A specific soluble protein from the catecholamine storage vesicles of bovine adrenal medulla II. Physical characterization

BIOCHIMICA ET BIOPHYSICA ACTA

219

BBA 35369 A SPECIFIC SOLUBLE P R O T E I N FROM T H E CATECHOLAMINE STORAGE VESICLES OF BOVINE ADRENAL MEDULLA II. PHYSICAL CHARACTERIZATION

A. G. KIRSHNER AND •. KIRSHNER Department of Biochemistry, Duke University Medical Center, Durham, N.C. 27706 (U.S.A.)

(Received December I9th, 1968)

SUMMARY 50% of the soluble protein of the chromaffin granules consists of a homogeneous protein. The protein has a molecular weight of 81 200 in neutral salt solutions as measured by rapid equilibrium osmometry. Molecular weight measurement by sedimentation equilibrium in 6 M guanidine-HC1 with a reducing agent present shows that the protein is composed of two identical or nearly identical subunits of weight average mol. wt. 40 600. The protein is partially randomized at neutral pH at both ionic strengths 0.03 and 0.3, as indicated by a marked dependence of the sedimentation coefficient on protein concentration. However, at ionic strength 0.3, as the pH approaches the isoelectric pH (4.5), the protein is more compact and behaves in an almost globular manner.

INTRODUCTION Upon hypotonic lysisl, ~ of the catecholamine storage vesicles of bovine adrenal medulla, approx. 80% of the total vesicle proteins is found in the soluble fraction 1. When this protein was purified by chromatography and gel filtration as reported earlier, approx. 50% was obtained as a homogeneous protein. Reports on the purification and properties of this protein (since named chromogranin A by BLASCHKO et al. 4) have been previously published by this and other laboratories 3-9. The present paper is concerned with characterizing and clarifying some of the physical properties of chromogranin A. METHODS AND MATERIALS The protein was prepared as described previously a. The stock solutions were exhaustively dialyzed against the solvents used in the studies until the absorbance of the dialysate at 280 m/z was less than 0.025 units. The concentrations of the protein Biochim. Biophys. Acta, 181 (1969) 219-225

220

A . G . KIRSHNER, N. KIRSHNER

stock solutions were then determined from the absorbance at 280 m# in a Gilford Spectrophotometer using the molar extinction coefficient of 56 64o (ref. 3). The final dialysate solution was used to dilute the protein stock solutions to their appropriate concentrations and was also used as the solvent stock solutions. Reagents. Commercially available analytical grades of NaC1 and MgC12 were used without further purification. The appropriate solvent concentration was prepared, and the p H was adjusted to 6. 9 with NaOH for the neutral solvents. After dialysis, the p H of the final dialysate was again measured. The "acidic" solvent consisted of the NaC1 stock solutions buffered with o.ooi M sodium acetate (pH 4.95)- Again the protein was exhaustively dialyzed against the "acidic" solvent, and the p H of the final dialysate was remeasured. Guanidine •HC1 was purified b y the method of NOZAKI AND TANFORD10. Sedimentation velocity. Studies were performed on a Beckman Model E analytical ultracentrifuge equipped with Schlieren optics and a phase plate. The commercially available capillary-type synthetic-boundary epoxy centerpiece (12 ram, 2 ° double sector) was used for all runs. All runs were performed at 25 ° unless otherwise stated and at 42 040 rev./min. One sector was filled with 0.4 ml of solvent, and the other sector was filled with o.18 ml of protein solution to ensure that there was no protein solution above the capillary. Photographs were taken at 8- or I6-minute intervals on Eastman metallographic plates. Sedimentation equilibrium. Studies were performed on a Beckman Model E ultracentrifuge equipped with interference optics. The commercially available doublesectored (12 ram, 2 ° sector) interference cell was used for these studies. The method of YPHANTIS11 was modified b y increasing the column lengths for greater resolution. Fringe displacement on an Eastman I I - G plate was measured with a Gaertner microcomparator once it had been determined that equilibrium had been reached. For convenience, ~ was assumed to be o.717 cm3/g as calculated from the amino acid composition. Rapid equilibrium osmometry. Measurements were performed on a Mechrolab osmometer (model 503, F and M Division of Hewlett-Packard, Avondale, Pa. (U.S.A.)). All solutions were filtered through a fine scintered-glass filter. Schleicher and Schuell B I 9 membranes were soaked in the appropriate solvent for at least 24 h before using.

