- Email: [email protected]
Bovine serum amine oxidase: Some differences between chromatographically defined forms including sensitivity to inhibition by dithiomolybdate
Comp. Biochem. Physiol. Vol. 104B,No. 4, pp. 669-673, 1993
0305-0491/93 $6.00+ 0.00 Pergamon Press Ltd
Printed in Great Britain
BOVINE SERUM AMINE OXIDASE: SOME DIFFERENCES BETWEEN CHROMATOGRAPHICALLY DEFINED FORMS INCLUDING SENSITIVITY TO INHIBITION BY DITHIOMOLYBDATE GERALD MULRYAN and JAMESMASON* Biochemistry Department, Trinity College, Dublin, Ireland (Tel. 772-941; Fax 772-400) (Received 22 September 1992; accepted 6 November 1992)
Abstract--1. Bovine serum amine oxidase (BSAO) forms, defined by DEAE--cellulose and hydroxyapatite chromatography, were shown to have different kinetic characteristics with p-dimethylaminobenzylamine as a substrate; there was also some variation in heat sensitivity. 2. The forms showed similar sensitivity to inhibition by aminoguanidine but there were differences with respect to dithiomolybdate. 3. The results also provided some support for the view that both subunits were active catalytically. 4. It is concluded that while the forms were interconvertible, the practice of pooling different chromatographic fractions may not be acceptable in all circumstances. 5. While inhibition by dithiomolybdate is of biochemical interest it is concluded that inhibition of BSAO by thiomolybdates is unlikely to play a role in the clinical syndromes which are common in cattle exposed to Mo in the herbage.
INTRODUCTION
Bovine serum amine oxidase (BSAO) is a member of a widespread group of extramitochondrial copperdependent enzymes catalysing the oxidative deamination of biogenic amines, the cross-linking of collagen and elastin, as well as having a potential role in the regulation of intracellular polyamines (Bachrach, 1985). Substrate specificity varies, thus while benzylamine and heptylamine are readily oxidized by all plasma amine oxidases, non-ruminant origin enzymes (porcine, equine, human, etc.) show a preference for biogenic amines such as histamine, dopamine and serotonin, but have only a low activity towards diamines. By contrast, ruminant source enzymes (bovine and ovine) have been described as spermine oxidases (Petterson, 1985) because of their activity towards spermine and spermidine. However, it is probably somewhat simplistic to characterize amine oxidases by their substrate specificity (Bachrach, 1985) since this can be changed by the purification procedures or by immobilization, for example (Bachrach, 1985; Gorkin, 1985). The authors' interest in amine oxidases arose from studies of the widespread and economically significant "copper deficiency" syndromes of ruminants produced by thiomolybdates (Mason, 1986, 1990). The clinical problems follow the ingestion of herbage containing relatively modest levels (10-30 ppm Mo) of molybdenum compounds which are converted to thiomolybdates by interaction with sulphides in the *To whom correspondence should be addressed.
rumen (Mason, 1990). The compounds interfere with copper metabolism but also inhibit the oxidase activity of another type of copper enzyme, ceruloplasmin, at least in vitro (Kelleher and Mason, 1986; Lannon and Mason, 1986). Another group (Chidambaram et al., 1984) have reported that other copper enzymes, including cytochrome oxidase, superoxide dismutase and tyrosinase were inhibited by tetrathiomolybdate in vitro with I 50% values in the range 1-5/tM. The nature of the disease symptoms observed in cattle led the authors to examine the effect of thiomolybdates upon BSAO as a representative member of its group of enzymes, since depressed growth, as well as defective cross-linking of elastin and collagen, are the commonest symptoms. Moreover, plasma benzylamine oxidase activity has been shown to be depressed in primary copper deficiency in calves (Mills et al., 1976). BSAO and swine serum amine oxidase (SSAO) seem to be related closely enough to be considered as source variants of the same enzyme (Bachrach, 1985; Petterson, 1985) thus both are dimers consisting of two similar subunits and the overall amino acid compositions of both enzymes are similar, although not identical. The covalently bound redox factor of copper amine oxidases, previously believed to be pyrroloquinoline quinone (PQQ) has now been identified as 6-hydroxydopa (Janes et al., 1990) although nutritional studies have shown PQQ to be an essential dietary constituent for mice (Killgore et al., 1989) and deficiency to lead, inter alia, to increased collagen solubility and to depressed lysyl oxidase activity (Killgore et al., 1989). There is some debate as to whether both subunits of
669
670
GERALD MULRYAN and JAMES MASON
the dimeric enzyme, similar if not identical, are active catalytically. Preliminary studies with bovine serum in vitro revealed inhibition of amine oxidase activity by dithiomolybdate (Mulryan and Mason, 1987) and also complex non-linear kinetics. Scrutiny of the literature revealed that kinetic analyses had often been carried out on pooled heterogeneous material since the chromatographic procedures commonly employed to purify the enzyme yield multiple forms of both bovine and porcine enzyme (Falk et al., 1983). While it has been concluded that a pooled enzyme source is acceptable for SSAO (Falk et al., 1983) our studies show that this is not the case for BSAO and in particular with p-dimethylaminobenzylamine as substrate. This alternative substrate was employed since it yields an aldehyde product with a "]'maxat 355 nm whereas benzaldehyde, the product of benzylamine oxidation, absorbs in the same region (ca 255 nm) as thiomolybdate and especially dithiomolybdate. The usage of this alternative substrate appears to have revealed aspects which are not apparent with the more commonly employed benzylamine. Thus our studies reported here provide some support for the view that both subunits of the dimer are active. They also demonstrate that there are significant differences between the forms including inhibition by dithiomolybdate. They also show that the forms are interconvertible. It is apparent that for BSAO it cannot be assumed that pooled enzyme sources are suitable for all studies. MATERIALS AND METHODS
Separation o f the different forms o f BSAO The protocol used and the nomenclature adopted was based on that reported by Summers et al. (1979). Six BSAO forms were defined compared to the four described by Summers et al. The method generally, but not always, yielded sufficient amounts of each form to enable a comprehensive comparison in most respects although in common with previous reports for SSAO (Falk et al., 1983) the proportions varied from batch to batch. Three active BSAO fractions (A, B and C) were obtained from the DEAE-cellulose column chromatography step by elution, successively, with 0.3, 0.07 and 0.2 M phosphate buffer at pH 7. This contrasts to the two fractions (A and B) obtained by Summers et al., 1979 following elution with 0.05 and 0.07 M phosphate buffer. For the subsequent step, that is separation into two subfractions (1 and 2) by hydroxyapatite chromatography, Summers et al. used 0.01 and 0.06 M phosphate buffer at pH 6.8 for the two-step elution, whereas 0.006 and 0.1 M phosphate were employed in the present study. Since Yamada and Yasonuba (1962) had reported that the enzyme(s) was unstable at buffer concentrations lower than 10 mM the protocol employed, described in detail later, minimized exposure to low ionic strength buffers particularly
during dialysis proved overall proached 60% Summers et al.
steps. This resulted in a much imyield, which while variable, apcompared to the 20% reported by (1979).
