Purification and partial sequencing of human placental alkaline phosphatase

Vol. 116, No. 3, 1983

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1 076-I 083

November 15, 1983

PURIFICATION AND PARTIAL SEQUENCING OF HUMAN PLACENTAL ALKALINE PHOSPHATASE Elhanan Ezra*, Russell Blacher + and Sidney Udenfriend* Roche Institute of Molecular Biology* and Department of Molecular Genetics + Roche Research Center Nutley, New Jersey 07110 Received October 5, 1983 SUMMARY: Two forms of human placental alkaline phosphatase have been purified to homogeneity utilizing high performance liquid chromatography. Both have the same amino acid composition but they differ in their carbohydrate substituents. Sequence data indicate that the two forms are identical for the first forty two residues from the amino terminus are presented.

Mammalian alkaline phosphatases

(E.C. 3:1.31) have been known for

almost 70 years and have received intensive study because of their utility in clinical diagnosis

(i). Much is therefore known about their physical and

catalytic properties

(2). There appear to be a number of tissue-specific

forms of alkaline phosphatase, immunological

supposedly,

isozymes.

Based on genetic and

studies and on physical properties at least three forms of

human alkaline phosphatase have been identified, bone (liver and kidney)(3,4).

placental,

intestinal and

There have been a number of reports on the

purification of each of the recognized forms of alkaline phosphatase However, terized.

from a structural standpoint Alkaline phosphatases

to be quite heterogeneous

(5).

these enzymes have yet to be charac-

extracted from most tissues have been shown

(6) and it is not clear from most published reports

whether the methods used were adequate for resolving individual components of a given tissue form of the enzyme. The introduction of high performance purification

liquid chromatography

to protein

encouraged us to apply the technique to alkaline phosphatases.

In fact Chang et al.

(7) had already shown the practicability

exchange HPLC to calf intestinal alkaline phosphatase.

of applying ion

We have applied ion

exchange HPLC as well as reversed phase HPLC to the purification of human

0006-291X/83 $I .50 Copyright © 1983 by Academ~ Press, ~c. A fl r ~ h ~ o f reproduct~n m any form reserved.

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alkaline phosphatases.

In this report we present the purification

geneity of two forms of human placental alkaline phosphatase. weights,

to homo-

Molecular

amino acid analyses and partial sequence data are presented.

MATERIALS AND METHODS Commercial human placental alkaline phosphatase was obtained from Sigma, (Type XXIV; 15.6 U/mg) (St. Louis, MO) and the Green Cross Corp. (Code 3000) (Osaka, Japan). The enzyme was also extracted from freshly obtained human placenta by standard procedures (8). Diethanolamine and p-nitrophenylphosphate were purchased from Sigma. 4-Ethylmorpholine (Fluka A.G., Buchs, Switzerland), l-propanol, and acetone were distilled over ninhydrin. Materials for gel electrophoresis were obtained from BioRad Labs (Richmond, CA). The weak anion exchange column NU-Gel, 200 nm pore size (1.5 x 22 c m ) , was obtained from Separation Industries, Metuchen, NJ. The strong anion exchange column Mono Q (HR 5/5) was obtained from Pharmacia, Uppsala, Sweden. Cyanopropyl bonded silica support, 300 nm pore size, was obtained from J.T. Baker, Phillipsburg, NJ. The buffer used in all chromatography systems was 4-ethylmorpholine acetate, 20 mM, pH 7.4, which also contained 1 mM MgCI 2 and 20 ~M ZnSO4 to stabilize the enzyme. For ion exchange chromatography 5 percent l-propanol was added and a gradient of NaCI was superimposed on the buffer. For reversed phase HPLC i00 mM sodium acetate was added to the buffer solution prior to mixing with the l-propanol gradient. All chromatography was carried out at 4°C at flow rates of 30-40 ml/hr. Post-column fluorescence detection was carried out with fluorescamine (Hoffmann La Roche Inc., Nutley, NJ) as described previously (9). Three to four percent of the column effluent was directed toward the fluorescence monitoring system. Protein was assayed by the fluorescamine procedure (9) with bovine serum albumin as the standard. Phosphate activity was measured at pH 9.8 in micro titer plates using 1.0 M diethanolamine buffer and p-nitrophenyl-phosphate as substrate as described in the instructions in the Sigma catalog. One unit of enzyme activity represents one umol of substrate hydrolyzed per min at 37°C. Amino acid composition was determined by the fluorescamine procedure (i0) using approximately 1 ~g quantities o f p u r e enzyme. For cysteine analysis samples were reduced and treated with iodoacetic acid as described by Hirs (ii). Carboxymethylated proteins were purified by the HPLC procedure of Pan et al. (12). Neutral and amino sugars were assayed by the methods used by Kilpatrick et al. (13). Protein microsequencing was carried out on 400 pmols (25 ng) of enzyme using an Applied Biosystems model 470A sequencer (14). PTH amino acids at each cycle were identified by HPLC using a Beckman Ultrasphere ODS column and trifluoroacetic acid/acetonitrile buffer system (15). RESULTS AND DISCUSSION Three preparations tigated.

of human placental alkaline p h o s p h a t a s e w e r e

Two were commercial samples that had been partially purified.

other was from freshly obtained tissue. genity.

