Vesicles with lactate dehydrogenase and without alkaline phosphatase present in the resting zone of epiphyseal cartilage.

Matrix vesicles are membrane-invested vesicles that initiate mineralization in the extracellular matrix of calcifying tissues. The epiphyseal cartilages of young-rat rib bones were divided into the growth zone and the resting zone, followed by the isolation of matrix vesicles after collagenase treatment. Matrix vesicles with both alkaline phosphatase and lactate dehydrogenase were detected in the growth cartilage found in the epiphyseal growth plates of young rabbits [Hosokawa, Uchida, Fujiwara & Noguchi (1988) J. Biol. Chem. 263, 10045-10047], but were not detected in the resting zone. By contrast, and surprisingly, lactate dehydrogenase-containing vesicles without alkaline phosphatase were found in the resting zone, but not in the growth zone. In both the growth and resting zones, isoenzyme patterns of lactate dehydrogenase in the two different vesicles were identical with those of cytosolic lactate dehydrogenase of chondrocytes, suggesting the presence of a mechanism for specific uptake of cytosolic lactate dehydrogenase. The same results as for young-rat rib bones were obtained with the resting and growth cartilages of young-dog and monkey rib bones.     

Mechanism of biliary secretion of membranous enzymes: bile acids are important factors for biliary occurrence of gamma-glutamyltransferase and other hydrolases

To elucidate the mechanism of biliary occurrence of gamma-glutamyl transferase [EC 2.3.2.2] and alkaline phosphatase [EC 3.1.3.1], the effect of bile acids on the biliary level of these enzymes was studied in vivo and in vitro. Following intravenous administration of taurocholate, the activities of both enzymes in rat bile increased markedly with a concomitant increase in the excretion of the bile acid. The biliary levels of these enzymes increased to reach a maximum at 10-20 min after administration of the bile acid and decreased thereafter. Right-side-out oriented rat liver canalicular membrane vesicles which localize gamma-glutamyltransferase, aminopeptidase M and alkaline phosphatase on their outer surface (Inoue, M., Kinne, R., Tran, T., Biempica, L., & Arias, I.M. (1983) J. Biol. Chem. 258, 5183-5188) were prepared. Upon incubation of the vesicles with either intact or heat-treated bile samples, the membranous enzymes were released from the vesicles in a time-dependent manner. Incubation of these vesicles with physiological concentrations of taurocholate also solubilized these enzymes from the membranes. Affinity chromatographic analysis on concanavalin A-Sepharose revealed that the transferase thus solubilized retained the hydrophobic domain responsible for anchoring the enzyme to membrane/lipid bilayers. These results indicate that bile acid(s) excreted into the bile canalicular lumen solubilized these enzymes from the apical membrane surface of the biliary tract cells by their detergent action.     

Purification and partial characterization of ATP pyrophosphohydrolase from fetal bovine epiphyseal cartilage

ATP pyrophosphohydrolase was partially purified from fetal bovine epiphyseal cartilage. The purification was about 10- and 100-fold over the enzyme activities of matrix vesicle fraction and cell homogenate, respectively. The pyrophosphohydrolase and alkaline phosphatase were separated by a sequential application of Sepharose CL-6B and DEAE-cellulose column chromatographies. The purified enzyme migrated as a single band corresponding to the molecular weight of 230,000 in sodium dodecyl sulfate-polyacrylamide disc gel by electrophoresis. The enzyme absolutely required Zn2+ for its activity and appeared to bind Zn2+ strongly with an apparent affinity of p[Zn2+]0.5 = 13.4. The apparent Km for ATP was 0.18 mM. The enzyme was also reactive toward various nucleoside triphosphates including GTP, CTP, and UTP. In contrast, various phosphodiesters including RNA, UDP-glucose, NAD, and bis-p-nitrophenylphosphate were 5% or less as reactive as the nucleoside triphosphates. The pyrophosphohydrolase was inactive toward adenosine 3':5'-monophosphate or various phosphonates. UDP-glucose (1 mM), NAD (1 mM), or RNA (1 mg/ml) failed to inhibit the ATP pyrophosphohydrolase activity. These observations suggest that the ATP pyrophosphohydrolase of the cartilage is probably not a phosphodiesterase I. The matrix vesicle fraction, which probably also included some plasma membrane vesiculated during collagenase digestion, contained the highest specific activity of the enzyme as compared to other subcellular fractions of either epiphyseal or articular cartilage.     