Separation protocol employed Bovine blood was collected fresh at the abattoir. All steps after coagulation were carried out at 4~C. Crystalline ammonium sulphate was added, with stirring, to serum cleared by centrifugation to give a final saturation of 35%. After 12 hr the precipitate was removed by centrifugation at 10,000 g for 40 min in an RC-5B Sorvall refrigerated centrifuge. The supernatant was then taken to 55% saturation with ammonium sulphate and after 12 hr the precipitate containing BSAO activity was harvested by centrifugation as before. This was taken up in 0.01 M phosphate buffer at pH 7 and dialysed against a large volume of 0.015 M buffer (phosphate at pH 7) over 24 hr. After removal of the inactive precipitate by centrifugation aliquots of the clear supernatant were applied to a DEAE-cellulose column (28.4 x 4 cm) previously equilibrated with 0.003M phosphate buffer at pH7. Active fractions were obtained by stepwise elution with, respectively, 0.03, 0.07 and 0.2 M phosphate buffer pH7 in sufficient volumes (generally 11 of each) to elute all the activity emerging at each step. The individual pooled elutes of the different peaks (A, B and C) were then taken to 40% saturation by the addition of crystalline ammonium sulphate and the pH adjusted to neutrality by the addition of concentrated ammonia. After 12 hr the inactive precipitate was removed by centrifugation and the active supernatants taken to 55% saturation as before. After 12 hr the precipitate was harvested by centrifugation, redissolved in 0.02M phosphate buffer pH 6.8 and equilibrated with the same buffer by dialysis. Aliquots of the three post-dialysis fractions obtained were diluted 3-fold with distilled water and applied to a hydroxyapatite column (2 x 17 cm) pre-equilibrated with 0.006 M phosphate buffer pH 6.8. Two peaks of BSAO activity were eluted from the column with 0.006 and 0.1 M phosphate buffer. Elution continued until no further BSAO activity emerged. The main active fractions in each peak were then pooled and adjusted to 55% saturation with solid ammonium sulphate and the pH adjusted to neutrality with concentrated ammonia. After 12hr the precipitate obtained by centrifugation was redissolved in 0.1 M phosphate buffer pH8 and dialysed 12 hr against the same buffer. The samples could be stored at - 2 0 ° C for at least 12 months with no loss of activity. Measurement o f the P-dimethylaminobenzylamine oxidase activity o f BSAO The p-dimethylaminobenzylamine oxidase activity of the serum, the eluates at the various stages of the purification eluates procedure, as well as the separated forms was measured continuously in a Gilford
Bovine serum amine oxidase
°,.'°°fd j
Table 1. K~t and K,, values for the oxidation of p-dimethylaminobenzylamine for most of the BSAO forms. The data were subjected to a non-linear least-square fit analysis (Leatherbarrow, 1987) assuming the involvementof two non-interacting active sites, K~t values are expressed as Ao.d. 355 nm/min x 10.3
ct!°I Okl
3
!
i
3"5
4
-Log(M)
Fig. l. Inhibition of the p-dimethylaminobenzylamine oxidase activity of bovine serum (15 mg protein per 1 ml assay cocktail) by dithiomolybdate. Vertical axis residual BSAO activity. Horizontal axis--log of dithiomolybdate concentration (M). 2000 single beam spectrophotometer coupled to a Phillips 8251 a single pen recorder. The appearance of product was followed at 355 nm for at least 10 min at 25°C. The temperature of a solution of the substrate in 0.1 M potassium phosphate buffer at p H 8 was raised to 25°C before the reaction was started by the addition o f the various B S A O preparations. The B S A O preparations were kept on ice before use, and the final assay cocktail volume was 1 ml. F o r the kinetic constants, etc., final substrate concentrations between 0.0025 and 14.40 m M were employed. N o background rate was observed in the absence of either enzyme or substrate. RESULTS
Double reciprocal plots ofp-dimethylaminobenzylamine concentration and reaction velocity showed variability and marked deviations from linearity (not shown). The data were subjected to a non-linear least square fit analysis (Leatherbarrow 1987) and was best described (Chi2 < 0.02) by a model which assumed the involvement of two non-interacting sites with different affinities for p-dimethylaminobenzylamine.
2/,0"
120"
/
/~,
K ~-(raM)
K~t
K~ (mM)
Ai
5.91 -I- 0.26
0.04 + 0.01
6.71 + 0.43
4.94 + 1.19
A2 BI CI C2 Serum
3.12 + 0.12 1.70 + 0.20 9.75___1.18 3.01 _+0.20 2.20_+0.22
0.07+ 0.01 0.26_ 0.18 0.18_+0.05 0.05_+0.01 0.14+ 0.04
9.68 __.6.41 4.93 + 4.31 6.07+ 0.21 0.38 _ 0.07 9.18_+4.86 20.70_+1.96 4.90_+1.57 14.00_+9.14 2.08_+0.87 21.90_+17.40
Typical low and high Km values were 0.14 + 0.04 and 21.3 -I- 17.4 mM. Early studies also showed that while the BSAO p-dimethylaminobenzylamine oxidase activity o f serum was inhibited by dithiomolybdate the interaction was complex, see Fig. 1, and incomplete even at dithiomolybdate concentrations o f 668 ttM where 30% of the activity remained. There was no comparable inhibition with either tri- or tetrathiomolybdate.
