Vol. 188, No. 2, 1992 October
BIOCHEMICAL
AND BIOPHYSICAL
30, 1992
INTERACTION
Armine
OF DOPAMINE-fi-MONOOXYGENASE GRANULE MEMBRANE LIPIDS
WITH
G.Pogosyan, Anna S.Boyajian, Marietta and Konstantin G.Karagezyan
Institute of Molecular Armenian Republic, Received
RESEARCH COMMUNICATIONS Pages 678-683
September
9,
CHROMAFFIN
Y.Mkrtchyan
Biology, Academy of Sciences of the 375044, Yerevan, Armenian Republic
1992
The interaction between bovine adrenal medullary dopamine-fi-monooxygenase and liposomes from chromaffin granule and salt concentration membrane lipids as a function of pH, lipid Efficient adsorption of was studied by ultracentrifugation. to liposomes occurs in the pH range dopamine-@-monooxygenase The adsorption was not detec5.0-6.5 and at low ionic strength. The membrane dopamine-P-monooxygeted in the case of apoenzyme. nase forms acomplex with liposomes more effective than soluble The data obtained lead to certain conclusions about the does. 0 1992Academic specificity of complex between the enzyme and liposomes. Press,Inc. Dopamine-fl-monooxygenase (EC 1.14.17.1) catalyzes the conversion of dopamine to noradrenaline [l]. The enzyme is present in catecholamine secretory vesicles (chromaffin granules) of cells [2] and adrenergic neurons [3]. both adrenal medullary There are two forms of the enzyme in the granules, a membrane form and a soluble form. The membrane form, being one of the peripheral proteins of the granular membrane and localized on its inner surface, can be extracted by detergents and separated in a water-soluble form. In vitro experiments have shown that in a hydrophilic medium, homogeneous preparations of both forms are practically identical in structure, catalytic activity and immunological characteristics. However, membrane dopamine-fl-monooxygenase contains an additional hydrophobic peptide, absent from the soluble form, which does not influence its physicochemical characteristics when in a hydrophilic medium [4-71. The functional significance of the location of the enzyme in both the membrane and the soluble fraction of granules is not clear. The structural basis for the attachment of membrane 0006-291X/92 Copyright All rights
$4.00
0 1992 by Academic Press, of reproduction in any form
Inc. reserved.
678
Vol.
188,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
dopamine-fl-monooxygenase to biological and model membranes as well as the role of lipid surrounding in this process and possible influence of membrane lipids on the enzyme structural and catalytic properties have been difficult to ascertain. Recently we were investigated the interaction of the membrane and soluble forms of dopamine-@-monooxygenase with the vesicles made of an individual phospholipids [8,9]. The ability of the enzyme to form the reversible complex with some of these vesicles accompanied by the modification of the enzyme catalytic properties was observed. In this work we have investigated the association of dopamine-@-monooxygenase to liposomes made of total chromaffin granule membrane lipids and specificity of this process. A comparison of the efficiency of the association for membrane and soluble dopamine-@-monooxygenase is also presented.
