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C L IN . C HE M . 2 2/ 7 , 9 7 2- 97 6( 1 97 6)
972 CLIN ICA LCHEM ISTRY,V o l. 2 2 , No . 7 , 1 976
A lkalin e P ho sphatase. I. K in etics and In hib itio n b
yLevam iso le of P urified lsoenzym es from HumansHerman V an B
elle
I s tud ied the kin etic s a nd s en sitiv ity to wa rd inh
ibitio n b ylevam iso ie and R 8231 of the m ost im portant hum an
al-k alin e p ho sp ha ta se is oe nz ym es . N -E th ylamin oe th
an olp ro ve d su pe rio r to th e n ow w id ely us ed d ie th an
ola mineb uffe r, e sp ecia lly fo r th e e nzym es from th e inte
stine an dplacenta, behaving as an uncom petitive activator. T heo
ptim um pH Ia rg ely de pe nd s o n th e s ub stra te c on ce ntra
-tion . T he a dd ition o f M g2 + h as n o e ffe ct o n th e a
ctivities .T he m eaning of K m-values for a lka line phosphatases
isq ue stio ne d. ls oe nz ym es from h uman liv er, b on e, k id
ne y,and sp leen are s tro ng ly in hib ite d b y le vamis ole o r
R 8 23 1a t co nc entra tio ns th at b arely a ffe ct th e e nzym
es fro m in -te stine or p la ce nta. T he in hib ition is ste re
osp ecific, u n-competitive, and not changed by Mg2. Inh ib ition
isco un te ra cte d b y in cre asing co nce ntra tio ns o f N -e th
yl-am in oe th an ol. T h e mech an ism o f in hib itio n is s ug
ge ste d tob e fo rm atio n o f a co mp le x w ith th e p hos pho
en zym e.Add it iona l Keyphrases: n itr op he no llP , r ele as e
r atio #{149}N-eth yla min oeth an ol b uffe r vs . d ieth an olam
ine b uffe r #{149}soen-zym es of h um an o rig in #{149}Km va lues
, and i nt erp re ta ti on .mech an ism and o rg an s pe cific ity
o f in hib itio n
Although there are many reports on human alkalinephosphatases
(for a review , see 1) , few of them areconcerned w ith the
kinetics of the isoenzymes isolatedfrom liver, kidney, bone,
placenta, and intestine si-multaneously-i.e., under identical
conditions (2). Theknowledge of their k inetics, espcially in the
phos-phorylatable (3 ) buffers NEAE (N-ethylam i-noethanol) or DEA
(diethanolam ine), which are nowwidely used for the measurement of
serum alkalinephosphatase activ ity , may contribute in defining
theoptim al conditions of a referee method for this enzymein s eru
m.
Levam isole and R 8231 are known to be potent,organ-specific
inhibitors of alkaline phosphatases in thedog (4) and the rat (5).
A study on the sensitiv ity of theisoenzymes of human origin could
provide useful in-formation on the potentia l application of this
class ofinhibitors to the discrim ination of the isoenzymes inserum
. Such a study is the subject of this paper.- Laborato ry of B
iochem istry , Janssen Pharm aceu tica, ResearchL aborato ries ,
2340-B eerse, B elg ium .Nonstandard a bbre via tio ns us ed : N EA
E, N -e thy la min oe th ano l;DEA , d iethano lam ine; and P,
inorganic phosphate. In accord w ithconvention, brackets signify
concen tration; e.g., [SI m eans concen-t ra ti on o f s ub str at
e. R eceived Jan. 27, 1976; accepted A pril 6, 1976
In the accompanying paper, the applicability toserum of the
findings on isolated isoenzymes w ill bedescribed.M ateria ls and M
ethods
The human tissues w ere obtained from autopsy ma-terial, excep t
the placenta, w hich w as u se d imm ed ia te lyafter delivery. The
enzyme from bone was purified fromthe tibia and femur of a
five-month-old fetus aftersp on ta ne ou s ab ortio n.
