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.     

Purification and characterization of alkaline phosphatase from rat kidney.

Alkaline phosphatase [EC 3.1.3.1.] was purified about 250-fold from rat kidney, and its enzymological properties were studied. Kidney homogenate was extracted with n-butanol, passed through Sephadex G-200 and chromatographed on a DEAE-cellulose column. The peak from the DEAE-cellulose column was subjected to isoelectric focusing, and the alkaline phosphatase activity was separated into two peaks. The molecular weights of alkaline phosphatase in these peaks were 4.8.X10(4) and 1.0X10(5), as determined by SDS-polyacrylamide gel electrophoresis. Anti-serum against alkaline phosphatase from rat kidney was prepared, and was shown to neutralize the activity from kidney, liver or bone, but not that from intestine.     

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.     

An in vitro production of bone specific alkaline phosphatase

Induced alkaline phosphatase has been extracted from osteosarcoma cells grown in tissue culture medium. The extracted enzyme has been purified. Using electrophoresis, inhibition studies, and thermolability, the enzyme was categorized as alkaline phosphatase of osseous origin. Antibodies to this enzyme were reacted against alkaline phosphatase extracted from cadaveric bone, liver, intestine, kidney and fresh placenta. The antibodies were specific against alkaline phosphatase of osseous origin only. No cross-reaction occurred with the enzyme extracted from other sources. The data derived from these studies indicate that alkaline phosphatase of bone is a specific enzyme of osseous tissue. Furthermore, the enzyme has specific antigenic and other properties which distinguish it from alkaline phosphatases from other sources. A model for in vitro production of a specific alkaline phosphatase of bone is presented.     

Nucleotide sequence of the gene for alkaline phosphatase of Thermus caldophilus GK24 and characteristics of the deduced primary structure of the enzyme

The gene encoding Thermus caldophilus GK24 (Tca) alkaline phosphatase was cloned into Escherichia coli. The primary structure of Tca alkaline phosphatase was deduced from its nucleotide sequence. The Tca alkaline phosphatase precursor, including the signal peptide sequence, was comprised of 501 amino acid residues. Its molecular mass was determined to be 54? omitted?760 Da. On the alignment of the amino acid sequence, Tca alkaline phosphatase showed sequence homology with the microbial alkaline phosphatases, 20% identity with E. coli alkaline phosphatase and 22% Bacillus subtilis (Bsu) alkaline phosphatases. High sequence identity was observed in the regions containing the Ser-102 residue of the active site, the zinc and magnesium binding sites of E. coli alkaline phosphatase. Comparison of Tca alkaline phosphatase and E. coli alkaline phosphatase structures suggests that the reduced activity of the Tca alkaline phosphatase, in the presence of zinc, is directly involved in some of the different metal binding sites. Heat-stable Tca alkaline phosphatase activity was detected in E. coli YK537, harboring pJRAP.     

Alkaline phosphatase of <Emphasis Type="Italic">Physarum polycephalum</Emphasis> is insoluble

The plasmodia of Physarum polycephalum grow as multinucleated cells in the presence of sufficient humidity and nutriment. Under non-illuminating conditions, stresses such as low temperature or high concentrations of salts transform the plasmodia into spherules whereas dehydration induces sclerotization. Some phosphatases including protein phosphatase and acid phosphatase have been purified from the plasmodia, but alkaline phosphatase remains to be elucidated. Phosphatase of the plasmodia, spherules and sclerotia was visualized by electrophoresis gel-staining assay using 5-bromo-4-chloro-3-indolyl phosphate. Insoluble fractions of the sclerotia were abundant in phosphatase activity. The phosphatase which was extracted by nonionic detergent was subjected to column chromatography and preparative electrophoresis. Purified phosphatase showed the highest activity at pH 8.8, indicating that this enzyme belongs to alkaline phosphatase. The apparent molecular mass from sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing condition was estimated to be 100 kDa whereas that under reducing was 105 kDa. An amount of 1% sodium dodecyl sulfate or 0.5 M NaCl had no effects on the activity although the phosphatase showed heat instability, Mg²⁺-dependency and sensitivity to 2-glycerophosphate or NaF. The extracting conditions and enzymatic properties suggest that this alkaline phosphatase which is in a membrane-bound form plays important roles in phosphate metabolism.     

