Isolation and characterization of ovine serum alkaline phosphatases

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BIOCHIMICA ET BIOPHYSICA ACTA BBA

65277

ISOLATION AND CHARACTERIZATION OF OVINE SERUM ALKALINE PHOSPHATASES

OLE AALUND', JAN RENDEU* AND R. A. FREEDLAND Department of Veterinary Microbiology and Department of Physiological Sciences, School of Veterinary Medicine, University of California, Davis, Calif. (U.S.A.) (Received March 30th, Ig6S)

SUMMARY

The ovine serum alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) isozymes A, B, and C were fractionated by gel filtration on Sephadex G-zoo and by anion-exchange chromatography on DEAE-Sephadex. Alkaline phosphatase activity was confined to the middle peak of the Sephadex G-200 chromatogram. Sephadex G-200 had an exclusion value of 200 000, and it is concluded that the alkaline phosphatases have a molecular size not exceeding the average molecular size of molecules with molecular weight 200000. The Bphosphatase was almost entirely confined to sera from sheep of blood group O. However, the majority (68.5 %) of blood-group 0 substance in O-serum was found in the first peak of the Sephadex G-zoo chromatogram, whereas only I7.9% of this substance was found in the second peak. It is likely, that therefore, the alkaline phosphatase isozymes in serum do not exist in a complex with blood-group 0 substance. The K m ' values for p-nitrophenyl phosphate were found to be quite different for the A- and B-phosphatases. The P0 43--inhibition was non-competitive, and the mechanism involved in this phenomenon is discussed; it is suggested that the inhibition might be due to reactivity at an allosteric site which would be consonant with the observation of non-competitive inhibition by phosphate in the presence of excess Mg 2+. The L-phenylalanine inhibition was higher for O-serum than for Rserum, 70% and 56% inhibition respectively, suggesting an intestinal origin of the B-phosphatase. EDTA inhibited both 0- and R-serum IOO%. Some S042--inhibition (non-competitive) was noted with both 0- and R-seni..

INTRODUCTION Using starch-gel electrophoresis RENDEL AND STORMONTl detected three Abbreviation: NPP, p-nitrophenyl phosphate. * Present address: Royal Veterinary and Agricultural College, Department of Hygiene

and Bacteriology, Biilowsvej I3, Copenhagen V (Denmark) . •• Present address: Agricultural College, Uppsala (Sweden).

Biochim; Biophys. Acta,

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O.AALUN D,

J. RE NDEL,R. A. FREEDLAND

alkaline phosphatase (orthophosphoric monoester phosphohyd rolase, EC 3.1.3.1) isozymes in the serum of sheep. The phosphatase patterns were found to be closely correlate d wit h the blood t ype wit hin the R-O-i sys tem. Phosphatase B was almost entirely confined to sera from sheep of blood gro up 0 (or R), while phosphatase A occurred in all sh eep . The intensity of the slowest phosphatase zone (C) varied considerably . In a later report >the total alkaline phosphatase activity was shown to be much higher in sera from shee p of blood group 0 t han in anim als of groups R or i. The fact that the ovin e phosphatase isozym es had different mobilities in starch-gel electroph oresis suggests that it might be possibl e to separate th ese isozyrnes by anion ex change colum n ch romat ograp hy . PETERSON AND CHIAZZE 3 fractionated human serum on DEAE-cellulose and demonstrated " a progr essive increase in the electrophoretic migration of the proteins in successive chromat ographic fra ctions, until the end of the major albumin peak is reached" . In the present study the ovine serum alkaline phosphatases were fractionated on DEAE-Sephadex. The phosphatases were id entified by starch-gel electrophoresis . Gel-filtration on Sephadex G-200 was used to obtain an approximate estimate of the molecular size of the pho sphatases. They were also subjected to comparative kinetic studies. MATERIALS AND METHODS Three different ser a from adul t sheep (1-012 , blood group 0 ; 2392, blood group R ; and 3-I21 , blo od group 0 ) representing the three different phosphatase phenotypes (A-B-C-, A-C, and UX " ) were chromatographed, and a number of 0- and R-sera subjected to kinetic studies. Chromatography Anion-exchan ge column chromatography w as performed as described by SOBER AND PETERSON 4 and by FAHEY et al:". DEAE-Sephadex was used instead of DEAE cellulos e, and the elution was performed wit h the cone-cone phosphate gradient described by AALUND et al.a DEAE-Sephadex A-so (Pharmacia) (2.8 g dry wt .), particle size IOO-270 mesh, cap acity 3.5 mequivJg was used in a 1.1 X 38.0 em column. Gel filtration Gel filtration on Sephadex G-200 was done on a Sephadex preparation with a particle size of I4o-400 mesh using the technique des cribed by FLODIN' and b y GELOTTE et al», A 7.0 X 70.0 ern column was used and 'Iri s-buffer (pH 8.0) (0.1 M Tris (hydroxymethyl) aminomethan e 1.0 M NaCl) was used as eluent.

