Vol. 91, No. 4, 1979 December

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

28, 1979

Pages 1390-1398

AMINO ACID SEQUENCE OF YEAST PROTEINASE B INHIBITOR COMPARISON WITH INHIBITOR 2 Konrad

Hannelore

Maier,*

Miiller,** and Helmut

Rudolf Holzer*

Tesch,+

1

Irene

Witt,+

*Institut fiir Toxikologie und Biochemie der Gesellschaft fiir und Umweltforschung m.b.H. Miinchen, D-8042 Neuherberg, Strahlen**Biochemisches Institut und +Biochemisches LaborderKinderklinik, UniversitZt Freiburg, D-78 Freiburg, Germany Received November

5,1979

Summary: The amino acid sequence of proteinase B inhibitor 1 (IB1) from bakers' yeasthas been established by automated Edman degradation upto posithon 42. A comparisonwiththesequence ofproteinase B inhibitor 2 (I 2) revealed two differences: LEU-32 and GLU-34 in IB2 are replaced by VAL-32 and LYS-34 in IB1. Identity of the COOHterminalregionof IB1 with that of IB2 was proved by degradation with the carboxypeptidases A and Y. Furthermore, a chymotryptic peptide was isolated from each of the 74 residues containinginhibitors. The two fragments, ranging from position 42 totheCOOH termini of the inhibitors, were found to be identical with respect to electrophoretical mobility, end groups, aminoacidcomposition and peptide pattern after tryptic digestion. It is concluded, &hat theBtwo inhibitor sequences are identical beyond position 42. I 1 and I 2 are isoinhibitors, because they are coded by different genes. Two specific bakers' Both

yeast

inhibitors have

lated,

have

hibit

the

been

identical

same

whether

the

diploid

yeast

very

specific

*To whom requests

cell,

reprints

are

acid

activity.

are

coded

the should

1Abbreviations:

from (1,2).

with

different

immunologically

composed

Against

inhibitory

or whether for

amino

similar.

two polypeptides

polypeptides

and are

their

and IB2)

and characterized

which

end groups

maps are

B (IB1

purified

The inhibitors,

Furthermore,

peptide

proteinase

and TCAlresistant

points.

respectively. tic

recently

are heat-stable

isoelectric

for

of 74 residues,

compositions proteinase

and trypB they

The question

by different

multiplicity

re-

genes

arises in

X/79/241390-09$01.00/0

Copyright @ I979 by Academic Press. Inc. All rights of reproduction in anyform reserved.

1390

the

is due to an epi-

be addressed.

TCA, trichloroacetic acid; TLCK, tosyl-l-lysylchloromethyl ketone: DFP, diisopropyl fluorophosphate; TPCK, tosyl-L-phenylalanylchloromethyl ketone: dansyl, 5-dimethylaminonaphthalene-I-sulfonyl; DMBA, dimethylbenzylamine; PTH, phenylthiohydantoin.

0006-291

ex-

BIOCHEMICAL

Vol. 91, No. 4, 1979

genetic ready

been elucidated

answer this IB1

quence of

IB2.

quenced

This

parts

the primary structural

Furthermore,

fills

structure

of

IB2 has alon IB1

and the COOH-terminal and compared

a chymotryptic

and compared with peptide

RESEARCH COMMUNICATIONS

investigations

The NH2-terminal

have been established

been characterized 1*2.

(3),

question.

quence of

from

Since

modification.