Studies with the purified forms Data, for example the results illustrated in Figs 2 for forms AI and A2, when subjected to a non-linear least-square fit analysis (Leatherbarrow, 1987) were consistent with the involvement of two active sites. The two Km and K~t values for the various purified forms are shown in Table 1.
Inhibition studies
Preliminary studies on serum
360-
Studies using aminoguanidine, the classic carbonyl group inhibitor of plasma amine oxidases revealed no apparent differences between the forms (Fig 3a). By
1/,oo[(a) . /
0L~
750"
2:6
500" / A1
671
250" 0
A2
o lb 2b
tIoL, a
-A
- Log. amg.(M)
ac;[Ic)i%
2.8 3-1 313 3~5 0 - Log.dith.(M) Fig. 2. Lineweaver-Burk, double reciprocal plots for the oxidation ofp-dimethylaminobenzylamine by BSAO forms A1 and A2 at 25°C in 0.1 M phosphate buffer at pH 8.0. Each point is the average _ standard deviation of at least five determinations of reaction rate. The data are consistent with the involvement of two non-interacting active sites whose kinetic constants are given in the table.
/-, 8 12 In c.(min)
Fig. 3. Inhibition and heat inactivation of some of the forms of BSAO. (3a) Inhibition by aminoguanidine (amg). (3b) Inhibition by dithiomolybdate (dith). (3c) Heat inactivation. Vertical axis--residual activity. Horizontal axis---duration of preincubation at 70°C. Symbols; O, A1;/% A2; 0 , B1; A, B2. Each point is the average + standard deviation of at least five determinations.
672
GERALD MULRYAN a n d JAMES MASON
1.2 Act.
C
12
that the enzyme fractions obtained by DEAE and hydroxyapatite chromatography have some differences in kinetic characteristics with respect to pdimethylaminobenzylamine oxidation and the forms examined show some variations in sensitivity to ZXOD mho dithiomolybdate and to heat inactivation. Nevertheless as has been concluded already for SSAO (Falk et al., 1983) they do appear to be forms of the same C , , 0 enzyme, since they were shown to be interconvertible 0 5 10 15 when resubjected to DEAE-cellulose chromatogfraction no. raphy after dialysis against low ionic strength buffers. Fig. 4. Interconversion of BSAO forms. Rapid rechroThe sensitivity of the forms to aminoguanidine is the matography of BSAO form AI on a DEAE--cellulose column. Elution was step-wise with successively,0.05, 0.07 same. There have been previous indications of this and 0.2 M phosphate buffer at pH7. All the BSAO activity interconversion; thus Yamada et al. (1976) showed applied was recovered in the eluate. that there was conformational isomerism at least between two forms (B and C). This does not seem to contrast with dithiomolybdate, there were clear have been pursued further. The interconversion may differences between the two forms examined in detail be one explanation for the variability in the A l and B2 (Fig. 3b) with respect to dithio as inhibitor. proportions of the forms from preparation to In contrast to serum the results for each form were preparation encountered by the authors in the present linear and linear regression analysis of the data gave study and by Falk et al. (1983) for SSAO. There has been some discussion as to whether there I 50% values of 1.42 and 33.3 mM, respectively. Heat inactivation studies also revealed differences are one or two active sites, and/or whether the two between forms and the results are shown in Fig. 3(c). subunits of the dimeric molecule are in fact identical. Residual activity was measured at a substrate concen- Several authors, for example Summers et al. (1979) tration of 3.6 mM after the different enzyme prep- and Berg and Abeles (1980) have put forward the hypothesis of two distinct active sites to explain, for arations had been heated for different times at 70°C. example, non-stereospecific oxidation by the A (and A t ~/2of 2.5 min was calculated by linear analysis for B) form of the enzyme since benzylamine (Battersby forms A~ and A2, compared to 1.1 min for B l and B2. et al., 1979) and p-hydroxbenzylamine (Suva and lnterconvertability o f the f o r m s Abeles, 1978) are oxidized stereospecifically. The use The forms were reexamined after dialysis at 4°C of p-dimethylaminobenzylamine as a substrate may against low ionic strength buffers (phosphate 0.003 M have revealed activities of both subunits not obvious with the more commonly used benzylamine. Thus at pH7 and 0.05 M Tris-HC1 containing 0.01 mM CuSO4. Repassage through DEAE-cellulose of the simple kinetics, that is linear double reciprocal plots BSAO forms revealed extensive interconversion, thus and single KmS, have generally been reported for Fig. 4 illustrates a typical elution profile, in this case benzylamine (e.g. Costa et al., 1978, Battersby et al., 1979) and p-hydroxybenzylamine (Suva and Abeles, for form A~. Forms A, B and C are apparent. All the 1978; Dooley and Cot6, 1984). By contrast the preenzyme activity put on the column could be sent studies consistently give non-linear (two-slope) accounted for. Results for all the forms examined are summarized in Table 2. It is evident for all forms that plots compatible with the hypothesis of two distinct active sites, one per subunit. significant redistribution has occurred. Benzylamine appears to be a better substrate in that at saturating conditions for the low Km comDISCUSSION ponent of the reaction about 10 times the rate of oxidation was observed with benzylamine compared The results highlight the difficulty of making simple to p-dimethylaminobenzylamine. There appeared to conclusions with respect to BSAO and its (their) be no significant differences between the ratios of inhibition by thiomolybdates nevertheless the studies activities of the two substrates between the different of BSAO as an enzyme(s) are of interest. They show forms (not shown). Thus the two substrates appear to be being oxidized by the same enzyme, and the forms Table 2. Redistribution (percentage of each form eluted _+SD) of appear to be variants of the same enzyme. The BSAO activity following repassage of each form on a heterogeneity of SSAO has been noted by Falk et al. DEAE-cellulose column after dialysis against 0.003 M phosphate buffer pH 7 (1983), and indeed these authors conclude that "it Each form (%) No. would not be useful in any circumstances to employ BSAO form A B C of runs enzymes that have been resolved chromatographiA~ 12.7_+4 40.8_+6.7 46.1_+3.7 5 cally since the species themselves (forms) are not A2 40.5 _+ 3.5 22.5 _+ 2.1 42.0 _+ 1.4 2 homogeneous". These authors, while reporting that B~ 34.7_+27.4 26.9_+ 13.3 38.4_+ 14.2 3 C~ 41.5 8.5 50 1 with benzylamine as a substrate, the catalytic properA~ 41.2 12.5 46.3 1 ties were similar, did nevertheless also caution that
..-'.......... Cond.
