Jounrol
OJ
.~~,~,~~,~l~,,,,,,,f~, Vol 31. pp 1403-l4lO
003-30.12 7X 1201- I$03SO? 00 0
Pergamon Pres, Ltd 1978 Printed in Great Britain Society for Neurochemi3try Ltd
6 lnternaiional
THE STABILITY OF VITAMIN B, ACCUMULATION AND PYRIDOXAL KINASE ACTIVITY IN RABBIT BRAIN AND CHOROID PLEXUS REYNOLDSPECTOR and STANLEY N . SHIKUMA Division of Clinical Pharmacology, Department of Medicine, University of Iowa Hospitals and Clinics, Iowa City, IA 52242, U.S.A. (Rrctiioed
4 April 1978. Accepted 9 Jurir 1978)
Abstract-The effects of changes in the concentrations of pyridoxal phosphate and blogenic amines in brain on: ( I ) pyridoxal kinase (EC 2.7.1.35) activity in brain and choroid plexus; and (2) vitamin B, accumulation by brain slices and isolated, intact choroid plexuses were studied. New Zealand white rabbits were treated parenterally with 200 mg/kg pyridoxine-HCl for 3 days or 120mgjkg 4-deoxypyridoxine HCI or 5 mg/kg reserpine I day before death. After these treatments the mean concentration of pyridoxal phosphate in brain was elevated by 39% by pyridoxine and decreased by 570~by 4'-deoxypyridoxine. Reserpine had no effect. However, the ability of brain slices and isolated. intact choroid plexuses from the treated rabbits to accumulate [3H] vitamin B, (with [3H]pyridoxine in the medium) was not different from untreated controls. Also, the specific activity of pyridoxal kinase in brain and choroid plexus of treated rabbits was not different from controls. These results show that vitamin B, accumulation and pyridoxal kinase activity in brain and choroid plexus are independent of both pyridoxal phosphate and reserpine-sensitive biogenic amine concentrations in brain. Itr cifro studies with pyridoxal kinase showed that. in both choroid plexus and brain. pyridoxal kinase was a single enzyme with a molecular weight of 43.000 and a K, for pyridoxine of 2 . 0 ~ Crude ~ . and partially-purified pyridoxal kinase from brain was not inhibited by biogenic amines ( 1 mM) or pyridoxal phosphate ( 5 p ~ )These . if1 citro data are consistent with the lack of effect of changes in pyridoxal phosphate and biogenic amine concentrations (in brain) on pyridoxal kinase activity in brain iii cico.
THETOTAL vitamin B, (B,)' concentration in mam- doxine in the diet) and pyridoxamine-P can be oximalian brain is determined by the balance between dized t o pyridoxal-P by pyridoxamine phosphate oxi1 9 7 8 ~ )Excessive . concensaturable transport, binding and metabolic intercon- dase (EC 1.4.3.5) (SPECTOR. 1978a. trations of the phosphorylated B, vitamers are versions of the various B, vitamers (SPECTOR, & GREENWALD. 1978). Although B, in dephosphorylated intracellularly and transported out b; SPECTOR brain exists predominantly in the enzymatically active of brain cells (SPECTOR, 197%). No metabolism of phosphorylated forms (pyridoxal-P and pyridoxa- E3H]B6 to thc inactive [3H]pyridoxic acid (or its lacmine-P). the nonphosphorylated B, vitamers readily tone) was detected in brain or choroid plexus (SPECcross the blood-CSF barrier (the choroid plexus) and TOR. 197%; SPECTOR & G R E E K W A L D . 1978). possibly the blood--brain barrier by a saturable transIri uiuo, rabbit brain pyridoxal kinase activity has 1973; SPECTOR. been reported t o be affected by various drugs, diet port system (Loo. 1972; TISELIUS. 1978a, b). Once within the C S F and extracellular and development ( E B A D I et al., 1968, 1970. 1973; space of brain, the three nonphosphorylated B, EBADI.1972; McCoy et al., 1972). For example. reservitamers readily enter brain cells by diffusion (or faci- pine, amphetamine and vitamin B, deficiency induced litated diffusion) and are trapped intracellularly after by 4'-deoxypyridoxine were reported to increase pyriphosphorylation by a single enzyme, pyridoxal kinase doxal kinase activity and decrease pyridoxal phos(EC 2.7.1.35) (SPECTOR, 19786; SPECTOR& GREEN- phate levels in rabbit brain, whereas chlorpromazine WALD. 1978). Intracellular pyridoxine-P (from pyriand pyridoxine had the opposite effects (EBADIet a]., 1968, 1969, 1970, 1973). During development the con' The nomenclature for the 6 active B, vitamers (Be) centration of pyridoxal-P in brain was reported to and related compounds is that of the IUPAC-IUB Com- decrease, whereas pyridoxal kinase activity increased mission of Biochemical Nomenclature (Errr. J . Biochrm. (EBADI,1972). These results led to the hypothesis that (1973) 40. 325 327). The 6 active forms are pyridoxine. brain pyridoxal kinase activity is inversely related to pyridoxal. pyridoxamine, pyridoxine 5'-phosphate (pyriboth brain pyridoxal-P and biogenic amine levels doxine-P), pyridoxal 5'-phosphate (pyridoxal-P), and pyridoxamine S'-phosphate (pyridoxamine-P). The principal in- ( E B A D I et d., 1968, 1970, 1973; EBADI. 1972). H O W active metabolites of B, are pyridoxic acid and pyridoxic ever, some of these results may have been due to methodological artifacts (NEARY et al.. 1972). Moreacid lactone. Abbreoiarions used: B,, B, vitamers; T/M. tissue-to- over. other investigators have shown either no relationship between brain pyridoxal kinase activities and medium. 1403 \.r 31 6
ii
1404
REYNOLDSptcroR and STANLEY N. SHIKUMA
measured by the method of Loo & BADGER(1969). The concentration (pmol/kg wet weight) of pyridoxal-P was calculated as the difference between the concentration of the total pyridoxal and the concentration of endogenous pyridoxal. The ability of slices of rabbit cortex and medulla and of choroid plexus to accumulate C3H]B, after pretreatment of the rabbits with parenteral4'-deoxypyridoxine, reserpine and pyridoxine (as described above) was investigated by methods previously described in detail (SPECTOR,1978h). Briefly, about 25mg of doubly-washed 0.25mm slices of cortex or medulla from each rabbit were incubated in 3 ml artificial CSF containing 5 mM-glucose. 40 n~-['H]pyridoxine and 0.0, 0.3, 0.8, 2.0 or 200 phi-pyridoxine for 30 min at 37°C in a metabolic shaker under 95% 0 2 :5% C 0 2 (SPECTOR.1978h). From each rabbit four 25mg samples from each brain region at each concentration were individually incubated in 3 m l medium and then filtered and washed. The 'H radioactivity in the slices (minus the appropriate blanks) and in the medium was determined. A Hofstee transformation of the saturable accumulation MATERIALS AND METHODS (by the brain slices) of B, from the medium (pmol per ['HIPyridoxine (2.8 Ci/mmol), prepared by an exchange 30 min per kg wet weight) and the concentration of ['HIprocedure, was obtained from Amersham-Searle, Arlington pyridoxine plus unlabeled pyridoxine ( p ~ in) the medium Heights, IL. All 6 B, vitamers, except pyridoxine-P which was performed (using linear regression analysis) t o obtain was synthesized (SPECTOR.1978h). 