AND BIOPHYSICAL

completely

the

se-

to the total

peptide

from IB1

corresponding the

should

gap between

sehas

fragment the

se-

of IB1. MATERIALS AND METHODS

Chemicals. Amino acid calibration mixture and chemicals forautomated Edman degradation were from Beckman Instruments GmbH; bovine serum albumin was from Behringwerke; Azocoll was from Calbiochem; Bio-Gel P-2 (>400 mesh) was from Bio-Rad; blue dextran 2000was from Deutsche Pharmacia, a-chymotrypsin (TLCK-treated) was from Merck) carboxypeptidase A from bovine pancreas (DFP-treated), dansyl-Cl and trypsin (TPCK-treated) were from Serva; carboxypeptidase Y from yeast was a gift from Dr. Hasilik. All other chemicals were of analytical grade and were obtained from Roth GmbH. Commercially grown bakers' yeast (Pleser-Hefe, Yeast strain. Darmstadt-Eberstadt) was used for isolation of the proteinase B inhibitors. The purification procedure of the yeast Inhibitor purification. proteinase B inhibitors 1 and 2 has been described in a previous communication (2). Inhibitory activity was determined by measuring the inhibition of Azocoll hydrolysis by proteinase B (I). It should that the specific activity of the proteinaseBinhibe mentioned, tors depends very strongly on the sample of Azocoll used as substrate for the proteinase. The inhibitor concentrations were routinely determined according to the method of Lowry et al. (4) with crystalline bovine serum albumin as standard. Isolation of chymotryptic peptides. Two milligrams of each inhibitor were dissolved in 0.4 ml of 0.2 M ammonium bicarbonate. DH 8.0, respectively. Hydrolysis at 25'C was performed by additioh of a-chymotrypsin at an enzyme-substrate ratio of 1:600 (mol/mol). Digestion was stopped after 20 min by heating for 10 min in a boiling water bath. The peptide mixtures were frozen and lyophilized. Separation of the chymotryptic peptides was performed by preparative disc-gel electrophoresis at pH 9.0 in 12 % polyacrylamidegels. After electrophoresis, the gels were sliced for extraction with The supernatants were lyophilized and desalted twice on a water. Bio-Gel P-2 column (1 x 10 cm) in the presence of 0.05 M ammonium bicarbonate, pH 8.0. Peptide mappihg. For pe tide mapping, 10 mg of the chymotryptic 10 plof 0.5Mammonium peptides CHS-IBI and CHS-I ii 2 weredissolvedin bicarbonate, pH8.0, and digested with3 % trypsin (w/w) at 25OC for 5 h. The obtained peptidemixtureswere dansylated and separated by thinlayerchromatography as recently described byMaieret al. (2). Amino acid analysis was carried out with a Amino acid analysis. The chymotryptic peptides were hydroBiotronik LC 6000 analyzer.

1391

Vol. 91, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

lyzed with 6 N HCl at 105'C for 4% h and analyzed according to the method of Spackmanetal. (5) by separation with a single column (6). Concentrations of IB1 and of the peptides, used for COOH-terminal sequence analysis, were determined by analyzing aliquots, which had been hydrolyzed with 6 N HCl at 105'C for 24 h. Automated Edman degradation. Automated Edman degradation of IB1 was performed in a Spinco 890 C sequencer, BeckmanInstruments Inc., Palo Alto, Calif., using a DMBA-program as described by Hermodson et al. (7). PTH-amino acids were identified by gas liquid chromatography and thin layer chromatography. Additionally, aliquots of the PTH-amino acid containing fractionswerehydrolyzed with 6 N HCl at105'C for 48hand subjected toaminoacid analysis. Determination of end groups. NH -terminal residues weredetermined bv the dansvl-Cl method (8). ZOOH-terminal residues were released enzymaticaily and determined by the amino acid analyzer. Degradation of native IB1 with carboxypeptidase A from bovine pancreas has been described in detail previously (2). As reported, four residues are split off by the enzyme at pH 8.5. A further residue is released stoichiometrically, when the pH is lowered to 4.2. The carboxypeptidase A-modified IB1 could be isolated from the incubation mixture by ethanol precipitation (85 % v/v). Two milligrams of the modified inhibitor were dissolved in 0.4 ml of 0.1 M pyridine acetate, pH 4.2 and 80 ~1 therefrom were taken to determine protein concentration by the analyzer after hydrolysis. Degradation by carboxypeptidase Y from yeast at 25“C was performed at an enzyme-substrate ratio of 1:720 (mol/mol). Samples (20 ~1) were taken after 1, 2, 4, 8, 12, 16, 20, 25, 3Q, 35, 40, 45 and 50 min to stop hydrolysis by ethanol precipitation (85 % v/v). The supernatants were analyzed for free amino acids. A plot was prepared showing the time dependent release of amino acids from the carboxypeptidase A-modified inhibitor. COOH-terminal studies on the peptides CH5-IB1 and CH5-IB2 were performed by hydrolysis with carboxypeptidase A from bovine pancreas as described for the native inhibitors (2).