Bovine serum amine oxidase the practice of pooling the various chromatographic fractions could yield misleading results under some circumstances. Our data show that this is so, and not surprisingly, remixing forms gives complex kinetics analagous to serum (not shown). Overall the results reported here show that pdimethylaminobenzylamine is a more interesting substrate than benzylamine in that its usage reveals features of BSAO which are not apparent with benzylamine. Thus the non-linear kinetics would fit an hypothesis of two subunits both of which are active catalytically. Thus the subunits may be similar if not identical but could differ in other respects, possibly access of the substrate. In addition the forms defined by chromatography show different affinities for p-dimethylaminobenzylamine. All this is worthy of further study. Dithiomolybdate appears to reveal differences at least between some forms which are not apparent with the classic carbonyl reagents aminoguanidine, and semicarbazide. As for the implications with respect to the pathogenesis of the bovine molybdenosis syndrome the complexity of the interactions makes definitive conclusions difficult, although the in vitro inhibitory concentrations are relatively high compared to dietary Mo levels and it is hard to envisage concentrations of this order being attained in plasma in vivo. Furthermore the authors have shown that dithiomolybdate is not an important plasma constituent. Moreover the half-life of dithiomolybdate in plasma is quite short ( < 10hr) compared to tri- and tetrathiomolybdate (35-45 hr) and for a number of reasons trithiomolybdate is likely to be the most significant form in the biochemical pathogenesis of the syndrome (Mason, 1986; Mason, 1990). These higher forms did not inhibit at the concentrations used. Finally field studies (Mulryan and Mason, 1992) do not reveal loss of BSAO activity in hypocuprotic and hypocupraemic cattle, most of which would have been exposed to significant levels of pasture Mo. Thus, although it is possible the accumulation of dithiomolybdate could occur locally, present evidence suggests that inhibition of BSAO is unlikely to be part of the pathogenesis of the various syndromes. Acknowledgements--We are grateful to the WellcomeTrust
for financial support.
REFERENCES
Bachrach, U. (1985) Copper amine oxidases and amines as regulators of cellular processes. In Structure and Functions of Amine Oxidases (Edited by Mondovi B.), pp. 5-20. CRC Press, Boca Raton, Florida.
673
Battersby, A. A., Staunton, J., Klinman, J. and Summers, M. C. (1979) Stereochemistryof oxidation of benzylamine by the amine oxidase from beef plasma. FEBS Left 99, 297-298. Chidambaram, M. V. Barnes, G. and Frieden, E. (1984) Inhibition of ceruloplasmin and other copper oxidases by thiomolybdate. J. of Inorg. Biochem. 22, 231-241. Costa, M. T., Sabatini, S., Turini P. and Mondovi, B. (1978) Inhibition of beef plasma amine oxidase activity by glycine. FEBS Letts. 94, 113-114. Dooley, D. M. and Cot~, C. E. (1984) Inactivation of beef plasma amine oxidase by sulfide J. biol. Chem. 259, 2923-2926. Falk, M. C., Staton, A. J. and Williams, T. J. (1983) Heterogeneity of pig plasma amine oxidase: molecular and catalytic properties of chromatographically isolated forms. Biochemistry 22, 3746-3751. Gorkin, V. Z. (1985) Qualitative alteration (transformation) in catalytic activity of amine oxidases. In Structure and Functions of Amine Oxidases (Edited by Mondovi B.), pp. 205-208. CRC Press, Boca Raton, Florida. Janes, S. M., Mu, D., Wemmer, D., Smith, A. J. Kaurs, S., Maltby, D., Burlingame, A. L. and Klinman J. P. (1990) A new redox cofactor in eukaryotic enzymes: 6-hydroxydopa at the active site of bovine serum amine oxidase. Science 248, 981-987. Kelleher, C. A. and Mason, J. (1986) Reversible inhibition of ovine ceruloplasmin by thiomolybdates. Int. J. Biochem. 18, 629-635. Killgore, J., Schidt, C., Duich, L., Romero-Chapman, N. and Rucker, R. B. (1989) Nutritional importance of pyrroloquinoline quinone. Science 245, 850-852. Lannon, B. and Mason, J. (1986) The inhibition of bovine ceruloplasmin oxidase activity by thiomolybdates in vivo and in vitro: A reversible interaction. J. Inorg. Biochem. 26, 107-115. Leatherbarrow, R. J. (1987) Enzfitter, a non-linear regression analysis program for the IBPC: Elsevier Biosoft, The Netherlands. Mason, J., (1986) Thiomolybdates: Mediators of molybdenum toxicity and enzyme inhibitors. Toxicology 42, 99-109. Mason, J., (1990). The biochemical pathogenesis of molybdenum-induced copper deficiency syndromes in ruminants--towards the final chapters. Irish Veter. J. 43, 18-21. Mills, C. F., Delgarno, A. C. and Wenham, G. (1976) Biochemical and pathological changes in the tissues of Friesian cattle during the experimental induction of copper deficiency. Br. J. Nutri. 35, 309-329. Mulryan, G. and Mason, J. (1987) The inhibition of bovine serum amine oxidase by thiomolybdates in vivo and in vitro. Biochem. Soc. Trans. 15, 248-249. Mulryan, G. and Mason, J. (1992) Assessment of liver copper status in cattle from plasma copper and plasma copper enzymes. Ann. Rech. Vkt~r. 23, 233-238. Petterson G. (1985) Plasma amine oxidase. In Structure and Functions of Amine Oxidases. (Edited by Mondovi B.) pp. 105-120. CRC Press, Boca Raton, Florida. Summers, M. C., Markovic, R., and Klinman, J. P. (1979) Spectrochemistry and kinetic isotope effects in the bovine plasma amine oxidase catalysed oxidation of dopamine. Biochemistry 18, 1969-1978. Suva, R. H. and Abeles, R. M. (1978) Studies on the mechanism of action of plasma amine oxidase. Biochemistry 17, 3538-3545. Yamada, H. and Yasonubu, K. T. (1962) Monoamine oxidase II. J. biol. Chem. 237, 3077-3082.
