Clinica Chimica Acta, 186 (1989) 249-254 Elsevier

CCA 04586

Short Communication

Alkaline phosphatase releasing activity in human tissues Brad A. Hamilton, Jennifer L. McPhee, Ken Hawrylak and Robert A. Stinson Division of Medical Laboratory Science and Clinical Pathology, Department University of Alberta, Edmonton, Alberta (Canada) (Received

of Pathology,

14 June 1989; accepted 8 August 1989)

Key words: Phosphatidylinositol;


Alkaline phosphatase

Introduction Alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC has been shown to be released from many mammalian tissues by phosphatidylinositol phospholipase C enzymes from bacterial sources such as Bacillus cereus [l-5]. The enzyme released is dimeric, the same as that which results from a butanol extraction of these tissues at acidic pH [6]. On the other hand, butanol extraction at alkaline pH results in tetrameric or aggregated alkaline phosphatase being solubilized [l]. This is believed to be due to butanol disruption of the membrane and solubilization of the alkaline phosphatase in its native form. It may be that an endogenous phospholipase, activated by low pH and possibly by butanol, is cleaving the phosphatidylinositol membrane anchor of alkaline phosphatase and releasing the enzyme [7]. The present communication assesses the alkaline phosphatase releasing activity of a number of human tissues, cells, and fluids. Materials and methods Materials 1-Butanol was purchased from BDH Inc., Darmstadt, FRG. Octyl-Sepharose CL4B was purchased from Pharmacia Fine Chemicals, Uppsala, Sweden. Leupeptin, pepstatin A, phenylmethylsulfonyl fluoride (PMSF), p-nitrophenylphosphate, boric acid, napthol AS-MX phosphate, Triton X-100, 2-( N-morpholino)ethanosulfonic acid (MES), 3-( N-morpholino)propanesulfonic acid (MOPS), and Tris were purchased from Sigma Chemical Company, St. Louis, MO, USA. Magnesium

Correspondence to: Dr. R.A. Stinson, Rm B-117, Clinical Edmonton, Alberta, Canada, T6G 2G3.


0 1989 Ekvier



Science Publishers B.V. (Biomedical



of Alberta,


chloride was from Fisher Scientific Company, Fair Lawn, NJ, USA. The buffer 2-(ethylamino)ethano1 was purchased from the Aldrich Chemical Company, Inc., Milwaukee, WI, USA. Polyacrylamide gradient gels in rods (2.5-278) were from Isolab Inc., Akron, OH, USA. Alkaline phosphatase assay Alkaline phosphatase activity was assayed in 1 ml of medium containing 10 mmol/l p-nitrophenyl phosphate, 1.5 mmol/l Mg*+, and 1.0 mol/l ethylaminoethanol, pH 10.3 at 30” C. The increase in absorbance at 404 nm was monitored with a spectrophotometer (Varian, model 2200), and enzyme activity was expressed as p-nitrophenol released (pmol/min per 1) [8]. Aborption to octyl-Sepharose CL-4B This material was washed in 50 mmol/l Tris, 1.0 mmol/l MgCl,, 0.1 mmol/l ZnCl,, pH 8.0, and recovered by centrifugation at 14000 x g for 2 min. An equal volume of 50 mmol/l Tris, 1.0 mmol/l MgCl,, 0.1 mmol/l ZnCl,, pH 8.0, was used to resuspend the packed octyl-Sepharose. A 100 ~1 aliquot of sample was applied to 50 ~1 of resuspended octyl-Sepharose CL4B and mixed on a rotating shaker for 5 min and then centrifuged at 14000 x g for 2 min. The alkaline phosphatase assayed in the supematant was expressed as a percentage of the total alkaline phosphatase activity before addition to the octyl-Sepharose, and gave the percent of dimeric enzyme. The percent tetramer was calculated by difference. Pure dimeric and tetrameric enzymes, and known mixtures of both forms were used as standards. Butanol extraction of liver plasma membranes Liver plasma membranes were prepared from healthy human liver and stored at - 20 ’ C [9]. Butanol extractions were performed at various pH values by resuspending the membranes in 50 mmol/l Tris, 50 mmol/l MOPS, and 50 mmol/l MES for pH values of 8.0, 7.0, and 6.0 respectively. Equal volumes of 1-butanol and membranes (alkaline phosphatase activity approximately 600 U/l) were mixed and shaken at low speed on a wrist action shaker for 1 h at room temperature. The mixture was then centrifuged at 10000 X g for 10 ruin at room temperature. The aqueous layer was removed and 100 ~1 (alkaline phosphatase activity approximately 250 U/l) applied to a gradient gel (2.5-27% polyacrylamide) and electrophoresed for 18 h at 150 V in 0.09 mol/l Tris, 0.08 mol/l boric acid, pH 8.4 [lo]. This procedure allowed separation of the enzyme based on its native M,. The gels were stained using 3.0 mg/ml napthol AS-MX phosphate, 1.5 mmol/l Mg*+, and 1.0 mol/l ethylaminoethanol, pH 10.3, as substrate medium. Fluorescent bands of alkaline phosphatase activity were photographed under UV light [6]. The controls were treated in the same manner except that 1-butanol was not added to them. Measurement of endogenous alkaline phosphatase activity in human tissues The osteosarcoma cell line, Saos-2, was grown in monolayer in a-modified Eagle’s minimum essential medium [ll]. Neutrophils were isolated as described


