Brain Phosphatase Research Articles

Identification of Soluble Protein Phosphatases That Dephosphorylate Voltage-sensitive Sodium Channels in Rat Brain

Rat brain sodium channels are phosphorylated at multiple serine residues by cAMP-dependent protein kinase. We have identified soluble rat brain phosphatases that dephosphorylate purified sodium channels. Five separable forms of sodium channel phosphatase activity were observed. Three forms (two, approximately 234 kDa and one, 192 kDa) are identical or related to phosphatase 2A, since they were 85-100% inhibited by 10 nM okadaic acid and contained a 36-kDa polypeptide recognized by a monoclonal antibody directed against the catalytic subunit of phosphatase 2A. Immunoblots performed using antibodies specific for isoforms of the B subunit of phosphatase 2A indicate that the two major peaks of phosphatase 2A-like activity, A1 and B1, are enriched in either B' or B alpha. The remaining two activities (approximately 100 kDa each) probably represent calcineurin. Each was relatively insensitive to okadaic acid, was active only in the presence of CaCl2 and calmodulin, and contained a 19-kDa polypeptide recognized by a monoclonal antibody raised against the B subunit of calcinerurin. Treatment of synaptosomes with okadaic acid to inhibit phosphatase 2A or cyclosporin A to inhibit calcineurin increased apparent phosphorylation of sodium channels at cAMP-dependent phosphorylation sites, as assayed by back phosphorylation. These results indicate that phosphatase 2A and calcineurin dephosphorylate sodium channels in brain, and thus may counteract the effect of cAMP-dependent phosphorylation on sodium channel activity.

The phosphorylation state of tau in the developing rat brain is regulated by phosphoprotein phosphatases.

The paired helical filaments (PHFs) in Alzheimer's disease neurofibrillary tangles are composed of PHF-tau which is thought to be hyperphosphorylated because several residues in postmortem samples of PHF-tau and human fetal tau are phosphorylated while the corresponding sites are not phosphorylated in autopsy-derived normal adult human brain tau. To determine how the phosphorylation of these sites is regulated, we isolated tau from rat brains at different embryonic and postnatal ages in the presence of okadaic acid to obtain tau in its most native in situ phosphorylation state. Fetal tau was highly phosphorylated from embryonic day 18 (E18) until postnatal day 11 (P11). Thereafter, the levels of fetal tau diminished as did its phosphorylation state concomitant with the appearance of the five adult tau isoforms. Several phosphorylation-dependent antibodies (i.e. AT270, AT8, AT180, T3P, and PHF1) that recognize PHF-tau also recognized these tau isoforms, albeit at reduced levels in the mature rat brain. This suggests that Thr172, Ser193, Thr222, Ser387, and Ser395 are normal sites of phosphorylation in rat brain tau. The inclusion of OK in the microtubule assembly buffers did not alter the ability of tau to bind microtubules at any age. However, phosphatases were activated and kinases were down-regulated in the rat brain after P12 since adult tau proteins were partially dephosphorylated at and beyond this time in the absence of OK. Protein phosphatase 2A (PP2A) and 2B (PP2B) activities in the adult rat brain extracts dephosphorylated tau efficiently, but protein phosphatases in extracts of the P6 rat brain did not have a similar effect. This suggests that the sensitivity of tau to OK after P12 may be regulated by the de novo induction of adult brain phosphatases. Finally, PP2A and/or PP2B in adult rat brain extracts dephosphorylated tau in a site-specific manner. Thus, PP2A and PP2B (or closely related phosphatases) may regulate the phosphorylation state of adult tau isoforms in vivo, and the generation of PHF-tau in the AD brain may result from the abnormal inactivation of similar phosphatases.

Accumulation of phosphorylated neurofilament in rat brain phosphatase inhibitor injection

Accumulation of phosphorylated neurofilament in rat brain phosphatase inhibitor injection

Kainic acid induced alterations in antibody recognition of connexin43 and loss of astrocytic gap junctions in rat brain