4'-deoxypyridoxine the transport constants, K , and V,,,,,, (SPFCTOR& GRfES(deoxypyridoxine),4'-pyridoxic acid, L-norepinephrine, D.LWALD. 1978; SPECTOR 19786). The K , can be interpreted norephedrine, reserpine and serotonin creatinine sulfate as the concentration of pyridoxine in the medium required were obtained from Sigma Chemical Co., St. Louis, MO. to half-saturate the intracellular accumulation of B,, and 5'-Deoxypyridoxine was a gift from Dr. C. C. PORTERthe b',,, as the maximal number of pmoles of B, per (Merck). 30 min per kg wet weight that can be accumulated by the All studies were performed on 1.4-2.0kg New Zealand saturable accumulation system (SPECTOR& GREENWALD. White rabbits. All rabbits were maintained on Purina Rab1978). bit Chow Checkers R and H,O ad lib. until about 2 h Choroid plexuses from these and other similarly-treated before death. All injected rabbits were killed 18 h after the rabbits were incubated in 3 ml artificial CSF containing final injection. Eighteen rabbits were injected once with 5 mM-glucose and 40 nM-['H]pyridoxine at 37°C for 30 5 mg/kg reserpine intravenously, 17 rabbits were injected min under 95% 0,: 5% CO,. At the end of the incubation once subcutaneously with 4'-deoxypyridoxineeHCI, and 16 the concentration of ['H]B6 in the choroid plexus divided rabbits were injected at 9:OO a.m. and 4:OO p.m. for 3 days by the concentration of ['Hlpyridoxine in the media (T,'M) with 100 mg/kg pyridoxine-HCl intravenously. Twenty- was determined (SPECTOR& GREENWALD, 1978). one control rabbits were injected once with buffer. The activity of pyridoxal kinase in brain cortex and Beside whole rabbit brain, 4 regions were utilized in the choroid plexus after the injection of 4'-deoxypyridoxine, following studies: the region of the brain above the lateral reserpine and pyridoxine as well as the regional distribuventricle including cortex, white matter, and periventricu- tion of pyridoxal kinase was assayed by measuring the lar grey matter (henceforth termed 'cortex.') the medulla, conversion of ['Hlpyridoxine to [3H]pyridoxine-P by the lateral one-fourth of the cerebellum (henceforth termed methods previously described in detail (CHERN & BEUTLER, 'cerebellum,') and the lateral and fourth ventricular choroid 1 9 7 5 ~ ;SOLOMON & HILLMAN, 1976; SPECTOR& GREENplexuses WALD,1978). Briefly, 0.251111 of the chilled supernatant The effect of 4'-deoxypyridoxine, reserpine and pyri- (30,OOOg for 20min)' of about 5 mg brain or 1 mg choroid doxine injections on the concentrations of pyridoxal and plexus, previously homogenized in 50: 50 artificial CSFpyridoxal-P in cortex or whole brain was measured by 0.1 M-potassium phosphate, pH 6.5 (henceforth termed methods previously described in detail (SRIVASTAVA & 'phosphate-CSF'), was added to 0.05 ml 10 mM-MgC1,. BEUTLER. 1973; SPECTOR, 1978a). Briefly, immediately after 0. I ml 10 mM-sodium ATP in 0.05 M potassium phosphate death, the total amount of pyridoxal-P and pyridoxal in buffer (pH 6.5). and 0.35ml artificial CSF (SPECTOR& homogenates of about lOOmg weighed samples of brain GREENWALD, 1978). After the solution had warmed to was measured in duplicate (SPECTOR,1978~).The pyridox- 3 7 T , the reaction was begun by adding 40 pmol C3H]pyrial-P in the homogenates was first dephosphorylated with doxine and 0, 2, 4, or 8 nmol pyridoxine in 0.05 ml potasacid phosphatase, and the total pyridoxal (endogenous pyr- sium phosphate buffer, pH 6.5 (SPECTOR& GREENWALD. idoxal and pyridoxal from dephosphorylated pyridoxal-P) 1978). After 1Omin at 37°C in a shaker, the reaction was was measured as the highly fluorescent pyridoxal semicar- stopped by adding 0.3 ml of chilled 0.6 M-metaphosphoric barsone (SPECTOR, 1978~). Endogenous pyridoxal was acid. The ['Hlpyridoxine-P and any [3H]pyridoxal-P was then separated from the C3H]pyridoxine on Amberlite CG-120 columns (SPECTOR.1978a). This assay without Centrifugation of the homogenates at 50,OOOg for 1 hr added unlabeled pyridoxine is termed the standard pyridoxal kinase assay. In some assays no artificial CSF was at 4°C did not alter the results of the standard pyridoxal used; instead the tissues were homogenized in 0.07 Mkinase assay.
pyridoxal-P (MCCORMICKet al., 1961) or a direct rather than an inverse relationship (LOO, 1972; TAPIA rt al., 1975; BHAGAVAN et al.. 1977). The purpose of the present studies was t o quantify the relationships between pyridoxal kinase activity, pyridoxal-P levels, a n d the recently-described vitamin B, accumulation systems in brain a n d choroid plexus (SPECTOR, 1 9 7 8 ~ .h ; SPECTOR& GRFENWALD,1978). Alterations in the concentration of pyridoxal-P and/or biogenic amines were effected by treatment of rabbits with various drugs a s well a s pyridoxine. When the activity of pyridoxal kinase as well as the accumulation of B, by brain and choroid plexus were found t o be unrelated t o the concentrations of pyridoxal-P or biogenic amines in brain, further biochemical characterization of both rabbit brain a n d choroid plexus pyridoxal kinase was performed.
'
1405
B, accumulation and pyridoxal kinase activity in brain potassium phosphate buffer (pH 6.5). and all the reagents were prepared in the same buffer. The nature of the product derived from [3H]pyridoxine incubated with brain supernatant in the standard pyridoxal kinase assay was checked by a column chromatographic method (TISE.LI~,S. 1973). As the conditions in the standard pyridoxal kinase assay were chosen to stay on the linear portion of the velocity curve. Michaelis-Menten kinetic constants for the conversion of pyridokinc to pyridoxine-P were obtained by using linear regression analysis and standard Hofstce transformations (COLQI,HOLY. 1971) in units of /LM (K,) and pmol per kg wet weight of original tissue per IOmin In some assays. the effects of various amines. B, vitamers and B, analogues on pyridoxal kinase activity werc measured by the standard pyridoxal kinase assay except that the pH of the assay mixture was adjusted to 6.4. The molecular weight of pyridoxal kinase in 0.3 ml supernatants (30.OOO g for 20 min) of 25 mg choroid plexus or 50mg hrain cortes. previously homogenized in chilled phosphate~CSF. was determined by the method of AUDRI.WS (1965). Briefly. the chilled supernatants (0.3 ml) were applied to a 36cm Sephadex G I 0 0 column equilibrated with phosphate-CSF (in a 4-C room) and standardized with Blue Dextran, bovine transferrin. ovalbumin and horse heart cytochrome c (ANDREWS.1965).0.41111 fractions were collected. and the pyridoxal kinase activity of each fraction was determined as described above in the standard pyridoxal kinase assay. The molecular weight of the pyridoxal kinase was interpolated from the standard curve (AADKLWS. 1965).
(v,,,tJ.
