ANALYTICAL
67, 97-109 (1975)
BIOCHEMISTRY
Assay
for
Human
CHING Division
Erythrocyte
Pyridoxine
Kinase
J. CHERN AND ERNESTBEUTLER
of Medicine,
City of Hope Duarte, California
National 91010
Medical
Center,
Received September 13, 1974; accepted February 24, 1975 An accurate and rapid method for the assay of pyridoxine kinase in human erythrocytes has been developed. The procedure involves the separation of the radioactive product from the substrate with Dowex 50 resin in a test tube. Using the assay designed, we found that human red blood cells have a pyridoxine kinase activity of 1.381 nmolelminlg of hemoglobin (n = 25, SE = 0.051), and the enzyme has a K, of approximately 1.72 X 10-l; M for pyridoxine. Pyridoxine phosphate was identified as the main product of the assay reaction catalyzed by human erythrocyte pyridoxine kinase in crude hemolysates.
Pyridoxine is one of three forms of vitamin B,. In its active coenzyme form, pyridoxal phosphate is recognized as important in the function of many enzyme systems in the body (1). The pathway for the conversion of pyridoxine to its active forms has not yet been fully established. The possible route for conversion of pyridoxine to pyridoxal phosphate in mammalian systems is as follows (2): Pyridoxine
%
pyridoxine
phosphate oxidase pyridoxal
phosphate.
Phosphorylation of pyridoxine to pyridoxine phosphate has been demonstrated in human red blood cells (3) and other tissues (4,5). The enzyme pyridoxal kinase (EC 2.7.1.35), which catalyzes the phosphorylation of pyridoxal and, less effectively, of pyridoxine, has been partially purified and characterized from several different sources (6). Pyridoxine kinases from yeast and Escherichia coli B (7), on the other hand, were found to have higher affinity for pyridoxine than for pyridoxal. Although pyridoxal kinase activity has also been demonstrated in human red blood cells (8), no information about the characteristics of the enzyme in red blood cells is available, probably due to the lack of a convenient and sensitive assay system. Whether pyridoxine kinase is the same enzyme as pyridoxal kinase in human red cells remains to be determined. Sideroblastic anemias are characterized by defective hemoglobin synthesis with accumulation of iron in mitochondria. Available evidence indicates the production of &aminolevulinic acid is impaired in erythroblasts in pyridoxine-responsive anemia (9), which is one of sideroblastic 97 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form resewed.
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BEUTLER
anemias. &Aminolevulinic acid synthetase, which requires pyridoxal phosphate as an active coenzyme (lo), is responsible for the condensation of succinyl CoA and glycine to &aminolevulinic acid. Deficiency of pyridoxal phosphate caused by a deficiency of pyridoxine kinase or pyridoxal kinase would be expected to result in decreasing heme synthesis by marrow erythroblasts, and this could lead to mitochondrial ironoverload. Pyridoxal phosphate may also be involved in mobilization of iron from mitochondria (11). For this reason, quantitation of pyridoxal kinase activity in erythrocytes is of considerable interest. Ordinarily used assays for pyridoxal kinase (12- 16) consist of preparing an acid extract and measuring pyridoxal phosphate activity by coupling to a pyridoxal phosphate-requiring apoenzyme system. Such assays are not applicable to red cell systems, however, because of the binding of pyridoxal phosphate to hemoglobin through a Schiffs base linkage and the difficulty of extracting small amounts of pyridoxal phosphate from excess protein and therefore its loss during the extraction procedure (17). It occurred to us that this difficulty would not be encountered if pyridoxine, rather than pyridoxal, was used as a substrate for assay of the kinase. If the same enzyme phosphorylated both vitamers, as has turned out to be the case, the technique could be used for the assay of pyridoxal kinase. We now describe a method for the assay of pyridoxine kinase in human erythrocytes. The procedure involves the separation of the radioactive product from the substrate, which can be achieved with Dowex 50 resin in a test tube. The convenience and rapidity of the assay permits accurate quantitation of the enzyme in normal human red blood cells for the first time. The Michaelis-Menten constant and identification of the reaction product of pyridoxine kinase are also presented. MATERIALS
AND
METHODS
Reagents. [“HI pyridoxine hydrochloride (1.7 Ci/mmole) was obtained from AmershamlSearle Co. Dowex 50-X8 (hydrogen form, 200-400 mesh) was purchased from Bio-Rad Laboratories, Dowex 1 (chloride form, 8% cross-linked, 20-50 mesh), pyridoxine hydrochloride, pyridoxal hydrochloride, pyridoxamine hydrochloride, pyridoxal-5’-phosphate (PLP), ATP and alkaline phosphatase (from calf mucosa, Type I) were obtained from Sigma Chemical Co. All other reagents were analytical quality. At an early stage of this study, [4,5-‘-LC,]pyridoxine (142 &i/mg), a gift of Hoffmann-La Roche Inc., Nutley, NJ, was used as the substrate for pyridoxine kinase. However, we found that commercially available [“Hlpyridoxine could be employed as a substrate for the enzyme as efficiently as [‘“Cl pyridoxine. Thereafter, all studies were performed using [“HI pyridoxine as substrate.
HUMAN
ERYTHROCYTE
PYRIDOXINE
KINASE
99
All experiments involving vitamin B6 compounds were conducted under conditions of reduced light. Chromatographic experiments were carried out at room temperature. Preparation of hemolysates. Venous blood from adults was collected in acid-citrate-dextrose (ACD) solution, filtered on a sulphoethyl cellulose-Sephadex G-25 column according to the procedure of Nakao et al. (18) to remove the leukocytes and platelets. The red blood cells were further washed twice with 0.154 M saline. Hemolysates were prepared by making a 1 to 10 dilution of washed red cells in water. Unless otherwise indicated, all experiments were performed using freshly drawn blood. Determination of pyridoxine kinase activity in hemolysates. The assay system consisted of 100 ~1 of 1 M KH,PO,/K,HPO, buffer, pH 7.0, 50 ~1 of 10 mM ATP (neutralized), 50 ~1 of 10 mM MgCl?, 100 ~1 of 5 X lo-” M [“HI pyridoxine (specific activity, 39.06 &i/pmole), an appropriate amount of hemolysate and water to a volume of 1.2 ml. A blank without the substrate, pyridoxine, was included for each hemolysate. The reaction mixture was incubated in the dark at 37°C for 60 min with shaking, and the reaction was terminated by the addition of 0.7 ml of 12% perchloric acid (PCA) in an ice bath. One hundred microliters of 5 x lo-” M [:‘H] pyridoxine was added to the blank. After filtration through Whatman No. 2 filter paper, 0.6 ml of filtrate was neutralized with 0.225 ml of 1 M K&O,. The mixture was allowed to stand for 5 min, and 0.5 ml of clear supernatant was adjusted to a pH of 4.0-4.5 with one to two drops of 0.5 M formic acid, using pH paper as indicator. Two-tenths milliliter of Dowex 50 resin-suspension (one volume of settled resin, previously equilibrated with 0.05 M potassium formate buffer, pH 4.3, and two volumes of the buffer) was transferred to each solution. Each tube was mixed on a rotatory mixer for 30 min. During this time, the free pyridoxine was adsorbed on the resin beads. The beads were allowed to settle out, and 0.5 ml of the clear supernatant was removed and counted in 10 ml of Bray’s solution (19) with a Packard scintillation counter. A [“Hlpyridoxine standard was also subjected to the same treatment except that the Dowex 50 resin-suspension was replaced with an equal volume of formate buffer. In converting radioactivity to nanomoles of product formed, the volume occupied by the resin was also taken into account. The equation for conversion is as follows: P, = (P, x S,)/(S,
x 1.049),
where P, is the concentration of the product (pyridoxine phosphate); P, is the radioactivity of the product: S, is the concentration of the standard ([“Hlpyridoxine); S, is the radioactivity of the standard; and 1.049 is the correction factor for the volume occupied by the resin.