0305-0491/93 $6.00+ 0.00 Pergamon Press Ltd
Printed in Great Britain
BOVINE SERUM AMINE OXIDASE: SOME DIFFERENCES BETWEEN CHROMATOGRAPHICALLY DEFINED FORMS INCLUDING SENSITIVITY TO INHIBITION BY DITHIOMOLYBDATE GERALD MULRYAN and JAMESMASON* Biochemistry Department, Trinity College, Dublin, Ireland (Tel. 772-941; Fax 772-400) (Received 22 September 1992; accepted 6 November 1992)
Abstract--1. Bovine serum amine oxidase (BSAO) forms, defined by DEAE--cellulose and hydroxyapatite chromatography, were shown to have different kinetic characteristics with p-dimethylaminobenzylamine as a substrate; there was also some variation in heat sensitivity. 2. The forms showed similar sensitivity to inhibition by aminoguanidine but there were differences with respect to dithiomolybdate. 3. The results also provided some support for the view that both subunits were active catalytically. 4. It is concluded that while the forms were interconvertible, the practice of pooling different chromatographic fractions may not be acceptable in all circumstances. 5. While inhibition by dithiomolybdate is of biochemical interest it is concluded that inhibition of BSAO by thiomolybdates is unlikely to play a role in the clinical syndromes which are common in cattle exposed to Mo in the herbage.
INTRODUCTION
Bovine serum amine oxidase (BSAO) is a member of a widespread group of extramitochondrial copperdependent enzymes catalysing the oxidative deamination of biogenic amines, the cross-linking of collagen and elastin, as well as having a potential role in the regulation of intracellular polyamines (Bachrach, 1985). Substrate specificity varies, thus while benzylamine and heptylamine are readily oxidized by all plasma amine oxidases, non-ruminant origin enzymes (porcine, equine, human, etc.) show a preference for biogenic amines such as histamine, dopamine and serotonin, but have only a low activity towards diamines. By contrast, ruminant source enzymes (bovine and ovine) have been described as spermine oxidases (Petterson, 1985) because of their activity towards spermine and spermidine. However, it is probably somewhat simplistic to characterize amine oxidases by their substrate specificity (Bachrach, 1985) since this can be changed by the purification procedures or by immobilization, for example (Bachrach, 1985; Gorkin, 1985). The authors' interest in amine oxidases arose from studies of the widespread and economically significant "copper deficiency" syndromes of ruminants produced by thiomolybdates (Mason, 1986, 1990). The clinical problems follow the ingestion of herbage containing relatively modest levels (10-30 ppm Mo) of molybdenum compounds which are converted to thiomolybdates by interaction with sulphides in the *To whom correspondence should be addressed.
rumen (Mason, 1990). The compounds interfere with copper metabolism but also inhibit the oxidase activity of another type of copper enzyme, ceruloplasmin, at least in vitro (Kelleher and Mason, 1986; Lannon and Mason, 1986). Another group (Chidambaram et al., 1984) have reported that other copper enzymes, including cytochrome oxidase, superoxide dismutase and tyrosinase were inhibited by tetrathiomolybdate in vitro with I 50% values in the range 1-5/tM. The nature of the disease symptoms observed in cattle led the authors to examine the effect of thiomolybdates upon BSAO as a representative member of its group of enzymes, since depressed growth, as well as defective cross-linking of elastin and collagen, are the commonest symptoms. Moreover, plasma benzylamine oxidase activity has been shown to be depressed in primary copper deficiency in calves (Mills et al., 1976). BSAO and swine serum amine oxidase (SSAO) seem to be related closely enough to be considered as source variants of the same enzyme (Bachrach, 1985; Petterson, 1985) thus both are dimers consisting of two similar subunits and the overall amino acid compositions of both enzymes are similar, although not identical. The covalently bound redox factor of copper amine oxidases, previously believed to be pyrroloquinoline quinone (PQQ) has now been identified as 6-hydroxydopa (Janes et al., 1990) although nutritional studies have shown PQQ to be an essential dietary constituent for mice (Killgore et al., 1989) and deficiency to lead, inter alia, to increased collagen solubility and to depressed lysyl oxidase activity (Killgore et al., 1989). There is some debate as to whether both subunits of
669
670
GERALD MULRYAN and JAMES MASON
the dimeric enzyme, similar if not identical, are active catalytically. Preliminary studies with bovine serum in vitro revealed inhibition of amine oxidase activity by dithiomolybdate (Mulryan and Mason, 1987) and also complex non-linear kinetics. Scrutiny of the literature revealed that kinetic analyses had often been carried out on pooled heterogeneous material since the chromatographic procedures commonly employed to purify the enzyme yield multiple forms of both bovine and porcine enzyme (Falk et al., 1983). While it has been concluded that a pooled enzyme source is acceptable for SSAO (Falk et al., 1983) our studies show that this is not the case for BSAO and in particular with p-dimethylaminobenzylamine as substrate. This alternative substrate was employed since it yields an aldehyde product with a "]'maxat 355 nm whereas benzaldehyde, the product of benzylamine oxidation, absorbs in the same region (ca 255 nm) as thiomolybdate and especially dithiomolybdate. The usage of this alternative substrate appears to have revealed aspects which are not apparent with the more commonly employed benzylamine. Thus our studies reported here provide some support for the view that both subunits of the dimer are active. They also demonstrate that there are significant differences between the forms including inhibition by dithiomolybdate. They also show that the forms are interconvertible. It is apparent that for BSAO it cannot be assumed that pooled enzyme sources are suitable for all studies. MATERIALS AND METHODS
Separation o f the different forms o f BSAO The protocol used and the nomenclature adopted was based on that reported by Summers et al. (1979). Six BSAO forms were defined compared to the four described by Summers et al. The method generally, but not always, yielded sufficient amounts of each form to enable a comprehensive comparison in most respects although in common with previous reports for SSAO (Falk et al., 1983) the proportions varied from batch to batch. Three active BSAO fractions (A, B and C) were obtained from the DEAE-cellulose column chromatography step by elution, successively, with 0.3, 0.07 and 0.2 M phosphate buffer at pH 7. This contrasts to the two fractions (A and B) obtained by Summers et al., 1979 following elution with 0.05 and 0.07 M phosphate buffer. For the subsequent step, that is separation into two subfractions (1 and 2) by hydroxyapatite chromatography, Summers et al. used 0.01 and 0.06 M phosphate buffer at pH 6.8 for the two-step elution, whereas 0.006 and 0.1 M phosphate were employed in the present study. Since Yamada and Yasonuba (1962) had reported that the enzyme(s) was unstable at buffer concentrations lower than 10 mM the protocol employed, described in detail later, minimized exposure to low ionic strength buffers particularly
during dialysis proved overall proached 60% Summers et al.
steps. This resulted in a much imyield, which while variable, apcompared to the 20% reported by (1979).