Studies on matrix vesicles isolated from chick epiphyseal cartilage. Association of pyrophosphatase and ATPase activities with alkaline phosphatase.

Fractions composed primarily of cells (Fraction I), membrane fragments (Fraction II) and matrix vesicles (Fraction III) were isolated from chick epiphyseal cartilage. The characteristics of the alkaline phosphatase (EC 3.1.3.1), pyrophosphatase (EC 3.6.1.1) and ATPase (EC 3.6.1.3) activities in the matrix vesicle fraction were studied in detail. Mg-2-+ was not absolutely essential to any of the activities, but at low levels was stimulatory in all cases. Higher concentrations inhibited both pyrophosphatase and ATPase activities. Both the stimulatory and inhibitory effects were pH-dependent. Ca-2-+ stimulated all activities weakly in the absence of Mg-2-+. However, when Mg-2-+ was present, Ca-2-+ was slightly inhibitory. Thus, none of the activities appear to have a requirement for Ca-2-+, and hence would not seem to be involved with active Ca-2-+ transport in the typical manner. The distribution of alkaline phosphatase, pyrophosphatase, and Mg-2-+ ATPase activities among the various cartilage fractions was identical, and concentrated primarily in the matrix vesicles. Conversely, the highest level of (Na-+ + K-+)-ATPase activity was found in the cell fraction. All activites showed nearly identical sensitivities to levamisole (4 - 10-3 M) which caused nearly complete inhibition of alkaline phosphatase and pyrophosphatase. About 10-15% of the ATPase activity was levamisole-insensitive. The data are consistent with the concept that the Mg-2-+-ATPase and pyrophosphatase activities of matrix vesicles stem from one enzyme, namely, alkaline phosphatase.     

The role of nucleoside triphosphate pyrophosphohydrolase in in vitro nucleoside triphosphate-dependent matrix vesicle calcification

Nucleoside triphosphate pyrophosphohydrolase (EC 3.6.1.8) activity is associated with matrix vesicles purified from collagenase digests of fetal calf epiphyseal cartilage. This enzyme hydrolyzes nucleoside triphosphates to nucleotides and PPi, the latter inducing precipitation in the presence of Ca2+ and Pi. An assay for matrix vesicle nucleoside triphosphate pyrophosphohydrolase is developed using beta, gamma-methylene ATP as substrate. The assay is effective in the presence of matrix vesicle-associated ATPase, pyrophosphatase, and alkaline phosphatase activities. A soluble nucleoside triphosphate pyrophosphohydrolase is obtained from matrix vesicles by treatment with 5 mM sodium deoxycholate. The solubilized enzyme induced the precipitation of calcium phosphate in the presence of ATP, Ca2+, and Pi. Extraction of deoxycholate-solubilized enzymes from matrix vesicles with 1-butanol destroys nucleoside triphosphate pyrophosphohydrolase activity while enhancing the specific activities of ATPase, pyrophosphatase, and alkaline phosphatase. In solutions devoid of ATP and matrix vesicles, concentrations of PPi between 10 and 100 microM induce calcification in mixtures containing initial Ca2+ X P ion products of 3.5 to 7.9 mM2. This finding plus the discovery of nucleoside triphosphate pyrophosphohydrolase in matrix vesicles supports the view that these extracellular organelles induce calcium precipitation by the enzymatic production of PPi. Nucleoside triphosphate pyrophosphohydrolase is more active against pyrimidine nucleoside triphosphates than the corresponding purine derivatives. The pH optimum is 10.0 and the enzyme is neither activated nor inhibited by Mg2+ or Ca2+ ions or mixtures of the two. Vmax at pH 7.5 for beta, gamma-methylene ATP is 0.012 mumol of substrate hydrolyzed per min per mg of protein and Km is below 10 microM. The enzyme is irreversibly destroyed at pH 4 and is stable at pH 10.5.     

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.     