MATERIALS
AND
METHODS
Chromaffin granules from bovine adrenal medulla were prepared by the method of Hoffman et al [lo]. Preparations of membrane and soluble dopamine-fl-monooxygenase were obtained from the corresponding fractions of the granules in the electrophoretically homogeneous forms by a method published previously [ll]. Electrophoretically homogeneous preparations included membrane and soluble forms of dopamine-b-monooxygenase were obtained from bovine adrenal medulla by the procedure of Ljones et al [12]. On the initial steps of both the purification procedures a catalase was added as described by Colombo et al [13]. The specific activity of dopamine-fl-monooxygenase preparations assayed by the method of Kuzuya and Nagatsu [14] with tyramine as substrate, was such that 25f5 Jnmol of hydroxytyramine were formed miri' md of protein. Protein concentration was determined according to Lowry et al [15], using bovine serum albumin as standard. The apoenzyme was prepared by dialysis against EDTA [lG].Lipids were extracted from chromaffin granule membrane according to the method described by Bligh and Dayer[l7].The lipid mixture was evaporated to dryness under nitrogen stream. To the resulted thin film the appropriate amount of 20mM potassium phosphate solution at corresponding pH (see results) was added and the liposome suspension was produced by mechanical shaking for 30 min at room were not contaminated by temperature. Preparations of liposomes protein. The concentration of lipids was calculated from the weight of dried matter. The enzyme and liposome mixture were infor 60 cubated at room temperature for 60 min, then centrifuged min at 100 OOOg in a Beckman (USA) ultracentrifuge (model SpinCO L-2).After centrifugation the protein contents of the pellet were calculated from the difference between the initial concentraControl centrifugation of the tion and that in the supernatant. enzyme alone did not give any pellet.Spectrophotometric measurements were made on a Zeiss (Germany) spectrophotometer (model The data presented below are the results of three Spekord M-40). Each experimental point is the average independent experiments. of five measurements. 679
Vol.
188, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
RESULTS Centrifugation sence
of
with
mes adsorb of
about the
lipid
40% of
binding
the
the
enzyme
enzyme
strength lished form of enzyme.
pH 5.5
total
the
pellet
decreased
enzyme.
the
was four No binding
times
the
enzyme
The
prepellet-
abscissa
in
in the suspension beconcentration the liposoof the
was also
enzyme.
A depen-
observed
above with
(fig.3). An NaCl concentration the adsorption. The extent of the
in
in a very narrow range at pH 5.5. Practically
in
liposomes
that
(fig.l).
amount
pH value
occurs optimum
dopamine-b-monooxygenase
suspension
concentration In saturating
the
was found
to
at
on the
Efficient adsorption es:5.0-6.5,with the of
liposome showed
liposomes
represents the the centrifugation.
dence
the
dopamine-fl-monooxygenase
ed together fig.1 fore
of
pH 7.0.
(fig.2). of
pH valuno amount
Binding
increasing
of
ionic
of 0.1 M completely aboadsorption for membrane higher,
was observed
in
than
for
the
case
prove
the
soluble of
apo-
DISCUSSION
ence
of
The results
which
interaction
between
lipid
concentration
are
presented
above
dopamine-fl-monooxygenase
existand
chro-
(mg/ml)
Fig.1. Dependence of the d opamine-fl-monooxygenase liposomes on lipid concentration (pH 5.5).Protein in all samples was O.01mg/ml;sample volume 7 ml.
adsorption to concentration
Fig.2. Dependence of the dopamine-@monooxygenase adsorption to liposomes on pH values. Protein and lipid concentrations were 0.04 mg/ml and 0.18 mg/ml, respectively; sample volume 7 ml. 680
Vol.
188, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
a4.
a3-
a2-
CL1_
L
I
1
M25 w50 NoCl concentmfion
a075 fMI
Ffg.3. Dependence of the dopamine-fi-monooxygenase liposomes on NaCl concentration (pH 7.0). Other conditions as in fig.1.
maffin
granule
membrane
pheral
proteins
to
lipids.
lipids
multielectrostatic
Usually,
bilayer
or
attraction.
Also
ing effect of salt concentration (fig.3) suggests that interactions The decrease for the adsorption. liposomes
surface
results
in
brought
liposome main
fraction line,
has
components are
of
the
zwitterionic
in
this
explained
case,
by increasing
chromaffin
phosphatidylethanolamine
is
of peri-
the
by
diminish-
on the adsorption to liposomes of ionic type are responsible in electric potential of the
a negative
or
adsorption
membranes
the decrease in the coulombic surface and ionic groups
Dopamine-fl-monooxygenase The
about
the
adsorption to experimental
salt
attraction of
the
charge
at
granule
neutral
concentration between protein. pH 5.5
membrane
lipids
the [18].