Separation protocol employed Bovine blood was collected fresh at the abattoir. All steps after coagulation were carried out at 4~C. Crystalline ammonium sulphate was added, with stirring, to serum cleared by centrifugation to give a final saturation of 35%. After 12 hr the precipitate was removed by centrifugation at 10,000 g for 40 min in an RC-5B Sorvall refrigerated centrifuge. The supernatant was then taken to 55% saturation with ammonium sulphate and after 12 hr the precipitate containing BSAO activity was harvested by centrifugation as before. This was taken up in 0.01 M phosphate buffer at pH 7 and dialysed against a large volume of 0.015 M buffer (phosphate at pH 7) over 24 hr. After removal of the inactive precipitate by centrifugation aliquots of the clear supernatant were applied to a DEAE-cellulose column (28.4 x 4 cm) previously equilibrated with 0.003M phosphate buffer at pH7. Active fractions were obtained by stepwise elution with, respectively, 0.03, 0.07 and 0.2 M phosphate buffer pH7 in sufficient volumes (generally 11 of each) to elute all the activity emerging at each step. The individual pooled elutes of the different peaks (A, B and C) were then taken to 40% saturation by the addition of crystalline ammonium sulphate and the pH adjusted to neutrality by the addition of concentrated ammonia. After 12 hr the inactive precipitate was removed by centrifugation and the active supernatants taken to 55% saturation as before. After 12 hr the precipitate was harvested by centrifugation, redissolved in 0.02M phosphate buffer pH 6.8 and equilibrated with the same buffer by dialysis. Aliquots of the three post-dialysis fractions obtained were diluted 3-fold with distilled water and applied to a hydroxyapatite column (2 x 17 cm) pre-equilibrated with 0.006 M phosphate buffer pH 6.8. Two peaks of BSAO activity were eluted from the column with 0.006 and 0.1 M phosphate buffer. Elution continued until no further BSAO activity emerged. The main active fractions in each peak were then pooled and adjusted to 55% saturation with solid ammonium sulphate and the pH adjusted to neutrality with concentrated ammonia. After 12hr the precipitate obtained by centrifugation was redissolved in 0.1 M phosphate buffer pH8 and dialysed 12 hr against the same buffer. The samples could be stored at - 2 0 ° C for at least 12 months with no loss of activity. Measurement o f the P-dimethylaminobenzylamine oxidase activity o f BSAO The p-dimethylaminobenzylamine oxidase activity of the serum, the eluates at the various stages of the purification eluates procedure, as well as the separated forms was measured continuously in a Gilford
Bovine serum amine oxidase
°,.'°°fd j
Table 1. K~t and K,, values for the oxidation of p-dimethylaminobenzylamine for most of the BSAO forms. The data were subjected to a non-linear least-square fit analysis (Leatherbarrow, 1987) assuming the involvementof two non-interacting active sites, K~t values are expressed as Ao.d. 355 nm/min x 10.3
ct!°I Okl
3
!
i
3"5
4
-Log(M)
Fig. l. Inhibition of the p-dimethylaminobenzylamine oxidase activity of bovine serum (15 mg protein per 1 ml assay cocktail) by dithiomolybdate. Vertical axis residual BSAO activity. Horizontal axis--log of dithiomolybdate concentration (M). 2000 single beam spectrophotometer coupled to a Phillips 8251 a single pen recorder. The appearance of product was followed at 355 nm for at least 10 min at 25°C. The temperature of a solution of the substrate in 0.1 M potassium phosphate buffer at p H 8 was raised to 25°C before the reaction was started by the addition o f the various B S A O preparations. The B S A O preparations were kept on ice before use, and the final assay cocktail volume was 1 ml. F o r the kinetic constants, etc., final substrate concentrations between 0.0025 and 14.40 m M were employed. N o background rate was observed in the absence of either enzyme or substrate. RESULTS
Double reciprocal plots ofp-dimethylaminobenzylamine concentration and reaction velocity showed variability and marked deviations from linearity (not shown). The data were subjected to a non-linear least square fit analysis (Leatherbarrow 1987) and was best described (Chi2 < 0.02) by a model which assumed the involvement of two non-interacting sites with different affinities for p-dimethylaminobenzylamine.
2/,0"
120"
/
/~,
K ~-(raM)
K~t
K~ (mM)
Ai
5.91 -I- 0.26
0.04 + 0.01
6.71 + 0.43
4.94 + 1.19
A2 BI CI C2 Serum
3.12 + 0.12 1.70 + 0.20 9.75___1.18 3.01 _+0.20 2.20_+0.22
0.07+ 0.01 0.26_ 0.18 0.18_+0.05 0.05_+0.01 0.14+ 0.04
9.68 __.6.41 4.93 + 4.31 6.07+ 0.21 0.38 _ 0.07 9.18_+4.86 20.70_+1.96 4.90_+1.57 14.00_+9.14 2.08_+0.87 21.90_+17.40
Typical low and high Km values were 0.14 + 0.04 and 21.3 -I- 17.4 mM. Early studies also showed that while the BSAO p-dimethylaminobenzylamine oxidase activity o f serum was inhibited by dithiomolybdate the interaction was complex, see Fig. 1, and incomplete even at dithiomolybdate concentrations o f 668 ttM where 30% of the activity remained. There was no comparable inhibition with either tri- or tetrathiomolybdate.
Studies with the purified forms Data, for example the results illustrated in Figs 2 for forms AI and A2, when subjected to a non-linear least-square fit analysis (Leatherbarrow, 1987) were consistent with the involvement of two active sites. The two Km and K~t values for the various purified forms are shown in Table 1.
