Regulation of rat-liver glycoprotein: N-acetylneuraminic acid transferase activity by pyrimidine nucleotides.

CMP-N-acetylneuraminic acid: glycoprotein sialyltransferase activities were assayed in rat liver microsomal fractions using desialylated fetuin as the substrate acceptors for N-acetylneuraminic acid. It was found that cytidine nucleotides specifically depressed enzyme activities. CMP was shown to act as a competitive inhibitor with an apparent Ki of 0.62 mM. N-Acetylneuraminic acid at 1.15 mM had no effect on enzyme activities. Uridine nucleotides at 1.15 mM, especially UDP, increased enzyme activities. UDP may act as an allosteric activating agent increasing the apparent V. Other nucleotides, sugars and nucleotide-sugars at similar concentrations affected sialyltransferase activities only slightly. A general mechanism is proposed for the regulation of glycosyltransferase activities by free nucleotides.     

Purification and characterization of beta-galactoside (alpha 2 leads to 6)sialyltransferase from rat liver and hepatomas

Asialofetuin sialyltransferase from Triton X-100 extracts of rat liver was resolved by phosphocellulose chromatography into two fractions, designated I and II in order of elution. When previously treated with Arthrobacter ureafaciens neuraminidase, fraction I eluted at about the same position as II while no alteration occurred in II. Primary rat hepatomas contained only a single asialofetuin sialyltransferase, identical to fraction I in chromatographic behavior. Transferases I and II were purified to near homogeneity. Transferase II, as well as neuraminidase-treated I, could be sialylated auto-catalytically, indicating that the lack of sialic acid in II is not due to the lack of a sialic-acid-accepting site. Both enzymes formed an (alpha 2 leads to 6)sialylgalactoside linkage with asialo-glycoproteins of the glycosylamine-type and with lactose, and were indistinguishable immunologically. Nevertheless, the transferases exhibited different molecular weights of 37000 (I) and 43000 (II). When heated at 50 degrees C, transferase I lost half its original activity within 20 min while II was scarcely inactivated. Kinetically, transferase I showed three-times higher affinity than II for CMP-N-acetylneuraminic acid and for desialylated plasma membrane. Asialofetuin sialyltransferase was also purified from primary rat hepatoma. The purified enzyme was identical to transferase I in every respect examined. We conclude that hepatomas contain transferase I but lack transferase II.     

Improved synthesis of CMP-sialates using enzymes from frog liver and equine submandibular gland.

An efficient method for the preparation of CMP-N-acetylneuraminic acid using crude or partially purified CTP:N-acylneuraminate cytidylyltransferase from equine submandibular gland or frog liver is described. The yield of the sugar nucleotide after purification by ion-exchange chromatography and gel filtration was 95%. The compound was studied by 360 MHz 1H NMR spectroscopy in addition to the usual chemical and physical analyses. The preparation of radioactive or unlabelled CMP 4-O-methyl-N-acetylneuraminic acid, which is not known to occur in nature, was achieved in 17% yield with the aid of the equine enzyme.     

Cyclic CMP phosphodiesterase: isolation, specificity and kinetic properties

Cyclic CMP phosphodiesterase activity was demonstrated in rat liver, heart, brain, kidney, intestine, skeletal muscle, blood, testes, ovaries, spleen and lung; that present in the liver was purified to homogeneity by a sequential process of ammonium sulphate fractionation, gel filtration, two ion-exchange chromatographic steps, preparative electrophoresis and two affinity chromatographic stages with selection at each stage for maximum specificity. The final enzyme preparation was confirmed as a single protein by HPLC and isoelectric focussing; the total yield obtained was 1.5% and the final specific activity of 48.6 mumol cyclic CMP hydrolysed/min/mg reflected a 88,000 fold purification. The phosphodiesterase had a Mr of 2.8 X 10(4), pH optimum 7.2-7.4, isoelectric point between 4.2 and 4.4 and a Km of 9.0 mM cyclic CMP. This enzyme differs from a previously isolated cyclic CMP phosphodiesterase in its amino acid composition and specificity. The absolute specificity for 3',5'-cyclic CMP as substrate distinguishes this cyclic CMP phosphodiesterase from all other reported phosphodiesterases and shows it to be a novel enzyme. Its potential as a research tool and the significance of its occurrence are discussed.     

Nuclear poly(A) polymerase from rat liver and a hepatoma. Comparison of properties, molecular weights and amino acid compositions.

