A proper transmembrane Ca2+ gradient is essential for the higher enzymatic activity of adenylate cyclase

Vol.

175,

March

No.

2, 1991

BIOCHEMICAL

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RESEARCH

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Pages 366-371

15, 1991

A PROPER TRANSMEMBRANE Ca2+ GRADIENT IS ESSENTIAL FOR THE HIGHER ENZYMATIC ACTIVITY OF ADENYLATE CYCLASE F.Y.

Yang*

and Y.P.

Tu

National Laboratory of Biomacromolecules, Biophysics, Academia Sinica, 100080, Received

January

21,

Institute of Beijing, China

1991

Summary: Adenylate cyclase from bovine brain cortex was reconstituted into liposomes with (1000 fold) or without transmembrane Ca2+ gradient. The highest enzyme activity (the active center of enzyme exposing outside) was observed in the vesicles with lower Ca2+ concentration outside (~10~~ M, similar to physiological condition). If the transmembrane Ca2+ gradient was in the inverse direction (i.e. higher Ca2+ concentration outside, 1 mM), a lowest enzyme activity would appear. Sucha difference could be diminished following addition of A23187. Obtained results showed that a proper transmembrane Ca2+ gradient is essential for the optimal fluidity of phospholipid bilayer, favouring the formation of suitable conformation of adenylate cyclase with higher enzyme activity. 0 1991AcademicPrx55, Inc.

The cytosolic free Ca2+ in most cells is around 10-7-10'6 M, whereas the extracellular Ca2+ concentration is about 10m3 M. So, it results a 1000-10000 fold transmembrane Ca2+ gradient.[ll It is well known that the maintenance of such concentration gradient is of vital importance in the cell function.[21 What is the role of transmembrane Ca2+ gradient for lipid-protein interaction of biomembrane? Is it essential for the formation of a suitable conformation with higher activity of transmembrane enzymes? We have reported that divalent cations may play a role in altering the fluidity of the proteoliposomes, leading to a conformation change in the reconstituted H+-ATPase, which increase its activi* To whom correspondence

should

be addressed.

Abbreviations ACc: Catalytic unit of adenylate cyclase; ACc*Lca--: Twoside lower Ca2+ ( 1 MM Ca2+, similar to cytosolic concentration ) ACc-containing roteoliposomes. ACc*Lca++: Two-side higher Ca2+ ( 1 mM Ca if + ) ACc-containing proteoliposomes. ACc*Lca+-: Inside higher Ca2+ ACc-containing proteoliposomes. Outside higher Ca2+ ACcsLca-+: ACc-containing proteoliposomes. CD: Circular dichroism. DPH: Diphenyl hexatriene. 0006291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

366

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ty.r3,*l In the present study, adenylate cyclase from bovine brain cortex was reconstituted into soybean phospholipid vesicles with (1000 fold) or without transmembrane Ca2+ gradient. The enzyme activity, conformation and fluidity of four types of proteoliposomes (the active center of enzyme facing outside ) were compared. Obtained results showed that a proper transmembrane Ca2+ gradient is essential for the optimal fluidity of phospholipid bilayer and suitable conformation of adenylate cyclase with higher enzyme activity.