Inhibition studies
Preliminary studies on serum
360-
Studies using aminoguanidine, the classic carbonyl group inhibitor of plasma amine oxidases revealed no apparent differences between the forms (Fig 3a). By
1/,oo[(a) . /
0L~
750"
2:6
500" / A1
671
250" 0
A2
o lb 2b
tIoL, a
-A
- Log. amg.(M)
ac;[Ic)i%
2.8 3-1 313 3~5 0 - Log.dith.(M) Fig. 2. Lineweaver-Burk, double reciprocal plots for the oxidation ofp-dimethylaminobenzylamine by BSAO forms A1 and A2 at 25°C in 0.1 M phosphate buffer at pH 8.0. Each point is the average _ standard deviation of at least five determinations of reaction rate. The data are consistent with the involvement of two non-interacting active sites whose kinetic constants are given in the table.
/-, 8 12 In c.(min)
Fig. 3. Inhibition and heat inactivation of some of the forms of BSAO. (3a) Inhibition by aminoguanidine (amg). (3b) Inhibition by dithiomolybdate (dith). (3c) Heat inactivation. Vertical axis--residual activity. Horizontal axis---duration of preincubation at 70°C. Symbols; O, A1;/% A2; 0 , B1; A, B2. Each point is the average + standard deviation of at least five determinations.
672
GERALD MULRYAN a n d JAMES MASON
1.2 Act.
C
12
that the enzyme fractions obtained by DEAE and hydroxyapatite chromatography have some differences in kinetic characteristics with respect to pdimethylaminobenzylamine oxidation and the forms examined show some variations in sensitivity to ZXOD mho dithiomolybdate and to heat inactivation. Nevertheless as has been concluded already for SSAO (Falk et al., 1983) they do appear to be forms of the same C , , 0 enzyme, since they were shown to be interconvertible 0 5 10 15 when resubjected to DEAE-cellulose chromatogfraction no. raphy after dialysis against low ionic strength buffers. Fig. 4. Interconversion of BSAO forms. Rapid rechroThe sensitivity of the forms to aminoguanidine is the matography of BSAO form AI on a DEAE--cellulose column. Elution was step-wise with successively,0.05, 0.07 same. There have been previous indications of this and 0.2 M phosphate buffer at pH7. All the BSAO activity interconversion; thus Yamada et al. (1976) showed applied was recovered in the eluate. that there was conformational isomerism at least between two forms (B and C). This does not seem to contrast with dithiomolybdate, there were clear have been pursued further. The interconversion may differences between the two forms examined in detail be one explanation for the variability in the A l and B2 (Fig. 3b) with respect to dithio as inhibitor. proportions of the forms from preparation to In contrast to serum the results for each form were preparation encountered by the authors in the present linear and linear regression analysis of the data gave study and by Falk et al. (1983) for SSAO. There has been some discussion as to whether there I 50% values of 1.42 and 33.3 mM, respectively. Heat inactivation studies also revealed differences are one or two active sites, and/or whether the two between forms and the results are shown in Fig. 3(c). subunits of the dimeric molecule are in fact identical. Residual activity was measured at a substrate concen- Several authors, for example Summers et al. (1979) tration of 3.6 mM after the different enzyme prep- and Berg and Abeles (1980) have put forward the hypothesis of two distinct active sites to explain, for arations had been heated for different times at 70°C. example, non-stereospecific oxidation by the A (and A t ~/2of 2.5 min was calculated by linear analysis for B) form of the enzyme since benzylamine (Battersby forms A~ and A2, compared to 1.1 min for B l and B2. et al., 1979) and p-hydroxbenzylamine (Suva and lnterconvertability o f the f o r m s Abeles, 1978) are oxidized stereospecifically. The use The forms were reexamined after dialysis at 4°C of p-dimethylaminobenzylamine as a substrate may against low ionic strength buffers (phosphate 0.003 M have revealed activities of both subunits not obvious with the more commonly used benzylamine. Thus at pH7 and 0.05 M Tris-HC1 containing 0.01 mM CuSO4. Repassage through DEAE-cellulose of the simple kinetics, that is linear double reciprocal plots BSAO forms revealed extensive interconversion, thus and single KmS, have generally been reported for Fig. 4 illustrates a typical elution profile, in this case benzylamine (e.g. Costa et al., 1978, Battersby et al., 1979) and p-hydroxybenzylamine (Suva and Abeles, for form A~. Forms A, B and C are apparent. All the 1978; Dooley and Cot6, 1984). By contrast the preenzyme activity put on the column could be sent studies consistently give non-linear (two-slope) accounted for. Results for all the forms examined are summarized in Table 2. It is evident for all forms that plots compatible with the hypothesis of two distinct active sites, one per subunit. significant redistribution has occurred. Benzylamine appears to be a better substrate in that at saturating conditions for the low Km comDISCUSSION ponent of the reaction about 10 times the rate of oxidation was observed with benzylamine compared The results highlight the difficulty of making simple to p-dimethylaminobenzylamine. There appeared to conclusions with respect to BSAO and its (their) be no significant differences between the ratios of inhibition by thiomolybdates nevertheless the studies activities of the two substrates between the different of BSAO as an enzyme(s) are of interest. They show forms (not shown). Thus the two substrates appear to be being oxidized by the same enzyme, and the forms Table 2. Redistribution (percentage of each form eluted _+SD) of appear to be variants of the same enzyme. The BSAO activity following repassage of each form on a heterogeneity of SSAO has been noted by Falk et al. DEAE-cellulose column after dialysis against 0.003 M phosphate buffer pH 7 (1983), and indeed these authors conclude that "it Each form (%) No. would not be useful in any circumstances to employ BSAO form A B C of runs enzymes that have been resolved chromatographiA~ 12.7_+4 40.8_+6.7 46.1_+3.7 5 cally since the species themselves (forms) are not A2 40.5 _+ 3.5 22.5 _+ 2.1 42.0 _+ 1.4 2 homogeneous". These authors, while reporting that B~ 34.7_+27.4 26.9_+ 13.3 38.4_+ 14.2 3 C~ 41.5 8.5 50 1 with benzylamine as a substrate, the catalytic properA~ 41.2 12.5 46.3 1 ties were similar, did nevertheless also caution that
..-'.......... Cond.