+

Protein determinaiioa. Quantitativ e protein determination in the chromatographi c fractions was don e byread ing the absorb an ce at 280 m,u on a Beckman DB spectrophoto meter. Electrophoresis and alkaline ph osphatase stai ning Starch-gel electrophoresis and alkaline phosphatase st aining were p erformed as described by GAHNE9, and RENDEL et al. 2 • E ach gel was sliced into t wo halves B iochim, B iophy s. A cta, lIO (196 5) II3-1 23

OVINE SERUM ALKALINE PHOSPHATASES

II5

parallel to the surface of the gel. The top half was then stained for alkaline phosphatase and the bottom half for protein with arnidoblack. Q~tantitative alkaline

phosphatase analysis Quantitative alkaline phosphatase analysis was performed by a modification of the method described by GAREN AND LEVINTHAL 1 0 • The reaction mixture was as follows: 0.8 ml of Tris-buffer (pH g.o), 0.1 ml of NPP solution (IO ,umoles or Iess/ml in the above Tris-buffer), and O.I ml of serum or other sample. The rate of release of p-nitrophenol from NPP was determined following the increase in absorbance at 400 mfl using a Gilford Model 2000 Multiple Absorbance Recorder. Kinetic analysis The kinetic analysis was performed in the following way: 0.8 ml of 0.1 M Tris-buffer (pH 9.0), 0.1 ml of serum appropriately diluted according to the original activity, and varying amounts of NPP diluted in the Tris-buffer. All inhibitors and activators were dissolved in Tris-buffer and added to the reaction mixture at the expense of the Tris-buffer, Values of apparent K m (K m /) and apparent Kl (K1') were determined by the method of LINEWEAVER AND BURK ll. The following concentrations of substrate were used (final concentrations) : I ' IO- 3 M, 5' ro- 4 M, 3.33' IO- 4 M, and 2.5' 10-4 M NPP. The inhibitors were at the following final concentrations: P0 48- , I ' ro- 6 M; S04 2- , 1'10-1 M and 1'10-2 lVI; EDTA, 1'10-2 M; t-phenylalanine, I ' IO- 2 M; and Mg 2+ (1'10- 2 M) was used as an activator. The activity was measured by the increase in absorbance at 400 mfl using a Gilford Model 2000 Multiple Absorbance Recorder, and only the early rates were used for kinetic analysis. Blood-grcup 0 substance determination The amount of the soluble blood-group 0 substance in a serum or in a fraction thereof was measured by the inhibition titer, i.e. the highest dilution of the fluid which definitely inhibited a standard anti-O serum-".