The autorelease of alkaline phosphatase from the plasma membrane during the incubation of cultured liver cell homogenates

When a rat hepatoma cell (R-Y121B) homogenate was incubated at 37 degrees C, 30-70% of the total alkaline phosphatase was released into the supernatant fluid from the precipitate fractions. The release reached a plateau level after 10 h of incubation at 37 degrees C. The optimum pH value for the release was 7.4. Alkaline phosphatase activity increased during the incubation of the cell homogenates, but this increase was independent of the enzyme release. Serum increased not only alkaline phosphatase activity in the cultured cells but also enzyme release in their homogenates. In addition, we examined a rat liver homogenate and the following 11 cell lines: 3 hepatoma cell lines, including the R-Y121B cell line, 4 liver cell lines, 2 human urinary bladder carcinoma cell lines, a kidney cell line, and a mouse adrenal tumor cell line. Only in the cultured liver cell line and hepatoma cell lines, 30-60% of the total enzyme was released into the soluble fraction from the precipitate fractions; the release was not observed in the other cell lines, nor in the rat liver homogenate. The release of alkaline phosphatase took place in both heat-stable and heat-labile alkaline phosphatases. Alkaline phosphatase, extracted from cell homogenates, showed two bands during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The mobilities of the two bands changed inversely with or without sodium dodecyl sulfate. In general, the alkaline phosphatase which showed slow mobility with sodium dodecyl sulfate was more readily released from the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective Extraction of Alkaline Phosphatase and 51 -Nucleotidase from Milk Fat Globule Membranes by a Single Phase n-Butanol Procedure

ABSTRACT

A single phase extraction procedure employing 8% (v/v) n-butanol at room temperature extracted over 90% of alkaline phosphatase activity and over 60% of 5'-nucleotidase activity from bovine milk fat globule membranes (MFGM). For 5'-nucleotidase, higher n-butanol concentrations lead to loss of activity, while lower concentrations were ineffective in extracting the enzyme. When extractions were performed at 0°C, similar yields were obtained for alkaline phosphatase extraction with 8% (v/v) n-butanol, but 5¹- nucleotidase extraction required 10% (v/v) n-butanol for similar yields. However, 5'-nucleotidase was less susceptible to denaturation during extraction at 0°C. The Km values and substrate specificities for both alkaline phosphatase and 5'-nucleotidase were unchanged by extraction with 8% (v/v) n-butanol. The 8% (v/v) n-butanol extraction procedure provides a 3-fold purification step, and an enzyme preparation suitable for further purification.     

Subcellular localization of enterokinase (Enteropeptidase EC 3.4.21.9) in rat small intestine

The subcellular localization of enterokinase is controversial. In this study, enterokinase was extracted from a soluble fraction and a brush border fraction of rat small intestine by differential centrifugation. The soluble fraction contained 41% of the initial enterokinase activity while the brush border fraction contained only 4.6% of the initial activity. In contrast, alkaline phosphatase monitored as a brush border marker, yielded 26.3 in the brush border fraction and only 6% in the soluble fraction. Further separation of the soluble fraction on a Sepharose 4B column revealed three peaks of enterokinase activity. One small peak (3%) of a bound enzyme (M_r, 2·10^?6) and two larger peaks of free enzyme (M_r, 3·10⁵ and 9·10⁵). In contrast, alkaline phosphatase major fraction was in a high molecular weight peak of bound enzyme. When the brush border fraction was chromatographed only a single peak of bound enterokinase and alkaline phosphatase were found. In the lower part of the small intestine, no brush border-bound enterokinase was found, while the peak of alkaline phosphatase was the same as in the upper intestine. These data suggest that enterokinase activity in the rat intestine is mainly in a free form localized in the mucin and soluble fraction and to a negligible extent in the brush border.     

