Specific binding of tubulin to a guanine nucleotide-binding inhibitory regulatory protein in adenylate cyclase system, Ni

Vol.

132,

October

No. 15,

1, 1985

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1985

193-197

SPECIFIC BINDING OF TUBULIN TO A GUANINENUCLEOTIDE-BINDINGINHIBITORY REGULATORY PROTEIN IN ADENYLATECYCLASESYSTEM,Ni Kyoichiro Department

of Physiological

Higashi

and Sadahiko Ishibashi

Chemistry, Hiroshima University Hiroshima 734, Japan

School of Medicine

Received August 28, 1985 SUMMARY: A protein was isolated from the soluble fraction of rat brain by affinity chromatography with Sepharose to which guanine nucleotide-binding inhibitory regulatory protein in adenylate cyclase system, Ni, was immobilized. The molecular weight of this protein, specifically bound to the N-affinity column, was estimated as 54,000 on sodium dodecylsulfate-polyacry 1amide gel electrophoresis. Alternately prepared tubulin also bound to the N.-affinity column. The amino acid compositions of these proteins were also 14 entical. It is strongly suggested that this Ni-binding cytosolic protein is D 1985 Academic Press, Inc. tubulin.

Evidence has accumulated latory

protein

in adenylate

not only in the regulation cell-membrane etc (2,3). clarified

Molecular

rat brain here,

it

including

role

study,

(abbreviated

cyclase

likely

in adenylate

that

this

metabolism,

cyclase

Ca2+ influx, have not been

system (1).

a Ni-affinity

column in search of proin the soluble

in the column. Ni-binding

regu-

as Ni >, is involved

of Ni, however,

One of major proteins retained

inhibitory

system (1) but also in various

phosphoinositide

we prepared

with Ni.

was specifically is quite

cyclase system,

mechanisms of the actions

except for its

interacting

guanine nucleotide-binding

of adenylate

functions,

In the present teins

that

protein

fraction

From the results

of

obtained

is tubulin.

MATERIALSANDMETHODS acia,

3H-GppNHp and activated CH-Sepharose were obtained from Amersham and Pharmrespectively. All other chemicals were of reagent grade.

Abbreviations : EGTA, ethyleneglycol bis(B-aminoethylether)-N,N,N',N'-tetraGppNHp, guanylyl-5'imidodiphosphate; Ni , guanine nucleotide-binding inhibitory regulatory protein in adenylate cyclase system; SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis; Tris, tris(hydroxymethyl)aminomethane.

acetate;

0006-291X/85

193

$1.50

Copyright 0 1985 by Academic Press, Inc. AN rights oJ‘ reproducrion in any form reserved.

Vol.