RESULTS

All three sera were subjected to gel filtration on Sephadex G-200, and fractions from each chromatogram were analyzed by starch-gel electrophoresis. Phosphatase activity was found exclusively in the middle peak of the chromatograms. This observation was confirmed by quantitative analysis of the alkaline phosphatase activity in the chromatographic fractions from serum No. I-Ol:2 (Fig. I). The results of the starch-gel electrophoresis of Sephadex G-200 fractions from serum No. 1-012 are shown in Fig. 2. As seen from the figure both the A and B phosphatases were recovered in the second peak. The A-zone was somewhat weakened, and the C phosphatase was not visible at the sample concentration used. However, proof that the C phosphatase also occurred in the second peak was obtained when this peak was rechromatographed on DEAE-Sephadex. The amidoblack-stained half of the gel showed the presence of a-2M globulin in the :first peak of the Sephadex G-200 chromatogram and in the valley between the first and second peaks. Albumin occurred in the third peak, and a trace of protein Biochim. Biophys. Acta, 110 (1965) II3-123

II6

O. AALUND, J. RENDEL, R. A. FREEDLAND

= =

Alkaline phos phatase activ ity observed on starch gel

c

...u

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Fig. I . Gel filtration on Sephadex G·200 o f 30 ml of O-serum from sheep No. 1-012 . - - - , protein concentration (absorbance at 280 nuz}: - • - - -, alkaline phosphatase activity. Analysis for Ovblood group substance was done on four concentrated pools of fractions (I, II , III, and IV: concentrated 30, 20,25, and 17 times respectively) , and they were further subjected to starchgel electrophoresis (Fig. 2).

with the same migration rate as albumin was found in the rest of the chromatogram. Transferrin was present in the second and third peaks. The results of the gel filtration of the sera No. 2392 and No. 3-121 were principally the same as for No . 1-012 . However, as expected, the second peak of the former cont ained on ly A phosphat ase, whereas the same peak of the latt er contained the wide A pho sph at ase zone characterist ic of the " X" phosphatase t ype. There were

Fig. 2. Starch-gel electrophoresis of the concentrated pools of F ractions I-IV (Fig. I) from Sephadex G-200 chromatography of serum No . 1-012. Only the anode side of'the gel is shown. The upper part of the figure represents the ami doblack-stained bottom slice of the gel, and the lower part the phosphatase-stained top slice. Samples I , 4, 7, 10, and 13 = control serum (No. 1-012); 2 and 3, poo l I; 5 and 6, II ; 8 and 9, III; I I and 12 , IV. a, A-phosphat ase ; b, B-phosphatase ; c, C-phosphat ase; d, a-2M glo bulin; 0, transferrins; f, albumin. Start, start of elect r op horesis.

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(1965) 113-123

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OVlNE SERUM ALKALINE PHOSPHATASES

also individual differences in the transferrin patterns reflecting their genetic types-s, The concentration of the blood-group 0 substance was determined in serum No. 1-012 and in every fifth of its fractions in the Sephadex G-200 chromatogram. The whole serum had an inhibition titer of 7.8. 10-3 while the titers in the fractions ranged from I to 0.25, the latter values being encountered only within the limits of the first protein peak in the chromatogram. By comparing the inhibition titer in the whole serum with the titers in the chromatographic fractions it was calculated that 57% of the a-activity applied to the column was recovered. The precision of the inhibition test increases with the concentration of the blood-group substance in the fluid to be tested. Inhibition tests were therefore also made on four concentrated pools from the first peak, the valley between the first and second peaks, the second peak, and the third peak. The four pools covered 64.4 % of the whole chromatogram. The extension of the various pools (I, II, III, and IV) and the portion of the 0activity found in each pool is given in Fig. I. Of the total amount of O-activity 52 % was recovered in the four concentrated pools; and the distribution of the amount of recovered activity in I, II, III, and IV was 68.5%, 9.9%, 17.9%, and 3.7% respectively. Anion exchange column chromatography The fractions in the second peak of the Sephadex G-200 chromatogram (serum No. 1-012) were pooled and 0.2 of this pool (equivalent to 6 ml of serum) was concentrated to a volume of 6 rnl by dialysis against a 15% solution of polyvinylpyrrolidone in Tris-buffer (pH 8.0) (0.1 M Tris + 1.0 1\1: NaCl) then dialyzed against 0.02 M phosphate buffer (pH 8.0) and finally fractionated by anion exchange column chromatography on DEAE-Sephadex (Fig. 3). The chromatographic fractions were thereafter subjected to starch-gel electrophoresis. The positions of the alkaline phosA