Identification and Purification of a Derepressible Alkaline Phosphatase from Anacystis nidulans R2

We have examined the increase in alkaline phosphatase activity in the cyanobacterium Anacystis nidulans R2 upon phosphate deprivation. Much of the activity is released into the medium when A. nidulans is osmotically shocked, indicating that the enzyme is located either in the periplasmic space or is loosely bound to the cell wall. The polypeptide associated with phosphatase activity has been identified as a single species of M_r 160,000. Several lines of evidence demonstrate that this polypeptide is responsible for alkaline phosphatase activity: (a) It is absent when cells are grown in the presence of phosphate and specifically accumulates during phosphate deprivation. (b) It is the major periplasmic polypeptide extracted by osmotic shock. (c) It represents over 90% of the protein in a fraction of periplasmic polypeptides enriched for phosphatase activity. (d) Antibodies raised against the purified species of M_r 160,000 inhibit phosphatase activity by approximately 70%.     

Differential theophylline inhibition of alkaline phosphatase and 5'-nucleotidase of bovine milk fat globule membranes

1. The effects of theophylline (1,3-dimethylxanthine) on alkaline phosphatase and 5'-nucleotidase activities of bovine milk fat globule membranes (MFGM) were examined. 2. Theophylline inhibited MFGM alkaline phosphatase in a concentration-dependent manner with 50% inhibition produced by 99 +/- 28 microM theophylline. 3. The 5'-nucleotidase activity was resistant to theophylline inhibition with 50% inhibition produced by 33.9 +/- 3.1 mM theophylline. 4. Theophylline was an uncompetitive inhibitor of MFGM alkaline phosphatase with a Ki of 126 +/- 15 microM. 5. The extent of theophylline inhibition of alkaline phosphatase activity was independent of the substrate utilized in the assay. 6. The effect of theophylline on bovine MFGM alkaline phosphatase was similar to theophylline effects on other mammalian alkaline phosphatases of liver/bone isoenzyme origin.     

Alkaline phosphatase activity in whitefly salivary glands and saliva

Alkaline phosphatase activity was histochemically localized in adult whiteflies (Bemisia tabaci B biotype, syn. B. argentifolii) with a chromogenic substrate (5-bromo-4-chloro-3-indolylphosphate) and a fluorogenic substrate (ELF-97). The greatest amount of staining was in the basal regions of adult salivary glands with additional activity traced into the connecting salivary ducts. Other tissues that had alkaline phosphatase activity were the accessory salivary glands, the midgut, the portion of the ovariole surrounding the terminal oocyte, and the colleterial gland. Whitefly nymphs had activity in salivary ducts, whereas activity was not detected in two aphid species (Rhodobium porosum and Aphis gossypii). Whitefly diet (15% sucrose) was collected from whitefly feeding chambers and found to have alkaline phosphatase activity, indicating the enzyme was secreted in saliva. Further studies with salivary alkaline phosphatase collected from diet indicated that the enzyme had a pH optimum of 10.4 and was inhibited by 1 mM cysteine and to a lesser extent 1 mM histidine. Dithiothreitol, inorganic phosphate, and ethylenediaminetetraacetic acid (EDTA) also inhibited activity, whereas levamisole only partially inhibited salivary alkaline phosphatase. The enzyme was heat tolerant and retained approximately 50% activity after a 1-h treatment at 65 degrees C. The amount of alkaline phosphatase activity secreted by whiteflies increased under conditions that stimulate increased feeding. These observations indicate alkaline phosphatase may play a role during whitefly feeding.     