Poly(A) polymerase was extracted from isolated nuclei of rat liver and a rapidly growing solid tumor (Morris hepatoma 3924A). The enzyme from each tissue was purified by successive chromatography on DEAE-Sephadex, phosphoecllulose, hydroxyapatite and QAE-Sephadex. Purified enzyme from both liver and tumor was essentially homogeneous as judged by polyacrylamide gel electrophoresis. Under nondenaturing conditions, enzyme activity corresponded to visible protein and, upon denaturation, a single polypeptide was detected. The enzymes had absolute requirements for Mn2+ as the divalent ion, ATP as the substrate and an oligonucleotide or polynucleotide as the primer. Both enzymes were inhibited by sodium pyrophosphate, N-ethylmaleimide, Rose Bengal, cordycepin 5'-triphosphate and several rifamycin derivatives. The reactions were unaffected by potassium phosphate, alpha-amanitin and pancreatic ribonuclease. However, the liver and hepatoma enzymes differed from each other with respect to apparent Km, primer saturation levels and sensitivity to pH changes. The most striking differences between the enzymes were in their calculated molecular weights (liver, 48000; hepatoma, 60000) and amino acid compositions. Finally, the level of the hepatoma enzyme relative to that of the liver enzyme was at least 1.5-fold higher when expressed per mg DNA.     

Different reactivity to lysophosphatidylcholine, DIDS and trypsin of two brain sialyltransferases specific for O-glycans: a consequence of their topography in the endoplasmic membranes

Some properties of two distinct rat brain sialyltransferases, acting on fetuin and asialofetuin, respectively, were investigated. These two membrane-bound enzymes were both strongly inhibited by charged phospholipids. Neutral phospholipids were without effect except lysophosphatidylcholine (lysoPC) which modulated these two enzymes in a different way. At 5 mM lysoPC, the fetuin sialyltransferase was solubilized and highly activated while the asialofetuin sialyltransferase was inhibited. Preincubation of brain microsomes with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), known as a specific anion inhibitor and a non-penetrating probe, led to a moderate inhibition of the asialofetuin sialyltransferase just as in the case of the ovomucoid galactosyltransferase (used here as a marker for the luminal side of the Golgi membrane); under similar conditions, the fetuin sialyltransferase was strongly inhibited. In the presence of Triton X-100, which induced a disruption of membranes, all three enzymes were strongly inhibited by DIDS. Trypsin action on intact membranes showed that asialofetuin sialyltransferase, galactosyltransferase and fetuin sialyltransferase were all slightly inhibited. After membrane disruption by Triton X-100, the first two enzymes were completely inactivated by trypsin while the fetuin sialyltransferase was quite insensitive to trypsin treatment. From these data, we suggest that the fetuin sialyltransferase, accessible to DIDS, is an external enzyme, oriented closely towards the cytoplasmic side of the brain microsomal vesicles (endoplasmic and Golgi membranes), whereas the asialofetuin sialyltransferase is an internal enzyme, oriented in a similar manner to the galactosyltransferase. Moreover, the anion site (nucleotide sugar binding site) of the fetuin sialyltransferase must be different from its active site, as this enzyme, when solubilized, is strongly inhibited by DIDS while no degradation is observed in the presence of trypsin.     

Decreased activity of cytidine 3':5'-monophosphate (cyclic CMP) phosphodiesterase in the fast-growing Morris hepatoma 3924A, but not in the slow-growing Morris hepatoma 9618A.

The activity level of the newly-identified cyclic CMP phosphodiesterase in the fast-growing Morris hepatoma 3924A was found to be much lower than the control (normal or host) liver. Its level in the slow-growing Morris hepatoma 9618A (a minimal deviation tumor), on the other hand, was the same as the host liver. The level of cyclic AMP phosphodiesterase was higher, whereas that of cyclic GMP phosphodiesterase was lower, in hepatoma 3924A than the control liver. In comparison, the levels of the two enzymes were both depressed in hepatoma 9618A. These findings suggest that depression of cyclic CMP phosphodiesterase may be related to the process and the rate of malignant growth, and that metabolism of cyclic CMP may be more crucial than that of cyclic AMP or cyclic GMP in the neoplastic cell proliferation.     

Catalytic mechanism of CMP:2-keto-3-deoxy-manno-octonic acid synthetase as derived from complexes with reaction educt and product.