Calcification of in vitro developed hypertrophic cartilage

We have recently reported that dedifferentiated cells derived from stage 28-30 chick embryo tibiae, when transferred in suspension culture in the presence of ascorbic acid, develop in a tissue closely resembling hypertrophic cartilage. Ultrastructural examination of this in vitro formed cartilage showed numerous matrix vesicles associated with the extracellular matrix (C. Tacchetti, R. Quarto, L. Nitsch, D. J. Hartmann, and R. Cancedda, 1987, J. Cell Biol. 105, 999-1006). In the present article we report that the in vitro developed hypertrophic cartilage undergoes calcification. We indicate a correlation between the levels of alkaline phosphatase activity and calcium deposition at different times of development. Following the transfer of cells into suspension culture and an initial lag phase, the level of alkaline phosphatase activity rapidly increased. In most experiments the maximum of activity was reached after 5 days of culture. When alkaline phosphatase activity and 45Ca deposition were measured in the same experiment, we observed that the increase in alkaline phosphatase preceded the deposition of nonwashable calcium deposits in the cartilage.     

Extracellular alkaline phosphatase activity in mineralizing matrices of cartilage and bone: ultrastructural localization using a cerium-based method.

The ultrastructural localization of alkaline phosphatase (A1P) activity has been demonstrated in epiphyseal growth cartilage and metaphyseal bone of rats. Epiphyso-metaphyseal specimens were decalcified with EDTA and treated with MgCl2 to regenerate the enzymatic activity before incubation in a medium containing beta-glycerophosphate, MgCl2 and CeCl3. A1P activity was present on the outer surface of the plasmamembrane of maturing and hypertrophic chondrocytes and of osteoblasts. Moreover, the reaction product was present in chondrocyte lacunae, in matrix vesicles, and in cartilage matrix, as well as among uncalcified collagen fibrils of osteoid tissue in bone. The intensity of reaction was the lowest, or completely lacking, where the degree of matrix calcification was the highest. These results suggest that alkaline phosphatase is transported from the cells into the cartilage and bone matrix by its association with matrix vesicles and plasmamembrane components, and that its activity in cartilage and bone matrix is inhibited as it is incorporated in the mineral substance.     

Enzymatic removal of alkaline phosphatase from renal brush-border membranes. Effect on phosphate transport and on phosphate binding

Brush-border membrane vesicles prepared from rabbit kidney cortex were incubated at 37 degrees C for 30 min with phosphatidylinositol-specific phospholipase C. This maneuver resulted in a release of approx. 85% of the brush-border membrane-linked enzyme alkaline phosphatase as determined by its enzymatic activity. Transport of inorganic [32P]phosphate (100 microM) by the PI-specific phospholipase C-treated brush-border membrane vesicles was measured at 20-22 degrees C in the presence of an inwardly directed 100 mM Na+ gradient. Neither initial uptake rates, as estimated from 10-s uptake values (103.5 +/- 6.8%, n = 7 experiments), nor equilibrium uptake values, measured after 2 h (102 +/- 3.4%) were different from controls (100%). Control and PI-specific phospholipase C-treated brush-border membrane vesicles were extracted with chloroform/methanol to obtain a proteolipid fraction which has been shown to bind Pi with high affinity and specificity (Kessler, R.J., Vaughn, D.A. and Fanestil, D.D. (1982) J. Biol. Chem. 257, 14311-14317). Phosphate binding (at 10 microM Pi) by the extracted proteolipid was measured. No significant difference in binding was observed between the two types of preparations: 31.0 +/- 9.37 in controls and 29.8 +/- 8.3 nmol/mg protein in the proteolipid extracted from PI-specific phospholipase C-treated brush-border membrane vesicles. It appears therefore that alkaline phosphatase activity is essential neither for Pi transport by brush-border membrane vesicles nor for Pi binding by proteolipid extracted from brush-border membrane. These results dissociate alkaline phosphatase activity, but not brush-border membrane vesicle transport of phosphate, from phosphate binding by proteolipid.     

DNA polymerase III of Escherichia coli. Purification and identification of subunits.