Intracerebral administration of kainic acid (KA) in rats was previously shown to abolish immunohistochemical labelling for the astrocytic gap junction protein connexin43 (Cx43) at sites depleted of neurons (Vukelic et al: Neurosci Lett 130:120-124, 1991). This response of Cx43 has now been further investigated with a number of different sequence-specific anti-Cx43 antibodies. At lesion sites in the thalamus, striatum, and hippocampus examined immunohistochemically with an antibody against amino acids (aa's) 346-363 in the Cx43 sequence, the antibody used in the earlier study, Cx43-immunoreactivity was increased 5 h after KA injections, absent by 24 h and for up to 2 weeks post-injection, and began to return to less than normal levels by 2 to 3 weeks post-injection. Analyses of KA lesion sites with antibodies against other sequences of Cx43 (amino acids 283-298, 253-270, 241-260, 113-123, and 49-61) revealed not only the presence but in some cases an increased density of Cx43 immunoreactivity after a survival time of 1 week. Immunolabelling patterns at these sites consisted of relatively large, coarse profiles rather the fine punctate labelling typically seen in sections of normal brain. In homogenates of KA-injected striatum analyzed by Western blots, Cx43 was detected at near normal or slightly increased levels at various survival times examined. The 43 kDa phosphorylated form of Cx43 and its faster migrating 41 kDa dephosphorylated form which is generated post-mortem by a brain phosphatase were both present after standard methods of tissue preparation for Western blot analysis, while only the 43 kDa form was present in normal and KA-injected striatum after inactivation of brain metabolism by focused cranial microwave irradiation. Ultrastructural investigations of lesions sites within the thalamus revealed a virtual absence of astrocytic gap junctions. These results demonstrate that Cx43 levels initially increase after intracerebral KA treatment, that its molecular organization in resident astrocytes is altered such that epitopes that are normally accessible to antibody are hidden while those that may be hidden or relatively inaccessible are exposed, and that this molecular alteration in Cx43 is associated with loss of astrocytic gap junctions.

Dephosphorylation of tau protein and Alzheimer paired helical filaments by calcmeurin and phosphatase-2A

We have shown previously that brain tissue contains protein kinases which can phosphorylate tau protein to a state reminiscent of the pathological state of Alzheimer paired helical filaments (PHFs); these include proline-directed kinases which phosphorylate SP or TP motifs (such as MAP kinase and GSK-3) [Drewes et al. (1992); Mandelkow et al. (1992)], as well as a novel kinase which phosphorylates S262 of tau protein and thereby strongly reduces the binding of tau to imcrotubules [Biernat et al. (1993)]. Here we report on the corresponding phosphatases in brain which normally keep the ‘pathological’ sites free of phosphate. The major phosphatases acting on tau are calcineurin and PP-2A, but not PP-1. Both are present and active in brain extracts, they can dephosphorylate recombinant tau after prior phosphorylation with either MAP kinase, GSK-3, or brain extract, and the course of dephosphorylation can be monitored with antibodies diagnostic of the pathological state of tau. Both phosphatases also act directly on PHF tau isolated from Alzheimer brains.

Sequence specificity in recognition of the epidermal growth factor receptor by protein tyrosine phosphatase 1B.

Protein tyrosine phosphatases all contain a conserved cysteine that forms an intermediate thiophosphate ester bond during tyrosine phosphate hydrolysis. A bacterial glutathione S-transferase fusion protein containing rat brain phosphatase PTP1b was constructed in which this conserved cysteine was mutated to serine. The resulting catalytically inactive enzyme was labeled in vivo to high specific activity with 35S, and the binding of this labeled fusion protein to the immunoprecipitated epidermal growth factor (EGF) receptor was evaluated. The binding was ligand-dependent, and saturation analysis revealed a nonlinear Scatchard plot, with a Kd for high affinity binding of approximately 100 nM. A number of glutathione S-transferase fusion proteins containing src homology 2 (SH2) domains attenuated phosphatase binding in a concentration-dependent manner. Phospholipase C (PLC) gamma and the GTPase-activating protein of ras were the most potent inhibitors. Tyrosine-phosphorylated EGF receptor peptide fragments were evaluated for specific inhibition of PTP1b and PLC gamma SH2 binding to the activated receptor. One such peptide, modeled on EGF receptor tyrosine 992, blocked the binding of both fusion proteins. Another phosphopeptide, modeled on tyrosine 1148, inhibited the binding of PTP1b but not the PLC gamma fusion protein. This site specificity was confirmed by analysis of equilibrium binding of the fusion proteins to EGF receptors mutated in each of these phosphorylation sites. The results revealed clear sequence specificity in the binding of proteins involved in the regulation of intracellular signaling by receptor tyrosine kinases.