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We found that increase in pH or ionic strength during adsorption with Dowex 50 resins led to higher blank values as the result of release of substrates from the resins. It is also understandable that removal of substrates is necessarily incomplete in the test tube. Small amounts of the substrate (5-10% ; 1 ,OOO-2,000 cpm) remain in the supernatant fraction in equilibrium with the resin. We observed that the blank could be reduced considerably by repetitive adsorption with the resins. However, with subtraction of the blank, only one adsorption with resins was found to be sufficient for most purposes. Pyridoxine kinase activity was expressed as nanomoles of pyridoxine phosphate formed per min and specific activity as nanomoles of pyridoxine phosphate formed per min per gram of hemoglobin (g Hb). Hemoglobin content was determined spectrophotometrically as cyanmethemoglobin (20). RESULTS Recovery of Pyridoxine Added to Hemolysates
and Pyridoxine
Phosphate
In order to examine the recovery of pyridoxine, 1 nmole (=0.0284 &i) of [l”C]pyridoxine was incubated with 1 ml of hemolysate in the absence of ATP at 37°C for 1 hr. The mixture was extracted with PCA, filtered and neutralized as for the determination of pyridoxine kinase activity in human erythrocytes, except that the adsorption with Dowex 50 resin was not carried out. Duplicate results indicated that 96.4% of pyridoxine was recovered. Apparently only insignificant amounts were lost during extraction with PCA. [3H] pyridoxine phosphate was synthesized from [3H]pyridoxine by using highly purified human erythrocyte pyridoxine kinase. When 1.85 nmoles (=0.0024 &I) of [3H]pyridoxine phosphate was mixed with 0.5 ml of hemolysate in the presence of the assay components except for pyridoxine, followed by extraction with PCA, filtration and neutralization, approximately 99% of [“Hlpyridoxine phosphate was recovered. After 1 hr of incubation at 37°C with 3.7 nmoles of [3H]pyridoxine phosphate, 90% of the [3H] pyridoxine phosphate could still be recovered. Chromatographic Separation from the Substrate
of the Reaction
Product
Separation of pyridoxine phosphate from pyridoxine was performed on a Dowex 50 column at pH 4.3 on the basis of different pK, values of the two compounds. Two milliliters of hemolysate was incubated with
HUMAN
ERYTHROCYTE
PYRIDOXINE
KINASE
101
100 ~1 of [Wlpyridoxine (specific activity, 28.4 &i/pmole) and other components for the assay of pyridoxine kinase in a total volume of 2.4 ml for 1 hr, followed by perchloric acid precipitation, filtration, neutralization and titration as described for the assay of pyridoxine kinase in hemolysates. One milliliter of the titrated solution, was applied to a Dowex 50 column (0.5 X 4 cm), which had been equilibrated previously with 0.05 M potassium formate buffer, pH 4.3. The column was first eluted with the formate buffer and then washed with 0.05 M potassium citrate buffer, pH 6.8, containing 0.5 M KCl. Fractions were collected and counted in Bray’s solution. A blank was subjected to the same treatment. An authentic pyridoxal phosphate (PLP) and [‘“Cl pyridoxine mixture was also employed to verify the locations of the phosphorylated vitamin B6 and free vitamin B,. Pyridoxal phosphate was determined by measuring the fluorescence of the semicarbazone of PLP at 460 nm when excited at 380 nm in a Farrand spectrofluorometer as described previously (17). Figure 1 shows the separation of the reaction product from the substrate, and Fig. 2 demonstrates locations of the phosphorylated vitamin B, and free vitamin B,. The good resolution of the phosphorylated vitamin B, from vitamin B6 compounds is the foundation for the assay of pyridoxine kinase in human erythrocytes.
Froctlon
Number
1. The chromatographic separation of the reaction product of crude hemolysate pyridoxine kinase from the substrate, pyridoxine (PN). on a Dowex 50 column. The column was eluted with 0.05 M potassium formate buffer, pH 4.3, and then washed with 0.05 M potassium citrate buffer, pH 6.8, containing 0.5 M KC1 as indicated by the arrow ( 1 ). One-and-one-half-milliliter fractions were collected. Radioactivity of each fraction was determined (O-O). A control was also subjected to the same treatment (0-O). The experimental details were given in the text. FIG.
102
CHERN
AND BEUTLER
Frocf~on
Number
2. Resolution of a mixture of authentic pyridoxaf phosphate (PLP), and pyridoxine (PN) on a Dowex 50 column. One nanomole (=0.0284 &I) of ‘“C-labelled PN was mixed with 5 pg of PLP in 1 ml of 0.05 M potassium formate buffer, pH 4.3, and applied to a Dowex 50 column. The radioactivity of 2-ml fractions was determined (O-O), and the concentration of PLP was estimated by measuring the fluorescence of the semibarzone of PLP at 460 nm when excited at 380 nm (o-0). The conditions of chromatography were identical to those in Fig. 1. FIG.
Characterization
of the Reaction
Product
The differentiation of [W] pyridoxine phosphate from PLP was carried out on a Dowex 1 column. The pooled solution containing phosphorylated [‘“Cl pyridoxine, which was obtained from the first-peak tubes of Dowex 50 chromatography, as described in Fig. 1, was mixed with 20 pg of PLP and loaded on a Dowex 1 column (0.5 X 4 cm) previously equilibrated with 0.05 M potassium formate buffer, pH 4.3. The column was eluted with a linear 40-ml gradient from 0.05 M potassium formate buffer, pH 4.3, to 0.05 M potassium citrate buffer, pH 4.3, with 0.15 M KCl. The separation of the reaction product from pyridoxal phosphate is presented in Fig. 3. Direct verification that the product of the reaction was, in fact, pyridoxine phosphate was not possible, because of the lack of availability of a source of authentic material. A commercial lot of putative pyridoxine5-phosphate obtained from Schwarz-Mann, of New York, proved to be, in point of fact, pyridoxal-5-phosphate. Since authentic pyridoxine was available, however, it was possible to identify, indirectly, the product of the reaction by its degradation to pyridoxine by alkaline phosphatase. Accordingly, the reaction product (i.e., phosphorylated 3H-labeled vitamin B,J was first separated from the substrate, [3H]pyridoxine, on a small Dowex 50 column (0.5 X 4 cm) as described above. The solution containing the putative phosphorylated vitamin B, was then titrated to
HUMAN
ERYTHROCYTE
PYRIDOXINE
KINASE
103
FIG. 3. Differentiation of the reaction product of crude hemolysate pyridoxine kinase reaction from pyridoxal phosphate (PLP) on a Dowex 1 column. A mixture of W-labeled reaction product and PLP was transferred to a Dowex 1 column. A linear 40-ml gradient from 0.05 M potassium formate buffer, pH 4.3, to 0.05 M potassium citrate buffer, pH 4.3, with 0.15 M KC1 was started ( 4 ) to elute the column. Two-and-one-half-milliliter fractions were collected. Radioactivity (O-O) and fluorescence for PLP (0-O) of each fraction were measured as described in Fig. 2.