RESULTS
In Table 1 are shown thc effects of parenteral 4'-deoxypyridoxine, reserpine, and pyridoxine on pyridoxal-P concentrations in rabbit cortex. Treatment with pyridoxine significantly raised the pyridoxal-P concentration when compared with controls. Treatment with 4'-deoxypyridoxine significantly lowcred the pyridoxal-P concentration. Reserpine had no significant effect. In whole brain (Table I ) parenteral 4'-deoxypyridoxine also significantly lowered the concentration of pyridoxal-P. TAIILt I
C O l C t \TKATIOL
Treatment Control (cortex) 4-Deoxypyridoxine Reserpine Pyridoxine Control (whole brain) 4'-Deosypyridoxine
OF
TABLL 2 TRAYSPORT CO\STA\TS
B,
ACT( MI LATIOI. H \
KT (PM)
(PmoL kg per 30 min)
0.15 f 0.03 0.08 & 0.01 0.22 & 0.03 0.15
1.36 i 0.22 1.31 f 0.08 1.52 i 0.20 I .3x
1111 I\
Treatment Control (4) 4'-Deoxypyridoxrne (3) Reserpine (3) Pyridoxine (2)
Values are means +s.L.M. with the number of experiments in parentheses. Rabbit brain slices obtained from treated rabbits were incubated in artificial CSF containing 40 n~-['H]pyridouine with various amounts of unlabeled pyridoxine for 30 min. Hofstee transformations of the saturable 30 min accumulation data were performed to yield the K , and V,,,.,, for B, accumulation (COLQIHOIX. 1971). The K , and V , , , for slices of medulla ( N = 2) were 0.20 pM and 0.75 pmol kg (30 min)- I . ~
'
The transport constants K,. and V,,,,,, for the intracellular accumulation of B, by rabbit brain cortex slices are shown in Table 2. There was no significant difference from controls in the average K , or L:,,,,, in rabbits pretreated with 4'-deoxypyridoxine. reserpine. or pyridoxine. The effects of 4'-deoxypyridoxine. reserpine, and pyridoxine on the ability of isolated rabbit choroid plexuses t o accumulate r3H]B, with [3H]pyridoxine in the medium are shown in Table 3. The choroid plcxuses from the treated rabbits did not accumulate C3H]B, significantly differently from the controls. The tritiated product of the standard pyridoxal kinase assay cochromatographed with synthesized pyridoxine-P (TISELIUS. 1972). However. because of the imperfect separation of pyridoxine-P and pyridoxal-P, up to 10% of [3H]pyridoxine-P could have been oxidized to r3H]pyridoxal-P by pyridoxine phosphate & HILLMAN. 1976). This would not oxidase (SOLOMON affect the standard pyridoxal kinase assay as pyridoxal-P elutes with pyridoxine-P when the 'H-product is separated from [3H]pyridoxine on Amberlite CG120 columns (SPECTOR. 197th). No [3H]pyridoxic acid or [3H]pyridoxic acid lactone was detected.
PYRIDOXAL-P
ALD I'YWIDOXAL WHOLF BRAII.
Pyridoxal-P + Pyridoxal (pmol per kg)
FOR
RABBIT HRAII. CORTI Y
lh,
RAHHIT CORTf X A Z D
Pyridoxal-P (pmol per kg)
Cortex 6.5 f 0.5(4) 5.4 F 0.5 (4) 3.3 f 0.6* (3) 2.3 f 0.4* (3) 7.3 f 0.7 (4) 5.X f 0.8 (4) 8.8 f 0.3* (4) 7.5 f 0.4* (4) Whole brain 3.3 f 0.5(3) 4.5 f 0.7 ( 3 ) 2.9 f 0.2+(4) 1.9 & 0.11. (4)
1.1 i 0.2(4) 1.1 i 0.3(3) 1 5 f 0.1 (4)
1.4 f 0.2 (4) 1.2 f 0.7 (3) 1.0 f 0 I (4)
Values are means +s.E.M. with the number of determinations in parentheses. See methods for doses of drugs. * Values differ from respective controls with P < 0.05 [Dunnett's test. DLUXETT
(1955)l.
t Values differ from controls with P < 0.05 (Student's t-test, 2 tailed).
REYNOLDSPtCTOR and STANLEY N. SHIKUMA
1406
TABLE3. ACCUMULATIONOF [3H] B6 PLEXIJS
Treatment
BY RABBIT CHOROID
TABLE5. KINETICCONSTANTS
OF PYRIDOXAL KINASE CHOROID PLEXUS
in oitro
30-min TIM Treatment
Control 4'-Deox ypyridoxine
Reserpine Pyridoxine
5.39 f 0.63 (8) 6.26 f 0.57 (6) 5.51 f 0.48 (6) 5.86 f 0.75 (6)
K, ( b ~ )
Control (5) 4'-Deoxypyridoxine (5) Reserpine (3)
Pyridoxine ( 3 ) Values are means ~ s . F . . M .with the number of determinations in parentheses. Choroid plexuses were obtained from rabbits after various drug treatments and incubated in artificial CSF containing 40 n~-[~H]pyridoxinefor 30 min. The total ['HIB, concentration in the tissue divided by the total [3H]pyridoxine concentration in the media (T/M) was determined (SPECTOR & GRIXNWALD, 1978).
V",,, (PnoIfig
per 10 min)
2.0 f 0.3
121 f 20
*
144 f 10
1.8 f 0.2 2.0 0.2 2.0 f 0.2
ix
145 f 50 127 f I I
Values are means fs.t.M. with the number of determinations in parentheses. After various treatments choroid plexuses from each rabbit were removed and pooled. The K , and r/;,,,$, of pyridoxal kinase were then determined.
v,,,,
about 5 times and the by 2 times as compared Using the standard pyridoxal kinase assay with t o the K , and V,,,,, in the standard pyridoxal kinase (pyridoxal various amounts of unlabeled pyridoxine, the mean assay. Howcver, the ratio of the mean K , and V,,,.,, for the conversion of pyridoxine t o pyri- kinase) from choroid plexus of the reserpine-treated doxine-P by unpurified supernatants of brain cortex rabbits divided by the V,,,,, in the control was not were calculated and are shown in Table 4. There was significantly altered (1 2 0 in the standard pyridoxal no significant difference in the mean K , or V,,,,,, kinase assay; 1.30 in potassium phosphate buffer). between control and 4'-deoxypyridoxine-, reserpine- Similar results were obtained in cortex. The molecular weight of cortical pyridoxal kinase or pyridoxine-treated rabbits. Medulla had significantly less ( P < 0.05) pyridoxal kinase activity (per (from brain) as determined by Sephadex column chromatography (ANDKEWS,1965) is shown in Fig. g) than cortex (Table 4). Using unpurified choroid plexus supernatants, I . Pyridoxal kinase appeared as a sharp single peak there was also no significant difference in the mean that emerged just after ovalbumin. The extrapolated K , and V,,,,,, for pyridoxal kinase between control and molecular weight was 43,000. The pyridoxal kinase 4'-deoxypyridoxine-. reserpine- or pyridoxine-treated of choroid plexus and of rabbit red blood cells rabbits (Table 5). behaved identically, i.e. emerged from the column as In 2 control and 2 reserpine-treated rabbits pyri- a sharp single peak with a molecular weight of 43,000. doxal kinase activity was measured in cortex and The effects of several B, vitamers, B, analogues choroid plexus using only 0 . 0 7 ~potassium phos- and biogenic amines on unpurified pyridoxal kinase phate buffer (pH 6.5) instead of the buffers in the activity from cortex are shown in Table 6. Pyridoxine standard pyridoxal kinase assay. Concentrations of (5 p ~ ) , pyridoxal (5 p ~ )and 4'-deoxypyridoxine 5, 10 and 20pM unlabeled pyridoxine were used. In (10 p ~ significantly ) decreased pyridoxal kinase acthe control and reserpine-treated rabbits the mean K , tivity, whereas pyridoxal-P (5 PM), 5'-deoxypyridoxine and in cortex were 10.6 p~ and 148 pmol/kg per ( I O O ~ M )and the amines ( 1 . 0 m ~ )had n o significant IOmin and 1 4 . 8 and ~ ~ 199pmoljkg per IOmin. re- effect. Addition of sodium ascorbate (5mM) t o the spectively; in choroid plexus the mean K , and assay tubes containing serotonin (to prevent oxivalues (in the control and reserpine-treated rabbits) dation) did not alter the results. In one experiment were 9 . 7 and ~ ~242pmoljkg per IOmin and 1 0 . 7 ~ serotonin ~ (1.O mM) and norepinephrine (1 .O mM) did and 3 I7 pmol/kg per 10 min, respectively. Thus, per- not significantly affect the activity of partially purified forming the pyridoxal kinase assay in potassium pyridoxal kinase from cortex. The enzyme was puriphosphate buffer increased the K , in both tissues by fied by ammonium sulfate fractionation (MCCORMICK
v,,,,
v,,.,,
v,,,,
TABLL 4. KINETIC CONSTANTS Region Cortex (6) Cortex (4) Cortex ( 3 ) Cortex (3) Cerebellum (4) Medulla (4) Whole brain (2) Whole brain (2)
OF I ~ Y R I D O X A L KINASE FROM B R A I N
Treatment -
4-Deoxypyridoxine Reserpine Pyridoxine ~
~-
~
-
4-Deoxypridoxine
K,
01~)
2.2 f 0.2 2.1 f 0.1 2.4 f 0.3 2.2 & 0.2 1.9 f 0.1 1.9 f 0.1 2.0
2.2
(pmolfig per 10 min) 69 f 5 16 f 2 78 f 6 83 + 4 65 f 5 51 f 2
75 84
Values are means ~ s . E . Mwith . the number of determinations in parentheses. After various treatments rabbit brains were removed and the K , and V,,,, of pyridoxal kinase were determined in supernatants of brain cortex and of various regions of the brain.