Isozyme-Specific Monoclonal Antibodies of CaM-Stimulated Phosphatase: Identification of an Enzyme Domain Involved in Metal Ion Activation

Bovine brain and lung extracts each contain three forms of CaM-stimulated phosphatase, designated BPI, BPII, and BPIII, and LPI, LPII, and LPIII, respectively. The different forms show uniform immunoreactivity toward one a subunit-specific monoclonal antibody, VD3, but differential reactivity toward another antibody, VJ6. Using the procedure of limited clostripain digestion, the binding sites for mAb VD3 and mAb VJ6 have been localized to the carboxylterminal region of about 40 amino acid residues and to the carboxylterminal one-third of the a subunit, respectively. The result has substantiated our previous suggestion that the isolated multiple forms of the brain and lung phosphatases represent isozymes rather than in vitro proteolysis artifacts. The effects of the two monoclonal antibodies on p-nitrophenylphosphatase activities of the bovine brain phosphatase isozymes have been examined. BPI and BPIII, the two VJ6 reactive brain isozymes, can be inhibited by VJ6 antibody in a metal ion and CaM-dependent manner; none of the isozymes are inhibited by mAb VD3. The inhibition of BPI by mAb VJ6 is most pronounced when the assay is carried out in the presence of CaM and Ni2+, whereas little or ao effect is seen if Mg2+ plus Ca2+ are used as metal ion activators. Thus, VJ6 appears to identify an a-subunit domain important for the Ni2+-induced activation of CaM-stimulated phosphatase.

Two distinct membrane-bound phosphatidylinositol-4-phosphate phosphatases in bovine brain

Solubilized phosphatidylinositol-4-phosphate 4-phosphatase from bovine brain resolved into two peaks of activity by ion exchange chromatography. Both exhibited substantial detergent binding characteristic of integral membrane proteins, and both appear specific for phosphatidylinositol-4-phosphate, but their pH optima differ: the earlier eluting fraction (peak 1) is optimally active between pH 5.5 and 6, whereas the later eluting fraction (peak 2) is most active around pH 8.5. Detergent inhibition studies suggest that peak 2, but not peak 1, interacts with phosphatidylinositol-4-phosphate in the context of a single mixed micelle. Further characterization of these activities should help shed light on the biological function of polyphosphoinositide phosphatases.

Demonstration of calmodulin-stimulated phosphatase isozymes by monoclonal antibodies.

A procedure combining immunoprecipitation and immunotransblot employing subunit-specific monoclonal antibodies of the brain phosphatase, VJ6 and VA1, was used on tissues including heart, muscle, lung, spleen, pancreas, uterus, and liver. The various tissue extracts were subjected to immunoprecipitation by the beta subunit-specific VA1-immunoabsorbant, the immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunotransblot, using both the alpha and beta subunit-specific antibodies VJ6 and VA1, respectively. Protein bands corresponding to alpha and beta subunits and the immunostain of beta subunit were detected in all samples, whereas alpha subunit was strongly stained only in the brain extract, weakly in heart and muscle extracts, and essentially negatively in all the other samples. In contrast, a polyclonal antiserum of bovine brain calmodulin-stimulated phosphatase could immunostain both alpha and beta subunits from all tissues. Calmodulin-binding protein fractions from a number of bovine tissues were all shown to contain the immunoprecipitable alpha subunit, as well as calmodulin-stimulated p-nitrophenylphosphatase activity. Micropeptide mapping showed that alpha subunits of bovine brain and bovine lung calmodulin-stimulated phosphatase isozymes were distinct molecular species. These results provide direct evidences for the existence of calmodulin-stimulated phosphatase isozymes in mammalian tissues.

Pelvic pain: Problems in diagnosis and treatment

We have shown previously that brain tissue contains protein kinases which can phosphorylate tau protein to a state reminiscent of the pathological state of Alzheimer paired helical filaments (PHFs); these include proline-directed kinases which phosphorylate SP or TP motifs (such as MAP kinase and GSK-3) [Drewes et al. (1992); Mandelkow et al. (1992)], as well as a novel kinase which phosphorylates S262 of tau protein and thereby strongly reduces the binding of tau to imcrotubules [Biernat et al. (1993)]. Here we report on the corresponding phosphatases in brain which normally keep the ‘pathological’ sites free of phosphate. The major phosphatases acting on tau are calcineurin and PP-2A, but not PP-1. Both are present and active in brain extracts, they can dephosphorylate recombinant tau after prior phosphorylation with either MAP kinase, GSK-3, or brain extract, and the course of dephosphorylation can be monitored with antibodies diagnostic of the pathological state of tau. Both phosphatases also act directly on PHF tau isolated from Alzheimer brains.