previously by Gainer and Stinson [8]. An equal weight of 250 mmol/l Tris, pH 6.5, was added to tissues, cells or fluids. These were homogenized for 10 s on high speed in a Servall Omni mixer. Triton X-100 was added to 1% (v/v) and the suspensions were rocked for 1 h at 4O C in the presence of protease inhibitors (20 pmol/l leupeptin, 100 pmol/l pepstatin A, and 0.02% (w/v) phenylmethylsulfonyl fluoride). An equal volume of 1-butanol was added and the mixture was shaken for 30 min at 4 o C on a wrist action shaker at low speed. A sample from the aqueous layer was then electrophoresed on polyacrylamide gradient gels as described above and the relative amount of dimeric alkaline phosphatase was estimated visually. The aqueous layer was also assayed by adsorption to octyl-Sepharose CL4B. The percent dimer and tetramer was determined by adsorption to octyl-Sepharose as described above. The same experiments were also performed on tissues, cells, or fluids resuspended in 250 mmol/l Tris, pH 8.0. The native structure of alkaline phosphatase in each of the tissues, cells, or fluids was determined from the gradient gels at pH 8.0 and from adsorption to octyl-Sepharose (only tetrameric enzyme adsorbs). The amount of alkaline phosphatase present in each tissue was determined by performing butanol extractions on all of the tissues, fluids, or cells at both pH 6.5 and 8.0. The total amount of alkaline phosphatase released was approximately the same at both pH values and was reported as a relative amount.

TABLE I The presence of alkaline phosphatase and the endogenous phospholipase activity in various human tissues, cells and fluids Tissue or fluid


Plasma membranes Placenta Intestine Bone Neutrophil Saos-2 cells Serum Milk Bile MecolliWll Synovial flmd Amniotic fluid (early) Amniotic fluid (late)

Relative alkaline phosphatare activity 3+ 1+ 4+ 4+ 1+ 1+ 4+ 2+ 1+ 2+ 4+ 1+ 1+ 1+

Native WatermW structure a Tetramer Tetramer Tetramer Tetramer Tetramer Tetramer Tetramer Dimer Tetramer Tetramer Tetramer Dimer Tetramer Tetramer

Presence of endogenous phospholipase b 4+ 3+ 4+ 2+ 4+ 1+ 4+

and dimer

and dimer and dimer and dimer

4+ 1+ 3+ 2+ 4+

4b Determined from the relative amounts of the two species on polyacrylamide gradient gels stained for alkaline phosphatase activity and from the relative amounts of hydrophobic and hydrophilic alkaline phosphatase as determined by adsorption to octyl-Sepharose, following butanol extraction of the tissues, cells, and fluids at pH 8.0 ’ and pH 6.5 b.



Butanol extraction of liver plasma membranes at pH 8.0 resulted in the solubilization of alkaline phosphatase in a tetrameric form (Fig. 1). A mixture of dimeric and tetrameric alkaline phosphatase is released as a result of butanol extraction at pH 7.Q but only dimeric enzyme results when butanol extraction is performed at pH 6.0 (Fig. 1). Controls to which no butanol was added showed all alkaline phosphatase activity at the top of the gradient gels (Fig. 1). Protease inhibitors did not affect the solubilization of alkaline phosphatase during butanol extraction. As determined by gradient gel electrophoresis the quatemary structure of alkaline phosphatase was tetrameric in all tissues and cells, but was a mixture of tetramer and dimer, or exclusively dimer, in fluids (Table I). The relative amount of alkaline phosphatase varied with the tissue (Table I). The amount of dimeric alkaline phosphatase solubilization by butanol extraction at pH 6.5 was determined by gradient gel electrophoresis and by adsorption‘ to octyl-Sepharose (dimeric enzyme does not adsorb). The amount of dimer reflects the activity of the endogenous alkaline phosphatase releasing activity (presumably a phospholipase) at this slightly acidic pH. The values obtained were 2 + or higher for all tissues, cells, and fluids except neutrophils and bile (Table I).











Fig. 1. Gradient gel electrophoresis of extracted alkaline phosphatasu. Extractions of l&r plasma membranes were done at pH vahu% of 8.0, 7.0, and 6.0 in the absence (cohmms a, c, and e, respectively) and presence (columns b, d, and f, respectively) of butanol.