B, accumulation and pyridoxal kinase activity in brain
1407
lation by brain slices and choroid plexus are not changed significantly by altering the concentrations of pyridoxal-P or biogenic amines in brain and (2) In hi hi tor Per cent control that pyridoxal kinase from brain is similar, if not identical, to pyridoxal kinase from choroid plexus. Control I 00 (7) Pyridoxine. 5 PM 26 (2)* Before discussing these results. several technical Pyridoxal, 5 PM 15 (2)* matters require comment. First, the method employed Pyridoxal-P. 5 PM I 1 I (2) in this study to measure pyridoxal-P in brain is quan4'-Deoxypyridoxine. 10 PM 25 f 1 (6)* titative (SRIVASTAVA & BEUTLER.1973). This is impor5'-Deoxypyridoxine. 100 PM 104 k 3 (4) tant because previously employed methods using perSerotonin. 1.0 mM 85 f 2 ( 6 ) Norepinephrine. 1.0mM 97 f 3 (6) chloric or trichloroacetic acid d o not quantitatively Norephedrine. 1.0mM 103 f 3(4) extract the pyridoxal-P from tissue (SPECTOR.1 978a. Values are means k S.E.M. with the number of deter- h). Second, metaphosphoric acid was employed in our minations in parentheses. In each experiment duplicate pyridoxal kinase assay to maintain the [3H]pyridoxsupernatants from about 5 mg brain cortex were incubated ine-P intact. Use of other protein precipitating agents at 37°C in 0.8ml buffer (pH adjusted to 6.4) containing such as perchloric acid does not quantitatively mainATP, Mg'+. ['Hlpyridoxine and. in some cases, various & GREI~Sinhibitors as in the standard pyridoxal kinase assay. After tain [3H]pyridoxine-P intact (SPECTOR WALD. 1978). Third. the conversion of [3H]pyridoxine 10 min the d.p.m. converted to ['Hlpyridoxine-P per mg original tissue were determined. In the control assays to [3H]pyridoxine-P was employed to quantify pyri8094 i 234d.p.m. (s.E.M.; N = 7) per mg tissue were con- doxal kinase activity because neither pyridoxine nor verted to ['Hlpyridoxine-P. The control samples were pyridoxine-P bind significantly to or interact with tisarbitrarily set at lOOS/,. 1978). Pre* P < 0.05 by Scheffe's method for multiple comparisons sue components (SPECTOK& GKEESWALD. vious studies employing the conversion of pyridoxal in the Gaussian analysis of variance (COLOI~HOL'N. 197!). (as substrate) to pyridoxal-P as a measure of pyrirt a/.. 1961) and then passed through a Sephadex doxal kinase activity may be invalid because of interG-100 column as described above. The two most actions between pyridoxal-P and proteins or biogenic active fractions were pooled and used for the pyri- amines. NEAKYrt al. (1972) showed that pyridoxal-P doxal kinase assay as in Table 6. interacts with biogenic amines such as dopamine and norepinephrine and suggested that the previously reported inhibition of pyridoxal kinase activity by DISCUSSION biogenic amines (EBADIet al.. 1968, 1973) with the The principal findings reported herein are ( I ) that possible exception of serotonin was due to measurepyridoxal kinase activity and vitamin B, accumu- ment artifacts. i.e. interactions between the amines and the reaction product. pyridoxal-P, rather than an inhibition of pyridoxal-P production by a direct effect of biogenic amines on pyridoxal kinase. Measurement of pyridoxal kinase activity by the decline of the s u b strate concentration. thc appearance of ADP. or the conversion of pyridoxine to pyridoxine-P avoids the difficulties inherent in quantifying the appearance of >" Pvridoxal kinase pyridoxal-P as a measure of pyridoxal kinase activity (CHERN & BEC'TLEK. 197%; NEARYrt d.. 1972). Our results confirm the previously reported erects of 4'-deoxy-pyridoxine and pyridoxine on pyridoxal-P levels in whole brain and cortex (Eearx t't d..1970). 1 0 Reserpine did not alter pyridoxal-P levels (Table I ) . I I 1 10,000 25.000 50.000 75!000 100,000 Although we did not measure the levels of biogenic M o l e c u l a r Weight amines in the reserpine-treated rabbits. the behavioral FIG. 1. Determination of the molecular weight of brain effects (lethargy, weakness, etc.) were clear and consispyridoxal kinase by gel filtration. A 36 x 0.6 cm Sephadex tent with a severe depletion of biogenic amines (ERADI G-100 column was standardized with Blue Dextran (void et a/.. 1968). volume). bovine transferrin (68,000), ovalbumin (45.000). Previously. we have shown that both isolated cliorand horse heart cytochrome C (12,400) by the method of oid plexuses and brain cortex slices from rabbits ANDREWS(1965). After standardization. 0.3 ml of superna- accumulate ['HIB, whea [3H]pyridoxine is present tant of brain was applied to the column and fractions were in the medium (SPECTOR. 1978h). The accumulation collected. The pyridoxal kinase activity i n each fraction systems in both tissues are saturable and energywas determined by the standard pyridoxal kinase assay. Shown is the V, (elution volume) divided by the V, (void dependent (SPECTOR.197Xh). In both cortex and chorvolume) as a function of molecular weight ( A N D R ~ W S . oid plexus from 70 to 80:" of the ['HIB, within the 1965).The VJV0 for pyridoxal kinase was 1.23 which extra- tissues is phosphorylated after a 30 min incubation in 40-50 n~-[~H]pyridoxine. In both tissues the polates to a molecular weight of 43.000. TABLI6.
EFFrCT OF VARIOUS AMINtS. B, VITAMf RS A I D ANALOGL'ES OY BRAIN PYRIDOXAL K I \ A S t ACTIVITY
OLD SPECTORand STANLEY N. SHIKUMA
metabolism of vitamin B, in rabbit brain and choroid decarboxylase and pyridoxal phosphate in brain. J . plexus. J . biol. Chem. 253. 2373-2378. Nwrohiol. 6, 159- 170. SRIVASTAVA S. K. & BEUTLER E. (1973) A new fluorimetric TIS~LIIJS, H.-G. (1 972) A chromatographic separation of method for the determination of pyridoxal 5'-phosphate. the different forms of vitamin B,. Clirr. Chirm Acta 40, Biochim. hiophys. Acta 304. 765-773. 3 19-324. H., TABORDA E. & PEREZ TISELIUS,H.-G. (1973) Metabolism of tritium-labelled TAPIAR.. PASANTFS-MORALES DE LA MORA M. (1975) Seizure susceptibility in the pyridoxine and pyridoxine S'-phosphate in the central developing mouse and its relationship to glutamate nervous system. J . Netooclrem. 20, 937-946.