RESULTS

Sedimentation velocity studies The sedimentation of chromogranin A varied markedly with the protein concentration at neutral p H (Fig. I). A comparison of the effect of NaC1 and MgC12 on the sedimentation coefficient indicates that the protein preferentially binds the divalent cation. 0.03 M NaC1 at p H 6.95 could not stabilize the protein sufficiently for a consistent s vs. concentration study, whereas o.oi M MgCI~ could. It is also obvious from Fig. I that the effect of protein concentration on sedimentation was markedly diminished as the p H approached the isoelectric p H (4.5) of the protein 3. Since the protein precipitates at its isoelectric pH, a centrifugation below 4.95 was not tried. Tile values of s°25 presented in Table I were extrapolated from a plot of I]S vs. c Biochim. Biophys. Acta, I 8 I (1969) 219-225

PROTEIN FROM CATECHOLAMINE STORAGE VESICLES

221 0500

(SJ • [4}

o, A31 o /,,st# 2 )

40

/ / /4"

0 450

0 / ./. t / •/."/ //I

•

O40¢

"~ 3.5

/ \, '...~

"

(1)

•

I 0 • / I ."1

...."// •

"t •

/ ~"i

0 35C

30

l/•

0/0 •//:'S~II /"/ t //

7

.....a

• 11 ,./"I,IA • / //./[3 t

X IL,X,.

""\o~121

2.5

., t

0 30C ,

k"~D ,t

13l "b,=,e(4} 20 ~

L

I

I

I

I

I

I

I

I

1{5)1

0 25C

I

2 3 4 5 6 ? 8 9 I0 II 12 Prolein Concenlrolion(g/I)

I ~;~~,b,',,'2 Protein Concentrotioo(%)

Fig. I. S e d i m e n t a t i o n velocity e x p e r i m e n t a l d a t a . C u r v e I (m) p r o t e i n in 0. 3 M NaC1, p H 4.9; C u r v e 2 ([~), p r o t e i n in o.oi M MgCI,, p H 6.5 a n d 6. 7 (two sets of d a t a ) ; C u r v e 3 (A), p r o t e i n in 0. 3 M NaC1, p H 6.95; C u r v e 4 (0), p r o t e i n in o.I M MgCI= (pH 6.45); C u r v e 5 (©), p r o t e i n in o.I M NaC1 (pH 6.95). Fig. 2. T h e d a t a of Fig. i (except t h e first curve) p l o t t e d as i/s vs. c o n c e n t r a t i o n . T h e s y m b o l s are t h e s a m e as in Fig. I.

(Fig. 2) for the neutral pH studies. K for these studies was then calculated from the equation i / s = i / s ° (I + t i c )

(1)

The K value for the pH 4.95 study was calculated from the equation s = s o (i -

Kc)

(2)

and found to be 17.6 ml/g (when calculated from Equation I the value is 19.28 ml/g). K values for globular protein, when calculated from Equation 2 are in the range of TABLE I SEDIMENTATION

pH

V E L O C I T Y D A T A OF C H R O M O G R A N I N

Salt

Conch.

(M) 4.9 6.95 6.95 6.45 6.5-6. 7 Average

NaC1 NaC1 NaC1 MgC12 MgCt~

0. 3 o.I 0. 3 o.I o.oi

A

IN VARIOUS SOLVENTS

s°=s,w

I/S°25,w

K

3.87 3.52 3.68 3.93 3.88 3.7 ~"

0.284 0.275 0.254 o.258

i7.4"" 69.oi4"** 6o.25"** 74.3"** 64.77"*"

(mqg)

* s°=0,w = 3.78 × 0.88852 = 3.36. ** E q u a t i o n 2 in t e x t . *'* E q u a t i o n i in t e x t .

Biochim. Biophys. Acta, 181 (1969) 219-225

222

A . G . KIRSHNER, N. KIRSHNER

TABLE II MOLECULAR WEIGHT

D A T A ON C H R O M O G R A N I N A IN V A R I O U S S O L V E N T S

Salt

Conch.

pH

H/c

Mol. wt.

NaCI MgC12

0.3 o.i

6.95 6.25

0.325 o.294

77 200 85 3oo

(M)

Average

81 200

Guanidine.HC1 5.56 plus mercaptoethanol o.2

5.9

4 ° 600 × 2 = 81 200

5-1o ml/g. The significant difference in K values found in neutral salt solutions and in acidic salt solutions can be attributed to a partial randomization of the protein due to the change in pH.

Molecular weight When molecular weight studies were attempted on the protein in either o.I M NaCI or in o.I M MgC12 by a modification of the method of YPttANTISn, it became obvious from the interference patterns that the protein aggregated before equilibrium could be achieved. Normally equilibrium is achieved within 24 h, but the soluble protein appeared to be completely aggregated in lO-12 h at room temperature. Consequently, rapid equilibrium osmometry became the method of choice in salt solutions since the length of time the protein is exposed to room temperature is no more than that for a sedimentation velocity study. The sedimentation equilibrium method was, however, desirable for the 6 M guanidine-HCI-o.2 M mercaptoethanol studies, since the presence of the reducing agent prevented aggregation. Figs. 3 and 4 show the experimental data from which the molecular weights of the protein were calculated (Table II). Since the s°~5,wvalues in the various solvents are approximately the same (Table I) and since the molecular weight in some of these solvents averages

3000

040C

.~,2.00¢

,II. ................... .... ~ . ~ ....