The purification involved extraction w ith n -butanolof a m
icrosomal fraction of the tissues followed by gelchromatography
through Sephadex G -200. For con-centration we used Am icon-100
filters instead of theusual acetone or ammonium sulfate precip ita
tion steps.Phosphate buffer (20 mmol/liter, pH 7.8) containing
1mmol of M g2+ per liter was used throughout the wholeprocedure,
because it m arkedly increased the yields,either because of product
protection (6) or less ad-sorption to the column material. Before
assay , the en-zymes were diluted in tris(hydroxymethyl)am
ino-methane (10 mmol/liter, pH 8.0) containing 1 mmol ofMg2 per
liter. The specific activities-i.e., imol of ni-trophenol
released/mm per m illigram of protein inN EA E (100 m mol/liter, pH
10.2) w ith ES ] = 5 m mol/liter)-w ere: 31.2 (intestine), 7 .0
(liver), 3 .6 (kidney),2.4 (spleen), 100.6 (placenta), and 58.6
(bone).
The release of p -nitrophenol or P from p -nitro-phenylphosphate
was measured with either the Beck-man 25-K inetic System or the
Technicon AutoAnalyzeras described previously (4). Unless otherw
ise specified ,the experim ents were performed at room
temperature(22 1 #{176}C).
Levamisole [( - )-2,3,5,6-tetrahydro-6-phenylim-id az o(2 ,1 -b
)th ia zo le h yd ro ch lo rid e, M r 240.75] and R8231 [ ( ) -6
(m-b romophenyl )- 5 ,6 - di hyd ro im idazo -(2 ,1 -b )th iaz ole
o xala te, Mr 371.22] are products ofJanssen Pharmaceutica Research
Laboratories.ResultsE ffe ct of B uffe r C om po sitio n
Borate, glycine, and NEAE buffers, all at 50 mmol/liter, were
compared at 1.25 , 2 .5, 5, and 10 mmol/litersubstrate
concentrations and at pH 9.8 and 10.4. Underall these conditions
the NEAE buffer proved to be su-
8/2/2019 Levamisole (1976)
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aReLActivity10 0
so
a
[5 3V
a-
N P ra tior5
F .
1- 3
1- 2
I-I
9
, NEAE100mmo(/ti ter1 x lO6mot/ t iterR8231
100 200 #{163}00 600 600 1000[NEAEJmmot/t iter
NIP ra ti oi
-1.
Ret.Activity10 0
50
S
- a-
-a.-vS.
a I1- 3--a-
1-2
I-I
I -
[N AJmmol/t i ter-100
20 040 080 0
10.0[S] mmol / l ite r
Fig.2. Plot o f [ SI /V v s. E S] , sh ow in g u nc om pe ti ti
i nh ib it io nby R 8231 and uncom petitive activa tion by NEAE of
human l iveralkaline phosphatase
-5.0 0 1 .25 2.5 5.0
CLIN ICAL CHEM ISTRY, Vol. 22, No. 7 , 1976 973
100 200 #{163}00 600 60 0 1000[NEAEJmm0I/I iter
F ig . 1 . P lo ts o f th e re lativ e a ctiv itie s a nd th e
ra tio n itro ph en olre leased/P1 re leased (NIP) v s. t he
concentration o f N EA E fo r th eisoenzymes from (a ) intestine (S
) and placenta (0 ) a nd (b ) bonean d liver(mean for both isoenzym
es, w hich behae alm ostidentically)p H o f th e b uf fe r, 1 0 ; s
ub st ra te c on ce n tr at io n, 5 mmo l/ Il te r
perior, the enzyme activity being from two- to fivefoldgreater
than in the other buffers.E ffe ct o f B uffer C on ce ntra tio
n
At pH 10 and 5 mmol/liter substrate concentration,all isoenzyme
activ ities strongly depend on the NEAEconcentration (F igure 1).
This effect is largely due to thewell-known transphosphorylation,
as apparent from theratio nitrophenol released/Ps released . M
oreover, theeffect of increasing concentrations of the buffer is
in-fluenced by both pH and substrate concentration. D e-creasing
the pH enhances the activation while de-
creasing the substrate concentration lessens the effectinsofar
that, at the lowest substrate concentrationstested, increasing
concentrations of NEAE even seemto inhibit enzyme activity; that
is, the ratio
activ ity NEAE 1000 mmol/liter