OJ
.~~,~,~~,~l~,,,,,,,f~, Vol 31. pp 1403-l4lO
003-30.12 7X 1201- I$03SO? 00 0
Pergamon Pres, Ltd 1978 Printed in Great Britain Society for Neurochemi3try Ltd
6 lnternaiional
THE STABILITY OF VITAMIN B, ACCUMULATION AND PYRIDOXAL KINASE ACTIVITY IN RABBIT BRAIN AND CHOROID PLEXUS REYNOLDSPECTOR and STANLEY N . SHIKUMA Division of Clinical Pharmacology, Department of Medicine, University of Iowa Hospitals and Clinics, Iowa City, IA 52242, U.S.A. (Rrctiioed
4 April 1978. Accepted 9 Jurir 1978)
Abstract-The effects of changes in the concentrations of pyridoxal phosphate and blogenic amines in brain on: ( I ) pyridoxal kinase (EC 2.7.1.35) activity in brain and choroid plexus; and (2) vitamin B, accumulation by brain slices and isolated, intact choroid plexuses were studied. New Zealand white rabbits were treated parenterally with 200 mg/kg pyridoxine-HCl for 3 days or 120mgjkg 4-deoxypyridoxine HCI or 5 mg/kg reserpine I day before death. After these treatments the mean concentration of pyridoxal phosphate in brain was elevated by 39% by pyridoxine and decreased by 570~by 4'-deoxypyridoxine. Reserpine had no effect. However, the ability of brain slices and isolated. intact choroid plexuses from the treated rabbits to accumulate [3H] vitamin B, (with [3H]pyridoxine in the medium) was not different from untreated controls. Also, the specific activity of pyridoxal kinase in brain and choroid plexus of treated rabbits was not different from controls. These results show that vitamin B, accumulation and pyridoxal kinase activity in brain and choroid plexus are independent of both pyridoxal phosphate and reserpine-sensitive biogenic amine concentrations in brain. Itr cifro studies with pyridoxal kinase showed that. in both choroid plexus and brain. pyridoxal kinase was a single enzyme with a molecular weight of 43.000 and a K, for pyridoxine of 2 . 0 ~ Crude ~ . and partially-purified pyridoxal kinase from brain was not inhibited by biogenic amines ( 1 mM) or pyridoxal phosphate ( 5 p ~ )These . if1 citro data are consistent with the lack of effect of changes in pyridoxal phosphate and biogenic amine concentrations (in brain) on pyridoxal kinase activity in brain iii cico.
THETOTAL vitamin B, (B,)' concentration in mam- doxine in the diet) and pyridoxamine-P can be oximalian brain is determined by the balance between dized t o pyridoxal-P by pyridoxamine phosphate oxi1 9 7 8 ~ )Excessive . concensaturable transport, binding and metabolic intercon- dase (EC 1.4.3.5) (SPECTOR. 1978a. trations of the phosphorylated B, vitamers are versions of the various B, vitamers (SPECTOR, & GREENWALD. 1978). Although B, in dephosphorylated intracellularly and transported out b; SPECTOR brain exists predominantly in the enzymatically active of brain cells (SPECTOR, 197%). No metabolism of phosphorylated forms (pyridoxal-P and pyridoxa- E3H]B6 to thc inactive [3H]pyridoxic acid (or its lacmine-P). the nonphosphorylated B, vitamers readily tone) was detected in brain or choroid plexus (SPECcross the blood-CSF barrier (the choroid plexus) and TOR. 197%; SPECTOR & G R E E K W A L D . 1978). possibly the blood--brain barrier by a saturable transIri uiuo, rabbit brain pyridoxal kinase activity has 1973; SPECTOR. been reported t o be affected by various drugs, diet port system (Loo. 1972; TISELIUS. 1978a, b). Once within the C S F and extracellular and development ( E B A D I et al., 1968, 1970. 1973; space of brain, the three nonphosphorylated B, EBADI.1972; McCoy et al., 1972). For example. reservitamers readily enter brain cells by diffusion (or faci- pine, amphetamine and vitamin B, deficiency induced litated diffusion) and are trapped intracellularly after by 4'-deoxypyridoxine were reported to increase pyriphosphorylation by a single enzyme, pyridoxal kinase doxal kinase activity and decrease pyridoxal phos(EC 2.7.1.35) (SPECTOR, 19786; SPECTOR& GREEN- phate levels in rabbit brain, whereas chlorpromazine WALD. 1978). Intracellular pyridoxine-P (from pyriand pyridoxine had the opposite effects (EBADIet a]., 1968, 1969, 1970, 1973). During development the con' The nomenclature for the 6 active B, vitamers (Be) centration of pyridoxal-P in brain was reported to and related compounds is that of the IUPAC-IUB Com- decrease, whereas pyridoxal kinase activity increased mission of Biochemical Nomenclature (Errr. J . Biochrm. (EBADI,1972). These results led to the hypothesis that (1973) 40. 325 327). The 6 active forms are pyridoxine. brain pyridoxal kinase activity is inversely related to pyridoxal. pyridoxamine, pyridoxine 5'-phosphate (pyriboth brain pyridoxal-P and biogenic amine levels doxine-P), pyridoxal 5'-phosphate (pyridoxal-P), and pyridoxamine S'-phosphate (pyridoxamine-P). The principal in- ( E B A D I et d., 1968, 1970, 1973; EBADI. 1972). H O W active metabolites of B, are pyridoxic acid and pyridoxic ever, some of these results may have been due to methodological artifacts (NEARY et al.. 1972). Moreacid lactone. Abbreoiarions used: B,, B, vitamers; T/M. tissue-to- over. other investigators have shown either no relationship between brain pyridoxal kinase activities and medium. 1403 \.r 31 6
ii
1404
REYNOLDSptcroR and STANLEY N. SHIKUMA
measured by the method of Loo & BADGER(1969). The concentration (pmol/kg wet weight) of pyridoxal-P was calculated as the difference between the concentration of the total pyridoxal and the concentration of endogenous pyridoxal. The ability of slices of rabbit cortex and medulla and of choroid plexus to accumulate C3H]B, after pretreatment of the rabbits with parenteral4'-deoxypyridoxine, reserpine and pyridoxine (as described above) was investigated by methods previously described in detail (SPECTOR,1978h). Briefly, about 25mg of doubly-washed 0.25mm slices of cortex or medulla from each rabbit were incubated in 3 ml artificial CSF containing 5 mM-glucose. 40 n~-['H]pyridoxine and 0.0, 0.3, 0.8, 2.0 or 200 phi-pyridoxine for 30 min at 37°C in a metabolic shaker under 95% 0 2 :5% C 0 2 (SPECTOR.1978h). From each rabbit four 25mg samples from each brain region at each concentration were individually incubated in 3 m l medium and then filtered and washed. The 'H radioactivity in the slices (minus the appropriate blanks) and in the medium was determined. A Hofstee transformation of the saturable accumulation MATERIALS AND METHODS (by the brain slices) of B, from the medium (pmol per ['HIPyridoxine (2.8 Ci/mmol), prepared by an exchange 30 min per kg wet weight) and the concentration of ['HIprocedure, was obtained from Amersham-Searle, Arlington pyridoxine plus unlabeled pyridoxine ( p ~ in) the medium Heights, IL. All 6 B, vitamers, except pyridoxine-P which was performed (using linear regression analysis) t o obtain was synthesized (SPECTOR.1978h). 