lipids
(phosphatidylcho-
and sphingomyelin).A
smaller
amo-
unt of acidic phospholipids form the negative surface charge [19]. Therefore, there must be specific positively charged domain(s) on protein molecule acting as an adsorptive site. It is clear, may
that
the
diminishes
ed such
abolished
the
to
9.0
positive (fig.2).
decrease
adsorption 6.0
in
adsorption
a monotonic
drogenase pH from
decrease
to [20].
at pH value
in glyceraldehyde-3-phosphate
phospholipid Here
below
charge with increasing pH It was previously observliposomes
unexpectedly,
5.5.The
the
same dependence
dehy-
with
increasing
adsorption of
is
an adsor-
ption on pH value was observed by us earlier, when investigating the complex formation between dopamine-fl-monooxygenase and formed from neutral phospholipids, by the use of a vesicles, fluorescence anisotropy techniques [8]. The comparison of Kdiss 681
Vol.
188,
values
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
of
complex determined at different pH has shown that at times lower than that at pH 7.2. In PH 5.7 Kdiss is about twenty connection with this, it is important to note that dopamine-@-monooxygenase undergoes reversible tetramer to dimer dissociation and this dissociation is pH dependent [20 - 221. At pH 5.5-5.7 the tetrameric form dominantly exists in the enzyme population, whereas further increase in pH affects the decrease Thus the data presented in fig.2 in tetramer to dimer ratio. might also suggest that the affinity of phospholipid vesicles to tetramer is greater that to dimer. On the other hand the dependence of the association of dopamine-fi-monooxygenase to phoson the state of the enzyme subunit dissociapholipid vesicles tion suggests about the specific of complex formation process. It is interesting that both the membrane and the soluble forms of dopamine-fl-monooxygenase exist in the interior of the chromaffin granules which is at pH 5.5-5.7 [23]. In this pH range the enzyme is more active, because the tetrameric form has a lower Km for substrate than dimeric does [21]. So far the results of experiments that association of our suggest the dopamine-fl-monooxygenase to vesicles formed from chromaffin granule membrane lipids is favored under the pH, characteristic to the in vivo environment. Therefore it seems likely that, besides charge, other factors such as conformational changes can modify the adsorption properties of dopamine-fl-monooxygenase. This conclusion is confirmed by inability of apoenzyme to associate with liposomes, and also by comparing the efficiency of adsorption of membrane and soluble forms of the enzyme. On the other hand the higher extent of adsorption for the membrane dopamine-fl-monooxygenase compared to the soluble form also suggests that the extra hydrophobic peptide of membrane dopamine-fi-monooxygenase does participate in the interaction of the enzyme with chromaffin granule membrane lipids, and serves to provide firmer binding of membrane dopamine-P-monooxygenase to the granular membrane.
REFERENCES 1.
Friedman, S., and Kaufman, 4763- 4773. 2. Laduron, P. (1975) FEBS Lett. 3. Potter, L-T., and Axelrod, 142, 291-298. 4. Bjerrum, O.J., Helle, K.B., 181, 231-237.
S. 52, J.
(1965)
240,
132-134. (1963) J.Pharmacol.Exp.Ther.
and Bock,
682
J.Biol.Chem.
E.
(1979)
Biochem
J.
Vol.
188,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
5.