Formation of multiple forms of acid and alkaline phosphatases in relation to the culture age of Aspergillus niger

Acid phosphatase (EC 3.1.3.2 [EC] ) was extracted from mycelia ofAspergillus niger, then separated and purified into four fractions.These acid phosphatases, designated IA, IB, II and III, hadpH optima at 5.0, 4.5–5.0, 4.5 and 2.5, respectively.None required the presence of divalent cations, and all werestrongly inhibited by NaF. They were non-specific acid phosphatasesbut varied in their activities with various substrates. Thealkaline phosphatase (EG 3.1.3.1 [EC] ) of A. niger was also separatedinto two fractions, alkaline phosphatases I and II. Changes in the activity ratios of these acid and alkaline phosphataseswere studied during culture in a peptone medium. The activityof acid phosphatase II was higher than the others when the culturewas young. The activity of acid phosphatase III increased toa maximum in the actively growing phase, then decreased. Thatof acid phosphatase I became highest in the mature culture.In contrast, the activity of alkaline phosphatase I was higherthan the others in young cultures, while alkaline phosphataseII became dominant in the mature culture. Activities of the various acid and alkaline phosphatases indifferent regions of the growing colonies were also studied.The changing patterns of these enzymes in both liquid and surfacecultures were compared. When A. niger was cultured in a medium containing a low concentrationof phosphate, acid phosphatase activity greatly increased afterthe consumption of phosphate, but alkaline phosphatase activitydid not. ¹ The present experiments were carried out, for the most partat the Institute of Applied Microbiology of the University ofTokyo. (Received February 10, 1975; )     

Selective extraction of marker enzymes of bovine milk fat globule membrane by nonionic detergents.

Large amounts (66-97%) of marker enzymes such as alkaline phosphatase, 5'-nucleotidase, phosphodiesterase I, and gamma-glutamyl transpeptidase of bovine milk fat globule membrane (MFGM) were selectively solubilized by nonionic detergents such as Triton X-100, Tween 20, Nonidet P-40, Liponox NCK, and Emulgen 109-P. On the other hand, the extractability of MFGM protein with these detergents was less than 50%. Judging from the recovery of total activity, it is likely that alkaline phosphatase, phosphodiesterase I, and gamma-glutamyl transpeptidase are activated by nonionic detergents, whereas 5'-nucleotidase is somewhat inhibited by the detergents, except for Tween 20, and acid phosphatase is strongly inhibited by all detergents. In addition, solubilization of the protein with the nonionic detergents was found to be somewhat selective by SDS-polyacrylamide gel electrophoresis. There was no appreciable difference between the five brands of nonionic detergents used as regards the extractability of protein and the enzymatic activity of the extracted marker enzymes of MFGM, except that the solubilizing ability of Tween 20 was relatively low.     

Extraction and partial purification of mouse uterine alkaline phosphatase.

Alkaline phosphatase in uterine homogenates from day 7 pregnant mice was solubilized using 0.2% (v/v) Triton X-100 and extracted wtih 20% (v/v) n-butanol. The procedure, which resulted in 182-fold purification, included ammonium sulfate precipitation, DEAE-cellulose anion exchange chromatography and Sephadex G200 gel filtration. Solubilization with Triton X-100 was an important step in the procedure since extraction with n-butanol alone only partially solubilized the enzyme and gave low extraction yields, much of the enzyme activity remaining in association with negatively charged residues. However, butanol extraction of Triton X-100-treated homogenates gave high yields of enzyme and eliminated p-nitrophenyl phosphatases which displayed activity in the pH range 3.0--7.5, together with a large proportion of inactive protein. The activity of the purified enzyme preparations was electrophoretically homogeneous on cellulose acetate membranes, suggesting that the alkaline phosphatase in the mouse uterus exists in a single isozymic form. Polyacrylamide-gel electrophoresis revealed that the purified preparations contained at least one protein as an impurity. Attempts to further purify the alkaline phosphatase by isoelectric focusing were unsuccessful since the enzyme was found to have an isoelectric point of about 5.0 and at this pH it was rapidly inactivated.     

