homology of the /?-subunits of luteinizing hormone from various species and the ... Primary Structure of the Bovine and Porcine Luteinizing-Hormone ,!?-Subunit.
Eur. J. Biochem. 39,235-253 (1973)
Luteinizing Hormone The Primary Structures of the P-Subunit from Bovine and Porcine Species Guy MAGHUIN-ROGISTER and Georges HENNEN Section d'Endocrinologie, DApartement de Clinique et de SAmBiologie MAdicales, Institut de Mkdecine, Universitb de Libge (Received April 9/July 13, 1973)
The sequences of the 119 amino acids of bovine and porcine luteinizing hormone /?-subunits were determined, 17 amino acid replacements being observed between these species. Variability in the primary structure was observed forporcine/?-subunit,in that position 10 is occupied by both arginyl and glutamyl residues. No free amino terminal residue was detected for both porcine and bovine j3-polypeptide chains and both exhibited carboxy-terminal heterogeneity. The homology of the /?-subunits of luteinizing hormone from various species and the /?-subunit of the bovine thyroid-stimulating hormone is discussed as the subunit of both hormones can combine with a similar a-subunit.
Considerable progress has recently been made in the elucidation of the primary structures of pituitary glycoprotein hormones. I n 1970, Liao et al. [1,2] succeeded in establishingthe amino acid sequences of a and /?-subunits of bovine thyroid-stimulating hormone. Provisional sequences for ovin0 luteinizing hormone a and /?-subunits were presented by Liu et al. [3,4] which were subsequently revised [5] until completion [6, 71. Independently, we reported on the structure of bovine luteinizing hormone /Isubunit [8] and, in a preliminary communication, we recently compared the bovine j3 sequence with that of the porcine /? subunit [9]. The present study describes all methodological details and structural data which permitted the elucidation ofthe complete sequences of bovine and porcine luteinizing hormone /?-subunits with the assignment of the amide forms of glutamyl and aspartyl residues. MATERIALS AND METHODS
Preparation of Luteinizing Hormone and of Its Subunits Bovine and porcine luteinizing hormones were pursed as previously described [10,111. As determined by the ovarian ascorbic acid depletion test of Parlow [12], the potencies of bovine and porcine Abbrevktion. QAE-Sephadex, quaternary diethyl-(2-hydroxypropyl) ethylenediamine. Enzymes. Trypsin (EC 3.4.4.4); pepsin (EC 3.4.4.2); or-chymotrypsin (EC 3.4.4.5); carboxypeptidase A (EC 3.4.2.1); carboxypeptidaseB (EC 3.4.2.2); leucine aminopeptidase (EC 3.4.1.1); prolidase (EC 3.4.3.7). Eur. J. Biochem. 39 (1973)
luteinizing hormones were 3 and 1.2 times that of National Institutes of Health luteinizing hormone 8.15, respectively. Their b~ and /? subunits were obtained by chromatography on SE-(or SP-)Sephadex C-25 (Pharmacia) in 8 M urea [13,11]. The properties of our subunit preparations were described elsewhere [lo, 11,131.
Protein Derivatives Reduction of disulfide bridges followed by alkylation was carried out as described by Crestfield et al. [141. Iodoacetic acid, sodium salt (Calbiochem, iodine-free grade, recrystallized), was used as the alkylating agent. Maleylation of lysine residues was performed a t pH 9.4 in borate buffer [15]. Chemical Fragmentation and Enzymatic Hydrolysis The cyanogen bromide fragments of the reduced and alkylated protein were prepared as described by Samy et al. [16]. Tryptic (Worthington, treated with L-1-tosylamido-2-phenylethyl chloromethyl ketone) hydrolysis was performed as detailed in an earlier study [13]. Further cleavage of the large tryptic peptides was done by pepsin (Worthington)or chymotrypsin (Mann Research Laboratories, a-chymotrypsin) as described [8,9]. Thermolysin (Calbiochem, containing 650/, enzyme protein and 31 calcium acetate) digestions were carried out in 0.125 M ammonium bicarbonate pH 8.5, for 2 or 4 h at 37 "C, with a weight ratio of enzyme to peptide of 1:100.