Slater, E-P., Zaremba, C., and Hoque-Angeletti, R.A. (1981) Arch.Biochem.Biophys. 211, 288-293. 6. Taljanidisz, J., Stewart, K., Smith, A.J., and Klinman, J.P. (1989) Biochemistry 28, 10054-10061. 7. Stewart, L.C., and Klinman, J.P. (1988) Ann.Rev.Biochem. 57, 551-592. (1990) Biomedical 8. Boyadzhyan, A.S., and Karagezyan, K.G. Science 1, 379-383. 9. Pogosyan, A.G., Boyadzhyan A.S., Mkrtchyan M.Y., and Karagezyan, K.G. (1992) Biomedical Science (in press). 10. Hoffman, P.G., Zinder, O., Bonner, W.M., and Pollard, H.B. (1976) Arch.Biochem.Biophys. 176, 375-388. 11. Foldes, A., Jeffrey, P.L., Preston, B.N., and Austin, L. (1972) Bi0chem.J. 126, 1209-1217. 12. Ljones, T., Skotland, T., and Flatmark, T. (1976) Eur.J.Biochem. 61, 525-533. 13. Colombo, G., Papadopoulos, N.J., Ash, D.E., and Villafranca, J.J. (1987) Arch.Biochem.Biophys. 252, 71-80. T. (1969) Enzymologia 36, 31-38. 14. Kuzuya, H., and Nagatsu, 15. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J.Biol.Chem. 193, 265-275. 16. Skotland, T., and Ljones, T. (1979) Eur.J.Biochem. 94, 145151. 17. Bligh, E.G., and Dyer, W-1. (1959) Can.J.Biochem.Physiol. 37,
911-917.
Skotland, T., and Ljones, T. (1979) Inorg.Persp. Biol.and 2, 151-180. Medic., 19. Dreyfus, H., Aunis, D., Harth, S., and Mandel, P. (1977) Biochem. Biophys.Acta 489, 89-97. 20. Gutowicz, J., and Madrzycka, T. (1978) Biochem.Biophys.Acta 554, 358-363. 21. Saxena, A., Hensley, P., Osborne, J.C., Fleming, J., and Fleming, P.J. (1985) J.Biol.Chem. 260, 3386-3392. 22. Dhawan, S., Hensley, P., Osborne, J. C., Fleming, Jr.P., and Fleming, P.J. (1986) J.Biol.Chem. 261, 7680-7684. 23. Winkler, H., and Westhead, H. (1980) Neuroscience 51, 18031823. 18.
683
BIOCHEMICAL
AND BIOPHYSICAL
30, 1992
INTERACTION
Armine
OF DOPAMINE-fi-MONOOXYGENASE GRANULE MEMBRANE LIPIDS
WITH
G.Pogosyan, Anna S.Boyajian, Marietta and Konstantin G.Karagezyan
Institute of Molecular Armenian Republic, Received
RESEARCH COMMUNICATIONS Pages 678-683
September
9,
CHROMAFFIN
Y.Mkrtchyan
Biology, Academy of Sciences of the 375044, Yerevan, Armenian Republic
1992
The interaction between bovine adrenal medullary dopamine-fi-monooxygenase and liposomes from chromaffin granule and salt concentration membrane lipids as a function of pH, lipid Efficient adsorption of was studied by ultracentrifugation. to liposomes occurs in the pH range dopamine-@-monooxygenase The adsorption was not detec5.0-6.5 and at low ionic strength. The membrane dopamine-P-monooxygeted in the case of apoenzyme. nase forms acomplex with liposomes more effective than soluble The data obtained lead to certain conclusions about the does. 0 1992Academic specificity of complex between the enzyme and liposomes. Press,Inc. Dopamine-fl-monooxygenase (EC 1.14.17.1) catalyzes the conversion of dopamine to noradrenaline [l]. The enzyme is present in catecholamine secretory vesicles (chromaffin granules) of cells [2] and adrenergic neurons [3]. both adrenal medullary There are two forms of the enzyme in the granules, a membrane form and a soluble form. The membrane form, being one of the peripheral proteins of the granular membrane and localized on its inner surface, can be extracted by detergents and separated in a water-soluble form. In vitro experiments have shown that in a hydrophilic medium, homogeneous preparations of both forms are practically identical in structure, catalytic activity and immunological characteristics. However, membrane dopamine-fl-monooxygenase contains an additional hydrophobic peptide, absent from the soluble form, which does not influence its physicochemical characteristics when in a hydrophilic medium [4-71. The functional significance of the location of the enzyme in both the membrane and the soluble fraction of granules is not clear. The structural basis for the attachment of membrane 0006-291X/92 Copyright All rights
$4.00
0 1992 by Academic Press, of reproduction in any form
Inc. reserved.