pH 9.3 with 1 M K,CO,. One milligram of alkaline phosphatase was added to the mixture and incubated at 37°C for 2 hr. The pH of the solution was then adjusted to 4.25 with 0.5 M formic acid and mixed with approximately 1 mg each of pyridoxine, pyridoxal and pyridoxamine as markers. The mixture was applied to a column of Dowex 50 (0.9 X 44 cm) equilibrated previously with 0.05 M ammonium formate buffer, pH 4.25 (21). A linear gradient consisting of 200 ml of 0.05 M ammonium formate buffer, pH 4.25, and 200 ml of 0.5 M ammonium formate buffer, pH 7.5, in the reservoir was used to elute the column. After the completion of the gradient, additional ammonium formate buffer, 0.5 M, pH 7.5, was required to elute pyridoxamine from the column. The resolution of the free forms of vitamin B, markers in addition to the identification of pyridoxine as the main vitamin B, component of the reaction product of pyridoxal kinase is presented in Fig. 4A. The column was also calibrated with a mixture consisting of [“Hlpyridoxine and the three vitamin B, compounds in order to correct for impurities present in commercial preparations of those reagents (Fig. 4B). The small quantity of radioactive pyridoxal observed in Fig. 4A obviously is attributable to the contamination by such a compound in the commercial preparation. The procedure described to resolve free forms of vitamin B, offers a simple method to purify the specific vitamin B, when the purity of reagent is critical in the study. Determination
of Optimum
Conditions
for Pyridoxine
Kinase Assay
Hemofysate volume. Under the assay conditions for human erythrocyte pyridoxine kinase as described in Materials and Methods, the rela-
104
CHERN AND
. r
BEUTLER
PL
.
.
I ;
Fraction
Fraction
Number
Number
4. (A) Dowex 50 chromatographic analysis of the reaction product treated with alkaline phosphatase. The reaction product, “H-labeled, was hydrolyzed with alkaline phosphatase and loaded with three vitamin B, compounds (pyridoxine (PN), pyridoxal (PL) and pyridoxamine (PM)) on a Dowex 50 column. The column was developed with a linear ammonium formate buffer gradient. Eluate fractions (5.7 ml each) were measured for radioactivity (O-O). Three B, vitamin markers were detected spectrophotometrically by absorbance at 295 nm (0-O). (B) Calibration of the same column with a mixture consisting of [3H]pyridoxine and the three Bs vitaminers. See text for details. FIG.
HUMAN !
ERYTHROCYTE
PYRIDOXINE
KINASE
105
4.0 -
x -2 E \ L 3.0 E
l -.
c ;; E z0 92 2.0z I .o : r f E 5 /, 0’
.
Ij_/_
.i
’
. , 0.1
0.2
0.3
,
,
,
,
0.4 0.5 0.6 Hemolysates(ml)
,
,
0.7
0.8
0.9
FIG. 5. The relationship between the concentration of pyridoxine kinase in different volumes of hemolysate and the rate of formation of pyridoxine phosphate.
tionship between the concentration of hemolysate pyridoxine kinase and the formation of pyridoxine phosphate was linear over a range of hemolysate volumes which produced from 0 to more than 0.026 nmolelmin of pyridoxine phosphate (Fig. 5). Routinely, two volumes, 0.2 and 0.4 ml, of hemolysate were employed to quantitate pyridoxine kinase activity in erythrocytes. Time course. The rate of formation of pyridoxine phosphate under these conditions was linear for at least 3 hr (Fig. 6). A 60-min incubation period was adopted as a convenient interval that would ensure measurement of the reaction in a linear portion of the curve. Phosphate buffer concentration. The effect of phosphate buffer concentration on pyridoxal kinase activity is shown in Table 1. An optimum concentration of 80- 100 mrvr was found.
TimeCMinutes)
FIG. 6. The relationship between the rate of formation of pyridoxine phosphate and time.
106
CHERN
AND
BEUTLER
TABLE THE EFFECT
OF PHOSPHATE FORMATION
Phosphate concentration
1
BUFFER CONCENTRATION ON THE RATE OF PYRIDOXINE PHOSPHATE
OF
% of maximum activity
(ITIM)
83.9 86.9 100.0 96.8 83.6
41.7
58.3 83.3 125.0 166.7
Substrafe concentration. The rate of formation of pyridoxine phosphate was studied as a function of the substrate concentration (Fig. 7A). Lineweaver-But-k plots (Fig. 7B) revealed a K, of 1.72 X IOWfi M for human erythrocyte pyridoxine kinase. On the basis of these data a pyridoxine concentration of 4.16 X lo-” M was selected for the assay. Higher concentrations of pyridoxine with the same specific radioactivity (which gives a high substrate background) or dilution with nonradioactive pyridoxine (which decreases the specific radioactivity) cause a decrease in the sensitivity of assay. Requirements for ATP and a divalent cation. Human erythrocyte pyridoxine kinase was found to require both ATP and a divalent cation for maximum activities. By using partially purified enzyme, we observed that the maximum velocity at the optimum Mg?+ concentration (4.16 X 1O+ M) is the same as that at the optimum Zn”+ concentration (3.3 X IO-” M), although Zn2+ was superior to Mg2+ below the optimum
I - 1.0
-0.5
0
0.5
1.0
1.5
2.0
2.5
;x10-6 FIG. 7. (Al Substrate velocity curve of human erythrocyte pyridoxine kinase in crude hemolysates. (B) Lineweaver-Burk plots giving a K, of approximately 1.72 X lo-” M for pyridoxine. The units of l/V are expressed as min/nmoles of pyridoxine phosphate (PNP) formed and l/S as llnmoles of pyridoxine.