4'-deoxypyridoxine the transport constants, K , and V,,,,,, (SPFCTOR& GRfES(deoxypyridoxine),4'-pyridoxic acid, L-norepinephrine, D.LWALD. 1978; SPECTOR 19786). The K , can be interpreted norephedrine, reserpine and serotonin creatinine sulfate as the concentration of pyridoxine in the medium required were obtained from Sigma Chemical Co., St. Louis, MO. to half-saturate the intracellular accumulation of B,, and 5'-Deoxypyridoxine was a gift from Dr. C. C. PORTERthe b',,, as the maximal number of pmoles of B, per (Merck). 30 min per kg wet weight that can be accumulated by the All studies were performed on 1.4-2.0kg New Zealand saturable accumulation system (SPECTOR& GREENWALD. White rabbits. All rabbits were maintained on Purina Rab1978). bit Chow Checkers R and H,O ad lib. until about 2 h Choroid plexuses from these and other similarly-treated before death. All injected rabbits were killed 18 h after the rabbits were incubated in 3 ml artificial CSF containing final injection. Eighteen rabbits were injected once with 5 mM-glucose and 40 nM-['H]pyridoxine at 37°C for 30 5 mg/kg reserpine intravenously, 17 rabbits were injected min under 95% 0,: 5% CO,. At the end of the incubation once subcutaneously with 4'-deoxypyridoxineeHCI, and 16 the concentration of ['H]B6 in the choroid plexus divided rabbits were injected at 9:OO a.m. and 4:OO p.m. for 3 days by the concentration of ['Hlpyridoxine in the media (T,'M) with 100 mg/kg pyridoxine-HCl intravenously. Twenty- was determined (SPECTOR& GREENWALD, 1978). one control rabbits were injected once with buffer. The activity of pyridoxal kinase in brain cortex and Beside whole rabbit brain, 4 regions were utilized in the choroid plexus after the injection of 4'-deoxypyridoxine, following studies: the region of the brain above the lateral reserpine and pyridoxine as well as the regional distribuventricle including cortex, white matter, and periventricu- tion of pyridoxal kinase was assayed by measuring the lar grey matter (henceforth termed 'cortex.') the medulla, conversion of ['Hlpyridoxine to [3H]pyridoxine-P by the lateral one-fourth of the cerebellum (henceforth termed methods previously described in detail (CHERN & BEUTLER, 'cerebellum,') and the lateral and fourth ventricular choroid 1 9 7 5 ~ ;SOLOMON & HILLMAN, 1976; SPECTOR& GREENplexuses WALD,1978). Briefly, 0.251111 of the chilled supernatant The effect of 4'-deoxypyridoxine, reserpine and pyri- (30,OOOg for 20min)' of about 5 mg brain or 1 mg choroid doxine injections on the concentrations of pyridoxal and plexus, previously homogenized in 50: 50 artificial CSFpyridoxal-P in cortex or whole brain was measured by 0.1 M-potassium phosphate, pH 6.5 (henceforth termed methods previously described in detail (SRIVASTAVA & 'phosphate-CSF'), was added to 0.05 ml 10 mM-MgC1,. BEUTLER. 1973; SPECTOR, 1978a). Briefly, immediately after 0. I ml 10 mM-sodium ATP in 0.05 M potassium phosphate death, the total amount of pyridoxal-P and pyridoxal in buffer (pH 6.5). and 0.35ml artificial CSF (SPECTOR& homogenates of about lOOmg weighed samples of brain GREENWALD, 1978). After the solution had warmed to was measured in duplicate (SPECTOR,1978~).The pyridox- 3 7 T , the reaction was begun by adding 40 pmol C3H]pyrial-P in the homogenates was first dephosphorylated with doxine and 0, 2, 4, or 8 nmol pyridoxine in 0.05 ml potasacid phosphatase, and the total pyridoxal (endogenous pyr- sium phosphate buffer, pH 6.5 (SPECTOR& GREENWALD. idoxal and pyridoxal from dephosphorylated pyridoxal-P) 1978). After 1Omin at 37°C in a shaker, the reaction was was measured as the highly fluorescent pyridoxal semicar- stopped by adding 0.3 ml of chilled 0.6 M-metaphosphoric barsone (SPECTOR, 1978~). Endogenous pyridoxal was acid. The ['Hlpyridoxine-P and any [3H]pyridoxal-P was then separated from the C3H]pyridoxine on Amberlite CG-120 columns (SPECTOR.1978a). This assay without Centrifugation of the homogenates at 50,OOOg for 1 hr added unlabeled pyridoxine is termed the standard pyridoxal kinase assay. In some assays no artificial CSF was at 4°C did not alter the results of the standard pyridoxal used; instead the tissues were homogenized in 0.07 Mkinase assay.
pyridoxal-P (MCCORMICKet al., 1961) or a direct rather than an inverse relationship (LOO, 1972; TAPIA rt al., 1975; BHAGAVAN et al.. 1977). The purpose of the present studies was t o quantify the relationships between pyridoxal kinase activity, pyridoxal-P levels, a n d the recently-described vitamin B, accumulation systems in brain a n d choroid plexus (SPECTOR, 1 9 7 8 ~ .h ; SPECTOR& GRFENWALD,1978). Alterations in the concentration of pyridoxal-P and/or biogenic amines were effected by treatment of rabbits with various drugs a s well a s pyridoxine. When the activity of pyridoxal kinase as well as the accumulation of B, by brain and choroid plexus were found t o be unrelated t o the concentrations of pyridoxal-P or biogenic amines in brain, further biochemical characterization of both rabbit brain a n d choroid plexus pyridoxal kinase was performed.
'
1405
B, accumulation and pyridoxal kinase activity in brain potassium phosphate buffer (pH 6.5). and all the reagents were prepared in the same buffer. The nature of the product derived from [3H]pyridoxine incubated with brain supernatant in the standard pyridoxal kinase assay was checked by a column chromatographic method (TISE.LI~,S. 1973). As the conditions in the standard pyridoxal kinase assay were chosen to stay on the linear portion of the velocity curve. Michaelis-Menten kinetic constants for the conversion of pyridokinc to pyridoxine-P were obtained by using linear regression analysis and standard Hofstce transformations (COLQI,HOLY. 1971) in units of /LM (K,) and pmol per kg wet weight of original tissue per IOmin In some assays. the effects of various amines. B, vitamers and B, analogues on pyridoxal kinase activity werc measured by the standard pyridoxal kinase assay except that the pH of the assay mixture was adjusted to 6.4. The molecular weight of pyridoxal kinase in 0.3 ml supernatants (30.OOO g for 20 min) of 25 mg choroid plexus or 50mg hrain cortes. previously homogenized in chilled phosphate~CSF. was determined by the method of AUDRI.WS (1965). Briefly. the chilled supernatants (0.3 ml) were applied to a 36cm Sephadex G I 0 0 column equilibrated with phosphate-CSF (in a 4-C room) and standardized with Blue Dextran, bovine transferrin. ovalbumin and horse heart cytochrome c (ANDREWS.1965).0.41111 fractions were collected. and the pyridoxal kinase activity of each fraction was determined as described above in the standard pyridoxal kinase assay. The molecular weight of the pyridoxal kinase was interpolated from the standard curve (AADKLWS. 1965).
(v,,,tJ.