678
Vol.
188,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
dopamine-fl-monooxygenase to biological and model membranes as well as the role of lipid surrounding in this process and possible influence of membrane lipids on the enzyme structural and catalytic properties have been difficult to ascertain. Recently we were investigated the interaction of the membrane and soluble forms of dopamine-@-monooxygenase with the vesicles made of an individual phospholipids [8,9]. The ability of the enzyme to form the reversible complex with some of these vesicles accompanied by the modification of the enzyme catalytic properties was observed. In this work we have investigated the association of dopamine-@-monooxygenase to liposomes made of total chromaffin granule membrane lipids and specificity of this process. A comparison of the efficiency of the association for membrane and soluble dopamine-@-monooxygenase is also presented.
MATERIALS
AND
METHODS
Chromaffin granules from bovine adrenal medulla were prepared by the method of Hoffman et al [lo]. Preparations of membrane and soluble dopamine-fl-monooxygenase were obtained from the corresponding fractions of the granules in the electrophoretically homogeneous forms by a method published previously [ll]. Electrophoretically homogeneous preparations included membrane and soluble forms of dopamine-b-monooxygenase were obtained from bovine adrenal medulla by the procedure of Ljones et al [12]. On the initial steps of both the purification procedures a catalase was added as described by Colombo et al [13]. The specific activity of dopamine-fl-monooxygenase preparations assayed by the method of Kuzuya and Nagatsu [14] with tyramine as substrate, was such that 25f5 Jnmol of hydroxytyramine were formed miri' md of protein. Protein concentration was determined according to Lowry et al [15], using bovine serum albumin as standard. The apoenzyme was prepared by dialysis against EDTA [lG].Lipids were extracted from chromaffin granule membrane according to the method described by Bligh and Dayer[l7].The lipid mixture was evaporated to dryness under nitrogen stream. To the resulted thin film the appropriate amount of 20mM potassium phosphate solution at corresponding pH (see results) was added and the liposome suspension was produced by mechanical shaking for 30 min at room were not contaminated by temperature. Preparations of liposomes protein. The concentration of lipids was calculated from the weight of dried matter. The enzyme and liposome mixture were infor 60 cubated at room temperature for 60 min, then centrifuged min at 100 OOOg in a Beckman (USA) ultracentrifuge (model SpinCO L-2).After centrifugation the protein contents of the pellet were calculated from the difference between the initial concentraControl centrifugation of the tion and that in the supernatant. enzyme alone did not give any pellet.Spectrophotometric measurements were made on a Zeiss (Germany) spectrophotometer (model The data presented below are the results of three Spekord M-40). Each experimental point is the average independent experiments. of five measurements. 679
Vol.
188, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
RESULTS Centrifugation sence
of
with
mes adsorb of
about the
lipid
40% of
binding
the
the
enzyme
enzyme
strength lished form of enzyme.
pH 5.5
total
the
pellet
decreased
enzyme.
the
was four No binding
times
the
enzyme
The
prepellet-
abscissa
in
in the suspension beconcentration the liposoof the
was also
enzyme.
A depen-
observed
above with
(fig.3). An NaCl concentration the adsorption. The extent of the
in
in a very narrow range at pH 5.5. Practically
in
liposomes
that
(fig.l).
amount
pH value
occurs optimum
dopamine-b-monooxygenase
suspension
concentration In saturating
the
was found
to
at
on the
Efficient adsorption es:5.0-6.5,with the of
liposome showed
liposomes
represents the the centrifugation.
dence
the
dopamine-fl-monooxygenase
ed together fig.1 fore
of
pH 7.0.