6

1¸00(i

Q

~

~..~.~..jl~..~..

~

~

~ - ~

0.30(

e

436

8

440

2

4

6 8 r 2 (cmz }

450

2

4

6

0200

i

~

~

~

~

~

~

,b --~-

Protein Concenffotio, ( g/I )

Fig. 3. Fringe displacement (logarithmic scale), in microns, at equilibrium, vs. r 2 for c h r o m o g r a n i n A in 5.56 M guanidine .HC1, 0.2 M m e r c a p t o e t h a n o l (pH 5.9), 25 °. Fig. 4. E x p e r i m e n t a l d a t a for rapid equilibrium o s m o m e t r y . The units for osmotic pressure are centimeters of solvent. The value of R T in the units employed (at 25 °) is 2. 5 • lO 4. • . . . . . • , c h r o m o g r a n i n A in 0. 3 M NaC1 (pH 6.95), 25°; 0 - - 0 , protein in o.i M MgCI~ (pH 6.95), 25 °.

Biochim. Biophys. Acta, 181 (1969) 219-225

PROTEIN FROM CATECHOLAMINE STORAGE VESICLES

223

to 81 200, the obvious conclusion is t h a t there is no molecular weight change in a n y of these solvents except i n 5.56 M g u a n i d i n e • HC1, 0.2 M mercaptoethanol. DISCUSSION Chromogranin A, isolated a n d purified in our l a b o r a t o r y a n d b y SMITH AND WINKLER7, agrees r e m a r k a b l y well in a m i n o acid analysis on the basis of a c o m m o n s u b u n i t tool. wt. of 4o ooo (Table III) a n d is in close agreement on the mol. wt. 77 39 ° reported b y SMITH AND WINKLER9 a n d 81 200 reported here. I n 6 M g u a n i d i n e • HC10.2 M m e r c a p t o e t h a n o l a tool. wt. of 4 ° 600 has been f o u n d for the protein. Previous studies of t r y p t i c digests indicated t h a t the protein c o n t a i n e d a repeating s u b u n i t structure h a v i n g a tool. wt. of a b o u t 40 ooo. The molecular weight d e t e r m i n a t i o n s b y o s m o m e t r y a n d in 6 M g u a n i d i n e .HCI-o.2 M m e r c a p t o e t h a n o l indicate t h a t the p r o t e i n is composed of two identical or near identical s u b u n i t s held together b y one or two disulfide bonds. TABLE III COMPARISON OF AMINO ACID ANALYSES

A. Analyses from

S M I T H AND K I R S H N E R 3. B .

A I* Lys His Arg Asp Thr Ser Glu Pro Sly Ala Val Met Ile Leu Tyr Phe Cy~ Try Average

Analyses from

S M I T H AND W I N K L I ~ R 9.

B II*

32 6 18 3o io 28 85 33 25 3° 14 7 5 23 3 5 2 5

30 7 22 29 io 28 81 35 27 28 13 7 4 26 4 6 2

361

359

III**

IV*

57 7 42 56 19 55 156 68 52 55 25 I3 7 5° 8 II 3

3° 7 22 29 io 28 81 35 27 28 13 7 4 26 4 6 2

* Residues/4 o ooo g. ** Residues/77 390 g. The K values reported in T a b l e I i n d i c a t e t h a t the p r o t e i n is p a r t i a l l y r a n d o m i z e d in n e u t r a l salt solutions. A t p H 4.95, with sufficient salt present to obliterate a n y charge effects on the protein, the structure is more compact approaching t h a t of a globular protein. The S°2o,w value of t h e protein in 0.3 M T r i s - s u c c i n a t e buffer was reported b y SMITH AND WINKLER9 to be 3.05 S, which is I o % lower t h a n our value (Table II). This discrepany is n o t due t o the 5 ° t e m p e r a t u r e difference i n our studies, Biochim. Biophys. Acta, I81 (1969) 219-225