236
Primary Structure of the Bovine and Porcine Luteinizing-Hormone ,!?-Subunit
Isolation of Peptides Tryptic peptides and cyanogen bromide fragments from the reduced and 8-carboxymethylated proteins were fractionated first by gel filtration on Sephadex 6-50 fine (Pharmacia) or on Biogel P6 (Calbiochem) in 0.05 M ammonium bicarbonate buffer. The peptides yielded by enzymatic cleavage of large tryptic peptides were separated by gel filtration on Sephadex G-25 ( h e ) or 6-15 (Pharmacia) in 0.05 M ammonium bicarbonate buffer. Further purifications were achieved by ionexchange chromatography, paper electrophoresis and paper chromatography. Peptides first chromatographed on QAE-Sephadex A-25 (Pharmacia) were subsequently desalted on Biogel P2 (Calbiochem) in 0.05 M ammonium bicarbonate buffer. Experimental conditions of chromatography for each experiment are indicated in figure legends. Paper electrophoresis was carried out on Whatman 3-MM paper in a buffer p H 3.7 (pyridine-acetic acid-water ;3 :20 :77, v/v/v) at 75 V x cm-l for 1 h or in a buffer p H 1.9 (formic acid-acetic acid-water; 5: 15:80, v/v/v) a t 50 V x cm-l for 90 min; the apparatus was a Pherograph Hormuth. Paper chromatography was done with Whatman 3-MM paper; the solvent was the upper phase of a n-butanol-acetic acid-water (4: 1:5, v/v/v) mixture. Purity of the peptides was assessed by paper electrophoresis, under the conditions described above, NH,-terminal analysis (dansyl method) and amino acid composition. Analytical Methods The amino acid compositions were determined according to Spackman et al. [I71 on a Beckman model 121 amino acid analyzer. Hydrolyses were performed on 20 to 50 nanomoles peptides, in 1ml distilled, constant-boiling hydrochloric acid, a t 110 "C for 24 h in evacuated ampules. Sodium thioglycolate (0.2 mg in 20p1, 6 N hydrochloric acid) was added to prevent destruction of carboxymethylcysteine. The detection of glycopeptides after column chromatography was performed by the phenolsulfuric acid procedure [I81 or by the detection of amino sugars during their amino acid analysis. Carboxy-terminal amino acids were identified on the amino acid analyzer after hydrolysis with carboxypeptidases A and B (Worthington) as described earlier [19] or by hydrazinolysis [20]. The sequential degradation of peptides were performed according to Gray [21]. The hydrolyses of dansyl-peptides were performed under the conditions described by Gros and Labouesse [22]. The aminoterminal residues were identified as their dansyl derivatives after thin-layer chromatography [23]
on polyamide sheets (Cheng-Chin Trading Co). Dansyl-arginine was identified by electrophoresis (30 min, 40 V X cm-l) on a polyamide layer in a buffer p H 1.9 (acetic acid-formic acid-water ; 120 :28: 1852, v/v/v) with dansyl-arginine (Calbiochem) as reference.