HUMAN
ERYTHROCYTE
PYRIDOXINE
KINASE
107
concentration of Zn2+, and slight inhibition was found when the concentration of Zn2+ exceeded its optimum (22). Routinely, ATP and MgCl, at a concentration of 4.16 X 1O-” M each were included in the assay mixture. No significant effect of dialysis on the pyridoxine kinase activity with the complete assay system was observed, indicating that the quantitation of pyridoxal kinase activity is not affected by dialyzable endogenous factors in hemolysates. The ATP and Mg”+ concentrations were varied from 0.25-5 mu each. Over this range, the activity of the enzyme was essentially the same. Pyridoxine
Kinase Activity
in Human
Red Blood Cells
By using the pyridoxine kinase assay designed for erythrocytes, a specific activity of 1.381 nmole/min/g Hb (n = 25, SE = 0.05 1) was determined for the pyridoxine kinase in human red blood cells obtained from healthy Caucasian adults. DISCUSSION
Three requirements have to be fulfilled for the development of a meaningful assay of pyridoxine kinase in red blood cells. These are (a) no binding of the reaction product to hemoglobin, (b) sensitivity and (c) rapidity. Pyridoxine is the alcohol form of the vitamin complex and has a -CH,OH group at the 4 position of the pyridine ring, rather than an aldehyde group as does pyridoxal. Since the aldehyde is the functional group required for the formation of a Schiff s base, one would expect no binding of pyridoxine or its phosphorylated derivative to hemoglobin or other proteins through a Schiff’s-base linkage. This was proven to be the case by nearly complete recovery of [3H]pyridoxine and [3H]pyridoxine phosphate added to hemolysates. By using this specific property of pyridoxine, the activity of pyridoxine kinase in erythrocytes is measured employing commercially available [3H] pyridoxine as substrate and estimating the amount of radioactivity of the product. The separation of products from substrates was accomplished by adsorption with Dowex 50 resin in a test tube instead of tedious column chromatographic separation. The effect of phosphate buffer concentration on pyridoxine kinase activity was studied in regard to the possibly inhibitory effect of phosphate on phosphatase activity in human erythrocytes (3). Whether the slightly lower levels of pyridoxine kinase found at lower phosphate buffer concentration is due to the action of a phosphate-inhibitable phosphatase still remains to be established. Preliminary studies revealed that stroma-free hemolysates contain no phosphatase activity when [3H]pyridoxine phosphate is used as substrate under the assay conditions employed for pyridoxal kinase. The finding is consistent with the
108
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observation by Lumeng et al. (23) that phosphatase, which hydrolyzes phosphorylated B6 vitamers, is associated with erythrocyte ghosts. Studies of pyridoxal kinase activities in erythrocytes from normal subjects and alcoholic patients with a pyridoxal phosphate-requiring system have been reported by Hines (24) and Lumeng and Li (23). No difference of erythrocyte pyridoxal kinase activities in alcoholic and nonalcoholic subjects was found by Lumeng and Li. The result is in contrast to that of Hines, who reported that the erythrocyte pyridoxal kinase activity was decreased in alcoholics. The contradictory data reported by these two groups of investigators could be the result of the fact that the binding of pyridoxal phosphate to hemoglobin through a Schiffs-base linkage and therefore its loss during the extraction procedure was not taken into account. Red blood cells contain only a small amount of pyridoxine kinase activity (1.381 nmoles/min/g Hb) compared with those in liver, kidney and brain found by McCormick et al. (6). The specific activities of human erythrocyte pyridoxal kinase reported in the literature (8,23) are one-fourth to one-half of the mean value we found in normal Caucasians. We have observed that highly purified human erythrocyte pyridoxine kinase phosphorylated pyridoxal approximately as actively as pyridoxine (22). It is, therefore, recommended that pyridoxine instead of pyridoxal be used as the substrate for measuring the pyridoxal kinase activity in erythrocytes. The product of the assay reaction catalyzed by human erythrocyte pyridoxine kinase was identified as pyridoxine phosphate. No conversion of pyridoxine phosphate to pyridoxal phosphate was observed, although the ability of red cells to transform pyridoxine phosphate to pyridoxal phosphate has been noted (3). It may be that oxidase activity is inhibited under our assay conditions for pyridoxine kinase. On the other hand, a small percentage of conversion might escape detection either due to the binding of pyridoxal phosphate to proteins or due to photolysis of pyridoxal phosphate. ACKNOWLEDGMENT This research was supported in part by Grant No. HL 07449 from the National Institutes of Health.
REFERENCES 1. Braunstein, A. E. (1960) in The Enzymes (Boyer, P. D., Lardy, H.. and Myrback. K., eds.), Vol. 2, pp. 133-184, Academic Press, New York and London. 2. Rodwell, V. W. (1970) in Metabolic Pathways (Greenberg, D. M., ed.), 3rd ed., Vol. IV, pp. 41 l-436, Academic Press, New York and London. 3. Anderson. B. B., Fulford-Jones, C. E., Child, J. A., Beard. M. E., and Bateman. C. J. T. (1971)J. C/in. Invest. 50, 1901-1909.
HUMAN
ERYTHROCYTE
PYRIDOXINE
KINASE
109
4. Wada, H., Morisue, T., Nishimura, Y., Sakamoto, Y., and Ichihara. K. (1959) Proc. Jap. Acad. 35, 299. 5. Gaut, Z. N., and Solomon, H. M. (1972) Biochem. Pharmacol. 21, 2395-2400. 6. McCormick, D. B., Gregory, M. E., and Snell, E. E. (1961) J. Biol. Chem. 236, 2076-2084. I. White, R. S., and Dempsey, W. 9. (1970) Biochemistry 9, 4057-4064. 8. Hamfelt, A. (1967) Clin. Chim. Acta 16, 7-18. 9. Aoki. Y., Urata. G.. Wada, 0.. and Takaku, F. (1974) J. Clin. freest. 53, 1326-1334. 10. Schulman, M. P., and Richart, D. A. (1957) J. Biol. Chem. 226, 181-189. 11. Cooper, R. G.. Webster, L. T.. Jr., and Harris, J. W. (1963) J. C/in. Invest. 42, 926. 12. Storvick, C. A., Banson. E. M., Edwards, M. A., and Woodring, M. J. (1964) in Methods of Biochemical Analysis (Glick. D. ed.). Vol. 12, pp. 183-276, Interscience, New York. 13. Chabner, B. and Livingston, D. (1970) Anul. Biochem. 34, 413-423. 14. Walsh, M. P. (1966) Amer. J. C&z. Pathol. 46, 282-285. 15. Wada. H., Morisue. T., Sakamoto. Y., and Ichihara, K. (1957) J. Vifuminol. (Kyoto) 3, 183-188. 16. Hines, J. D.. and Love, D. S. (1969) J. Lab. C/in. Med. 73, 343-349. 17. Srivastava, S. K.. and Beutler, E. (1973) Biorhim. Biophys. Acta 304, 765-773. 18. Nakao, M., Nakayama, T., and Kankura, T. (1973) Nature (London) 246, 94. 19. Bray, G. A. (1960) Anal. Biochem. 1, 279-285. 20. Beutler, E. (1971) Red Cell Metabolism-A Manual of Biochemical Methods, pp. 13. Grune and Stratton, New York. 21. Tiselius, H. G. (1972) C/in. Chim. Acta’40, 319-324. 22. Chern. C. J., and Beutler, E., C/in. Chim. Acta, in press. 23. Lumeng, L., and Li, T-K. (1974) J. C&I. freest. 53, 693-704. 24. Hines, J. D. (1970) N. Engl. 1. Med. 283, 1173.