What is most important

the purification another.

invesThe

All three demonstrated marked hetero-

is that patterns of elution at each step of

Shown in Table I differed markedly from one preparation

to

What appeared as a major peak in one preparation was either absent

or a minor peak in another.

A most probable explanation of this hetero-

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Vol. 116, No. 3, 1983 Table I. Step

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Purification of Human Placental Alkaline Phosphatase Specific Fold Activity Protein Activity Purification Recovery units mg units/mg %

Starting Material DEA

NU-Gel I II III IV

7806

287

27

1

i00

-

Mono Q M-I M-2 M-3 Cyanopropyl C-I C-2

407 2630 2351 1431

9.3 13.2 18.8 37.8

44 199 125 38

1.6 7.4 4.6 1.4

5.2 33.7 30.1 18.3

1541 514 108

5.4 2.9 1.9

285 177 56

10.6 6.6 2.1

19.7 6.6 1.4

678 465

0.38 0.25

1784 1860

66 69

8.7 6.0

geneity is differences in the degree of glycosylation of the enzyme in each preparation.

Some heterogeneity in a given sample is probably inherent in

the post-translational processing of the enzyme (16).

However,

the large

differences that we observed from sample to sample could only represent artifacts introduced by the different methods of sample preparation.

Com-

mercial preparations may also contain mixtures of several genetic forms of the enzyme (17).

For practical reasons we selected the Sigma preparation for

further study. The conditions for purification of calf intestinal alkaline phosphatase on ion exchange columns that had been worked out by Chang et al. well for the human placental enzyme.

(7) worked

The instability of the enzyme at high

concentrations of organic solvent and at pH values below 5.5 put limitations on the types of reversed phase HPLC systems that could be used. l-propanol concentrations

Since high

(>40%) were required for eluting the enzyme from

RP-8 columns this system was discarded.

With Cyanopropyl columns elution was

achieved in the range of 20 to 30% l-propanol at pH 7.4

This was therefore

adopted for the final step in purification. The alkaline phosphatase preparation N-ethyl morpholine acetate buffer,

(Sigma) was dissolved

(i0 mg/ml) in

25 mM, pH 7.4. Twenty-five ml (250 mg)

were applied to a DEAE NU-Gel column and the chromatogram was developed with the same buffer superimposed on a gradient of NaCI.

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As shown in Fig. IA

Vol. 116, N o . 3, 1983

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BIOCHEMICAL AND

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Fig i. Stages of HPLC purification of human placental alkaline phosphatase. (A) Chromatography on a DEAE NU-Gel column. (B) Chromatography of area II (Fig. IA) on a Mono Q column. (C) Chromatography of M-I (Fraction 34-43, Fig. IB) on a cyanopropyl column.

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alkaline phosphatase

activity was eluted over a broad area (fractions 50-

i00), clear evidence of heterogeneity. fractions was almost quantitative

The total recovery of enzyme in all

(Table I). The material eluted from the

chromatogram was arbitrarily divided into five peaks and peaks II and III, that contained the bulk of the activity, were purified further. when chromatographed

on a Mono Q column,

was resolved into at least five

components with alkaline phosphatase activity the complexity of the chromatographic tion of this material.

(data not shown).

Peak II when subjected to chromatography on a Mono Q

activity was in area M-I

When the material in M-I was

on a reversed phase cyanopropyl

column two peaks of protein

that coincided with enzyme activity were clearly resolved two peaks are designated C-I and C-2.

(Fig. IC). These

It should be noted that although

overall recoveries of enzyme activity were excellent the procedure

Most of the

(fractions 34-43) which appeared to contain two

resolved peaks of activity.

rechromatographed

Because of

pattern we did not continue purifica-

column was resolved into three areas, as shown in Fig. IB.

incompletely

Peak III,

(Table I), due to the heterogeneity

(75-90%) at each step of

only microgram quantities

of the final products were obtained. Both C-I and C-2 when

subjected to SDS gel electrophoresis,

were found

to be over 95% pure (Fig. 2) and migrated essentially as single bands with identical M r values.

Amino acid analyses of C-I and C-2

the two differed in sugar composition

were identical but

(Table II). The combination of data

from SDS gels and amino acid assay gave a subunit molecular weight of 70,000, assuming identical subunits. values

(5).