Materials

and Methods

Materials: ATP, Lubrol, A23187 were purchased from Sigma Chemical Co. Octyl glucoside, Dithiothreitol, Aresenazo III and cholic acid from SERVA. DPH was product of Molecular Probes Co. Asolectin was purchased from Nanyuan Plant Oil Factory in Beijing, China and purified before use. Preparation of resolved catalytic unit of adenylate cyclase. The catalytic unit of adenylate cyclase ( ACc ) from bovine brain cortex was solubilized and resolved from B-adrenegic receptor and GTP regulatory protein by the method of Cerione et a1.151 The specific enzyme activity, assayed in the presence of 15mM Mg2+, was 200-400 pmol cAMP/min/mg protein. Reconstitution of catalytic unit of adenylate cyclase into asolectin vesicles by Sephadex G50 qel filtration. 1 mg resolved ACc was incubated with sonicated asolectin (10mg in 0.6 ml buffer A: 10 mM Tris-HCl, pH7.4, 100 mM NaCl, 1pM Ca2+, 1 mM DTT) and o&y1 glucoside (0.85%) for 30 min at O°C. Removal of detergent was accomplished by Sephadex G50 methodL61. The eluates were centrifuged at 100,OOOxg for 1 hr at 4OC, and the protein-lipid pellets were suspended. Four types of proteoliposomes, incorporating ACc were reconstituted separately as follows: Two-side lower Ca2+-containing (1pM Ca2+,similar to cytosolic concentration) proteoliposomes ( ACcmLca-): The eluate in buffer A was centrifuged and the pellet was suspended in the same buffer. outside higher Ca2+- containing proteoliposomes (ACceLca-+): the pellet obtained in (a) was suspended in the buffer B (identical to the buffer A except that 1 mM Ca2+ replace the l/.&M Ca2+). cc> inside higher Ca2+ -containing proteoliposomes (ACc*Lca+-): 100 mM EGTA was added to the eluate in buffer B, and the pellet was sedimented by centrifugation.The sediment obtained was suspended in the buffer A. Two-side higher Ca2+- containing proteoliposomes (ACc.Lca++): the sediment obtained in (c) was suspended in the buffer B. Adenvlate cvclase and protein assay. Adenylate cyclase activity was measured by the method of Cerion et al-r51 with slight modification. The enzyme activity shown was basal activity. Protein concentration was determined by the method of Lowry et a1.[71 Fluorescence measurement.[81 Fluorescence was measured using Hatachi F4010 spectrofluorimeter equipped with temperature control. Measurement was made in the ratio mode with excitation and emission band widths of 5 nm. Enzyme concentration was 50 pg/ml. Samples were excited at 285 nm, and the emission light monitored at 300-380 nm. 367

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were recorded on a JASCO Circular dichroism.[91 CD spectra All measurements were done in 10 mM J-500 spectropolarimeter. To eliminate Tris-HCl ( pH 7.4 ), 100 mM NaCl at 25 OC. scattering due to liposomes, each CD spectrum of liposomes was The subtracted from that of the proteoliposomes containing ACc. ellipticity curve was calculated by averaging 3 records. The e-helix content were estimated by the method of Chen et alil"l 10 ~1 DPH tetrahyFluorescence polarization study using DPH. drofurane solution ( 22 mM ) was added to 5 ml of 10 mM Trismixed for 15 min. To 1.5 ml of the solution, 1.0 mg HCl (pH 7.6), 10 mM Tris-HCl ( pH 7.6 ) phospholipid of proteoliposomes and were added to make a total volume of 2.0 ml. After vigorous stirring, this solution was incubated at 30 OC for 15 min, then at room temperature for 2 hrs. Fluorescence was measured by means of a Hitachi 650-60 fluorescence spectrophotometer fitted with a polarization attachment at 30 OC. The wavelengths of excitation and emission were 360 nm and 430 nm, respectively. Results

and Discussion

The reconstituted activity of ACc in asolectin vesicles could be markedly stimulated by forskolin ( 6 fold ) or Mn2+ (5 fold), but no stimulation was observed with guanidine nucleotide. This may deduce the ACc used for incorporating into asolecthat tin vesicles was highly resolved from GTP regulatory protein. The efficiency of reconstitution of ACc ranged from 40-60% as estimated from protein determination. The proteoliposomes were prepared with sufficiently low Ca2+ permeability. Addition of ionophore A23187 would lead to a release of Ca2+ outward, and hence a rapid increase of absorbance (675-680 nm) using Aresenazo III as Ca2+ indicator ( data not shown ). Following treatment with sodium cholate, no obvious difference in enzymatic activity of reconstituted proteoliposomes would be detected. So, it could be deduced that ACc was inserted in a highly oriented manner with most of the active sites facing outside, similar to inside-out cell preparations. Then, the enzymatic activities of four types of proteoliposomes with or without Ca2+ gradient were determined and compared. From Table 1, it could be seen that the highest activity was observed in the case of ACc*Lca+(lower Ca2+ outside) which Table 1. enzymatic