The alkaline phosphatase releasing activity is not a proteolytic enzyme but is probably a phospholipase of the C or D type specific for phosphatidylinositol [7,12,13]. Butanol extraction at pH values below 7.0 appears to activate (stimulate) this endogenous phospholipase activity that releases alkaline phosphatase from liver plasma membranes as a dimer (Fig. 1). Whether the alcohol stimulates the endogenous phospholipase or is necessary just to disrupt the membrane to allow the phospholipase to work is not known [14]. If the alkaline phosphatase is solubilized from membranes or cells under conditions where phospholipases are not active (alkaline pH), then tetrameric enzyme results (Fig. 1). Alkaline phosphatase appears to exist as a tetramer in membranes whereas it is a mixture of dimer and tetramer, or solely dimer, in fltids and secretions. This suggests that an endogenous phospholipase is releasing alkaline phosphatase from cells as dimers. Butanol extraction of the tissues, cells and fluids at acidic pH and subsequent analysis of the quaternary structure of alkaline phosphatase by both gradient gel electrophoresis and adsorption to octyl-Sepharose CG4B indicates that the endogenous phospholipase is present in varying activity in most of the tissues, cells, and fluids. There was good correlation between the amount of hydrophilic (dimeric) alkaline phosphatase as measured by adsorption to octyl-Sepharose and the presence of the endogenous phospholipase as estimated from gradient gels. These results indicate that there is an endogenous enzyme in most tissues, cells, and fluids which releases alkaline phosphatase as hydrophilic dimers. To ascertain whether this releasing activity can be a factor contributing to raised levels of alkaline phosphatase in serum will require further study. Acknowledgements

We thank Dorothy Rutkowski for technical assistance. This work was supported by a grant to RAS from the Medical Research Council of Canada and by studentships to BAH and RI-I from the Alberta Heritage Foundation for Medical Research. References 1 Hawrylak K, Stinson RA. The solubilization of tetrameric alkaline phosphatase from human liver and its conversion into various forms by phosphatidylinositol phospholipase C or proteolysis. J Biol Chem 1988;263:14368-14373. 2 Hawrylak K, Stinson RA. Tetramexic alkaline phosphatase from human liver is converted to dimers by phosphatidylinositol-phospholipase C. FEBS Lett 1987;212:289-291. 3 Low MG, Finean JB. Release of alkaline phosphatase from membranes by a phosphatidylinositolspecific phospholipase C. Biochem J 1977;67:281-284. 4 Taguchi R, Ikezawa H. Phosphatidylinositol-specific phospholipase C from Ciostridium nouyi type A. Arch Biochem Biophys 1978;186:196-201.

254 5 Taguchi R, Asahi Y, Ikexawa H. Purification and properties

6 7 8 9 10 11

12 13 14

of phosphatidylinositol-specific phospholipase C of Bacillus thtuingiensis. Biochem Biophys Acta 1980;619:48-57. T&panier JM, Seargeant LE, Stinson RA. Affinity purification and some molecular properties of human liver alkaline phosphatase. Biochem J 1976;155:653-660. Malik AS, Low MG. Conversion of human placental alkaline phosphatase from a high A4, form to a low It4, form during butanol extraction. Biochem J 1986;240:519-527. Gainer AL, Stinson RA. Evideuce that alkaline phosphatase from human neutrophils is the same gene product as the liver/kidney/bone isoenxyme. Clin Chim Acta 1982;123:11-17. Chakrabartty A, Stinson RA. Properties of membrane-bound and solubilized forms of alkaline phosphatase from human liver. Biochim Biophys Acta 1985;839:174-180. Stinson RA. Sire and stability to sodium dodecyl sulfate of alkaline phosphatases from their three established human genes. Biochim Biophys Acta 1984;790:268-274. Nakamura T, Nakamura K, Stinson R4. Release of alkaline phosphatase from human osteosarcoma cells by phosphatidylinositol phosphohpase C: effect of tunicamycin. Arch Biochem Biophys 1988;265:190-196. Low MG. Biochemistry of the glycosyl-phosphatidylinositol membrane protein anchors. Biochem J 1987;244:1-13. Low MG, Prasad RS. A phospholipase D specific for the phosphatidyhnositol anchor of cell surface proteins is abundant in plasma. Proc Natl Acad Sci USA 1988;85:980-984. Miki A, Kominami T, Ikehara Y. pH dependent conversion of liver-membranous alkaline phosphatase to a serum-soluble form by n-butanol extraction. Biochem Biophys Res Commun 1985;126:89-95.

Alkaline phosphatase releasing activity in human tissues.

249 Clinica Chimica Acta, 186 (1989) 249-254 Elsevier CCA 04586 Short Communication Alkaline phosphatase releasing activity in human tissues Brad...
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