RESULTS
In Table 1 are shown thc effects of parenteral 4'-deoxypyridoxine, reserpine, and pyridoxine on pyridoxal-P concentrations in rabbit cortex. Treatment with pyridoxine significantly raised the pyridoxal-P concentration when compared with controls. Treatment with 4'-deoxypyridoxine significantly lowcred the pyridoxal-P concentration. Reserpine had no significant effect. In whole brain (Table I ) parenteral 4'-deoxypyridoxine also significantly lowered the concentration of pyridoxal-P. TAIILt I
C O l C t \TKATIOL
Treatment Control (cortex) 4-Deoxypyridoxine Reserpine Pyridoxine Control (whole brain) 4'-Deosypyridoxine
OF
TABLL 2 TRAYSPORT CO\STA\TS
B,
ACT( MI LATIOI. H \
KT (PM)
(PmoL kg per 30 min)
0.15 f 0.03 0.08 & 0.01 0.22 & 0.03 0.15
1.36 i 0.22 1.31 f 0.08 1.52 i 0.20 I .3x
1111 I\
Treatment Control (4) 4'-Deoxypyridoxrne (3) Reserpine (3) Pyridoxine (2)
Values are means +s.L.M. with the number of experiments in parentheses. Rabbit brain slices obtained from treated rabbits were incubated in artificial CSF containing 40 n~-['H]pyridouine with various amounts of unlabeled pyridoxine for 30 min. Hofstee transformations of the saturable 30 min accumulation data were performed to yield the K , and V,,,.,, for B, accumulation (COLQIHOIX. 1971). The K , and V , , , for slices of medulla ( N = 2) were 0.20 pM and 0.75 pmol kg (30 min)- I . ~
'
The transport constants K,. and V,,,,,, for the intracellular accumulation of B, by rabbit brain cortex slices are shown in Table 2. There was no significant difference from controls in the average K , or L:,,,,, in rabbits pretreated with 4'-deoxypyridoxine. reserpine. or pyridoxine. The effects of 4'-deoxypyridoxine. reserpine, and pyridoxine on the ability of isolated rabbit choroid plexuses t o accumulate r3H]B, with [3H]pyridoxine in the medium are shown in Table 3. The choroid plcxuses from the treated rabbits did not accumulate C3H]B, significantly differently from the controls. The tritiated product of the standard pyridoxal kinase assay cochromatographed with synthesized pyridoxine-P (TISELIUS. 1972). However. because of the imperfect separation of pyridoxine-P and pyridoxal-P, up to 10% of [3H]pyridoxine-P could have been oxidized to r3H]pyridoxal-P by pyridoxine phosphate & HILLMAN. 1976). This would not oxidase (SOLOMON affect the standard pyridoxal kinase assay as pyridoxal-P elutes with pyridoxine-P when the 'H-product is separated from [3H]pyridoxine on Amberlite CG120 columns (SPECTOR. 197th). No [3H]pyridoxic acid or [3H]pyridoxic acid lactone was detected.
PYRIDOXAL-P
ALD I'YWIDOXAL WHOLF BRAII.
Pyridoxal-P + Pyridoxal (pmol per kg)
FOR
RABBIT HRAII. CORTI Y
lh,
RAHHIT CORTf X A Z D
Pyridoxal-P (pmol per kg)
Cortex 6.5 f 0.5(4) 5.4 F 0.5 (4) 3.3 f 0.6* (3) 2.3 f 0.4* (3) 7.3 f 0.7 (4) 5.X f 0.8 (4) 8.8 f 0.3* (4) 7.5 f 0.4* (4) Whole brain 3.3 f 0.5(3) 4.5 f 0.7 ( 3 ) 2.9 f 0.2+(4) 1.9 & 0.11. (4)
1.1 i 0.2(4) 1.1 i 0.3(3) 1 5 f 0.1 (4)
1.4 f 0.2 (4) 1.2 f 0.7 (3) 1.0 f 0 I (4)
Values are means +s.E.M. with the number of determinations in parentheses. See methods for doses of drugs. * Values differ from respective controls with P < 0.05 [Dunnett's test. DLUXETT
(1955)l.
t Values differ from controls with P < 0.05 (Student's t-test, 2 tailed).
REYNOLDSPtCTOR and STANLEY N. SHIKUMA
1406
TABLE3. ACCUMULATIONOF [3H] B6 PLEXIJS
Treatment
BY RABBIT CHOROID
TABLE5. KINETICCONSTANTS
OF PYRIDOXAL KINASE CHOROID PLEXUS
in oitro
30-min TIM Treatment
Control 4'-Deox ypyridoxine
Reserpine Pyridoxine
5.39 f 0.63 (8) 6.26 f 0.57 (6) 5.51 f 0.48 (6) 5.86 f 0.75 (6)
K, ( b ~ )
Control (5) 4'-Deoxypyridoxine (5) Reserpine (3)
Pyridoxine ( 3 ) Values are means ~ s . F . . M .with the number of determinations in parentheses. Choroid plexuses were obtained from rabbits after various drug treatments and incubated in artificial CSF containing 40 n~-[~H]pyridoxinefor 30 min. The total ['HIB, concentration in the tissue divided by the total [3H]pyridoxine concentration in the media (T/M) was determined (SPECTOR & GRIXNWALD, 1978).
V",,, (PnoIfig
per 10 min)
2.0 f 0.3
121 f 20
*
144 f 10
1.8 f 0.2 2.0 0.2 2.0 f 0.2
ix
145 f 50 127 f I I
Values are means fs.t.M. with the number of determinations in parentheses. After various treatments choroid plexuses from each rabbit were removed and pooled. The K , and r/;,,,$, of pyridoxal kinase were then determined.
v,,,,
about 5 times and the by 2 times as compared Using the standard pyridoxal kinase assay with t o the K , and V,,,,, in the standard pyridoxal kinase (pyridoxal various amounts of unlabeled pyridoxine, the mean assay. Howcver, the ratio of the mean K , and V,,,.,, for the conversion of pyridoxine t o pyri- kinase) from choroid plexus of the reserpine-treated doxine-P by unpurified supernatants of brain cortex rabbits divided by the V,,,,, in the control was not were calculated and are shown in Table 4. There was significantly altered (1 2 0 in the standard pyridoxal no significant difference in the mean K , or V,,,,,, kinase assay; 1.30 in potassium phosphate buffer). between control and 4'-deoxypyridoxine-, reserpine- Similar results were obtained in cortex. The molecular weight of cortical pyridoxal kinase or pyridoxine-treated rabbits. Medulla had significantly less ( P < 0.05) pyridoxal kinase activity (per (from brain) as determined by Sephadex column chromatography (ANDKEWS,1965) is shown in Fig. g) than cortex (Table 4). Using unpurified choroid plexus supernatants, I . Pyridoxal kinase appeared as a sharp single peak there was also no significant difference in the mean that emerged just after ovalbumin. The extrapolated K , and V,,,,,, for pyridoxal kinase between control and molecular weight was 43,000. The pyridoxal kinase 4'-deoxypyridoxine-. reserpine- or pyridoxine-treated of choroid plexus and of rabbit red blood cells rabbits (Table 5). behaved identically, i.e. emerged from the column as In 2 control and 2 reserpine-treated rabbits pyri- a sharp single peak with a molecular weight of 43,000. doxal kinase activity was measured in cortex and The effects of several B, vitamers, B, analogues choroid plexus using only 0 . 0 7 ~potassium phos- and biogenic amines on unpurified pyridoxal kinase phate buffer (pH 6.5) instead of the buffers in the activity from cortex are shown in Table 6. Pyridoxine standard pyridoxal kinase assay. Concentrations of (5 p ~ ) , pyridoxal (5 p ~ )and 4'-deoxypyridoxine 5, 10 and 20pM unlabeled pyridoxine were used. In (10 p ~ significantly ) decreased pyridoxal kinase acthe control and reserpine-treated rabbits the mean K , tivity, whereas pyridoxal-P (5 PM), 5'-deoxypyridoxine and in cortex were 10.6 p~ and 148 pmol/kg per ( I O O ~ M )and the amines ( 1 . 0 m ~ )had n o significant IOmin and 1 4 . 8 and ~ ~ 199pmoljkg per IOmin. re- effect. Addition of sodium ascorbate (5mM) t o the spectively; in choroid plexus the mean K , and assay tubes containing serotonin (to prevent oxivalues (in the control and reserpine-treated rabbits) dation) did not alter the results. In one experiment were 9 . 7 and ~ ~242pmoljkg per IOmin and 1 0 . 7 ~ serotonin ~ (1.O mM) and norepinephrine (1 .O mM) did and 3 I7 pmol/kg per 10 min, respectively. Thus, per- not significantly affect the activity of partially purified forming the pyridoxal kinase assay in potassium pyridoxal kinase from cortex. The enzyme was puriphosphate buffer increased the K , in both tissues by fied by ammonium sulfate fractionation (MCCORMICK
v,,,,
v,,.,,
v,,,,
TABLL 4. KINETIC CONSTANTS Region Cortex (6) Cortex (4) Cortex ( 3 ) Cortex (3) Cerebellum (4) Medulla (4) Whole brain (2) Whole brain (2)
OF I ~ Y R I D O X A L KINASE FROM B R A I N
Treatment -
4-Deoxypyridoxine Reserpine Pyridoxine ~
~-
~
-
4-Deoxypridoxine
K,
01~)
2.2 f 0.2 2.1 f 0.1 2.4 f 0.3 2.2 & 0.2 1.9 f 0.1 1.9 f 0.1 2.0
2.2
(pmolfig per 10 min) 69 f 5 16 f 2 78 f 6 83 + 4 65 f 5 51 f 2
75 84
Values are means ~ s . E . Mwith . the number of determinations in parentheses. After various treatments rabbit brains were removed and the K , and V,,,, of pyridoxal kinase were determined in supernatants of brain cortex and of various regions of the brain.