DNA polymerase III, the core of the DNA polymerase III holoenzyme, has been purified 28,000-fold to 97% homogeneity from Escherichia coli HMS-83. The enzyme contains subunits: alpha, epsilon, and theta of 140,000, 25,000, and 10,000 daltons, respectively. The alpha subunit has been previously shown to be a component of both DNA polymerase III and the more complex DNA polymerase III holoenzyme (Livingston, D.M., Hinkle, D., and Richardson, C. (1975) J. Biol. Chem. 250, 461-469; McHenry, C., and Kornberg, A. (1977) J. Biol. Chem. 252, 6478-6484). It is demonstrated here that the epsilon and theta subunits are also subunits of the DNA polymerase III holoenzyme. Thus, the DNA polymerase III holoenzyme contains at least six different subunits. Our preparation has both the 3' leads to 5' and 5' leads to 3' exonuclease activities previously assigned to DNA polymerase III (Livingston, D., and Richardson, C. (1975) J. Biol. Chem. 250, 470-478).     

Purification and properties of the manganese superoxide dismutase from the liver of bullfrog, Rana catesbeiana

A manganese superoxide dismutase (Mn-SOD) from the liver of bullfrog, Rana catesbeiana, was purified to electrophoretic homogeneity. The enzyme has a molecular weight of about 84,000 and is composed of four identical subunits, each containing one manganese atom. The amino acid composition of the enzyme is similar to that of Mn-SODs isolated from human and chicken livers, but differs considerably from that of the Escherichia coli enzyme (D. Barra et al. (1984) J. Biol. Chem. 259, 12595-12601; R. A. Weisiger and I. Fridovich (1973) J. Biol. Chem. 248, 3582-3592; H. M. Steinman (1978) J. Biol. Chem. 253, 8708-8720). The N-terminal amino acid is lysine. The sequence of 23 amino acid residues in the N-terminal region was determined. It shows excellent homologies with those of the human and chicken enzymes (H. M. Steinmam and R. L. Hill (1973) Proc. Natl. Acad. Sci. USA 70, 3725-3729; C. Ditlow et al. (1982) Carlsberg Res. Commun. 47, 81-91). The frog liver enzyme is also located exclusively in the mitochondrial matrix. Immunologically the same enzyme is also found in the tadpole liver, in an amount of about one-half of that in the adult bullfrog.     

Phosphatidylserine modulates calf intestine alkaline phosphatase activity towards low and high molecular weight substrate

Pretreatment of calf intestine alkaline phosphatase with phosphatidylserine resulted in an inhibition of the phosphatase activity towards low - (p-nitrophenylphosphate) and high (phosphohistone) molecular weight substrate. Phosphatidylcholine, irrespectively of the substrate used did not cause enzyme modulation. 12-O-tetradecanoylphorbol-13-acetate, 1,2-diolein as well certain retinoids, known to effect phosphatidylserine-sensitive enzyme systems (Castagna, M. et al. 1982, J. Biol. Chem. 257, 7847-7851; Gmeiner, B. 1986, Biochim. Biophys. Acta 856, 392-394) had no influence on the modulated phosphatase. The lipid interacting drug trifluoperazine inhibited the enzyme activity towards phosphohistone, but not towards p-nitrophenylphosphate as a substrate. The results indicate that acidic phospholipid may play a role in activity modulation of calf intestine membranous alkaline phosphatase activity.     

Extracellular alkaline phosphatase activity in mineralizing matrices of cartilage and bone: ultrastructural localization using a cerium-based method

Summary The ultrastructural localization of alkaline phosphatase (AlP) activity has been demonstrated in epiphyseal growth cartilage and metaphyseal bone of rats. Epiphyso-metaphyseal specimens were decalcified with EDTA and treated with MgCl₂ to regenerate the enzymatic activity before incubation in a medium containing beta-glycerophosphate, MgCl₂ and CeCl₃. AlP activity was present on the outer surface of the plasmamembrane of maturing and hypertrophic chondrocytes and of osteoblasts. Moreover, the reaction product was present in chondrocyte lacunae, in matrix vesicles, and in cartilage matrix, as well as among uncalcified collagen fibrils of osteoid tissue in bone. The intensity of reaction was the lowest, or completely lacking, where the degree of matrix calcification was the highest. These results suggest that alkaline phosphatase is transported from the cells into the cartilage and bone matrix by its association with matrix vesicles and plasmamembrane components, and that its activity in cartilage and bone matrix is inhibited as it is incorporated in the mineral substance.     