This is in the range of previously reported

However, because of the marked heterogeneity

alkaline phosphatase preparations,

of starting

more precise comparisions

of M r values

from one study to another cannot be made. When subjected to sequencing both C-I and C-2 yielded a single identical amino terminal residue Ile. III,

Sequencing

through residue 42, Shown in Table

was identical for both C-I and C-2. Green and

Sussman

(20) had pre-

viously sequenced the first four residues of placental alkaline phosphatase.

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

94K

-

68K

-

43K

-

30K

-

21K

-

14K

-

-AP

1

2

3

4

5

Fig. 2. SDS page gel electrophoresis of human placental alkaline phosphatase at stages of purification shown in Fig. i. Electrophoresis was carried out on 8% gels as described (18). Silver staining was used for visualization (19): Lane 1 - 125 ng of protein standards; Lane 2 - 700 ng of the starting material; Lane 3 - 170 ng of M-I (Table I); Lane 4 - 170 ng of peak C-I (Table I); Lane 5 - 170 ng of peak C-2 (Table I). Table II.

Amino Acid and Carbohydrate Composition of Human Placental Alkaline Phosphate

Amino Acids and Sugars

C-I

C-2

Residues/mole

55 Aspartic acid 9.42 9.37 40 Threonine 6.95 6.65 32 Serine 6.37 6.40 60 Glutamic acid 10.19 10.30 Proline 5.66 5.71 33 54 Glycine 9.12 9.68 66 Alanine 10.72 10.61 35 Valine 6.15 6.12 14 Methionine 2.72 2.62 18 Isoleucine 3.09 3.12 48 Leucine 8.07 7.99 22 Tyrosine 3.68 3.68 22 Phenylalanine 3.74 3.65 Histidine 2.75 2.68 16 Lysine 4.02 4.12 24 Arginine 5.93 5.84 40 Cysteine* 0.71 0.72 5 Tryptophan 0.71 0.72 4 Glucosamine 2.42 3.88 14"* 22*** 0 0 Galactosamine 0 0 Mannose 1.61 0.49 i0 3 Galactose 0.65 0.49 4 3 Fucose 0 0 0 0 Xylose 0.48 0 3 0 N-acetyl$1ucosamine *Cysteine was determined after carboxymethylation. N-acetylglucosamine is present but cannot be measured by the procedures that were used. ** = C-I *** = C-2.

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Table III. Amino Terminal Sequence of Alkaline Phosphatase From Human Placenta i

1o

Ile-Ile-Pro-Val-Glu-Glu-Gtu-Asn-Pro-Asp-Phe-Trp-Asn-Arg-Glu-Ala-Ala-Glu20 30 Ala-Leu-Gly-Ala-Ala-Lys-Lys-Leu-Gln-Pro-Ala-Gln-Thr-Ala-Ala-Lys-Asn-Leu40

(Ile)-Ile-Phe-Leu-Gly-AspThe same sequence was obtained on three individual runs using Peak C-l, C-2 and a mixture of C-I and C-2 that had been carboxymethylated and further purified by HPLC. Average initial yields were better than 80% and the repetitive yield at each cycle averaged about 90%. There is some uncertainty as to the lie at position 37.

Our findings of lle-lle-Pro-Val- at the amino terminus are in accord with their report.

Since alkaline phosphatase is ~ dimer the finding of a single

end group and a single migrating species on SDS gels suggest that the two monomers are identical.

Since C-I and C-2 are identical at their amino

termini and have the same amino acid composition they most likely represent the same polypeptide chain with different carbohydrate components

(Table

III). It should be noted that placental alkaline phosphatase in no way resembles bacterial alkaline phosphatase (21), at least at its amino terminus. We were originally hesitant to subject such a large and highly glycosylated protein to sequencing.

The fact that no problems were encountered in

sequencing the first 42 residues indicates that the sugar residues are not near the amino terminus. The partial sequence of human alkaline phosphatase provides information that can be used to obtain the entire sequence by cloning procedures.

Similar

procedures may be used to purify and characterize the other tissue forms of alkaline phosphatase. ACKNOWLEDGEMENTS We thank L o u i s e G e r b e r and Larry Brink for their excellent technical assistance in these studies and to Roy Sun for the carbohydrate analysis. REFERENCES i. 2. 3.

Moss, D.W. (1982) Clin. Chem. 28, 2007-2016. Fernley, H.N. (1971) in The Enzymes, 4, (Boyer, P.D. ed) pp. 417-447, Academic Press, New York. Badger, K.S. and Sussman, H.H. (1976) Proc. Natl. Acad. Sci. USA 73, 2201-2205.

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4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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