B, accumulation and pyridoxal kinase activity in brain
1407
lation by brain slices and choroid plexus are not changed significantly by altering the concentrations of pyridoxal-P or biogenic amines in brain and (2) In hi hi tor Per cent control that pyridoxal kinase from brain is similar, if not identical, to pyridoxal kinase from choroid plexus. Control I 00 (7) Pyridoxine. 5 PM 26 (2)* Before discussing these results. several technical Pyridoxal, 5 PM 15 (2)* matters require comment. First, the method employed Pyridoxal-P. 5 PM I 1 I (2) in this study to measure pyridoxal-P in brain is quan4'-Deoxypyridoxine. 10 PM 25 f 1 (6)* titative (SRIVASTAVA & BEUTLER.1973). This is impor5'-Deoxypyridoxine. 100 PM 104 k 3 (4) tant because previously employed methods using perSerotonin. 1.0 mM 85 f 2 ( 6 ) Norepinephrine. 1.0mM 97 f 3 (6) chloric or trichloroacetic acid d o not quantitatively Norephedrine. 1.0mM 103 f 3(4) extract the pyridoxal-P from tissue (SPECTOR.1 978a. Values are means k S.E.M. with the number of deter- h). Second, metaphosphoric acid was employed in our minations in parentheses. In each experiment duplicate pyridoxal kinase assay to maintain the [3H]pyridoxsupernatants from about 5 mg brain cortex were incubated ine-P intact. Use of other protein precipitating agents at 37°C in 0.8ml buffer (pH adjusted to 6.4) containing such as perchloric acid does not quantitatively mainATP, Mg'+. ['Hlpyridoxine and. in some cases, various & GREI~Sinhibitors as in the standard pyridoxal kinase assay. After tain [3H]pyridoxine-P intact (SPECTOR WALD. 1978). Third. the conversion of [3H]pyridoxine 10 min the d.p.m. converted to ['Hlpyridoxine-P per mg original tissue were determined. In the control assays to [3H]pyridoxine-P was employed to quantify pyri8094 i 234d.p.m. (s.E.M.; N = 7) per mg tissue were con- doxal kinase activity because neither pyridoxine nor verted to ['Hlpyridoxine-P. The control samples were pyridoxine-P bind significantly to or interact with tisarbitrarily set at lOOS/,. 1978). Pre* P < 0.05 by Scheffe's method for multiple comparisons sue components (SPECTOK& GKEESWALD. vious studies employing the conversion of pyridoxal in the Gaussian analysis of variance (COLOI~HOL'N. 197!). (as substrate) to pyridoxal-P as a measure of pyrirt a/.. 1961) and then passed through a Sephadex doxal kinase activity may be invalid because of interG-100 column as described above. The two most actions between pyridoxal-P and proteins or biogenic active fractions were pooled and used for the pyri- amines. NEAKYrt al. (1972) showed that pyridoxal-P doxal kinase assay as in Table 6. interacts with biogenic amines such as dopamine and norepinephrine and suggested that the previously reported inhibition of pyridoxal kinase activity by DISCUSSION biogenic amines (EBADIet al.. 1968, 1973) with the The principal findings reported herein are ( I ) that possible exception of serotonin was due to measurepyridoxal kinase activity and vitamin B, accumu- ment artifacts. i.e. interactions between the amines and the reaction product. pyridoxal-P, rather than an inhibition of pyridoxal-P production by a direct effect of biogenic amines on pyridoxal kinase. Measurement of pyridoxal kinase activity by the decline of the s u b strate concentration. thc appearance of ADP. or the conversion of pyridoxine to pyridoxine-P avoids the difficulties inherent in quantifying the appearance of >" Pvridoxal kinase pyridoxal-P as a measure of pyridoxal kinase activity (CHERN & BEC'TLEK. 197%; NEARYrt d.. 1972). Our results confirm the previously reported erects of 4'-deoxy-pyridoxine and pyridoxine on pyridoxal-P levels in whole brain and cortex (Eearx t't d..1970). 1 0 Reserpine did not alter pyridoxal-P levels (Table I ) . I I 1 10,000 25.000 50.000 75!000 100,000 Although we did not measure the levels of biogenic M o l e c u l a r Weight amines in the reserpine-treated rabbits. the behavioral FIG. 1. Determination of the molecular weight of brain effects (lethargy, weakness, etc.) were clear and consispyridoxal kinase by gel filtration. A 36 x 0.6 cm Sephadex tent with a severe depletion of biogenic amines (ERADI G-100 column was standardized with Blue Dextran (void et a/.. 1968). volume). bovine transferrin (68,000), ovalbumin (45.000). Previously. we have shown that both isolated cliorand horse heart cytochrome C (12,400) by the method of oid plexuses and brain cortex slices from rabbits ANDREWS(1965). After standardization. 0.3 ml of superna- accumulate ['HIB, whea [3H]pyridoxine is present tant of brain was applied to the column and fractions were in the medium (SPECTOR. 1978h). The accumulation collected. The pyridoxal kinase activity i n each fraction systems in both tissues are saturable and energywas determined by the standard pyridoxal kinase assay. Shown is the V, (elution volume) divided by the V, (void dependent (SPECTOR.197Xh). In both cortex and chorvolume) as a function of molecular weight ( A N D R ~ W S . oid plexus from 70 to 80:" of the ['HIB, within the 1965).The VJV0 for pyridoxal kinase was 1.23 which extra- tissues is phosphorylated after a 30 min incubation in 40-50 n~-[~H]pyridoxine. In both tissues the polates to a molecular weight of 43.000. TABLI6.
EFFrCT OF VARIOUS AMINtS. B, VITAMf RS A I D ANALOGL'ES OY BRAIN PYRIDOXAL K I \ A S t ACTIVITY
OLD SPECTORand STANLEY N. SHIKUMA
metabolism of vitamin B, in rabbit brain and choroid decarboxylase and pyridoxal phosphate in brain. J . plexus. J . biol. Chem. 253. 2373-2378. Nwrohiol. 6, 159- 170. SRIVASTAVA S. K. & BEUTLER E. (1973) A new fluorimetric TIS~LIIJS, H.-G. (1 972) A chromatographic separation of method for the determination of pyridoxal 5'-phosphate. the different forms of vitamin B,. Clirr. Chirm Acta 40, Biochim. hiophys. Acta 304. 765-773. 3 19-324. H., TABORDA E. & PEREZ TISELIUS,H.-G. (1973) Metabolism of tritium-labelled TAPIAR.. PASANTFS-MORALES DE LA MORA M. (1975) Seizure susceptibility in the pyridoxine and pyridoxine S'-phosphate in the central developing mouse and its relationship to glutamate nervous system. J . Netooclrem. 20, 937-946.
