The amino acid sequence of the calmodulin obtained from sea anemone (Metridium senile) muscle

Vol.

96, No.

September

BIOCHEMICAL

1, 1980

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages

16, 1980

377-381

THE AMINO ACID SEQUENCE OF THE CALMODULIN OBTAINED FROM SEA ANEMONE (M&&m Takashi

TAKAGI,

Biological

Takayuki

Department Hokkaido

July

MUSCLE

NEMOTO and Kazuhiko

KONISHI

Institute, Faculty of Science, Tohoku University, Sendai 980

Michio

Received

he&e)

YAZAWA and Koichi

YAGI

of Chemistry, Faculty of Science, University, Sapporo 060, Japan

28,1980 Summary

The amino acid sequence of the calmodulin obtained from sea anemone muscle was determined. The calmodulin was composed of 148 amino acid residues and its amino terminal was blocked. When compared with bovine brain calmodulin, the number of amino acid residues per molecule was the same and there were 3 replacements at residues 99 (Tyr * Phe), 143 (Gln + Lys) and 147 (Ala -f Ser). Introduction The calcium-dependent in vertebrates and plants

(1,Z).

modulator Recently,

(3 - 12),

protein

calmodulin

suggesting

that

this

(calmodulin) is

found

protein

is

was first

widely

dicovered

in invertebrates

ubiquitous

among the

eucaryotes. The amino rat

testis

(14)

sequences

are

sea anemone the

acid

and bovine very

(16),

sea anemone

sequences

similar. and attempt

muscle

of calmodulins

uterus

from

have been already

I?re isolated

a calmodulin

was made to establish

bovine

reported from marine

the amino

acid

brain

(13),

and the invertebrate, sequence

of

calmodulin. Materials

Sea purification previous NH4HC03 Tryptic (superfine) mapping

(15)

isolated

and Methods

anemone (MetrLidiwn neniee) was obtained from the Sea of Okhotsk. The procedure of this calmodulin was described in detail in the Tryptic digestion of calmodulin was performed in 0.1 M paper (16). at 37°C for 7 h without any prior modification of the calmodulin. peptides were fractionated by gel filtration of Sephadex G-25 and were further separated using the two-dimensional peptide (1st dimension, high voltage paper electrophoresis at pH 5.5; 2nd

0006-291X/80/170377-05$01.00/0 377

Copyright 0 I980 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

96, No.

BIOCHEMICAL

1, 1980

AND

BIOPHYSICAL

RESEARCH

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dimension, descending paper chromatography using the solvent (n-butanol/ pyridinelacetic acid/water = 15/10/3/12)). Some of the peptides were further digested by thermolysin or Stiphy,Cococcae V-8 protease and the resulting peptides were separated by the peptide mapping. Overlapping peptides were obtained by the cyanogen bromide cleavage of calmodulin. Isolation procedures of the peptides include Sephadex G-50 chromatography and the peptide mapping. The amino acid analysis was performed by Hitachi 835-50 single column system amino acid analyzer after acid hydrolysis. The amino acid sequence of the peptides was determined by dansylEdman method (17).

Results The peptides calmodulin Fig.

were

1.

number

48-54)

mapping, determined

Methods.

to three

four

(tube

primarily

The amino

further 55-67)

fractions

amino

acid

sequence

filtration divided

eluting

were

to early

isolated

of the

isolated

as described

of the

as shown

calmodulin

Peptides by the peptides

in Materials

was deduced

in

(tube

fractions.

further

sequences

by the procedure

acid

of sea anemone muscle

by gel

were

numbers

separated

and the

digestion peaks

peak components

and later

in the

peptide

by tryptic

separated

The first

included

were

produced

and Discussion

and

from

0.6 -

0.5 -

0.4 E = ii Q

0.3 -

0.2 -

0.1 -

0’

40

”

I

50

I

60

70

Tube Fig.

1

60

90

100

number

Chromatography of tryptic digest of sea anemone calmodulin. The digest (ca. 11 mg in 0.1 M NH4HC03) was applied to a column (2.5 x 140 cm) of Sephadex G-25 equibrated with 0.05 M NH4HC03 and eluted with the same buffer at a rate of 12 ml/hr.

378

Vol. 96, No. 1, 1980

analyses

of these

overlapping

which

brain

(13),

amino

terminal

glutamic

testis

were

of glutamic

determined

As shown the amino

terminal

of aspartic

calmodulin

will

the

acid

residues

acid

sequence

the

different

primary

amide

acid

sequence

acid

of bovine The

Some residues

acid on paper

of

by V-8 protease, and asparagine electrophoresis.

unequivocally

except

assignment

of nearly

description

of the

of sea anemone

one half

muscle

of bovine

brain

calmodulin

was compared

sea anemone

muscle

calmodulin

and the

from

structure

those

of sea anemone

in Fig.