NH,-terminal quence

of

Identical

is

34 in

IBl

amide

groups,

minal

sequences

are

established

sequence

with

exchanged however,

COOH-terminal inhibitors

were

differences

compared

have

of the

The NH2-terminal

by two sequencer

results

The NH2 -terminal

Two significant quence

analysis.

was established

IB1

inhibitor. steps.

sequence

obtained of IBl

become the

total

for

LEU-32

over is

sequence

and GLU-34

two'inhibitors

(Fig.

analysis.

with

1392

IB2:

part

of

VAL-32 IB2. in

1.

se-

and LYS-

The number the

of

NH2-ter-

I).

As previously

reported,

as COOH-terminal

carboxypeptidase

native

in Fig.

this

in

to be unchanged

the

se-

42 degradation

if of

acid

with

summarized

obvious,

-VAL-HIS-THR-ASN-COOH

by hydrolysis

runs

was found

sequence

amino

A from

both

sequence bovine

pan-

5

10

45

50

70

74

55

15

60

20

3 e q uence ofIBl in comparison with the total aminoacid sequence chymotryptic peptides from IB1-and IB2. &, auto2 represent NH2-terminal amino acids determined with the dansylation amino acids determined after hydrolysis with carboxypeptidase 8.5 (2); -, COOH-terminal amino acid obtained by hydrolysis bovine pancreas after lowering the pH from 8.5 to 4.2; L-, the carboxypeptidase A-modified inhibitor established by hydrolfrom yeast at pH 4.2 (see also Fig. 2).

-(VAL,GLU)GLU-ASP-LYS-GLU-VAL-HIS-THR-ASN-COO-----7-G---77 -VAL-HIS-THR-ASN-COO-r-c--F--VAL-HIS-THR-ASN-COO\--VAL-ILE-GLU-ASN-VAL-GLU-GLU-ASP-LYS-GLU-VAL-HIS-THR-ASN-COO-

65

TYR-THR-ILE-LYS-VAL-PRO-ASP-VAL-LEU-HIS-LEU-ASN-LYS-LEU-LYS-GLU-LYS-HIS-ASN-ASP-

TYR-THR-> --+ N;H-THRN;H-THR7

H~N-THR-LYS-ASN-PHE-ILE-VAL-THR-LEU-LYS-LYS-ASN-THR-PRO-ASP-VAL-GLU-ALA-LYS-LYS-PHE+--+++--+--+++4++++-+~+44+ H:N-THR-LYS-ASN-PHE-ILE-VAL-THR-LEU-LYS-LYS-ASN-THR-PRO-ASP-VAL-GLU-ALA-LYS-LYS-PHE-

acid Figure 1. Partialgamin of IB2 (3). CHS-I 1 and CH5-I mated Edman degradation; ---+, COOH-terminal method (8); 7, A from bovine pancreas at pH with carboxypeptidase A from COOH-terminal amino acids of ysis with carboxypeptidase Y

IB2

CH5-IB2

CH5-IB1

IB1

IB2

CH5-IB2

CHS-IB1

IB1

IB2

IB1

IB2

IB1

Vol. 91, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL

Other

amino acids _----

__ __---l

RESEARCH COMMUNICATIONS

I

25 Digestion

time

50 Imin)

AFigure 2. COOH-terminal analysis of the carboxypeptidase modified IB1 by degradation with carboxypeptidase Y from yeast at pH 4.2, as described under "Materials and Methods".

cress tive to

at pH

8.5

release

of

(2).