(fig.2). of
pH valuno amount
Binding
increasing
of
ionic
of 0.1 M completely aboadsorption for membrane higher,
was observed
in
than
for
the
case
prove
the
soluble of
apo-
DISCUSSION
ence
of
The results
which
interaction
between
lipid
concentration
are
presented
above
dopamine-fl-monooxygenase
existand
chro-
(mg/ml)
Fig.1. Dependence of the d opamine-fl-monooxygenase liposomes on lipid concentration (pH 5.5).Protein in all samples was O.01mg/ml;sample volume 7 ml.
adsorption to concentration
Fig.2. Dependence of the dopamine-@monooxygenase adsorption to liposomes on pH values. Protein and lipid concentrations were 0.04 mg/ml and 0.18 mg/ml, respectively; sample volume 7 ml. 680
Vol.
188, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
a4.
a3-
a2-
CL1_
L
I
1
M25 w50 NoCl concentmfion
a075 fMI
Ffg.3. Dependence of the dopamine-fi-monooxygenase liposomes on NaCl concentration (pH 7.0). Other conditions as in fig.1.
maffin
granule
membrane
pheral
proteins
to
lipids.
lipids
multielectrostatic
Usually,
bilayer
or
attraction.
Also
ing effect of salt concentration (fig.3) suggests that interactions The decrease for the adsorption. liposomes
surface
results
in
brought
liposome main
fraction line,
has
components are
of
the
zwitterionic
in
this
explained
case,
by increasing
chromaffin
phosphatidylethanolamine
is
of peri-
the
by
diminish-
on the adsorption to liposomes of ionic type are responsible in electric potential of the
a negative
or
adsorption
membranes
the decrease in the coulombic surface and ionic groups
Dopamine-fl-monooxygenase The
about
the
adsorption to experimental
salt
attraction of
the
charge
at
granule
neutral
concentration between protein. pH 5.5
membrane
lipids
the [18].
lipids
(phosphatidylcho-
and sphingomyelin).A
smaller
amo-
unt of acidic phospholipids form the negative surface charge [19]. Therefore, there must be specific positively charged domain(s) on protein molecule acting as an adsorptive site. It is clear, may
that
the
diminishes
ed such
abolished
the
to
9.0
positive (fig.2).
decrease
adsorption 6.0
in
adsorption
a monotonic
drogenase pH from
decrease
to [20].
at pH value
in glyceraldehyde-3-phosphate
phospholipid Here
below
charge with increasing pH It was previously observliposomes
unexpectedly,
5.5.The
the
same dependence
dehy-
with
increasing
adsorption of
is
an adsor-
ption on pH value was observed by us earlier, when investigating the complex formation between dopamine-fl-monooxygenase and formed from neutral phospholipids, by the use of a vesicles, fluorescence anisotropy techniques [8]. The comparison of Kdiss 681
Vol.
188,
values
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
of
complex determined at different pH has shown that at times lower than that at pH 7.2. In PH 5.7 Kdiss is about twenty connection with this, it is important to note that dopamine-@-monooxygenase undergoes reversible tetramer to dimer dissociation and this dissociation is pH dependent [20 - 221. At pH 5.5-5.7 the tetrameric form dominantly exists in the enzyme population, whereas further increase in pH affects the decrease Thus the data presented in fig.2 in tetramer to dimer ratio. might also suggest that the affinity of phospholipid vesicles to tetramer is greater that to dimer. On the other hand the dependence of the association of dopamine-fi-monooxygenase to phoson the state of the enzyme subunit dissociapholipid vesicles tion suggests about the specific of complex formation process. It is interesting that both the membrane and the soluble forms of dopamine-fl-monooxygenase exist in the interior of the chromaffin granules which is at pH 5.5-5.7 [23]. In this pH range the enzyme is more active, because the tetrameric form has a lower Km for substrate than dimeric does [21]. So far the results of experiments that association of our suggest the dopamine-fl-monooxygenase to vesicles formed from chromaffin granule membrane lipids is favored under the pH, characteristic to the in vivo environment. Therefore it seems likely that, besides charge, other factors such as conformational changes can modify the adsorption properties of dopamine-fl-monooxygenase. This conclusion is confirmed by inability of apoenzyme to associate with liposomes, and also by comparing the efficiency of adsorption of membrane and soluble forms of the enzyme. On the other hand the higher extent of adsorption for the membrane dopamine-fl-monooxygenase compared to the soluble form also suggests that the extra hydrophobic peptide of membrane dopamine-fi-monooxygenase does participate in the interaction of the enzyme with chromaffin granule membrane lipids, and serves to provide firmer binding of membrane dopamine-P-monooxygenase to the granular membrane.