The activation of the sugar 2-keto-3-deoxy-manno-octonic acid (Kdo) is catalyzed by CMP-Kdo synthetase (EC 2.7.7.38) and results in a monophosphate diester with CMP. The enzyme is a pharmaceutical target because CMP-Kdo is required for the biosynthesis of lipopolysaccharides that are vital for Gram-negative bacteria. We have established the structures of an enzyme complex with the educt CTP and of a complex with the product CMP-Kdo by X-ray diffraction analyses at 100 K, both at 2.6 A resolution. The N-terminal domains of the dimeric enzyme bind CTP in a peculiar nucleotide-binding fold with the beta- and gamma-phosphates located at the so-called "PP-loop", whereas the C-terminal domains participate in Kdo binding and in the dimer interface. The unstable nucleotide-sugar CMP-Kdo was produced in a crystal and stabilized by freezing to 100 K. Its formation is accompanied by an induced fit involving mainchain displacements in the 2 A range. The observed binding conformations together with the amino acid conservation pattern during evolution and the putative location of the required Mg(2+) ion suggest a reaction pathway. The enzyme is structurally homologous to the CMP-N-acetylneuraminic acid synthetases in all parts except for the dimer interface. Moreover, the chainfold and the substrate-binding positions resemble those of other enzymes processing nucleotide sugars.     

Novel method for enzymatic synthesis of CMP-NeuAc

A novel method for synthesizing CMP-NeuAc was established. We first confirmed that the putative neuA gene of Haemophilus influenzae, identified by its whole genome sequence project, indeed encodes CMP-NeuAc synthetase (EC 2.7.7.43). The enzyme requires CTP as a cytidylyl donor for cytidylylation of NeuAc. The enzyme was coupled with an enzymatic CTP-generating system from CMP and inorganic polyphosphate as a sole phospho-donor driven by the combination of polyphosphate kinase and CMP kinase, where phosphorylation of CMP is done by the combined activity expressed by both enzymes, and subsequent phosphorylation of CDP by polyphosphate kinase itself occurred efficiently. When CMP-NeuAc synthetase of H. influenzae, polyphosphate kinase, and CMP kinase were added to the reaction mixture containing equimolar concentrations (15 mM) of CMP and NeuAc, and polyphosphate (150 mM in terms of phosphate), CMP-NeuAc was synthesized up to 10 mM in 67% yield.     

CMP-N-acetylneuraminic acid synthetase of Escherichia coli: high level expression, purification and use in the enzymatic synthesis of CMP-N-acetylneuraminic acid and CMP-neuraminic acid derivatives.

The gene encoding CMP-N-acetylneuraminic acid (CMP-NeuAc) synthetase (EC 2.7.7.43) in Escherichia coli serotype O7 K1 was isolated and overexpressed in E.coli W3110. Maximum expression of 8-10% of the soluble E.coli protein was achieved by placing the gene with an engineered 5'-terminus and Shine-Dalgarno sequence into a pKK223 vector derivative behind the tac promoter. The overexpressed synthetase was purified to greater than 95% homogeneity in a single step by chromatography on high titre Orange A Matrex dye resin. Enzyme purified by this method was used directly for the synthesis of CMP-NeuAc and derivatives. The enzymatic synthesis of CMP-NeuAc was carried out on a multigram scale using equimolar CTP and N-acetylneuraminic acid as substrates. The resultant CMP-NeuAc, isolated as its disodium salt by ethanol precipitation, was prepared in an overall yield of 94% and was judged to be greater than 95% pure by 1H NMR analysis. N-Carbomethoxyneuraminic acid and N-carbobenzyloxyneuraminic acid were also found to be substrates of the enzyme; 5-azidoneuraminic acid was not a substrate of the enzyme. N-Carbomethoxyneuraminic acid was coupled to CMP at a rate similar to that observed with NeuAc, whereas N-carbobenzyloxyneuraminic acid was coupled greater than 100-fold more slowly. The high level of expression achieved with the E.coli synthetase, together with the high degree of purity readily obtainable from crude cell extracts, make the recombinant bacterial enzyme the preferred catalyst for the enzymatic synthesis of CMP-N-acetylneuraminic acid.     

Purification, characterization and localization of serine protease of Morris hepatoma 8999.