Determination of Amide Forms of Glutamyl and Aspartyl Residues Leucine aminopeptidase was used for exhaustive hydrolysis of peptides under the following conditions t o 0.05 pmol peptide, dissolved in 100 pl 0.1 M TrisHC1 buffer p H 8.6 and containing 2.5 mM magnesium chloride, was added 5 p.l of a leucine aminopeptidase suspension (Worthington, 10.8 mg/ml, 100 units/mg) and the incubation kept at 37 "C for 4 or 24 h, according t o the size of peptide. Prolidase was required t o ensure hydrolysis of the imide bond between the glutamyl or aspartyl residues and proline. The enzyme was prepared from pig kidneys using the first two steps of the method described by Davis and Smith [24]. The specific activity [25] of this preparation was 26 units/ mg. The conditions used for cleavage with prolidase were the following: digestion of 0.05 pmol peptide dissolved in 100 pl 0.1 M Tris-HC1 buffer p H 8.6 containing 2.5 mM magnesium chloride and 10 mM manganese chloride, was initiated by incubation with 10 pl leucine aminopeptidase for 4 h. The prolidase solution (1 mg per ml of 0.05 M Tris-HC1 buffer p H 7.4, containing 10 mM manganese chloride and 0.03 M toluol) was left for activation I h a t 37 "C; 20 pl were added to the leucine aminopeptidase digest which was further incubated at 37 "C for 24 h. The hydrolysate was diluted directly with 1.3 ml 0.2M sodium citrate buffer p H 2.2, and analyzed on the amino acid analyzer. I n the conditions described by Spackman et al. [17], asparagine and glutamine emerge a t the serine position. RESULTS
Tryptic Peptides from the Reduced and S - Carboxymethylated Bovine Luteinizing Hormone /3-Xubunit The gel filtration pattern on Biogel P6 of a tryptic hydrolysate of reduced and 8-carboxymethylated bovine luteinizing hormone ,&subunit (25 pmol) in shown in Fig. I. Six fractions (A-F) were selected accordingly, each containing multiple peptides as revealed by analytical paper electrophoresis (Fig.2 ) . Fraction A, containing the glycopeptides was fractionated further by ion-exchange chromatography on QAE-Sephadex A-25 a t p H 8.0 (Fig.3). Fraction AI contained a pure glycopeptide, bovine 3't T,, while fraction A111 was a pure holopeptide bovine ,tI T,. As fraction I1 still appeared heterogeneous by anaEm. J. Biochem. 39 (1973)
237
G. Maghuin-Rogister and G. Hennen
1.5
'.4r $1.0 Ln m N c
m a,
c
R m L
0
n
Q
0.5
0.5
0.4 0.3 0.2
5 ...-. 0 I
0.1
0
0 20
40
60
Fraction no.
Pig.1. Gel filtration on Biogel P-6 of the tryptic hydrolysate of the reduced and carboxymethylated bovine p-subunit. Column, 5 x 95 cm; developing buffer, 0.05 M ammonium bicarbonate; flow rate, 35 ml/h; sample, 387 mg in 25 ml buffer; fraction size, 8 ml; temperature, 25 "C. The pooled fractions are indicated by solid bars
80 100 F r a c t i o n no.
140
120
Fig.3. Isolation of bovine B T , and T , peptides. Chromatography at 25 "C, on a column (2.5 x 15 cm) of QAE-Sephadex A-25 equilibrated with 0.05 M Tris-HC1 buffer pH 8.0; fraction size: 3.7 ml. The sample (peak A of Fig. 1, 93 mg) was applied in 5 ml of equilibration buffer. The slope of the linear gradient is indicated by the broken line
E
20 m
01 c
m
a, " LT
R m
L
$ 0
R
4
.2
2
-
.1 0 I
8T8b
w Taa
'
I
0 T2b A
o 0
C
D
E
F
Fig. 2. Paper electrophoresis of fractions obtained by gel filtration o n Biogel P-6 (Pig.1) of the tryptic hydrolysate of the reduced and earboxymethylated bovine B-subunit. Electrophoresis on Whatman 3 MM paper was in a pyridine-acetate buffer pH 3.7, a t 75 Vxcrn-l for 1h; temperature, 3 "C. The cathode is a t the top
Eur. J. Biochem. 39 (1973)
I
I
I
20
40
60
F r a c t i o n no.