REFERENCES 1.
Friedman, S., and Kaufman, 4763- 4773. 2. Laduron, P. (1975) FEBS Lett. 3. Potter, L-T., and Axelrod, 142, 291-298. 4. Bjerrum, O.J., Helle, K.B., 181, 231-237.
S. 52, J.
(1965)
240,
132-134. (1963) J.Pharmacol.Exp.Ther.
and Bock,
682
J.Biol.Chem.
E.
(1979)
Biochem
J.
Vol.
188,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
5.
Slater, E-P., Zaremba, C., and Hoque-Angeletti, R.A. (1981) Arch.Biochem.Biophys. 211, 288-293. 6. Taljanidisz, J., Stewart, K., Smith, A.J., and Klinman, J.P. (1989) Biochemistry 28, 10054-10061. 7. Stewart, L.C., and Klinman, J.P. (1988) Ann.Rev.Biochem. 57, 551-592. (1990) Biomedical 8. Boyadzhyan, A.S., and Karagezyan, K.G. Science 1, 379-383. 9. Pogosyan, A.G., Boyadzhyan A.S., Mkrtchyan M.Y., and Karagezyan, K.G. (1992) Biomedical Science (in press). 10. Hoffman, P.G., Zinder, O., Bonner, W.M., and Pollard, H.B. (1976) Arch.Biochem.Biophys. 176, 375-388. 11. Foldes, A., Jeffrey, P.L., Preston, B.N., and Austin, L. (1972) Bi0chem.J. 126, 1209-1217. 12. Ljones, T., Skotland, T., and Flatmark, T. (1976) Eur.J.Biochem. 61, 525-533. 13. Colombo, G., Papadopoulos, N.J., Ash, D.E., and Villafranca, J.J. (1987) Arch.Biochem.Biophys. 252, 71-80. T. (1969) Enzymologia 36, 31-38. 14. Kuzuya, H., and Nagatsu, 15. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J.Biol.Chem. 193, 265-275. 16. Skotland, T., and Ljones, T. (1979) Eur.J.Biochem. 94, 145151. 17. Bligh, E.G., and Dyer, W-1. (1959) Can.J.Biochem.Physiol. 37,
911-917.
Skotland, T., and Ljones, T. (1979) Inorg.Persp. Biol.and 2, 151-180. Medic., 19. Dreyfus, H., Aunis, D., Harth, S., and Mandel, P. (1977) Biochem. Biophys.Acta 489, 89-97. 20. Gutowicz, J., and Madrzycka, T. (1978) Biochem.Biophys.Acta 554, 358-363. 21. Saxena, A., Hensley, P., Osborne, J.C., Fleming, J., and Fleming, P.J. (1985) J.Biol.Chem. 260, 3386-3392. 22. Dhawan, S., Hensley, P., Osborne, J. C., Fleming, Jr.P., and Fleming, P.J. (1986) J.Biol.Chem. 261, 7680-7684. 23. Winkler, H., and Westhead, H. (1980) Neuroscience 51, 18031823. 18.
683