Influence of the phosphorylation state of neurofilament proteins on the interactions between purified filaments in vitro.

The extensive enzymic dephosphorylation of neurofilaments determined the progressive loss of their capacity to interconnect in vitro into a reticulated network, measured by the formation of highly viscous gels in purified preparations of neurofilaments [Leterrier & Eyer (1987) Biochem. J. 245, 93-101]. Conversely, a cyclic AMP-dependent activation of the gelation process was obtained by phosphorylation of the neurofilament proteins by the cyclic-nucleotide-dependent protein kinase added to the preparation. These findings argue for a direct relationship between the high phosphorylation level of the neurofilament subunits and the cross-bridging of the polymers in vitro. However, a transient stimulation of the neurofilament viscosity kinetics was also observed during the early steps of dephosphorylation with acid phosphatase, which, moreover, disappeared with longer incubation times before the net inhibition was obtained. In the same way, the calmodulin-dependent brain phosphatase, calcineurin, induced a permanent activation of the phenomenon, correlated with a low dephosphorylation capacity of the neurofilament molecules. Taken together, these results suggest a functional heterogeneity of the numerous phosphate groups of the neurofilament subunits and raise the hypothesis of a highly controlled regulation of the neurofilament cross-bridging by selective phosphorylation-dephosphorylation mechanisms.

A brain phosphatase with specificity for microtubule-associated protein-2.

A protein phosphatase has been isolated from brain using, as assay substrate throughout the purification, microtubule-associated protein-2 which had been phospho-labeled by its associated kinase. In contrast to other protein phosphatases, this phosphatase can effectively release phosphate from both the microtubule-binding and projection domains of microtubule-associated-protein-2. This enzyme appears to be a distinct, specific phosphatase that does not readily fit into previous classification schemes and is possibly the enzyme responsible for dephosphorylating microtubule-associated protein-2 in vivo.

Immunological characterization of phosphoprotein phosphatases

Phosphoprotein phosphatases regulate the biological activities of proteins through their involvement in cyclic phosphorylation/dephosphorylation cascades. A variety of multimeric phosphatases have been isolated and grouped into several classes, termed type 1 and types 2A, 2B, and 2C. To elucidate the relationship between the different phosphoprotein phosphatases, highly purified enzymes from soil amoebae, turkey gizzards, bovine heart and brain, and rabbit skeletal muscle and reticulocytes were tested for immunological antigenic relatedness. Two heterologous antibody preparations were employed for this purpose. One was made against an Acanthamoeba type 2A phosphatase and the other was made to bovine brain phosphatase type 2B (calcineurin, holoenzyme). Specific subunit cross-reactivity was examined by protein blot (“Western”) analysis. The antibody to the type 2A phosphatase reacted with the catalytic subunits of every type 2 enzyme tested, including both the catalytic and Ca 2+-binding subunits of the Ca 2+/calmodulin-dependent type 2B phosphatase (calcineurin), bovine cardiac type 2A phosphatase, and turkey gizzard smooth muscle phosphatase-1 (type 2A 1). It did not react with any type 1 phosphatase (catalytic subunit or ATP-Mg-dependent). The antigenic relatedness of calcineurin and the bovine cardiac type 2A phosphatase ( M r 38,000) was demonstrated further by protein blot analysis showing that the anti-calcineurin antibody cross-reacted with both enzymes. The mutual cross-reactivity poses an intriguing problem because these enzymes are so different in their molecular structures and modes of regulation. The degree of evolutionary conservation exhibited by the antigenic cross-reactivity of the type 2 enzymes from a broad range of species and tissues suggests a strong selective pressure on maintaining one or more features of these important regulatory enzymes.

Characterization of a calmodulin-dependent protein phosphatase from human platelets.