I
ao
I 100
Fig.4. Isolation of bovine B TI,, T8b and T2b peptides. Chromatography a t 25 "C, on a column (2 x 10 cm) of QAESephadex A-25 equilibrated with 0.01 M sodium acetate buffer in 8 M urea pH 5.0; fraction sizer: 3.6 ml. The sample (peak A11 of Fig. 3, 25 mg) was applied in 2 ml equilibration buffer. Ionic strength was increased in chloride. The slope of the linear gradient is indicated by the broken line
238
Primary Structure of the Bovine and Porcine Luteinizing-Hormone /%Subunit
lytical paper electrophoresis, the material was additionally chromatographed on QAE-Sephadex, in a dissociating buffer (Fig.4). Two holopeptides, bovine p TI,, and T8b and one glycopeptide T2b were thus obtained in pure form. Fraction B was fractionated by ion-exchange chromatography on QAE-Sephadex A-25 in a dissociating medium (Fig.5).Two peptides, bovine /IT, and Tsa, were isolated in pure form. Fraction C contained peptide bovine p TB(Fig.2) which was freed from its contaminants by ion-ex-
change chromatography in a dissociating buffer (Fig.6). Fraction D and E were fractionated by preparative high-voltage electrophoresis yielding peptides bovine p T, and T, respectively. Fraction F contained two peptides, bovine B TI and T, staining with Sakaguchi reagent, which were isolated by preparative paper electrophoresis a t pH 3.7.The faster one, T,, exhibiting a mobility identical to that of arginine. The compositions of the tryptic peptides are given in Table 1.
0.4
i
E 0
~t0.3 LD hl
-
0.3
m 0.2
m
c
$0.2
m
c m
2 0.1
2 0" Q
0.2
0.1
0.1
a, u
-z -
I
8
n
7
Q
0
0
0 I
I
0
20
I
I
40 60 F r a c t i o n no.
-A
0
0
I
I
80
100
Fig.5. Isolation of bovine T5and T,a peptides. Chromatography on a column ( 2 x 2 1 om) of QAE-Sephadex A-25. Conditions were as indicated for Fig.4. The sample (peak B of Fig. 1, 56 mg) was applied in 3 ml equilibration buffer
20
40 60 F r a c t i o n no.
80
Fig.6. Isolation of bovine T , peptide. Chromatography on column (2 x 20 cm) of QAE-Sephadex A-25. The experimental conditions were those used in Fig.4. The sample (peak C of Fig. 1, 33 mg) was applied in 3 ml of equilibration buffer
Table 1. Compsitions of bovine B tryptic peptides Data are expressed a8 molar ratios of the various amino acids. The numbers in parentheses are theoretical values. Hydrolysis time was 24 h and no correction for destruction during the hydrolysis was made Aminoacid Lysine Histidine Arginine Carboxymethylcysteine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Total residues Sugars
T,
1.0 (1)
T,
T,b
T,