2 in parentheses.

calmodulin

amino

are

Without

shown

regard

10 X(Ala,Asx,Glx)Le"-Thr-Glx-Glx-Glx-Ile-Ala-Gl"-Phe-Lys-G,"-A,a-Phe-ser-Le"-Phe-~~p30 Lys-Asp-Gly-Asx-G7y-Thr-I,e-Thr-Thr-Lys-G,"-Le"-Gly-Thr-"al-~,et-Ar~-ser-Le"-~~y-

Gl"-As"-Pro-Thr-Glu-*,a-Glu-Leu-Glx-~~x-Met-l,e-Asx-G,"-"a,-**x-*,a-*sx-G,y-~~x-

Gly-Thr-I1e-Asx-Phe-Pro-G1"-Phe-Le"-:~r-Met-Met-Ala-*r~-lys-Met-Lys-*sx-Thr-~~x90 Ser-Glx-Glx-Glu-lle-Arg-Glu-Ala-Phe-Arg-Val-Phe-Asp-lys-Asp-Gly-Asx-Gly-Phe-Ile-

100 Uyr)

110 Ser-Ala-Ala-Glu-Leu-Arg-His-Val-Met-Thr-Asx-Leu-Gly-Glu-Tml-Leu-Thr-Asx-Glx-Glu-

120

130 Val-Asp-Glu-#et-Ile-Arg-Glu-Ala-Asx-Ile-Asx-Gly-Asx-Gly-Glx-Val-Asx-Tyr-Glx-Glu-

140

Phe-Val-Lys-Met-Met-Thr-Ser-Lys-OH (Gin)

Fig.

2

Amino

acid

(Ala) sequence

for

elsewhere.

sequence of this

aspartic

A detailed

amino

be published

The amino with

and the

of

(15).

cleavage

was established

acids.

of complete

residues

group.

of the peptides

tripeptide

those

calmodulins

specific

glutamine,

sequence

and glutamic

determination

by the

acid,

2, the

uterus

with

COMMUNICATIONS

of 148 amino

acid

by an unidentified

by the mobility

in Fig.

combined

of amino

and bovine

determined

RESEARCH

was composed

same number (14)

BIOPHYSICAL

peptides

The calmodulin

was blocked

acid

AND

tryptic

was the

rat

and several

under

isolated

peptides.

residues,

were

BIOCHEMICAL

of

sea

anemone

calmodulin.

Tml

shows

E-N-

trimethyllysine. X indicates an unidentified blocking group. Residues which differ in bovine brain calmodulin (13) are shown underneath in parenthesis.

379

to

Vol. 96, No. 1, 1980

BIOCHEMICAL

the amides

left

undetermined,

recognized

out

of 148 amino

substituted

by Tyr

in calmodulin

of

experiment (T.

that

the residue

found

out

of 161 amino

conservative family

protein

than

of Ca2+-binding Bovine

138.

brain

Sea anemone

difference

region

hand,

+:"r located

calmodulin

C although

sepctrum

the

oxygen

suggest

that

that

(19),

99

was also

between

Ser

bovine

57 substitutions

were

is much more

they

only

to coordinate

but

C where

contained

ligands

at position

calmodulin

troponin

calmodulin

from

147

A preliminary

substitutions

2 tyrosines

weight

induced (16),

around

are

regarded

as a

bound

in between

Ca

change

be attributed

of parvalbumin

2+ to calmodulin is

terminal the

by Ca2+ was observed

280 nm should

of T$ , which

the amino

one,

at positions 99 and 138 Tyr. Since an identical

the conformation

EF hand model

one of the

binding

acid

residues

spectrum

carbonyl

residue

and Ser

proteins.

of equal

peptide

of amino

contained

If

99 was

observation).

calmodulin

to the difference 138 movement of Tyr. to calmodulin,

the

147 in scallop

troponin

UV absorption

two calmodulins

was

Phe at position

that

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substitution

was Phe (18).

muscle

acid

RESEARCH

Lys 143 was by Gln,

unpublished

skeletal

acids

reported

heni6ohma

and rabbit

3 amino

calmodulin,

to the number

BIOPHYSICAL

residues;

and Sharief

Honma and M. Yazawa,

cardiac

acid

Rem

showed

Comparing

only

in brain

Vanaman

was by Ala.

AND

involved

of this

residues

with

the

corresponding to the

(20)

can be applied

is coordinated‘by in the protein.

third On the

137 and 140 donating

Ca 2+ other the

Ca2'

at the fourth Ca2'-binding region. Our results 99 group of Tyr does not move by the binding of Ca 2+ ,

the phenol 138 of Tyr is displaced

to more hydrophilic

environment.

Acknowledgments This work was supported in part by a grant from the Ministry of Education, Science and Culture of Japan, and by a grant from the Muscle Dystrophy Association of America, Inc. We are indebted to Abashiri Fishermen's Cooperative Association and Shari Fishermen's Cooperative Association, Hokkaido for their help on the collection of sea anemone.

380

Vol.

96, No.

1, 1980

BIOCHEMICAL

AND

BlOPHYSlCAL

RESEARCH

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