1. A serine protease of hepatoma 8999, isolated in the mitochondrial fraction, was purified and crystallized. The purified enzyme was apparently homogeneous on ultracentrifugal analysis and polyacrylamide disc gel electrophoresis. The ratio of absorbance at 280 nm and 260 nm, A280/A260, was 1.90 and its absorption coefficient, A280 1% was 10.5 cm-1 estimated from dry weight measurements. Its S20, w value was 2.23 S and its molecular weight was estimated to be 24000 +/- 1000. The enzyme contained twice as much lysine, arginine and histidine as chymotrypsinogen did, but had a very similar amino acid composition to serine protease from skeletal muscle. Its isoelectric point was pH 10.6. 2. The substrate specificity of the enzyme was the same as that of chymotrypsin A. Its Km and kcat values for N-acetyl-L-tyrosine ethyl ester, N-acetyl-L-phenylalanine ethyl ester and N-acetyl-L-tryptophan ethyl ester were 0.35 mM and 10.69 s-1, 0.38 mM and 10.7 s-1, and 0.11 mM and 11.8 s-1, respectively. Its activity was completely inhibited by phenylmethylsulfonyl fluoride and partially inhibited with tosylphenylalanine chloromethyl ketone. 3. The enzyme was shown to be located in different granules from the intracellular particules (light and heavy mitochondrial fraction) by sucrose density gradient centrifugation, and it was stained in mast cells of the hepatoma 8999 by the immunofluorescent technique. 4. Serine protease is present in different amounts in various organs of rat and the enzyme from hepatoma 8999 gave a single band that fused completely with those of the enzymes from skeletal muscle, heart, liver and kidney, respectively, on Ouchterlony double-diffusion analysis using antiserum to the crystalline enzyme of hepatoma 8999, but the enzyme from small intestine did not react with the antiserum.     

Production of cytidine 5'-monophosphate N-acetylneuraminic acid using recombinant Escherichia coli as a biocatalyst

An Escherichia coli strain expressing three recombinant enzymes, i.e., cytidine 5'-monophosphate (CMP) kinase, sialic acid aldolase and cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase, was utilized as a biocatalyst for the production of CMP-NeuAc. Both recombinant E. coli extract and whole cells catalyzed the production of CMP-NeuAc from CMP (20 mM), N-acetylmannosamine (40 mM), pyruvate (60 mM), ATP (1 mM), and acetylphosphate (60 mM), resulting in 90% conversion yield based on initial CMP concentration used. It was confirmed that endogenous acetate kinase can catalyze not only the ATP regeneration in the conversion of CMP to CDP but also the conversion of CDP to CTP. On the other hand, endogenous pyruvate kinase and polyphosphate kinase could not regenerate ATP efficiently. The addition of exogenous acetate kinase to the reaction mixture containing the cell extract increased the conversion rate of CMP to CMP-NeuAc by about 1.5-fold, but the addition of exogenous inorganic pyrophosphatase had no influence on the reaction. This E. coli strain could also be employed as an enzyme source for in situ regeneration of CMP-NeuAc in a sialyltransferase catalyzed reaction. About 90% conversion yield of alpha2,3-sialyl-N-acetyllactosamine was obtained from N-acetyllactosamine (20 mM), CMP (2 mM), N-acetylmannosamine (40 mM), pyruvate (60 mM), ATP (1 mM), and acetyl phosphate (80 mM) using the recombinant E. coli extract and alpha2,3-sialyltransferase.     

Characterization of sialyltransferases from serum of normal and hepatoma Mc-29 bearing chickens

Sialyltransferase was measured in serum of normal and hepatoma Mc-29 bearing chickens. By preparative isoelectric focusing the multiple forms of sialyltransferase from both kind of serums was studied as well. By using influenza virus neuraminidase an attempt was made for partial structural characterization of the sialylation sites in asialofetuin applied as exogenous acceptor for sialyltransferase determination. It was established an elevated serum sialyltransferase activity in tumor bearing chickens with tumor an enzyme form was detected with pI-4.99 identical with an enzyme form described previously in solubilized plasma membrane preparations from hepatoma Mc-29. Monitoring of multiple forms of serum glycosyltransferases may be of value in answering the problem concerning the tissue origin of serum enzymes.     

Characterization of a nonhepatic alcohol dehydrogenase from rat hepatocellular carcinoma and stomach.