Induction of rat hepatic alkaline phosphatase and its appearance in serum: electrophoretic characterization of liver-membranous and serum-soluble forms

Simultaneous bile duct ligation and colchicine injection (2 mg/kg body weight) in rats caused a remarkable induction of alkaline phosphatase in the liver. Concomitantly, a marked elevation of the enzyme activity occurred in the serum, and three activity peaks (peaks I, II, and III) were separated by Sephadex G-200 gel filtration. By several criteria for alkaline phosphatase isoenzymes it was determined that the liver-derived enzyme was distributed in peak I (30% of total serum activity) as a vesicle-bound form and in peak II (65%) as a soluble form, while the intestinal enzyme was contained in peak III (5%). The serum alkaline phosphatase in peaks I and II was compared with the liver enzyme extracted from plasma membrane with n-butanol. Under non-reducing conditions, the soluble form of peak II showed an electrophoretic mobility different from that of the liver enzyme; in the presence of sodium dodecyl sulfate the serum-soluble form migrated a little more slowly than the liver one, while in the presence of Triton X-100 the former migrated much faster than the latter. The sedimentable fraction of peak I was found to contain two forms corresponding to the serum-soluble and liver-membranous forms. Neuraminidase treatment of these two forms reduced their mobilities but did not abolish the relative difference in their mobilities on gel electrophoresis in the presence of either Triton X-100 or sodium dodecyl sulfate. Under reducing conditions, however, each form (which was dissociated into single subunits) migrated with an identical mobility on sodium dodecyl sulfate gel electrophoresis. These results suggest that the hepatic alkaline phosphatase exists as conformationally different forms in the serum and the liver membrane (even solubilized), but the difference is no longer preserved after their denaturation into subunits.     

Induction of Rat Liver Alkaline Phosphatase by Bile Duct Ligation

Bile duct ligation causes a five- to sevenfold increase in the activity of rat liver alkaline phosphatase within 12 hours after ligation and a similar rise in the activity of alkaline phosphatase in serum. The increased serum activity is due entirely to the appearance of a new isoenzyme that has the properties of rat liver alkaline phosphatase. The increase in both serum and liver alkaline phosphatase is prevented by the prior administration of cycloheximide in a dose that inhibits protein synthesis by 70%. Rat liver alkaline phosphatase was then purified to homogeneity. Antibody was raised to purified rat liver alkaline phosphatase in rabbits. The antibody was coupled to sepharose 4B and affinity columns made. ³-H-leucine was then injected into the portal veins of sham operated rats and rats with bile duct ligation four hours after ligation. One hour after injection and five hours after ligation, animals were sacrificed. Liver alkaline phosphatase was purified by means of affinity chromatography and double immunoprecipitation with rabbit antibody to rat liver alkaline phosphatase and goat anti-rabbit gamma globulin. Bile duct ligation increased the incorporation of ³-H-leucine into liver alkaline phosphatase more than threefold compared with sham operated rats, 164 CPM/mg protein vs. 49 CPM/mg protein (p < .001). The data indicate that the increased activity of rat liver alkaline phosphatase after bile duct ligation is due to enzyme induction rather than to activation of a pre-existing, relatively inactive enzyme.     