B

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20

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40 50 60 Per cent effluent

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Fig. 3. Anion exchange chromatogram (DEAE-Sephadex) of the pool of fractions from the second peak in Fig. I. - - - , Protein concentration (absorbance at 280 mfl). The solid lines (A, B, B: upper part, and C) indicate the positions in the chromatogram of the alkaline phosphatase isozymes defined by starch-gel electrophoresis.

phatases (A, B, and C) are indicated in Fig. 3. Phosphatase C was found in the 5.07.5 % effluent. A split in the B-band as described by RENDEL et al» was observed. The slower part of the B-band was seen in the r6.3-26.2% effluent, while the upper (fast) part of the B-band was present in a pool of 31.2-42.5 % effluent. The A-band was found in two concentrated pools of fractions covering the range of 31.2-55.0% effluent. Biochim, Biopbys. Acta,

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(1965) 113-123

lIS

O. AALUND.J . RENDEL,R. A. FRE ED LAND , 3579 12 16 20 24 28 32 36 40 44

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Fig. 4. Anion-exchan ge ch r omatogr am (DEAE-Sephadex ) of 6 ml of serum No . 1-012 (blood gro up 0). - -- , protein con centration (absorbance at 280 mil) . . Th e solid lines (A, B and C) indicate the position s in the chromatogram of t he al kalin e phosphatase isozym es defin ed by starch-gel electrophoresis. The num bers an d positions of individual fractio ns subj ected t o starchgel ele ctrophoresis (Fig. 5) are give n at the top of the figure .

Fi g. 5. Starch-gel electrophoresis of fractions (concentrated approx. 7 times) fr om DEAESephadex chro matography of who le serum No . r- o r z (blo od group 0) . C, control ser um (ser um N o. r-or s ). The numbers refer to t he chromatographic fractions in Fig. 4. a, A-phosphatase ; b, B-phosphatase ; c, C-phosp hatase ; d. a -2M glob ulin; e, transferr in s; f, albumin. Start, st ar t of electro p h oresis.

B iochim , B iophys , A eta,

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OVI NE SE R UM ALKALINE PHOSPHATASES

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Fig. 6. Anion-exchange chrom a togram (DEAE -Sephad ex ) of 6 ml of serum No. 239 2 (blood gr oup R) . - - - , protein concent ration (abs orbance a t 280 mfl) . The solid Jines (r, 2, 3, an d 4) indicate pools of chromatographic fractions. These pool s were concentrated approx . 80 , 80, go, and r05 times respectively and then subjected to starch-gel electrophoresis (Fig. 7). The isozyme C was found in Pool I , t he isozyme A in Pools 2 and 3.

Who le serum (6 ml) from sheep No. I-OIZ and sheep No. 2392 were fractionated on DE AE -Seph aclex. The fractionation of serum No. I -OIZ acted as a control on t he alkaline phosphatas e elution pattern s obt ained by DEAE-Sephadex chromatography of Pe ak 2 in the Sephadex G-200 chromat ogr am . The positions of the alkaline ph osph atases in the chromat ograms are indicated in Figs . 4 and 6. Serum No. I-012 contained t he alkaline phosphat ases A, B , and C (Fig . 5), while serum No. 2392 contained only A and C (F ig. 7). A, B, an d C from 1-0 12 were found in exactly t he same positions as in t he DEAE-Seph adex chromatogram of Peak 2 from gel filtration of whole serum. The upper part of the B-zone described by R E NDEL et al.2 was eluted along with t he A-zo ne. In t he DEAE-Sephadex chr omato gram fr om serum No. 2392 four pools were m ade representing 0-I8%, 19-38%, 39-63 %, and 64-100 % effl uent respectively. These pools were concentrated 75-100 times by dialysis against