Ethanol oxidation by the soluble fraction of a rat hepatoma was compared to that of the liver. Ethanol oxidation by the hepatoma was NAD⁺-dependent and sensitive to pyrazole, suggesting the presence of alcohol dehydrogenase. At low concentrations of ethanol (10.8 mm) the alcohol dehydrogenase activities of hepatoma and liver supernatant fractions were comparable. When the concentration of ethanol was raised to 108 mm, the activity of the liver enzyme decreased, whereas the activity in hepatoma supernatant fractions was strikingly elevated. m-Nitrobenzaldehyde-reducing activity was also conspicuously higher in hepatoma supernatant fractions. By contrast the ability to metabolize steroids and cyclohexanone was less than that in supernatant fractions of the liver.Electrophoresis of the liver supernatant fractions on ionagar at pH 7.0 revealed only one component that oxidized ethanol. On the other hand, hepatoma supernatant fractions contained two components with alcohol dehydrogenase activity; one with the same electrophoretic mobility as the liver enzyme, the other showing a slower rate of migration. The latter component, which is absent in the liver, is referred to as hepatoma alcohol dehydrogenase. By electrophoresis on starch gels at pH 8.5, it could be demonstrated that the liver and hepatoma enzymes moved in opposite directions.The liver and hepatoma enzymes differ in electrophoretic mobility, susceptibility to heat treatment, pH activity optimum and some catalytic properties. The substrate specificity of the hepatoma enzyme is narrower than that of liver alcohol dehydrogenase; cyclohexanone or 3β-hydroxysteroids of A/B cis configuration and the corresponding 3-ketones are not substrates for the hepatoma enzyme. The overall substrate specificity characteristics are, however, similar to those of the liver enzyme in that the effectiveness of substrates increases with an increase in chain length and introduction of unsaturation or an aromatic group. Both liver and hepatoma alcohol dehydrogenase cross-react with antibody to horse liver alcohol dehydrogenase EE. The Michaelis constant for ethanol with the hepatoma enzyme is 223 mm, compared to 0.3 mm for liver alcohol dehydrogenase; at 1.0 m ethanol the hepatoma enzyme is not fully saturated with substrate. The Michaelis constant for 2-hexene-1-ol is 0.3 mm, indicating that the hepatoma enzyme is better suited for dehydrogenation of longer chain alcohols. Stomach alcohol dehydrogenase has kinetic properties comparable to those of the hepatoma enzyme, as well as similar electrophoretic mobility. The hepatoma enzyme can be detected in the serum of rats bearing hepatomas.     

Isolation and characterization of acylneuraminate cytidylyltransferase from frog liver

Frog liver (Rana esculenta) is a rich source of acylneuraminate cytidylyltransferase. The soluble enzyme was purified 250-fold almost to purity with 25% yield and a specific activity of 9 mkat/kg protein (0.54 U/mg protein) using DEAE Sephadex and Sepharose 6B chromatography, followed by preparative polyacrylamide gel electrophoresis. The molecular weight of the cytidylyltransferase was determined to be 163 000 with the aid of Sepharose 6B chromatography and gel electrophoresis, with or without dodecyl sulphate or urea. No subunits were found. The isoelectric point of the enzyme is at pH 6. Optimum reaction rate was observed at pH 9, 37 degrees C, 50mM Mg2 or Ca2 and ImM mercaptoethanol. The Km values for N-acetylneuraminic acid, N-glycoloylneuraminic acid and CTP are 1.6mM, 2.3 mM and 0.6mM, respectively. O-Acetylated sialic acids are inactive with the cytidylyltransferase from frog liver. Enzyme activity can be inhibited by SH reagents and CMP (Ki = 0.5mM).     

Chemical behaviour of cytidine 5'-monophospho-N-acetyl-beta-D-neuraminic acid under neutral and alkaline conditions

The chemical behaviour of CMP-N-acetylneuraminic acid under neutral and different alkaline conditions has been investigated. The products formed were isolated by ion-exchange chromatography and gel filtration and analysed by colorimetric methods, thin-layer chromatography, combined gas-liquid chromatography/mass spectrometry and/or 360-MHz 1H-NMR spectroscopy. A maximum stability of CMP-N-acetylneuraminic acid was observed at pH8-11. In the tested pH range of 6-13, CMP and N-acetylneuraminic acid were formed in variable amounts as decomposition products. 2-Deoxy-2,3-dehydro-N-acetylneuraminic acid was produced at pH greater than 7; the amount of this substance increased with increasing pH. In anhydrous triethylamine its yield was 50%. A new neuraminic acid derivative, N-acetyl-beta-D-neuraminic acid 2-phosphate, could be isolated from the mixture of alkaline decomposition products of CMP-N-acetylneuraminic acid. The yield of this compound was maximum 22% in anhydrous triethylamine. Because 2-deoxy-2,3-dehydro-N-acetylneuraminic acid was formed under simulated physiological conditions, it is assumed that this compound, which occurs in tissues and fluids of man and animals, is derived from CMP-N-acetylneuraminic acid non-enzymically also under conditions in vivo.     