Effect of activity

Proteoliposomes Enzyme ( pm01 /mg

ACc*Lca+-

activity cAMP/min protein )

* Number

of

transmembrane Ca2+ gradient on the of ACc containing proteoliposomes

880 ( 5 I*

ACc*Lca-370 ( 5 1

experiments. 368

ACc*Lca++ 270 (5 1

ACcsLca-+ 180 (7 1

Vol. 175, No. 2, 1991 Table 2. following

Proteoliposomes

BIOCHEMICAL

AND BIOPHYSICAL

Changes in adenylate the

dissipation

cyclase of

Ca2+ gradient

RESEARCH COMMUNICATIONS

activity

transmembrane

by A23187

Enzyme activity(pmo1

cAMP/min/mg) +A23178*

ACc*Lca+-

780

350

ACceLca-+

110

280

* Proteoliposomes were pretreated for 10 min at O°C before enzyme

with

A23187 (10 lg/ml)

assay.

If the transmembrane Ca2+ is similar to physiological situation. ( higher Ca2+ outside ), gradient was in the inverse direction a lowest enzyme activity would appear. Proteoliposomes without transmembrane Ca2+ gradient exhibited intermediate activities. It is interesting to note that following the dissipation of the difference in enzyme transmembrane Ca2+ gradient by A23187, activity between ACc*Lca+and ACc-Lea-+ would obviously be It leads to a decreasing in enzymatic diminished (Table 2). activity of the former ( close to ACcvLca-); but increasing in the latter case ( similar to that of ACc*Lca++ ). These results provided another indication that the enzymatic activity of reconstituted adenylate cyclase was markedly affected by the presence membrane

across of a Ca2+ gradient Ca2+ gradient is essential

membrane and a proper for the higher enzyme

transactivi-

ty. In these used.

order to compare the conformation of incorporated ACc in proteoliposomes, fluorescence spectroscopy and CD have been

The fluorescence emission spectra of ACc-incorporating proteoliposomes as compared with unreconstituted ACc are in Fig. 1. Fluorescence was measured with an excitation shown and difference in emission intensity was wavelength of 285 nm, recorded at 338 nm. As can been seen from Fig.1, reconstitutions of ACc in four types of asolection vesicles with (1000 fold) or without transmembrane Ca2+ gradient were accompanied by a decrease in intensity of intrinsic fluorescence with no marked shift in emission maximum at 338 nm. Their intrinsic protein fluorescence of four types of proteoliposomes decreased in the order: ACc*Lca+> ACcaLca-> ACc.Lca++ > ACcsLca-+. It may indicate that the microenvironment of tryptophanyl residues of 369

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70

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a

g 60. 3

320

340 Wavelength

360 (nm)

1_

!oa1

400

360

Fiq. 1. Intrinsic fluorescence ACc-incorporatin proteoliposomes S+ transmembrane Ca gradient. curve b, ACc*Lca+-; d, ACc.Lca++;

curve curve

Fiq. 2. CD spectra proteoliposomes. curve curve ACc

in

those

of

210

220 Wavelength

spectra with

230

240

250

(nm)

of ACc and or without

a, ACc; curve c, ACceLca--; curve e, ACcaLca-+.

of four

a, ACcvLca+-; c, ACc*Lca++;

types

d

types curve curve

asolectin

of ACc-containing b, ACcsLca--; d, ACcsLca-+.

vesicles

was

different

from

each

other. The

CD spectra

ACc-containing

in

the

vesicles

with

brane

Ca2+

tents

estimated

by

Chen's

interesting

to

note

It

is

helix with the zation

gradient

wavelength

were

contents of ACc that of increasing

range

(1000

shown

fold)

in

Fig.

method[IOl that

in in

four the

the

order

from or

without

2.

and the were listed of

200-250

transmeme-helix in Table

decreasing

in

types vesicles coincides enzymatic activities of

reported that lipid fluidity could cyclase activity. The degree of fluorescent DPH in these proteoliposomes decreased in

con3.

the

a-

exactly ACc.