132,

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from 200 g of frozen bovine brain by a procedure of SternNi was purified weis et al. (4) except for the use of phenyl-Sepharose instead of heptylamineSepharose in the final step. This hydrophobic chromatography was effective for the purification of Ni. The isolated Ni was assumed to be homogenous from the results of SDS-PAGEand specific activity of GppNHp binding. The purified Ni was concentrated with DEAE-Sephacel minicolumn (Vt=l ml). One ml of concentrated N. (about 30 nmol/ml) was mixed with 132 ul of GppNHp binding mixture and incubated for 2 hr at 3O'C. Final concentrations of the reaction mixture were as follows ; 26.5 uM Nit 17.7 mM Tr$s-HCl (pH 8.0), 0.9 mM EGTA, 0.9 mM dithiothreitol, 100 mM MgC12, and 39 ~.IM H-GppNHp (4 u Ci). At the end of the incubation, reaction was stopped by ice chilling. This GppNHp-bound Ni was passed through Sephadex G-25 equilibrated with 0.1 M NaHCO 5 mM MgCl 0.5 M NaCl, and 0.1 % Lubrol-PX (pH8.0), to separate from the conjugation with Tris- 2'1 and free 6'ppNHp, which are known to perturb activated CH-Sepharose. Then, 26 nmol of GppNHp-bound Ni were conjugated to 4 g of activated CH-sepharose . To determine the capacity of this affinity-column, an aliquot of the prepared Ni-Sepharose gel was charged into a mini-column (Vt=O.5 ml). Successive washing of this column with an equilibration buffer consisted of 20 mM TrisHCl (pH 8.QJ, 1 mM EGTA, 1 mM dithiothreitol, 11 mM MgC12, and 100 mM NaCl, eluted no H-GppNHp. El y tion with 6 M guanidium chloride, resulted in release of 1.32 nmol/ml gel of H-GppNHp from the column. This denatured affity-gel could easily be renatured by incubati n with equilibration buffer at room were estimated temperature within 30 min. Rebindable 9H-GppNHp binding-sites as more than 0.94 nmol/ml gel. Microtubule fraction was prepared from rat brain soluble fraction by an assembly-disassembly method of Shelanski et al. (5). SDS-PAGE revealed a major band (mol. wt. 54,OOO)of tubulin and minor bands (>200,00O)of microTubulin was further purified by tubule-associated proteins in this fraction. DEAE-cellulose column chromatography (6). Amino acid analysis were performed with a TOY0 amino acid analyzer using ortho-phthalaldehyde as the detection reagent (7). Hydrolysis of proteins was effected in 6 N of HCl (constant bioling grade) at 1lO'C for 20 h in vacua. SDS-PAGEwas carried out with a 11 % polyacrylamide gel according to the method of Laemmli (8). Protein concentration was determined by the method of Lowry, et al. (9) or by dye staining method with Bio-Rad protein assay kit and by micro-biuret reaction (10) in the case of amino acid analysis. RESULTSAND DISCUSSION Fig.

1

rat brain

shows the elution homogenate

profile

of

proteins

in the Ni -Sepharose

in the soluble

column (Vt=ll

fraction

of

ml) chromatography.

-A

ELUTION

Fig,

1.

imately

column Lubrol-PX.

VOLUME

(ml)

ApproxN.-affinity chromatography of rat brain soluble proteins. 12&mg of soluble protein from rat brain was put on Ni-Sepharose (Vt=llml).

An arrow

indicates

that

194

the

eluant

was

changed

to

0.2

%

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A

M

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B

Fig, 2. SDS-PAGE of the Ni-Sepharose eluate. Lanes A and B, fractions A and BinFig. 1. Fraction A was further rechromatographed on either Sepharose without N. (lanes 1 and 2) or Ni-Sepharose (lanes 3 and 4). Lanes A, 1, and 4, unadsor b ed fractions: and lanes B, 2, and 3, 0.2 % Lubrol-PX eluate fractions. Lane M, molecular weight markers; myosin-H chain (ZOO,OOO), B-galacto(92,500), serum albumin (66,200), obalbumin sidase (116,250), phosphorylase-b (45,000), and lactic dehydrogenase (30,000)

SDS-PAGEanalysis

revealed

column and was eluted with

a molecular

unadsorbed

of 54,000

(Fig.

( peak-A in Fig.

was rechromatographed weight

peak-B in Fig.

with 0.2 % Lubrol-PX,

weight

fraction

that

was mainly

2, lane 3).

on the same affinity

protein

in the first

Furthermore,

column was estimated that

binding

of immobilized protein,

compared protein arate

this

protein

experiment,

with

identification protein

of rechromatography was saturated

The capacity

binding

tubulin

of this

the prepared

tubulin

column.

alone,

but with

of this

protein,

Tubulin

could

from rat

solution

was eluted

0.2 % Lubrol-PX(data

195

purified

of the

be tubulin

brain.

and

The binding In a sep-

(0.13 uM) was put on

out from the column not not shown).

amino acid composition

(254 ng) which has been further

affinity

weight

on SDS-PAGE(Fig. 3).

tubulin

with

the value being compatible

protein

prepared

75 ml of the purified

a 10 ml of Ni-Sepharose the buffer

with

this

2 )

with the molecular

Ni. From abundance and the molecular

we assumed that

comigrated

thatNi-Sepharose

to be 1 - 2 nmol protein/gel,

when the

to lane A in Fig.

gel, the protein

chromatography.

in the

composed of a protein

of 54,000 was decreased in the unadsorbed fraction

the binding

with

which was retained

1; corresponding

(lane 4 in Fig. 2). Thus, it was likely

with

1,

through

of this

For the Ni-binding

DEAE-sephacel,

and

Vol.