Start

Fig. 7. Starch-gel elect r op h or esis of concentrated po ols of fra ct i ons (I. 2, 3. and 4 in Fig. 6) from DEAE-Sepha dex ch rom atograp hy of serum No. 2392 (blood gro up R). C. control sera. From left to ri ght : O -serum (No. r- on). R- serum (No. 293 2), a nd R -serum (No. 2392) . a, A-phosphatase ; b , Bvphcsphatase : c, Csphosph ataac . Start, start of ele ctrophor esis . C-band is seen in I . and A-band in 2 and 3 .

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polyvinylpyrrolidone. C-pho sphatase was found in the p ool representing 0 -18 % efflue nt , and A-phosphatase in the pool representing 19-38% effluent. No phosph at ases were found in the last two pools . Thus the alkaline phosphatases A and C in th is seru m were located in the same part s of the DEAE-Sephadex chroma t ogram as A and C from serum No. 1-01 2 . The amidob lack-stained bottom halves of t he starch-gels from serum No. 1-01 2 (F ig. 5) showed the presence of a-2M gl obu lin in the 48-IOO% effluent, of slow tran sferri n in the 33-45 % an d of fast transferrin in the 38-50 % effluent, and of albumin in the 70-100 % effluent. Kinetic analys is In order to det ermine the reliability of the determin ati on of the kinetic parameters, the K m ' was determined on one O-serum 5 times and on one R-serum 7 times. The values obtained and the st andard deviations were (1.78 ± 0.24) . 10- 4 M for th e R-serum and (3.29 ± 0 .19) . 10 - 4 M for the a-serum. The results of the studies 011 the kinetic properties ofthe R vsera, containing only type A and type C phosphatases, and the Ovsera, containing t yp e A, type B, and type C phosphatases are pr esented in Table I . The effect of variou s inhibitors and the activating effect of Mg2+ ar e also TA B L E I K I NETIC AN D I NHIBITION ST U DIES ON OVIN E AL KALINE PHOSPH AT ASE

The number of anim als is given in p are nt he ses after the K m and M easurement

[(1 values.

Mean, and standard error of mea1~ R eserwm

O-sen/ttl

App ar ent K m

«.: in presence of PO.'-

in p resen ce of M gH in presenc e of MgH and PO, 'Ap p are nt KI of PO,'[(I ' of P O.,· - in presenc e of Mg2+ P er cent inhibit ion b y EDTA P erc ent inh ibition b y L-phen ylal an in e K I ' of SO,2[(,n.'

R

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See t ext for m ethods.

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100 56

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1.2' 10-3

• These seven animals were among the nine and 10 an imals t est ed below . •• These seven animals were among the nine animals t est ed above.

shown in Table 1. The K rn ' values for 0- and R-sera were significantly different, thus indicating that the type B phosphatase has a K m ' different from the K m ' of t ype A and t ype C phosphatases. A highly purified B-phosphatase had a K m value of 1 2 .8· ro-·, which would account for the in cr eased K m ' value of th e a-serum. Th e inhi bition of act ivity by phosphate was examined (Table I and Fig. 8). Surprisingly, t he inhibition was f ound to be n on-competitive as can be seen in Fig. 8. Th e similar K m ' values obtained in the pr esence and absence of pho sphate are a strong confirmat ion that the inhibition is non-comp etitive. D espit e the different K m ' values for the tw o types of sera the K1' values for phosphate were similar and non-comp etitive in b oth cases. In order to check our syst em further , the kinet ics of purified Escherichia B iochi m , B iophys, Acta, IIO (19 65) II 3-I 23