0.9 (1)
0.9 (1)
0.9 (1)
0.9 (1) 1.1 (1)
0.6 (1)
1.0 (1) 0.9 (1)
1.1 (1) 0.9 (1)
1.9 2.8 1.0 2.9
2.0 (2) 1.0 (1)
1.0 (1) (2) (3) (1) (3)
0.9 (1) 2.7 (3)
2.9 (3) 0.9 (1) 1.0 (1)
T,
T,
T,
T,
1.0 (1)
1.0 (1)
0.8 (1) 0.9 (1)
1.0 (1)
3.6 (4)
1.0 (1)
2.6 (3) 1.6 (2)
0.9 (1)
1.2 (1) 1.6 (2) 1.0 (1) 1.8 (2j 1.0 (1) 0.8 (1) 1.6 (2)
1.0 (1) 3.6 (4)
1.1 (1)
1.6 0.8 0.6 1.9
1.0 (1)
(2) (1) (1) (2)
12
22
1.3 0.9
0.8 (1)
0.8 (1)
2.6
1.0 (1)
0.7 (1)
1.0 (1)
1.3
1.0 (1) 0.9 (1) 1.7 (2)
1.9 (2) 0.8 (1)
1.1 (1) 1.8 (2)
4.0 (4)
0.8 (1) 0.9 (1)
6.7 2.9 1.3 2.6 1.o
0.7 (1)
2.3
1.0 (1)
0.2 (0-1) 2.0 (2-3)
10
13-15
1.6
1.1 (1) 1.1 (1)
12
8
0.9 (1)
5
1.0 (1)
0.8
1.0 (1)
1
TI,
1.0 (1) 3.5 (4) 1.6 (2)
1.0 (1) 0.9 (1)
0.9 (1) 18
T,
0.9 (1)
0.6 (1)
2
T8b
Tsa
11
26
Present Present Eur. J. Biochem. 39 (1973)
239
G. Maghuin-Rogister and G. Hennen Table 2. Amino-mid sequence of bovine 9, t y p t i c peptides n.d., Not determined Tryptic peptide TI
Procedure
Amino acid sequence or composition
Complete Dansyl-Edman
Ser-Arg -a
See Table 3 See Table 4 Arginine T5
Val-Leu-Pro-Val-Ile-Leu-Pro-Pro-Met-Pro-GluArg
Complete Dansyl-Edman
Val-Leu-Pro-(Val-He)b-Leu-Pro-Pro-Met
+
Carboxypeptidases A B Leucine aminopeptidase prolidase
+
Ti?
Val-CmCys-Thr-Tyr-(His) a-Glx
+B
Leucine aminopeptidase T,
Tsa
+ d + + + +
5 min: Leu, 0.3; Arg, 1.0 1h: Leu, 0.8; Arg, 1.0 4 h: Val, 1.0; CmCys, 1.0; Thr, 0.7; Tyr, 0.7; His, n.d.; Glu, 0.6; Leu, 0.6; Arg, 0.5 residues/mole.
Complete Dansyl-Edman
Phe-Ale-Ser-Val-Arg Phe-Ala-Ser-Val
Complete Dansyl-Edman
Leu-Pro-Gly-CmCys-Pro-Pro-Gly-Val-Asp-Pro-Met Leu-Pro-Gly-CmCys-Pro-Pro-Gly-ValAsx
CarboxypeptidaseA Leucine aminopeptidase prolidase
4 h: Met, 1.0 residues/mole.
Complete
Leu-Pro-Gly-CmCys-Pro-Pro-Gly-ValAsp-Pro-Met-Val-Ser-Phe-Pro-ValAla-LeuSer-CmCys-(His,CmCys,Gly,Pro)-CmCys-Arg
Dansyl-Edman
Leu-Pro-Gly-CmCys-Pro-Pro
++++ -3
+
Tsb
Carboxypeptidases A To
+ 3 + +
Val-CmCys-Thr-Tyr-His-Glu-Leu-Arg
Complete Dansyl-Edman CarboxypeptidasesA
+++-+
4 h; Glu, 0.3; Arg, 1.0 residues/mole. 28 h; Val, 2.0; Leu, 1.7; Pro, 2.0; Ile, 1.0; Met, 0.5; Glu, 0.6; Arg, 0.9 residues/mole.
+B
+++-++
28 h: Leu, 1.0; Pro, 1.2; Gly, 1.2; CmCys, 1.0; Val, 0.2; Asp, 0.8; Met, 0.4 residues/mole.
-+++--++
4 h: CmCys, 0.5; Arg, 1.0 residues/mole.
Leu-Ser-Ser-Thr-Asp-CmCys-Gly-Pro-GlyArg
Complete Dansyl-Edman
Leu-Ser-Ser-Thr-Asx-CmCys-Gly
+
Carboxypeptidases A B Leucine aminopeptidase TI,
+--?.-
+++++-+
4 h: Gly, 0.9; Arg, 1.0 4 h: Leu, 1.0; Ser, 2.1; Thr, 1.0; Asp, 0.7; CmCys, 0.9; Gly, 0.6; Arg, 0.6 residues/mole
See Table 5.