67, 97-109 (1975)
BIOCHEMISTRY
Assay
for
Human
CHING Division
Erythrocyte
Pyridoxine
Kinase
J. CHERN AND ERNESTBEUTLER
of Medicine,
City of Hope Duarte, California
National 91010
Medical
Center,
Received September 13, 1974; accepted February 24, 1975 An accurate and rapid method for the assay of pyridoxine kinase in human erythrocytes has been developed. The procedure involves the separation of the radioactive product from the substrate with Dowex 50 resin in a test tube. Using the assay designed, we found that human red blood cells have a pyridoxine kinase activity of 1.381 nmolelminlg of hemoglobin (n = 25, SE = 0.051), and the enzyme has a K, of approximately 1.72 X 10-l; M for pyridoxine. Pyridoxine phosphate was identified as the main product of the assay reaction catalyzed by human erythrocyte pyridoxine kinase in crude hemolysates.
Pyridoxine is one of three forms of vitamin B,. In its active coenzyme form, pyridoxal phosphate is recognized as important in the function of many enzyme systems in the body (1). The pathway for the conversion of pyridoxine to its active forms has not yet been fully established. The possible route for conversion of pyridoxine to pyridoxal phosphate in mammalian systems is as follows (2): Pyridoxine
%
pyridoxine
phosphate oxidase pyridoxal
phosphate.
Phosphorylation of pyridoxine to pyridoxine phosphate has been demonstrated in human red blood cells (3) and other tissues (4,5). The enzyme pyridoxal kinase (EC 2.7.1.35), which catalyzes the phosphorylation of pyridoxal and, less effectively, of pyridoxine, has been partially purified and characterized from several different sources (6). Pyridoxine kinases from yeast and Escherichia coli B (7), on the other hand, were found to have higher affinity for pyridoxine than for pyridoxal. Although pyridoxal kinase activity has also been demonstrated in human red blood cells (8), no information about the characteristics of the enzyme in red blood cells is available, probably due to the lack of a convenient and sensitive assay system. Whether pyridoxine kinase is the same enzyme as pyridoxal kinase in human red cells remains to be determined. Sideroblastic anemias are characterized by defective hemoglobin synthesis with accumulation of iron in mitochondria. Available evidence indicates the production of &aminolevulinic acid is impaired in erythroblasts in pyridoxine-responsive anemia (9), which is one of sideroblastic 97 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form resewed.
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AND
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anemias. &Aminolevulinic acid synthetase, which requires pyridoxal phosphate as an active coenzyme (lo), is responsible for the condensation of succinyl CoA and glycine to &aminolevulinic acid. Deficiency of pyridoxal phosphate caused by a deficiency of pyridoxine kinase or pyridoxal kinase would be expected to result in decreasing heme synthesis by marrow erythroblasts, and this could lead to mitochondrial ironoverload. Pyridoxal phosphate may also be involved in mobilization of iron from mitochondria (11). For this reason, quantitation of pyridoxal kinase activity in erythrocytes is of considerable interest. Ordinarily used assays for pyridoxal kinase (12- 16) consist of preparing an acid extract and measuring pyridoxal phosphate activity by coupling to a pyridoxal phosphate-requiring apoenzyme system. Such assays are not applicable to red cell systems, however, because of the binding of pyridoxal phosphate to hemoglobin through a Schiffs base linkage and the difficulty of extracting small amounts of pyridoxal phosphate from excess protein and therefore its loss during the extraction procedure (17). It occurred to us that this difficulty would not be encountered if pyridoxine, rather than pyridoxal, was used as a substrate for assay of the kinase. If the same enzyme phosphorylated both vitamers, as has turned out to be the case, the technique could be used for the assay of pyridoxal kinase. We now describe a method for the assay of pyridoxine kinase in human erythrocytes. The procedure involves the separation of the radioactive product from the substrate, which can be achieved with Dowex 50 resin in a test tube. The convenience and rapidity of the assay permits accurate quantitation of the enzyme in normal human red blood cells for the first time. The Michaelis-Menten constant and identification of the reaction product of pyridoxine kinase are also presented. MATERIALS
AND
METHODS
Reagents. [“HI pyridoxine hydrochloride (1.7 Ci/mmole) was obtained from AmershamlSearle Co. Dowex 50-X8 (hydrogen form, 200-400 mesh) was purchased from Bio-Rad Laboratories, Dowex 1 (chloride form, 8% cross-linked, 20-50 mesh), pyridoxine hydrochloride, pyridoxal hydrochloride, pyridoxamine hydrochloride, pyridoxal-5’-phosphate (PLP), ATP and alkaline phosphatase (from calf mucosa, Type I) were obtained from Sigma Chemical Co. All other reagents were analytical quality. At an early stage of this study, [4,5-‘-LC,]pyridoxine (142 &i/mg), a gift of Hoffmann-La Roche Inc., Nutley, NJ, was used as the substrate for pyridoxine kinase. However, we found that commercially available [“Hlpyridoxine could be employed as a substrate for the enzyme as efficiently as [‘“Cl pyridoxine. Thereafter, all studies were performed using [“HI pyridoxine as substrate.
HUMAN
ERYTHROCYTE
PYRIDOXINE
KINASE
99
All experiments involving vitamin B6 compounds were conducted under conditions of reduced light. Chromatographic experiments were carried out at room temperature. Preparation of hemolysates. Venous blood from adults was collected in acid-citrate-dextrose (ACD) solution, filtered on a sulphoethyl cellulose-Sephadex G-25 column according to the procedure of Nakao et al. (18) to remove the leukocytes and platelets. The red blood cells were further washed twice with 0.154 M saline. Hemolysates were prepared by making a 1 to 10 dilution of washed red cells in water. Unless otherwise indicated, all experiments were performed using freshly drawn blood. Determination of pyridoxine kinase activity in hemolysates. The assay system consisted of 100 ~1 of 1 M KH,PO,/K,HPO, buffer, pH 7.0, 50 ~1 of 10 mM ATP (neutralized), 50 ~1 of 10 mM MgCl?, 100 ~1 of 5 X lo-” M [“HI pyridoxine (specific activity, 39.06 &i/pmole), an appropriate amount of hemolysate and water to a volume of 1.2 ml. A blank without the substrate, pyridoxine, was included for each hemolysate. The reaction mixture was incubated in the dark at 37°C for 60 min with shaking, and the reaction was terminated by the addition of 0.7 ml of 12% perchloric acid (PCA) in an ice bath. One hundred microliters of 5 x lo-” M [:‘H] pyridoxine was added to the blank. After filtration through Whatman No. 2 filter paper, 0.6 ml of filtrate was neutralized with 0.225 ml of 1 M K&O,. The mixture was allowed to stand for 5 min, and 0.5 ml of clear supernatant was adjusted to a pH of 4.0-4.5 with one to two drops of 0.5 M formic acid, using pH paper as indicator. Two-tenths milliliter of Dowex 50 resin-suspension (one volume of settled resin, previously equilibrated with 0.05 M potassium formate buffer, pH 4.3, and two volumes of the buffer) was transferred to each solution. Each tube was mixed on a rotatory mixer for 30 min. During this time, the free pyridoxine was adsorbed on the resin beads. The beads were allowed to settle out, and 0.5 ml of the clear supernatant was removed and counted in 10 ml of Bray’s solution (19) with a Packard scintillation counter. A [“Hlpyridoxine standard was also subjected to the same treatment except that the Dowex 50 resin-suspension was replaced with an equal volume of formate buffer. In converting radioactivity to nanomoles of product formed, the volume occupied by the resin was also taken into account. The equation for conversion is as follows: P, = (P, x S,)/(S,
x 1.049),
where P, is the concentration of the product (pyridoxine phosphate); P, is the radioactivity of the product: S, is the concentration of the standard ([“Hlpyridoxine); S, is the radioactivity of the standard; and 1.049 is the correction factor for the volume occupied by the resin.