Bovine serum amine oxidase the practice of pooling the various chromatographic fractions could yield misleading results under some circumstances. Our data show that this is so, and not surprisingly, remixing forms gives complex kinetics analagous to serum (not shown). Overall the results reported here show that pdimethylaminobenzylamine is a more interesting substrate than benzylamine in that its usage reveals features of BSAO which are not apparent with benzylamine. Thus the non-linear kinetics would fit an hypothesis of two subunits both of which are active catalytically. Thus the subunits may be similar if not identical but could differ in other respects, possibly access of the substrate. In addition the forms defined by chromatography show different affinities for p-dimethylaminobenzylamine. All this is worthy of further study. Dithiomolybdate appears to reveal differences at least between some forms which are not apparent with the classic carbonyl reagents aminoguanidine, and semicarbazide. As for the implications with respect to the pathogenesis of the bovine molybdenosis syndrome the complexity of the interactions makes definitive conclusions difficult, although the in vitro inhibitory concentrations are relatively high compared to dietary Mo levels and it is hard to envisage concentrations of this order being attained in plasma in vivo. Furthermore the authors have shown that dithiomolybdate is not an important plasma constituent. Moreover the half-life of dithiomolybdate in plasma is quite short ( < 10hr) compared to tri- and tetrathiomolybdate (35-45 hr) and for a number of reasons trithiomolybdate is likely to be the most significant form in the biochemical pathogenesis of the syndrome (Mason, 1986; Mason, 1990). These higher forms did not inhibit at the concentrations used. Finally field studies (Mulryan and Mason, 1992) do not reveal loss of BSAO activity in hypocuprotic and hypocupraemic cattle, most of which would have been exposed to significant levels of pasture Mo. Thus, although it is possible the accumulation of dithiomolybdate could occur locally, present evidence suggests that inhibition of BSAO is unlikely to be part of the pathogenesis of the various syndromes. Acknowledgements--We are grateful to the WellcomeTrust
for financial support.
REFERENCES
Bachrach, U. (1985) Copper amine oxidases and amines as regulators of cellular processes. In Structure and Functions of Amine Oxidases (Edited by Mondovi B.), pp. 5-20. CRC Press, Boca Raton, Florida.
673
Battersby, A. A., Staunton, J., Klinman, J. and Summers, M. C. (1979) Stereochemistryof oxidation of benzylamine by the amine oxidase from beef plasma. FEBS Left 99, 297-298. Chidambaram, M. V. Barnes, G. and Frieden, E. (1984) Inhibition of ceruloplasmin and other copper oxidases by thiomolybdate. J. of Inorg. Biochem. 22, 231-241. Costa, M. T., Sabatini, S., Turini P. and Mondovi, B. (1978) Inhibition of beef plasma amine oxidase activity by glycine. FEBS Letts. 94, 113-114. Dooley, D. M. and Cot~, C. E. (1984) Inactivation of beef plasma amine oxidase by sulfide J. biol. Chem. 259, 2923-2926. Falk, M. C., Staton, A. J. and Williams, T. J. (1983) Heterogeneity of pig plasma amine oxidase: molecular and catalytic properties of chromatographically isolated forms. Biochemistry 22, 3746-3751. Gorkin, V. Z. (1985) Qualitative alteration (transformation) in catalytic activity of amine oxidases. In Structure and Functions of Amine Oxidases (Edited by Mondovi B.), pp. 205-208. CRC Press, Boca Raton, Florida. Janes, S. M., Mu, D., Wemmer, D., Smith, A. J. Kaurs, S., Maltby, D., Burlingame, A. L. and Klinman J. P. (1990) A new redox cofactor in eukaryotic enzymes: 6-hydroxydopa at the active site of bovine serum amine oxidase. Science 248, 981-987. Kelleher, C. A. and Mason, J. (1986) Reversible inhibition of ovine ceruloplasmin by thiomolybdates. Int. J. Biochem. 18, 629-635. Killgore, J., Schidt, C., Duich, L., Romero-Chapman, N. and Rucker, R. B. (1989) Nutritional importance of pyrroloquinoline quinone. Science 245, 850-852. Lannon, B. and Mason, J. (1986) The inhibition of bovine ceruloplasmin oxidase activity by thiomolybdates in vivo and in vitro: A reversible interaction. J. Inorg. Biochem. 26, 107-115. Leatherbarrow, R. J. (1987) Enzfitter, a non-linear regression analysis program for the IBPC: Elsevier Biosoft, The Netherlands. Mason, J., (1986) Thiomolybdates: Mediators of molybdenum toxicity and enzyme inhibitors. Toxicology 42, 99-109. Mason, J., (1990). The biochemical pathogenesis of molybdenum-induced copper deficiency syndromes in ruminants--towards the final chapters. Irish Veter. J. 43, 18-21. Mills, C. F., Delgarno, A. C. and Wenham, G. (1976) Biochemical and pathological changes in the tissues of Friesian cattle during the experimental induction of copper deficiency. Br. J. Nutri. 35, 309-329. Mulryan, G. and Mason, J. (1987) The inhibition of bovine serum amine oxidase by thiomolybdates in vivo and in vitro. Biochem. Soc. Trans. 15, 248-249. Mulryan, G. and Mason, J. (1992) Assessment of liver copper status in cattle from plasma copper and plasma copper enzymes. Ann. Rech. Vkt~r. 23, 233-238. Petterson G. (1985) Plasma amine oxidase. In Structure and Functions of Amine Oxidases. (Edited by Mondovi B.) pp. 105-120. CRC Press, Boca Raton, Florida. Summers, M. C., Markovic, R., and Klinman, J. P. (1979) Spectrochemistry and kinetic isotope effects in the bovine plasma amine oxidase catalysed oxidation of dopamine. Biochemistry 18, 1969-1978. Suva, R. H. and Abeles, R. M. (1978) Studies on the mechanism of action of plasma amine oxidase. Biochemistry 17, 3538-3545. Yamada, H. and Yasonubu, K. T. (1962) Monoamine oxidase II. J. biol. Chem. 237, 3077-3082.