A calmodulin-dependent protein phosphatase has been identified in human platelets by its cross-reactivity with an antibody developed against a bovine brain calmodulin-dependent protein phosphatase and by its calmodulin-stimulated dephosphorylation of 32P-labeled substrates. The platelet enzyme was partially purified to separate it from calmodulin and calmodulin-independent phosphatases. The partially purified enzyme was stimulated by calmodulin, requiring 15 nM calmodulin for half-maximal activation. Calmodulin increased the Vmax of the phosphatase, with no significant effect on its Km. The enzyme was stimulated irreversibly and made calmodulin-independent by limited proteolysis. The optimal pH for the phosphatase was 7.5. After partial purification, phosphatase activity was significantly increased in the presence of Mn2+ and Ca2+ over that observed in the presence of Ca2+ alone. The enzyme effectively dephosphorylated casein, histone, protamine, and platelet actin. The holophosphatase was estimated to have a molecular weight of 76,900 as determined by sedimentation on sucrose gradients. Immunoblotting techniques using an antibody against the brain phosphatase suggests that the enzyme consists of 2 subunits of 60,000 and 16,500 daltons; the 60,000-dalton subunit co-migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a 60,000-dalton calmodulin-binding protein in the platelet suggesting that it is the calmodulin-binding subunit of the enzyme. The identification of a calmodulin-dependent protein phosphatase in human platelets suggests a role for Ca2+-dependent dephosphorylation in platelet activation.

Developmental studies of phospholipid-sensitive Ca2+-dependent protein kinase and its substrates and of phosphoprotein phosphatases in rat brain.

Ontogenetic changes in the protein phosphorylation/dephosphorylation systems in rat brain were investigated. It was found that the activity level of phospholipid-sensitive Ca2+-dependent protein kinase (PL-Ca-PK) in the particulate fraction of grey and white matter and the soluble fraction of grey matter increased rapidly and markedly after birth, reached the highest level at day 30, and declined slightly or remained unchanged thereafter. The enzyme level in the soluble fraction of white matter, in contrast, remained constant throughout the development and maturation of brain. Various ontogenetic changes in the substrate proteins for PL-Ca-PK were also noted. The levels of myelin basic protein and other substrates (notably the Mr 87,000, 58,000, 54,000, and 50,000 protein in grey matter) progressively increased during development, reaching the highest level at adulthood. The level of the Mr 66,000 protein from the particulate fraction of white and grey matter, on the other hand, increased rapidly after birth, reached a peak at day 18, and then declined to the initial neonatal level at the adult stage. The time scale for the increases in the levels of PL-Ca-PK and its many substrates paralleled that of brain development and maturation (synaptogenesis and myelinogenesis). The activity levels of phosphoprotein phosphatases (assayed using 32P-labeled myelin basic protein, histone, and protamine sulfate) were found to only slightly (up to 60%) increase or decrease in certain fractions from different brain regions during development, suggesting that phosphorylation, compared to dephosphorylation, may be more important in determining the phosphorylation state of cellular proteins.

Microtubule assembly using the microtubule-associated protein MAP-2 prepared in defined states of phosphorylation with protein kinase and phosphatase.

A microtubule-associated protein (the 270-kDa MAP-2) was prepared in two defined states of phosphorylation by (a) phosphorylation by associated kinase to the extent of 11-14 mol/mol, and (b) removal of 70-80% of this phosphate with a protein phosphatase purified from brain. The newly introduced phosphate was in addition to about 10 mol/mol already present in MAP-2 as isolated; these phosphates were not appreciably released by the phosphatase and did not exchange with ATP. In microtubules assembled with phosphorylated (24 mol/mol) MAP-2 the assembly rate was decreased, microtubule length and critical concentration for assembly were unaffected, and rates of loss of subunits were increased from both microtubule ends. Phosphorylation also reduced the binding of MAP-2 to taxol-stabilized microtubules. These changes were unequivocally due to phosphorylation, since phosphatase treatment reversed all of them. The brain phosphatase used in these experiments was purified 3000-fold towards histone, but only 100-fold towards MAP-2, suggesting brain may contain another enzyme more specific for MAP-2. Calcineurin, however, had only a low activity for MAP-2.