100
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We found that increase in pH or ionic strength during adsorption with Dowex 50 resins led to higher blank values as the result of release of substrates from the resins. It is also understandable that removal of substrates is necessarily incomplete in the test tube. Small amounts of the substrate (5-10% ; 1 ,OOO-2,000 cpm) remain in the supernatant fraction in equilibrium with the resin. We observed that the blank could be reduced considerably by repetitive adsorption with the resins. However, with subtraction of the blank, only one adsorption with resins was found to be sufficient for most purposes. Pyridoxine kinase activity was expressed as nanomoles of pyridoxine phosphate formed per min and specific activity as nanomoles of pyridoxine phosphate formed per min per gram of hemoglobin (g Hb). Hemoglobin content was determined spectrophotometrically as cyanmethemoglobin (20). RESULTS Recovery of Pyridoxine Added to Hemolysates
and Pyridoxine
Phosphate
In order to examine the recovery of pyridoxine, 1 nmole (=0.0284 &i) of [l”C]pyridoxine was incubated with 1 ml of hemolysate in the absence of ATP at 37°C for 1 hr. The mixture was extracted with PCA, filtered and neutralized as for the determination of pyridoxine kinase activity in human erythrocytes, except that the adsorption with Dowex 50 resin was not carried out. Duplicate results indicated that 96.4% of pyridoxine was recovered. Apparently only insignificant amounts were lost during extraction with PCA. [3H] pyridoxine phosphate was synthesized from [3H]pyridoxine by using highly purified human erythrocyte pyridoxine kinase. When 1.85 nmoles (=0.0024 &I) of [3H]pyridoxine phosphate was mixed with 0.5 ml of hemolysate in the presence of the assay components except for pyridoxine, followed by extraction with PCA, filtration and neutralization, approximately 99% of [“Hlpyridoxine phosphate was recovered. After 1 hr of incubation at 37°C with 3.7 nmoles of [3H]pyridoxine phosphate, 90% of the [3H] pyridoxine phosphate could still be recovered. Chromatographic Separation from the Substrate
of the Reaction
Product
Separation of pyridoxine phosphate from pyridoxine was performed on a Dowex 50 column at pH 4.3 on the basis of different pK, values of the two compounds. Two milliliters of hemolysate was incubated with
HUMAN
ERYTHROCYTE
PYRIDOXINE
KINASE
101
100 ~1 of [Wlpyridoxine (specific activity, 28.4 &i/pmole) and other components for the assay of pyridoxine kinase in a total volume of 2.4 ml for 1 hr, followed by perchloric acid precipitation, filtration, neutralization and titration as described for the assay of pyridoxine kinase in hemolysates. One milliliter of the titrated solution, was applied to a Dowex 50 column (0.5 X 4 cm), which had been equilibrated previously with 0.05 M potassium formate buffer, pH 4.3. The column was first eluted with the formate buffer and then washed with 0.05 M potassium citrate buffer, pH 6.8, containing 0.5 M KCl. Fractions were collected and counted in Bray’s solution. A blank was subjected to the same treatment. An authentic pyridoxal phosphate (PLP) and [‘“Cl pyridoxine mixture was also employed to verify the locations of the phosphorylated vitamin B6 and free vitamin B,. Pyridoxal phosphate was determined by measuring the fluorescence of the semicarbazone of PLP at 460 nm when excited at 380 nm in a Farrand spectrofluorometer as described previously (17). Figure 1 shows the separation of the reaction product from the substrate, and Fig. 2 demonstrates locations of the phosphorylated vitamin B, and free vitamin B,. The good resolution of the phosphorylated vitamin B, from vitamin B6 compounds is the foundation for the assay of pyridoxine kinase in human erythrocytes.
Froctlon
Number
1. The chromatographic separation of the reaction product of crude hemolysate pyridoxine kinase from the substrate, pyridoxine (PN). on a Dowex 50 column. The column was eluted with 0.05 M potassium formate buffer, pH 4.3, and then washed with 0.05 M potassium citrate buffer, pH 6.8, containing 0.5 M KC1 as indicated by the arrow ( 1 ). One-and-one-half-milliliter fractions were collected. Radioactivity of each fraction was determined (O-O). A control was also subjected to the same treatment (0-O). The experimental details were given in the text. FIG.
102
CHERN
AND BEUTLER
Frocf~on
Number
2. Resolution of a mixture of authentic pyridoxaf phosphate (PLP), and pyridoxine (PN) on a Dowex 50 column. One nanomole (=0.0284 &I) of ‘“C-labelled PN was mixed with 5 pg of PLP in 1 ml of 0.05 M potassium formate buffer, pH 4.3, and applied to a Dowex 50 column. The radioactivity of 2-ml fractions was determined (O-O), and the concentration of PLP was estimated by measuring the fluorescence of the semibarzone of PLP at 460 nm when excited at 380 nm (o-0). The conditions of chromatography were identical to those in Fig. 1. FIG.
Characterization
of the Reaction
Product
The differentiation of [W] pyridoxine phosphate from PLP was carried out on a Dowex 1 column. The pooled solution containing phosphorylated [‘“Cl pyridoxine, which was obtained from the first-peak tubes of Dowex 50 chromatography, as described in Fig. 1, was mixed with 20 pg of PLP and loaded on a Dowex 1 column (0.5 X 4 cm) previously equilibrated with 0.05 M potassium formate buffer, pH 4.3. The column was eluted with a linear 40-ml gradient from 0.05 M potassium formate buffer, pH 4.3, to 0.05 M potassium citrate buffer, pH 4.3, with 0.15 M KCl. The separation of the reaction product from pyridoxal phosphate is presented in Fig. 3. Direct verification that the product of the reaction was, in fact, pyridoxine phosphate was not possible, because of the lack of availability of a source of authentic material. A commercial lot of putative pyridoxine5-phosphate obtained from Schwarz-Mann, of New York, proved to be, in point of fact, pyridoxal-5-phosphate. Since authentic pyridoxine was available, however, it was possible to identify, indirectly, the product of the reaction by its degradation to pyridoxine by alkaline phosphatase. Accordingly, the reaction product (i.e., phosphorylated 3H-labeled vitamin B,J was first separated from the substrate, [3H]pyridoxine, on a small Dowex 50 column (0.5 X 4 cm) as described above. The solution containing the putative phosphorylated vitamin B, was then titrated to
HUMAN
ERYTHROCYTE
PYRIDOXINE
KINASE
103
FIG. 3. Differentiation of the reaction product of crude hemolysate pyridoxine kinase reaction from pyridoxal phosphate (PLP) on a Dowex 1 column. A mixture of W-labeled reaction product and PLP was transferred to a Dowex 1 column. A linear 40-ml gradient from 0.05 M potassium formate buffer, pH 4.3, to 0.05 M potassium citrate buffer, pH 4.3, with 0.15 M KC1 was started ( 4 ) to elute the column. Two-and-one-half-milliliter fractions were collected. Radioactivity (O-O) and fluorescence for PLP (0-O) of each fraction were measured as described in Fig. 2.