Friendland[ll] adenylate for

nm of

influence the

polariorder:

ACc*Lca-+ > ACc.Lca++ > ACcsLca-> ACc*Lca+(Table 4). In other words, the fluidity of four types of proteoliposomes followed the same order ( ACc*Lca+> ACc*Lca-> ACc.Lca++ > ACc*Lca-+ ) Table 3. Effect of transmembrane conformation of ACc-incorporating Proteoliposomes a-helix (

% 1

ACc*Lca+-

ACcsLca--

52.3 (

64.0 5

I*

(

* Number of experiments. 370

Ca2+ gradient on the proteoliposomes ACc*Lca++ 65.5

6 1

( 6 1

ACcsLca-+ 72.6 (

7

1

Vol.

175,

No.

BIOCHEMICAL

2, 1991

Table

4. Difference with or without

Proteoliposomes Polarization ( p )

AND

BIOPHYSICAL

in fluidity transmembrane

ACc.Lca+-

ACcsLca--

0.160f0.001

0.165+0.002

( 6 1 p < 0.02

RESEARCH

COMMUNICATIONS

of proteoliposomes Ca2+ gradient ACc.Lca++ 0.167+0.002

( 6 1

ACcqLca-+ 0.175f0.001

( 6 1 P < 0.02

( 6 1 p < 0.02

Experimental conditions are described in 'I Material and Methods". Values of fluorescence polarization are the mean value k S.E. with the number of experiments in parentheses. PiO.02 indicates that the difference is statistically significant comparing with ACceLca--. as the tration

enzymatic

activity.

( >1 PM ) would

results,

it

seems that

It

was known that

inhibit

adenylate

in addition

to

higher cyclase[l21.

direct

effect

Ca2+ concenFrom our Ca2+-mediated

lipid fluidity could also modulate the reconstituted change in adenylate cyclase activity. Based on the above-mentioned results, we tentatively suggest that a proper transmembrane Ca2+ gradient may offer both in the outer and inner layer a suitable fluidity of phospholipid, favouring the formation of an optimal conformation of the reconstituted adenylate cyclase with higher enzymatic activity. Whether the transmembrane Ca2+ gradient is also essential for the conformation

ther

and function

of

other

membrane proteins

would

require

fur-

investigation. Acknowledgments

of

This China

work was supported by National Nature and the Chinese Academy of Sciences.

Science

Foundation

REFERENCES 1. Hobbes, A. S. et al. (1980) Annu. Rev. Biophys. 9, 259-291 2. Carafoli, E. (1987) Annu. Rev. Biochem. 56, 395-433 3. Yang, F. Y. et al. (1983) Biochim. Biophys. Acta 779, 104-110 4. Yang, F. Y. and Zhang, X. F. (1985) FEBS Lett 182, 31-33 5. Cerion, R. A. et al. (1984) J. Biol. Chem. 259, 9979-9982 6. Feder, D. et al. (1986) EMBO J. 5, 1509-1514 7. Lowry, 0. H. et al. (1951) J. Biol. Chem. 193, 265-275 8. Kitchell, B. B. et al. (1979) Biochim. Biophys. Acta, 534,8998 9. One, S. et al. (1990) Biochim. Biophys. Acta 1022, 237-244 10. Chen, Y. H. et al. (1974) Biochemistry 13, 3350-3359 11. Friendland, G. et al. (1990) Biochim. Biophys. Acta 1022, l-7 12. Maceneli, S. (1984) Biochem. J. 224, 453-460

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