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No.

1, 1985

BIOCHEMICAL

AND

2

1

Comparison tubulin.

that

of a purified

composition rat

brain,

fication protein

3

Ni-Sepharose

RESEARCH

of

rat this

brain

adsorbed

protein

and from calf

protein

(molecular

Amino

reported

protein

1: acid

detergent

Amino

Data These

from values

that

(11).

composition

Calf

brain

of Rat

a)

Mr.

reference 11. were means of

54,000

brain

of tubulin

/

study) 54,000

50.5 30.4 27.6 64.8 40.8 38.4 36.6 24.0 39.5 15.9 21.4 14.3 22.7

___

------__

duplicates.

196

50.3 30.3 28.3 67.1 46.8 38.9 36.5 24.0 37.7 16.4 22.9 14.3 24.6

identi-

due either

protein 54,000

protein

from

to this

of elution

Mr.

tubulinb)

51 33 29 63 39 36 34 23 38 15 19 13 20

Amino acid

For further

(constituent

(this

a) b)

1).

colchicine-binding

Lubrol-PX

residues Aspartic acid Threonine Serine Glutamic acid Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Ilistidine Lysine

(Table

was not observed probably

acid

tubulin

weight

lane 3, purified

with

previously

as tubulin,

But the binding

the presence of non-ionic

Table

were compared

showed good agreement

brain

of the Ni-binding was examined.

tubulin

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M

Lanes 1 and 2, the adsorbed protein; and lane M, molecular weight markers.

and

tubulin;

of

BIOPHYSICAL

b,

to

buffer)

Vol.

132,

No.

BIOCHEMICAL

1, 1985

or to high lability

of colchicine-binding

could not be obtained Then, we tried to separate trial

even with

the colchicine-binding It is not certain

protein

prevented if

column,

in the cell.

It would be of interest,

two proteins

regulates

the function

protein

the result

however,

first,

and then

column.

But the

colchicine-bound

Ni is actually if

tubulin

seemed to indicate

between tubulin

oftubulinto

The binding

under the same condition.

by Ni-Sepharose

the interaction

such a binding

COMMUNICATIONS

of tubulin.

tubulin

Since the purified

to Ni-Sepharose

RESEARCH

to the cytoplasmic

the colchicine-bound

retained

BIOPHYSICAL

activity

the purified

to bind colchicine

was also unsuccessful.

neither

AND

the interaction

was that

and Ni. operating between the

of Ni, or vice versa.

Acknowledgement : We are grateful to Prof. Hisanobu Yoshida and Mr. Kouichi Kawazu for amino acid analysis of the protein. REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Katada, T., Bokoch, G.M., Northup, J.K., Ui, M., and Gilman, A.G. (1984) J. Biol. Chem. 259, 3568-3577. Okajima, F., and Ui, M.(1984) J. Biol.Chem. 259, 13863-13871. Nakamura, T., and Ui, M. (1985)J. Biol. Chem. 260, 3584-3593. Sternweis, P.C., and Robishaw, J.D. (1984) J. Biol. Chem. 259, 1380613813. Shelanski, M.L., Gaskin, F., and Cantor, C.R. (1973) Proc. Natl. Acad. Sci. USA. 70, 765-768. Murphy,.M.Li, and Borisy, G.G. (1975) Proc. Natl. Acad. Sci. USA, 72, 2696-2700. Benson, J.R., and Hare, P.E. (1975) Proc. Natl. Acad. Sci. USA, 72, 619622 Laemmli, U. (1970) Nature, 227, 680-685. Lowry, O.H., Rosebrough, N.G., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem.193, 265-275. Itzhaki, R.F., and Gill, D.M., (1964)Anal. Biochem, 9, 401-410 Lee, J.C., Frigon, R.P., and Timasheff, S. N. (1973) J. Biol. Chem. 248, 7253-7262

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