Biochemical Localization of Alkaline Phosphatase in the Cell Wall of a Marine Pseudomonad

The various layers of the cell envelope of marine pseudomonad B-16 (ATCC 19855) have been separated from the cells and assayed directly for alkaline phosphatase activity under conditions established previously to be optimum for maintenance of the activity of the enzyme. Under conditions known to lead to the release of the contents of the periplasmic space from the cells, over 90% of the alkaline phosphatase was released into the medium. Neither the loosely bound outer layer nor the outer double-track layer (cell wall membrane) showed significant activity. A small amount of the alkaline phosphatase activity of the cells remained associated with the mureinoplasts when the outer layers of the cell wall were removed. Upon treatment of the mureinoplasts with lysozyme, some alkaline phosphatase was released into the medium and some remained with the protoplasts formed. Cells washed and suspended in 0.5 M NaCl were lysed by treatment with 2% toluene, and 95% of the alkaline phosphatase in the cells was released into the medium. Cells washed and suspended in complete salts solution (0.3 M NaCl, 0.05 M MgSO(4), and 0.01 M KCl) or 0.05 M MgSO(4) appeared intact after treatment with toluene but lost 50 and 10%, respectively, of their alkaline phosphatase. The results suggest that the presence of Mg(2+) in the cell wall is necessary to prevent disruption of the cells by toluene and may also be required to prevent the release of alkaline phosphatase by toluene when disruption of the cells by toluene does not take place.     

Alkaline phosphatase activity does not mediate phosphate transport in the renal-cortical brush-border membrane.

We studied (1) the effect of primary modulators of phosphate transport, namely the hypophosphataemic mouse mutant (Hyp) and low-phosphorus diet, on alkaline phosphatase activity in mouse renal-cortex brush-border membrane vesicles and (2) the effect of several primary inhibitors of alkaline phosphatase on phosphate transport. Brush-border membrane vesicles from Hyp-mouse kidney had 50% loss of Na+-dependent phosphate transport, but only 18% decrease in alkaline phosphatase activity. The low-phosphorus diet effectively stimulated Na+/phosphate co-transport in brush-border membrane vesicles (+ 118%), but increased alkaline phosphatase activity only slightly (+13%). Levamisole (0.1 mM) and EDTA (1.0 mM) inhibited brush-border membrane-vesicle alkaline phosphatase activity of 82% and 93% respectively, but had no significant effect on Na+/phosphate co-transport. We conclude that alkaline phosphatase does not play a direct role in phosphate transport across the brush-border membrane of mouse kidney.     

Activation of alkaline phosphatase with Mg2+ and Zn2+ in rat hepatoma cells. Accumulation of apoenzyme

In Reuber rat hepatoma cells (R-Y121B), alkaline phosphatase activity increased without de novo enzyme synthesis (Sorimachi, K., and Yasumura, Y. (1986) Biochim. Biophys. Acta 885, 272-281). The enzyme was partially purified by butanol extraction from the particulate fractions. The incubation of the extracted alkaline phosphatase with the cytosol fraction induced a large increase in enzyme activity (5-10-fold of control). The dialyzed cytosol was more effective than the undialyzed cytosol during an early period of incubation at 37 degrees C. This difference between the dialyzed and the undialyzed cytosol fractions was due to endogenous Na+. For maximal activation of the enzyme, both Mg2+ above 1 mM and Zn2+ at low concentrations (below 0.01 mM) were needed, although Zn2+ at high concentrations (above 0.1 mM) showed an inhibitory effect. Zn2+ and Mg2+ alone slightly increased alkaline phosphatase activity. This activation of the enzyme was temperature dependent and was not observed at 0 or 4 degrees C. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the increase in alkaline phosphatase activity did not involve the fragmentation of the enzyme and that 65Zn2+ bound to it during enzyme activation with 65Zn2+ and Mg2+. The cytosol fraction not only supplied Zn2+ to the nascent enzyme but also increased the maximal enzyme activity more than did direct addition of metal ions. Ferritin and metallothionein contributed to the activation of alkaline phosphatase with the metal ions. Since the binding of Zn2+ and Mg2+ to the nascent alkaline phosphatase is disturbed in Reuber rat hepatoma cells (R-Y121B), the apoenzyme is accumulated inside the cells. The binding of Zn2+ and Mg2+ to the apoenzyme readily takes place in the cell homogenates accompanied by an increase in catalytic activity without new enzyme synthesis.