121

OVINE SERUM ALKALINE PHOSPHATASES 0- type serum

B

R - type serum B

560

320t

490

280r A

240

.i. v

200

c

210

140

1000 :2000 3000 4000

1/[SJ

1000 2000 3000 4000

1/[S]

Fig. 8. Lincweaver'-Burk plot of data from kinetic analysis. V, {tIDolcs PO.3- relcasedjljmin. 5, moles/I of NPP. A, uninhibited; B. PO.!3- at r : 10- 5 M; C. l\'[g2+ at I . IO-~ 1'11; D, MgH at 1'10- 3 M plus PO."- at 1'10-' M. Band D, noncompetitive inhibition of enzyme action as seen respectively in A and C. C. activation of enzyme action as seen in A.

coli alkaline phosphatase (Sigma Chemical Co., St. Louis, Mo. (U.S.A.)) was examined, and as previously reported with this enzyme!" we also found that phosphate inhibited this system completely. Since the enzyme requires divalent metal ions such' as Mg2+, it was thought to be important to examine the effects of this ion on the kinetics of the phosphatase. Therefore, the effect of Mg2+ and Mg2+ plus phosphate on the activity of the phosphatase was examined. Mg2+ at IO- 2 !VI strongly activated the enzyme without changing the K m' for NPP; furthermore, phosphate inhibited non-competitively despite the presence of Mg2+, and even high Mg2+ concentrations did not change the K i ' of phosphate. Attempts to fractionate this enzyme with (NH4)2S04 indicated that sulfate also inhibited these enzymes. This inhibition also appeared to be non-competitive, but sulfate was considerably less effective than phosphate as an inhibitor. L-phenylalanine, which has been shown specifically to inhibit intestinal alkaline phosphataset-, at 10- 2 M also inhibited the phosphatase activity of both 0- and R-serum. The greater inhibition of O-serum may indicate a greater sensitivity of the B-phosphatase than of the A- and C-phosphatases to this reagent; unfortunately there was only sufficient purified B-phosphatase to measure a few K m values and not enough for inhibitor studies. DISCUSSION

Experiments in vivo by RENDEL et al. 2 indicate that injections of O-blood group substance may induce the occurrence of B-phosphatase in the serum of sheep of blood group R. The mechanism involved in this phenomenon is unknown. Since Biochim, Biophys. Acta,