pH 9.3 with 1 M K,CO,. One milligram of alkaline phosphatase was added to the mixture and incubated at 37°C for 2 hr. The pH of the solution was then adjusted to 4.25 with 0.5 M formic acid and mixed with approximately 1 mg each of pyridoxine, pyridoxal and pyridoxamine as markers. The mixture was applied to a column of Dowex 50 (0.9 X 44 cm) equilibrated previously with 0.05 M ammonium formate buffer, pH 4.25 (21). A linear gradient consisting of 200 ml of 0.05 M ammonium formate buffer, pH 4.25, and 200 ml of 0.5 M ammonium formate buffer, pH 7.5, in the reservoir was used to elute the column. After the completion of the gradient, additional ammonium formate buffer, 0.5 M, pH 7.5, was required to elute pyridoxamine from the column. The resolution of the free forms of vitamin B, markers in addition to the identification of pyridoxine as the main vitamin B, component of the reaction product of pyridoxal kinase is presented in Fig. 4A. The column was also calibrated with a mixture consisting of [“Hlpyridoxine and the three vitamin B, compounds in order to correct for impurities present in commercial preparations of those reagents (Fig. 4B). The small quantity of radioactive pyridoxal observed in Fig. 4A obviously is attributable to the contamination by such a compound in the commercial preparation. The procedure described to resolve free forms of vitamin B, offers a simple method to purify the specific vitamin B, when the purity of reagent is critical in the study. Determination
of Optimum
Conditions
for Pyridoxine
Kinase Assay
Hemofysate volume. Under the assay conditions for human erythrocyte pyridoxine kinase as described in Materials and Methods, the rela-
104
CHERN AND
. r
BEUTLER
PL
.
.
I ;
Fraction
Fraction
Number
Number
4. (A) Dowex 50 chromatographic analysis of the reaction product treated with alkaline phosphatase. The reaction product, “H-labeled, was hydrolyzed with alkaline phosphatase and loaded with three vitamin B, compounds (pyridoxine (PN), pyridoxal (PL) and pyridoxamine (PM)) on a Dowex 50 column. The column was developed with a linear ammonium formate buffer gradient. Eluate fractions (5.7 ml each) were measured for radioactivity (O-O). Three B, vitamin markers were detected spectrophotometrically by absorbance at 295 nm (0-O). (B) Calibration of the same column with a mixture consisting of [3H]pyridoxine and the three Bs vitaminers. See text for details. FIG.
HUMAN !
ERYTHROCYTE
PYRIDOXINE
KINASE
105
4.0 -
x -2 E \ L 3.0 E
l -.
c ;; E z0 92 2.0z I .o : r f E 5 /, 0’
.
Ij_/_
.i
’
. , 0.1
0.2
0.3
,
,
,
,
0.4 0.5 0.6 Hemolysates(ml)
,
,
0.7
0.8
0.9
FIG. 5. The relationship between the concentration of pyridoxine kinase in different volumes of hemolysate and the rate of formation of pyridoxine phosphate.
tionship between the concentration of hemolysate pyridoxine kinase and the formation of pyridoxine phosphate was linear over a range of hemolysate volumes which produced from 0 to more than 0.026 nmolelmin of pyridoxine phosphate (Fig. 5). Routinely, two volumes, 0.2 and 0.4 ml, of hemolysate were employed to quantitate pyridoxine kinase activity in erythrocytes. Time course. The rate of formation of pyridoxine phosphate under these conditions was linear for at least 3 hr (Fig. 6). A 60-min incubation period was adopted as a convenient interval that would ensure measurement of the reaction in a linear portion of the curve. Phosphate buffer concentration. The effect of phosphate buffer concentration on pyridoxal kinase activity is shown in Table 1. An optimum concentration of 80- 100 mrvr was found.
TimeCMinutes)
FIG. 6. The relationship between the rate of formation of pyridoxine phosphate and time.
106
CHERN
AND
BEUTLER
TABLE THE EFFECT
OF PHOSPHATE FORMATION
Phosphate concentration
1
BUFFER CONCENTRATION ON THE RATE OF PYRIDOXINE PHOSPHATE
OF
% of maximum activity
(ITIM)
83.9 86.9 100.0 96.8 83.6
41.7
58.3 83.3 125.0 166.7
Substrafe concentration. The rate of formation of pyridoxine phosphate was studied as a function of the substrate concentration (Fig. 7A). Lineweaver-But-k plots (Fig. 7B) revealed a K, of 1.72 X IOWfi M for human erythrocyte pyridoxine kinase. On the basis of these data a pyridoxine concentration of 4.16 X lo-” M was selected for the assay. Higher concentrations of pyridoxine with the same specific radioactivity (which gives a high substrate background) or dilution with nonradioactive pyridoxine (which decreases the specific radioactivity) cause a decrease in the sensitivity of assay. Requirements for ATP and a divalent cation. Human erythrocyte pyridoxine kinase was found to require both ATP and a divalent cation for maximum activities. By using partially purified enzyme, we observed that the maximum velocity at the optimum Mg?+ concentration (4.16 X 1O+ M) is the same as that at the optimum Zn”+ concentration (3.3 X IO-” M), although Zn2+ was superior to Mg2+ below the optimum
I - 1.0
-0.5
0
0.5
1.0
1.5
2.0
2.5
;x10-6 FIG. 7. (Al Substrate velocity curve of human erythrocyte pyridoxine kinase in crude hemolysates. (B) Lineweaver-Burk plots giving a K, of approximately 1.72 X lo-” M for pyridoxine. The units of l/V are expressed as min/nmoles of pyridoxine phosphate (PNP) formed and l/S as llnmoles of pyridoxine.