A hybrid Escherichia coli alkaline phosphatase formed on proteolysis

Cleavage of bacterial alkaline phosphatase by trypsin at the R-11, A-12 bond of both subunits results in changes in the structure and function of the enzyme as previously reported (Roberts, C. H., and Chlebowski, J. F. (1984) J. Biol. Chem. 259, 729-733; Roberts, C. H., and Chlebowski, J. F. (1985) J. Biol. Chem. 260, 7557-7561). A hybrid dimer has been formed by cleaving the R-11, A-12 bond of only one of the two subunits. This enzyme species has been purified and characterized to investigate subunit interactions of this hybrid dimeric enzyme species. Subunit interactions were observed using various methods to study functional and structural properties of the enzyme. In a kinetic study the T-2/A-12 hybrid enzyme was found to have a Vmax similar to the A-12 fully trypsin-modified enzyme. On exposure to EDTA the hybrid was found to lose activity at essentially the same rate as the A-12 enzyme presumably as a consequence of loss of metal ions required for function. On adding metal ions back to the apoenzyme form, activity of the hybrid was reconstituted to a degree similar to that of the native enzyme whereas the activity of the A-12 enzyme was reconstituted to a much lesser extent. The Tm of the hybrid measured by differential scanning calorimetry was closer to the value obtained for the A-12 enzyme than the T-2 enzyme but circular dichroic spectra indicated secondary structural features of the hybrid different from both symmetrical forms of the enzyme. These results provide evidence for strong subunit interactions in the alkaline phosphatase dimer.     

Incorporation of bovine enterokinase in reconstituted soybean phospholipid vesicles

Bovine enterokinase was incorporated into vesicles reconstituted from a soybean phospholipid mixture. A thin film hydration procedure (MacDonald, R. I., and MacDonald, R. C. (1975) J. Biol. Chem. 250, 9206-9214) produced vesicles with 40% of the enterokinase activity bound in the membrane. The highest incorporation was observed when cholesterol or dimyristoylphosphatidylethanolamine was added to the soybean phospholipids. Crude and highly purified enterokinase preparations were incorporated to the same extent suggesting that other membrane components were not required for a successful reconstitution. The properties of enterokinase in phospholipid vesicles were compared with those of alkaline phosphatase, which was also added to the reconstitution system, and with the enzyme activities present in vesicles prepared from brush-border membranes. The enzyme activities were not released by solutions of high ionic strength and remained associated with the phospholipid vesicles on gel filtration, ultracentrifugation, and sucrose density centrifugation. Enterokinase and alkaline phosphatase had their active sites exposed to substrate in the brush-border membrane vesicles. In soybean phospholipid vesicles half of the active sites of both enzymes were on the outside, since release of the enzyme with Triton X-100 almost doubled the units of enzyme present. Incubation of the soybean phospholipid and brush-border membrane vesicles with papain released the exposed molecules of enterokinase. The released enzyme molecules were fully active but could not be reincorporated into phospholipid vesicles. This suggests that the structure imbedded in the lipid bilayer was essential for a successful reconstitution. We conclude that the reconstituted soybean phospholipid vesicles are a suitable membrane system for the further study of membrane-bound enterokinase.     

Structural evidence for a functional role of human tissue nonspecific alkaline phosphatase in bone mineralization