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FREEDLA~D

the majority (68.5%) of O-blood group substance in O-serum was found in the first peak of the Sephadex G-200 chromatogram, while all alkaline phosphatase activity and only 17.9% of the O-blood group substance was found in the second peak, it seems most likely that the alkaline phosphatase isozymes in serum do not exist in a complex with the blood-group 0 substance. If they did, such a substantial increase in molecular size would be expected that at least some of the alkaline phosphatase molecules should exceed the exclusion value for Sephadex G-zoo and hence be eluted in the first peak of the chromatogram. The exclusion value for Sephadex G-zoo is zoo 000. FLODIN AND KILLANDIm 15 found by analysis in the ultracentrifuge that the proteins in the second peak from the Sephadex G-200 chromatogram comprised one major and one minor component with sedimentation coefficients of about 7 Sand 4 S, respectively. The material in the third peak was homogeneous in the ultracentrifuge, and sedimented at a rate of about 4 S. Therefore it can be concluded that the molecular size of alkaline phosphatases in sheep serum does not exceed that of molecules with molecular weight 200000. Since no alkaline phosphatases were found in the third peak ofthe Sephadex G-zoo chromatogram, the alkaline phosphatases most probably have a sedimentation coefficient of 7 S (the major component in the second peak). FAHEY ei al.5 found two alkaline phosphatases in human serum. These were eluted in almost the same position as the phosphatases A and B in the DEAE-Sephadex chromatogram of the present study. AALUND et at.6 found the sedimentation coefficient (S20'W) for the proteins in this part of the chromatogram to be 7 S. No conclusions can be drawn about the molecular weight of the ovine alkaline phosphatases because the molecular size (not determined in the present study) influences both the sedimentation rate in the ultracentrifuge and the elution site in the Sephadex G-zoo chromatogram", It is evident from the kinetic analyses that the K m' values for NPP are different for the A- and B-phosphatases as indicated by studies with whole serum and purified B-phosphatase. It seems improbable that phosphate combines irreversibly with the enzyme, particularly at the active site, since the enzyme would inactivate itself rapidly during action. Thus the non-competitive inhibition by phosphate might be due to reactivity at an allosteric site!"; and, therefore, inhibition would be independent of substrate concentration. The non-competitive inhibition with a low K i ' value as observed in this study does not rule out the possibility of a competitive inhibition by phosphate with a much higher KIf value. The latter would be obscured under these conditions. The results with magnesium activation and EDTA inhibition indicate a metal requirement for these enzymes, which can be fulfilled by Mg2+. The maximum activation by Mg 2+ under our conditions was a 2-3 fold increase. The observation that the kinetic values remained unchanged in the presence of high Mg2+ concentrations indicates that the Mg2+ probably activates these two enzymes without altering the affinity for the substrate, although it is realized that K m values are only approximations for ES (enzyme-substrate complex) dissociation constants and not equal to these constants. The effect of phosphate, which is the same in the absence of excess Mg2+ and in the presence of a Mg2+jP0 4 ratio of 100, is indicative that the mode of phosphate inhibition is not the complexing and removal of Mg2+ from the reaction. The similar effect of non-competitive inhibition by phosphate in the presence of excess Mg2+ is consistent with an allosteric type inhibition. The very high K m ' values of the pure B-isozyme compared with the A-isozyme Biochim: Biopbys. A eta,

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OVINE SERUM ALKALINE PHOSPHATASES

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(as determined in R-serum) indicates that a small proportion ofthe B-isozyme in the serum (about 16% of total activity) could account for the observed changes in kinetics between R- and O-serum activities. It is interesting to note that despite moderately large variations in total serum alkaline phosphatase activity in different sheep of blood group 0 which all had a relatively high activity in comparison with sheep of blood group R2, the Km/values remained reasonably constant. This may indicate that the AlB isozyme ratio in the serum of sheep of blood group 0 remains relatively constant. The observed difference in sensitivity to L-phenylalanine of 0- and R-sera (Table I: 70% versus 56% inhibition) may be indicative of much greater sensitivity to this component of B-phosphatase than of A-phosphatase. t-phenylalanine was shown by FISHMAN et al.14 to be an organ-specific, stereospecific (no inhibitory effect of n-phenylalanine) inhibitor of human intestinal alkaline phosphatase (78% inhibition). Tentatively it might therefore be inferred that at least the B-phosphatase is an intestinal phosphatase. This would be consonant with ROBINSON AND PIERCE I B who found the human t-phenylalanine-sensitive (neuraminidase-resistant) alkaline phosphatase isozyme in the slow major zone of the starch-gel zymogram.

ACKNOWLEDGEMENT The authors express their sincere thanks to Professor C. STORMONT for encouragement and constructive criticism. H.EFERENCES I 2

J, RENDEL AND C. STORMONT, Proc, Soc, Exptl. Bioi. Med" lI5 (1964) 853. .J'. RENDEL, 0, AALUND, R. A. FREEDLAND AND F. MOLLER, Genetics, 50 (19 64)

973.

3 E, A, PETERSON AND E. A, CHIAZZE, Arch. Biochem, Biophys., 99 (1962) 136. 4 H. A. SOBER AND E, A. PETERSON, Federation Proc., 17 (1958) III6, 5

J. L.

FAHEY, P. F. McCoy AND M. GOULIAN,

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