Synthesis of N-acetylneuraminic acid and of CMP-N-acetylneuraminic acid in the rat liver cell.

Adult male rats, under starving and normal conditions, were injected intravenously with N-acetyl[3H]mannosamine and after various time intervals the specific radioactivities of free N-acetylneuraminic acid (NeuAc) and CMP-N-acetylneuraminic acid were determined in the liver. The specific radioactivity of free NeuAc was high even within 20s after injection; the maximum was reached between 7 and 10 min. The specific radioactivity of CMP-NeuAc showed a lag phase of approx. 1 min. Thereafter it increased quickly and rose above the specific radioactivity of free NeuAc, reaching a maximum about 20 min after injection. These results point to a channelling of the newly synthesized NeuAc molecules into a special compartment, from which they are preferentially used by the enzyme CMP-sialic acid synthetase. It is suggested that the cytosolic enzyme N-acetylneuraminic acid 9-phosphate phosphatase is working in concert with the nuclear localized enzyme CMP-N-acetylneuraminic acid synthetase. Incorporation of radioactive sialic acid into sialoglycoproteins in liver occurred 2 min after injection, and after 10 min bound radioactivity began to appear in the circulation, indicating a transport time of 8 min of sialoglycoproteins from the point of attachment of sialic acid to the point of excretion.     

Participation of cytochrome b5 in CMP-N-acetylneuraminic acid hydroxylation in mouse liver cytosol

The activity of CMP-N-acetylneuraminic acid hydroxylase, that converts CMP-N-acetylneuraminic acid (CMP-NeuAc) to CPM-N-glycolylneuraminic acid (CMP-NeuGc), in mouse liver was determined by a newly developed HPLC method using non-radioactive CMP-NeuAc as a substrate. The activity was detected in the cytosol fraction but not in the microsomal fraction. Either NADH or NADPH was used as an electron donor by the cytosol enzyme, but NADH was much more efficiently used than NADPH. An antibody against cytochrome b5 markedly reduced the CMP-NeuAc hydroxylase activity when added to incubation mixture containing either NADH or NADPH as an electron donor. These data led us to postulate the following electron transport system, which is involved in the CMP-NeuAc hydroxylation in mouse liver cytosol: (formula; see text) where X, Y, and Z are components supposedly involved.     

Selective preparation and characterization of membranous and soluble forms of alkaline phosphatase from rat tissues. A comparison with the serum enzyme

We developed a method for selective preparation of two forms of alkaline phosphatase from rat tissues. The enzyme was extracted by n-butanol treatment at pH 5.5 and pH 8.5 as soluble and aggregated (membranous) forms, respectively. The soluble form prepared from liver was found to be identical with the serum enzyme. Complete solubilization of the membrane-bound enzyme without detergents had a great advantage in its purification. Rat hepatoma AH-130 cells enriched in alkaline phosphatase were first used for purification of the liver-type enzyme. The hepatoma enzyme, purified by chromatographies on concanavalin-A-Sepharose, Sephacryl S-300 and hydroxyapatite was used for production of antibodies specific for the liver-type isozyme. An immunoaffinity column, prepared with anti-(hepatoma-enzyme) IgG was utilized for the enzyme purification from other tissues including the membranous form. Analyses of amino acid composition of the purified enzymes revealed that all the liver-type enzymes from hepatoma, liver, kidney and serum had the same composition, whereas the intestinal type consisted of the composition distinctly different from that in the liver type. In addition, there was no significant difference in amino acid composition between the soluble and membranous forms, suggesting a possible involvement in the membranous form of a hydrophobic component other than its polypeptide domain. The present method for selective preparation of the soluble and membranous forms of alkaline phosphatase will be useful for a further investigation on the interaction of the enzyme with membranes.