The human tissue nonspecific alkaline phosphatase (TNAP) is found in liver, kidney, and bone. Mutations in the TNAP gene can lead to Hypophosphatasia, a rare inborn disease that is characterized by defective bone mineralization. TNAP is 74% homologous to human placental alkaline phosphatase (PLAP) whose crystal structure has been recently determined at atomic resolution (Le Du, M. H., Stigbrand, T., Taussig, M. J., Ménez, A., and Stura, E. A. (2001) J. Biol. Chem, 276, 9158-9165). The degree of homology allowed us to build a reliable TNAP model to investigate the relationship between mutations associated with hypophosphatasia and their probable consequences on the activity or the structure of the enzyme. The mutations are clustered within five crucial regions, namely the active site and its vicinity, the active site valley, the homodimer interface, the crown domain, and the metal-binding site. The crown domain and the metal-binding domain are mammalian-specific and were observed for the first time in the PLAP structure. The crown domain contains a collagen binding loop. A synchrotron radiation x-ray fluorescence study confirms that the metal in the metal-binding site is a calcium ion. Several severe mutations in TNAP occur around this calcium site, suggesting that calcium may be of critical importance for the TNAP function. The presence of this extra metal-binding site gives new insights on the controversial role observed for calcium.     

Kinase FA-mediated regulation of rabbit skeletal muscle protein phosphatase. Reversible phosphorylation of the modulator subunit

A mechanism of activation of the ATP.Mg-dependent protein phosphatase (FC.M) has been proposed (Jurgensen, S., Shacter, E., Huang, C. Y., Chock, P. B., Yang, S.-D., Vandenheede, J. R., and Merlevede, W. (1984) J. Biol. Chem. 259, 5864-5870) in which a transient phosphorylation by the kinase FA of the modulator subunit (M) is the driving force for the transition of the inactive catalytic subunit (FC) into its active conformation. Incubation of FC.M with kinase FA and Mg2+ and adenosine 5'-(gamma-thio)triphosphate results in thiophosphorylation of M and also a conformational change in the phosphatase catalytic subunit; however, the enzyme remains inactive. Proteolysis of this inactive, thiophosphorylated complex causes proteolytic destruction of the modulator subunit and yields an active phosphorylase phosphatase species. Similar treatment of the native inactive enzyme does not yield active phosphatase. Evidence is presented, suggesting that a molecule of modulator is bound at an "inhibitory site" on the native enzyme. This modulator does not prevent the conformational change in the phosphatase catalytic subunit upon incubation with kinase FA and ATP.Mg but does partially inhibit the expression of the phosphorylase phosphatase activity.     

Solubility properties of alkaline phosphatase from matrix vesicles

Alkaline phosphatase has been extracted from matrix vesicles of a calcifying cartilage with 0.15 M KCl, 0.4 M guanidinium chloride and 0.05 M deoxycholate/50% butanol mixture. The catalytic properties of the three extracts have been compared. Although the highest amount of enzyme activity is extracted with the latter reagent (55%), some of it is also extracted with KCl (11%) and guanidinium (7%). By submitting isolated matrix vesicles to a short time sonication the distribution pattern of the alkaline phosphatase activity in the extracts is clearly modified, as the amount extracted with KCl increases from 14 to 50% and the portion extracted with deoxycholate decreases from 55 to 27% of the total enzyme activity of matrix vesicles. The enzymatic preparations were comparable on the basis of specific activities, affinity for the substrates (p-nitrophenylphosphate, ATP), thermostability, sensitivity to inhibitors and activators. By electrofocusing a value of pI = 4.15 was found for the alkaline phosphatase of matrix vesicles independently of the extraction medium. These results contradict the concept that alkaline phosphatase is exclusively an intrinsic membrane protein.     

Occlusion of Rb+ after extensive tryptic digestion of shark rectal gland Na,K-ATPase.

Na,K-ATPase from rectal glands of Squalus acanthias has been subjected to proteolysis with trypsin. The E1- and E2-forms of the enzyme can be distinguished from the inactivation patterns at low trypsin concentrations, as previously seen with kidney enzyme. Extensive degradation by trypsin in the presence of 5 mM Rb+ yields membrane fragments with a 19 kDa peptide as the major proteolytic fragment of the alpha-subunit. The sequence of the N-terminal 40 residues of this peptide is almost identical to that of a similar proteolytic fragment isolated by Capasso et al. (Capasso, J.M., Hoving, S., Tal, D.M., Goldshleger, R. and Karlish, S.J.D. (1992) J. Biol. Chem. 267, 1150-1158) using kidney Na,K-ATPase. Rb+ occlusion can be fully retained under these circumstances, supporting the findings with kidney enzyme that only minor parts of the alpha-subunit are required to form a functional occlusion-site.