224

A . G . KIRSHNER, N. KIRSHNER

since sedimentation velocity studies in o.oi M MgC12 (pH 6.7) at IO° gave the same s values found at 25 ° when corrected to standard conditions. The differences m a y be due to the different buffer systems employed, the succinate ion having a greater effect on the frictional coefficient of the protein than the chloride ion. Under their conditions the protein structure is partially randomized as it is in our more neutral p H studies. The conclusion by SMITH AND WINKLER9 that chromogranin A approaches a random coil in nature is not supported by our data of the p H effect on the protein and of the existence of two chains nor by their own intrinsic viscosity data. Their value of [~J = 42. 4 ml/g at low ionic strength (I : O.Ol5, p H 5.9) suggests a random coil, but the conditions are such that electrostatic charge effects have not been neutralized on the protein. When the ionic strength is raised to 0.3, at pH 5-9, so that electrostatic effects on the protein are diminished, the intrinsic viscosity drops to 18. 9 ml/g, a value which is too low for a random coil of either 80 ooo or 40 ooo tool. wt. Consequently, at pH 5.9, where there m a y be partial randomization due possibly to a pH effect alone, at the low ionic strength the electrostatic effects on those partially randomized regions of the protein m a y mask the presence of considerable structure in the rest of the molecule. In a study of proteins as random coils, TANFOED et al. 1~ give values in the range of E~I : 52.2 ml/g for serum albumin (tool. wt. 69 ooo) and 31.5 ml/g and 35.3 ml/g for pepsinogen and aldolase (tool. wt. 4 ° ooo) under conditions where randomization is complete (6 M guanidine .HCI-o.I M mercaptoethanol, 25°), values which are much higher than that of 18. 9 ml/g measured by SMITH AND WINKLER9. NOELKEN AND REIBSTEIN 1~ have also shown that bovine fl-casein B (tool. wt. 24 ooo) has an intrinsic viscosity of 22.2 ml/g in 6 M guanidine. HC1, o.I M potassium phosphate (pH 7), at 25 ° and an [~1 ~ 23.2 ml/g in o.04 M NaC1, 0.02 M EDTA (pH 7), at 25 °, therefore, even under mild solvent conditions, fl-casein B is completely randomized, fl-Casein B also has an unusually high proline content (I out of 6 residues), whereas the most common genetic variant of casein B (as1) has only half that number of prolines. Ho AND CHEN14 found that the intrinsic viscosity of the asl monomer in o.oi M KC1 (pH 7) was lO-12 ml/g depending on the temperature and increased to 19.2 ml/g in 6 M guanidine.HC1, o.i M KC1 (pH 7.I), and 25 °, indicating a more tightly folded structure. Neither casein variant has any disulfide bonds. By comparison, chromogranin A, in which lO% of the amino acid residues are proline and in which at least one, if not two, disulfide bridges connect two chains of approx. 4 ° ooo molecular weights each, approaches a globular structure under certain solvent conditions (low pH), and even under solvent conditions where there is partial randomization, considerable structure is obviously maintained by the protein. ACKNOWLEDGEMENTS The authors wish to acknowledge the helpful discussion with Dr. Charles Tanford during the progress of this work and in the preparation of the manuscript. This work was supported b y a research grant from the National Institutes of Health, AM-o5427 and a grant from the National Science Foundation, GB 5812.

Biochim. Biophys. Acta. 181 (1969) 219-225

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REFERENCES I N.-~-. HILLARP, S. LANGERSTEDT AND B. NILSON, Acta Physiol. Scand., 29 (1953) 251. 2 N . - ~ . HILLARP, in J. R. VANE, G. E. W. WOLSTENHOLME AND M. O'CONNOR, Adrenergic Mechanisms, Churchill, London, I96O, p. 481. 3 W. J. SMITH AND N. KIRSHNER, Mol. Pharmacol., 3 (1967) 52. 4 H. BLASCHKO, R. S. COMLINE, F. H. SCHNEIDER, M. SILVER AND A. D. SMITH, Nature, 215 (1967) 58 • 5 H. BLASCHKO AND K. B. HELLE, J. Physiol., London, 169 (1963) I2oP. 6 W. J. SMITH, A. KIRSHNER AND N. KIRSHNER, Federation Proc., 23 (1964) 35 o. 7 A. D. SMITH AND H. WINKLER, Biochem. J., 95 (1965) 42P8 N. KIRSHNER, C. HOLLOWAY, Vq. J. SMITH AND A. t{IRSHNER, in U. S. y o n EULER, S. ROSELL AND B. UVNAS, Mechanism of Release of Biogenic Amines, Pergamon, London, 1966, p. lO9. 9 A. D. SMITH AND H. WINKLER, Biochem. J., lO 3 (1967) 483 . IO Y. NOZAKI AND C. TANFORD, J. Am. Chem. Soc., 89 (1967) 736. i i D. A. YPHANTIS, Biochemistry, 3 (1964) 297. 12 C. TANFORD, K. KAWAHARA AND S. LAPANJE, J. Am. Chem. Soc., 89 (1967) 729. 13 M. NOELKEN AND M. REIBSTEIN, Arch. Biochem. Biophys., 123 (1968) 397. 14 C. H o AND A. H. CHEN, J. Biol. Chem., 242 (1967) 551.

Biochim. Biophys. Acta, 181 (1969) 219-225