Enzymatic dephosphorylation of endogenous and exogenous dolichyl monophosphate by calf brain membranes

Calf brain membranes have been shown to enzymatically dephosphorylate endogenous and partially purified, exogenous dolichyl [ 32P]monophosphate. The properties and specificity of the dolichyl monophosphatase activity have been studied by following the release of [ 32P]phosphate from exogenous dolichyl [ 32P]monophosphate added in a dispersion with Triton X-100. The calf brain phosphatase (1) is inhibited by Mn 2+, Mg 2+, Ca 2+, fluoride, and phosphate; (2) exhibits a neutral pH optimum; and (3) has an apparent K m of 200 μ m for dolichyl monophosphate. Dolichyl monophosphatase activity can be distinguished from phosphatidate phosphatase on the basis of their responses to fluoride and phosphate. Based on differential thermolability and the effects of divalent cations and EDTA, the calf brain dolichyl monophosphatase can also be discriminated from the general phosphatase activity assayed with p-nitrophenyl phosphate. Dolichyl monophosphatase activity can be solubilized by treating microsomes with Triton X-100. The enzymatic dephosphorylation of exogenous dolichyl [ 32P]monophosphate catalyzed by particulate and detergent-solubilized preparations is negligibly affected by equimolar concentrations of ATP and an assortment of phosphomonoesters, including phosphatidic acid and hexadecyl phosphate. A reduction of approximately 40% in dolichyl monophosphatase activity is observed in the presence of equimolar amounts of retinyl monophosphate. Overall, these results represent good evidence for the presence of a neutral polyisoprenyl monophosphatase in central nervous tissue.

Phosphatase alcaline du cerveau de boeuf Role du magnésium sur l'action de la l-phénylalanine et d'autres acides amines

1. 1. Alkaline phosphatase from beef brain (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) is not very sensitive to l-phenylalanine. The stereospecific effect is only 38%, compared to 65% and 90% for the calf intestinal and human placental enzymes, respectively. For all three enzymes, the phenomenon is uncompetitive and not allosteric in nature. 2. 2. At pH < 9.5, in the presence of Mg 2+, the d-isomer activates the brain phosphatase and has no effect on the two other enzymes. l-Phenylalanine is also an activator with respect to the control and its stereospecific effect is only evident when compared with the d-isomer. 3. 3. This activation is closely related to the activation by Mg 2+. Both phenomena are pH dependent, practically identical from pH 8.5 to 10.0 and their effect on the enzyme is noncompetitive. Mg 2+ is a possible site for the binding of l-phenylalanine to the brain enzyme. 4. 4. All the natural α-amino acids have an identical behaviour. The d-isomers produce the same activation as glycine, whereas the effect of l-isomers, themselves activators when compared with the control, is dependent on the molecular weight of the amino acid (alanine, serine have no effect, maximum effect is observed with tryptophan and on its conformation (the effects are different with leucine, isoleucine, norleucine). For the intestinal and placental phosphatases, only l-phenylalanine and l-tryptophan have an important effect. 5. 5. The mode of action of phenylalanine and the other amino acids capable of causing a stereospecific effect on the brain enzymes seems to be different from the one described previously in the literature for the rat intestinal and human placental alkaline phosphatases. It is suggested that the strong or the weak sensitivity, of these two kinds of enzymes, to Mg 2+ may be responsible for the observed differences. The role of Mg 2+ on the reactivity of alkaline phosphatases is also discussed.

5′-Nucleotidase and phosphatases in human brain

The activities of five enzymes catalysing release of phosphate have been determined in homogenates from different regions of human brain and cord; all enzymes showed activities within a small range of absolute values. The enzymes showed similar activities in autopsy and biopsy material. Activities of 5′-nucleotidase were higher in cortex than in white matter and compared closely with other mammalian brains. Activities of adenosine triphosphatase were usually 2–3 times higher in cortex than in white matter, Mg 2+-adenosine triphosphatase was at least twice as active as Ca 2+-adenosine triphosphatase; activities were lower than in animals. Alkaline phosphatase in human tissue was generally lower than in animal tissue whilst acid phosphatase was mainly higher. Probable functions of these enzymes in neural tissue were briefly discussed.

ALKALINE PHOSPHATASES IN BRAIN AND SPINAL CORD.

THERE has been much controversy as to whether alkaline phosphatase is a single enzyme or whether a number of such enzymes are present in tissues. The evidence derived from inhibition and pH studies suggests the existence of a plurality of alkaline phosphatases1–3. The subject has been studied histochemically by Glick and Fischer4, Dempsey5, Friedenwald and Maengwyn-Davies6 and Bourne7. The results of these authors also suggest a multiplicity of alkaline phosphatases. Sandier and Bourne8 separated out a number of substrate specific phosphatases by starch-gel electrophoresis, thus again indicating the plurality of alkaline phosphatase.