HUMAN
ERYTHROCYTE
PYRIDOXINE
KINASE
107
concentration of Zn2+, and slight inhibition was found when the concentration of Zn2+ exceeded its optimum (22). Routinely, ATP and MgCl, at a concentration of 4.16 X 1O-” M each were included in the assay mixture. No significant effect of dialysis on the pyridoxine kinase activity with the complete assay system was observed, indicating that the quantitation of pyridoxal kinase activity is not affected by dialyzable endogenous factors in hemolysates. The ATP and Mg”+ concentrations were varied from 0.25-5 mu each. Over this range, the activity of the enzyme was essentially the same. Pyridoxine
Kinase Activity
in Human
Red Blood Cells
By using the pyridoxine kinase assay designed for erythrocytes, a specific activity of 1.381 nmole/min/g Hb (n = 25, SE = 0.05 1) was determined for the pyridoxine kinase in human red blood cells obtained from healthy Caucasian adults. DISCUSSION
Three requirements have to be fulfilled for the development of a meaningful assay of pyridoxine kinase in red blood cells. These are (a) no binding of the reaction product to hemoglobin, (b) sensitivity and (c) rapidity. Pyridoxine is the alcohol form of the vitamin complex and has a -CH,OH group at the 4 position of the pyridine ring, rather than an aldehyde group as does pyridoxal. Since the aldehyde is the functional group required for the formation of a Schiff s base, one would expect no binding of pyridoxine or its phosphorylated derivative to hemoglobin or other proteins through a Schiff’s-base linkage. This was proven to be the case by nearly complete recovery of [3H]pyridoxine and [3H]pyridoxine phosphate added to hemolysates. By using this specific property of pyridoxine, the activity of pyridoxine kinase in erythrocytes is measured employing commercially available [3H] pyridoxine as substrate and estimating the amount of radioactivity of the product. The separation of products from substrates was accomplished by adsorption with Dowex 50 resin in a test tube instead of tedious column chromatographic separation. The effect of phosphate buffer concentration on pyridoxine kinase activity was studied in regard to the possibly inhibitory effect of phosphate on phosphatase activity in human erythrocytes (3). Whether the slightly lower levels of pyridoxine kinase found at lower phosphate buffer concentration is due to the action of a phosphate-inhibitable phosphatase still remains to be established. Preliminary studies revealed that stroma-free hemolysates contain no phosphatase activity when [3H]pyridoxine phosphate is used as substrate under the assay conditions employed for pyridoxal kinase. The finding is consistent with the
108
CHERN
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observation by Lumeng et al. (23) that phosphatase, which hydrolyzes phosphorylated B6 vitamers, is associated with erythrocyte ghosts. Studies of pyridoxal kinase activities in erythrocytes from normal subjects and alcoholic patients with a pyridoxal phosphate-requiring system have been reported by Hines (24) and Lumeng and Li (23). No difference of erythrocyte pyridoxal kinase activities in alcoholic and nonalcoholic subjects was found by Lumeng and Li. The result is in contrast to that of Hines, who reported that the erythrocyte pyridoxal kinase activity was decreased in alcoholics. The contradictory data reported by these two groups of investigators could be the result of the fact that the binding of pyridoxal phosphate to hemoglobin through a Schiffs-base linkage and therefore its loss during the extraction procedure was not taken into account. Red blood cells contain only a small amount of pyridoxine kinase activity (1.381 nmoles/min/g Hb) compared with those in liver, kidney and brain found by McCormick et al. (6). The specific activities of human erythrocyte pyridoxal kinase reported in the literature (8,23) are one-fourth to one-half of the mean value we found in normal Caucasians. We have observed that highly purified human erythrocyte pyridoxine kinase phosphorylated pyridoxal approximately as actively as pyridoxine (22). It is, therefore, recommended that pyridoxine instead of pyridoxal be used as the substrate for measuring the pyridoxal kinase activity in erythrocytes. The product of the assay reaction catalyzed by human erythrocyte pyridoxine kinase was identified as pyridoxine phosphate. No conversion of pyridoxine phosphate to pyridoxal phosphate was observed, although the ability of red cells to transform pyridoxine phosphate to pyridoxal phosphate has been noted (3). It may be that oxidase activity is inhibited under our assay conditions for pyridoxine kinase. On the other hand, a small percentage of conversion might escape detection either due to the binding of pyridoxal phosphate to proteins or due to photolysis of pyridoxal phosphate. ACKNOWLEDGMENT This research was supported in part by Grant No. HL 07449 from the National Institutes of Health.
REFERENCES 1. Braunstein, A. E. (1960) in The Enzymes (Boyer, P. D., Lardy, H.. and Myrback. K., eds.), Vol. 2, pp. 133-184, Academic Press, New York and London. 2. Rodwell, V. W. (1970) in Metabolic Pathways (Greenberg, D. M., ed.), 3rd ed., Vol. IV, pp. 41 l-436, Academic Press, New York and London. 3. Anderson. B. B., Fulford-Jones, C. E., Child, J. A., Beard. M. E., and Bateman. C. J. T. (1971)J. C/in. Invest. 50, 1901-1909.
HUMAN
ERYTHROCYTE
PYRIDOXINE
KINASE
109
4. Wada, H., Morisue, T., Nishimura, Y., Sakamoto, Y., and Ichihara. K. (1959) Proc. Jap. Acad. 35, 299. 5. Gaut, Z. N., and Solomon, H. M. (1972) Biochem. Pharmacol. 21, 2395-2400. 6. McCormick, D. B., Gregory, M. E., and Snell, E. E. (1961) J. Biol. Chem. 236, 2076-2084. I. White, R. S., and Dempsey, W. 9. (1970) Biochemistry 9, 4057-4064. 8. Hamfelt, A. (1967) Clin. Chim. Acta 16, 7-18. 9. Aoki. Y., Urata. G.. Wada, 0.. and Takaku, F. (1974) J. Clin. freest. 53, 1326-1334. 10. Schulman, M. P., and Richart, D. A. (1957) J. Biol. Chem. 226, 181-189. 11. Cooper, R. G.. Webster, L. T.. Jr., and Harris, J. W. (1963) J. C/in. Invest. 42, 926. 12. Storvick, C. A., Banson. E. M., Edwards, M. A., and Woodring, M. J. (1964) in Methods of Biochemical Analysis (Glick. D. ed.). Vol. 12, pp. 183-276, Interscience, New York. 13. Chabner, B. and Livingston, D. (1970) Anul. Biochem. 34, 413-423. 14. Walsh, M. P. (1966) Amer. J. C&z. Pathol. 46, 282-285. 15. Wada. H., Morisue. T., Sakamoto. Y., and Ichihara, K. (1957) J. Vifuminol. (Kyoto) 3, 183-188. 16. Hines, J. D.. and Love, D. S. (1969) J. Lab. C/in. Med. 73, 343-349. 17. Srivastava, S. K.. and Beutler, E. (1973) Biorhim. Biophys. Acta 304, 765-773. 18. Nakao, M., Nakayama, T., and Kankura, T. (1973) Nature (London) 246, 94. 19. Bray, G. A. (1960) Anal. Biochem. 1, 279-285. 20. Beutler, E. (1971) Red Cell Metabolism-A Manual of Biochemical Methods, pp. 13. Grune and Stratton, New York. 21. Tiselius, H. G. (1972) C/in. Chim. Acta’40, 319-324. 22. Chern. C. J., and Beutler, E., C/in. Chim. Acta, in press. 23. Lumeng, L., and Li, T-K. (1974) J. C&I. freest. 53, 693-704. 24. Hines, J. D. (1970) N. Engl. 1. Med. 283, 1173.