Hydrolysis

four

residues

a GLU residue

4.2,

is

boxypeptidase

A-modified

form.

Further

degradation

yeast

at pH

hibitor

is

signed

ued incubation

2),

known from

with

from

isolated

in a homogeneous

the

first

Y from

from the modified

tow residues

can be attributed

pH

The car-

by carboxypeptidase

amino acids

and characterization amino acid

TYR residue

(2)

preferentially the

IB1.

the

inbe as-

in Fig. after

The

1.

contin-

to a second GLU residue

66.

Isolation

trypsin

of

lowering

2. LYS, ASP and GLU can definitely

exceeds

single

was then

After

quantita-

released

was achieved

amount of GLU, which

in position

enzyme.

after

69, 68 and 67 as summarized

positions

(Fig.

completely

additionally IB1

shown in Fig.

to the

by the

The release

4.2.

stops

enzyme,

of

compositionr

chymotryptic

that

located

in position

attacks

(Fig.

a fragment

1).

was isolated

1394

both 41,

After

peptides.

inhibitors at which limited

It

is

contain

a

a-chymoproteolysis

from each inhibitor,

CH5-

Vol. 91, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

b

C

Figure 3. Disc-gel electrophoresis of the peptides CHS-IB1 and CH5-IB2 with 12 % polyacrylamide gels at pH 9.0. Gel a, 15 pg CH5-IB1; gel b, 15 kg CH5-IB2; gel c, 15 Kg c~5-IDl plus 15 kg CH5-IB2.

IB1

and CH5-IB2,

peptides

(8),

determined shown).

degradation

CH5-IB1

at pH 9.0 I,

gruent

(Fig.

33 residues, residue

and -VAL-HIS-THR-ASN-COOH

after

(Table

the

composed of

have THR as NH2-terminal

Cl method

ity

being

and CH5-IB2 (Fig.

right).

shown).

two chymotryptic

been sequenced

show the

peptide

in the

It

peptides course

established

is concluded identical.

of sequence

1395

sequence

A (Fig.

not

same electrophoretical amino acid

maps from tryptic

are

Both

bythedansyl-

as COOH-terminal

carboxypeptidase

3) and have identical Their

not

with

respectively.

compositions

digests

from these CHS-IB2, analysis

mobil-

of

are con-

results, which IB2

that had (3),

Vol. 91, No. 4, 1979

TABLE I:

Amino

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

AMINO ACID COMPOSITIONSOFTHEYEAST PROTEINASEB INHIBITORS 1 AND 2 (LEFT) AND OF THE CHYMOTRYPTICPEPTIDES CHS-IB1 AND CH5-IB2 (RIGHT) IB2 a Residues/molecule

acid

Asparagin/Aspartic Threonine Serine Glutamine/Glutamic Proline Glycine Alanine l/2-Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophan

acid

11.9

11.8

6.7

(7)

6.8

(7)

5.0

5.1

2.1

(2)

2.0

(2)

2.2

2.0

-6.6 2.2

7.3

5.3

(5)

5.1

(5)

2.1

1.0

(1)

1.0

(1)

3.3

3.1

(0.4

(0)

co.2

(0)

2.1

2.1

X0.2

(0)

x0.2

(0)

acid

(0.2 8.8

1.9

from position

Consequently, from

(5)

4.6

(0)

x0.2

residues

differences

between

quence analysis. compositions

1.0

-5.7 0.9

to.2

(0)

x0.2

(0)

3.0

3.0

(0.2

(0)

CO.2

(0)

11.4

10.7

4.7

(5)

5.0

(5)

1.8

(2)

3.0

(3)

crepancies

in the

1.7

(2)

3.0

(3)

6.0

6.0

2.9

(3)

3.0

(3)

x0.2