicates that ZmPP1 may belong to a family of related genes in maize. ZmPP1 RNA was present in all maize tissues examined, indicating that it may play a fundamental role in cellular homeostasis. To demonstrate that ZmPP1 encodes an active protein phosphatase and, in an effort to characterize this gene product biochemically, high levels of ZmPP1 were expressed in Escherichia coli. Active ZmPP1 enzyme dephosphorylates rabbit phosphorylase a and is strongly inhibited by okadaic acid and by the mammalian inhibitor-2. These data show that ZmPP1 is structurally and biochemically very similar to the corresponding enzyme in animal cells. These results also suggest that the function and regulation of the higher plant type 1 protein phosphatases may be similar to the mammalian protein phosphatases.