No dansyl-amino acid was identified. Released as a dansyl = dipeptideby the dansyl-Edman procedure, the order Val-Ile was determined by application of the subtractive procedure [30]. 8
b
Amino acid sequence data concerning those tryptic peptides are given in Tables 2, 3, 4 and 5. The sequence of bovine # Tgb l could not be completely established because of the large size of this peptide and its poor yield. Peptide Tsa resulting from a partial non-specific splitting of the Met-Val bond in Eur.J. Biochem. 39 (1973)
Tsb was easily isolated while the carboxy-terminal moiety resulting from this cleavage could not be recovered. Nevertheless, as described below, this portion of the sequence was determined by study of the corresponding cyanogen bromide fragment bovine t3 CNBr IV.
240
Primary Structure of the Bovine and Porcine Luteinizing-Hormone p-Subunit
Table 3. Amino-acid sequence of bovine?!, T2 peptide Amino acid compositions are expressed as molar ratios of the various amino acids. The number in parentheses are theoretical values. CHO symbolizes the polysaccharide prosthetic group Peptide
Procedure
Amino acid sequence and composition CHO I Gly-Pro-Leu-Ag-Pro-Leu-CmCys-Glna-Pro-Ile- Asn- Ala-Thr-Leu- Ala- Als-Glu-Lys < Tho Tha +Th,+ +-Thd-+ Present Gly-Pro-Leu-Arg-Pro-Leu-CmCys-Glx
Complete
--f
Sugars Dansyl-Edman Carboxypeptidases A Thermolysin peptides Tha
+B
--*+--+---+
- -
-+A+
4 h: Glu, 0.3; L p , 1.0 residues/mole
Sugars Dansyl-Edman
Leu, 1.0 (1); CmCys, 0.9 (1); Glu, 0.9 (1); Pro, 0.8 (1); Ile, 0.9 (1); Asp, 1.0 (1); Ala, 0.9 (1); Thr, 0.9 (1) Present Leu-CmCys-Glx
Dansyl
Leu, 0.9 (1); Ala, 2.0 (2); Glu, 1.1 (1); Lys, 1.0 (1) Leu
Thb
---
+ -+
--f
The Dansyl Thd Dansyl
Leu, 1.1 (1); Ala, 0.9 (1) Leu
-+
Ah, 1.0 (1);Glu, 1.0(1); LYS,1.0 (1) Ala -+
Tzb Complete SUgUS Dansyl-Edman
CHO I
CmCys-Gln-Pro-Ile-Asn-Ala-Thr-Leu-Ala-Ala-Glu-Lys Present --f 3
a As amino acid analysis of the enzymatic digest (leucine amino peptidase in the peptide, the identification of Gln is only tentative.
The determination of the complete amino acid sequence of the glycopeptides bovine B T, and T2b and of the large holopeptides T, and TI,, needed further enzymatic cleavage. Glycopeptide bovine B T, was digested with thermolysin. Fig. 7, shows the gel filtration pattern of the thermolytic digest. The glycopeptide Tha (Table 3) was obtained in pure form in the first peak, the second peak contained three shorter peptides which were isolated by paper electrophoresis at pH 3.7. The one nearest the origin was the dipeptide Th, (Table 3), the second was the pentapeptide Thb (Table 3), the tripeptide Thd having the highest cathodic mobility. The thermolytic N-terminal fragment of bovine T, was not recovered. Data from carboxypeptidase digestion of T, together with the
-
CmCys-Glx-Pro-Ile-Asx-Ala-Thr-Leu ----t
-+ --+ ---+--+
+ prolidase, 48 h) revealed residues, not present
compositions of Tha,b,c,d permitted the complete elucidation of bovine T, structure (Table 3). Comparison of T, and T2b sequences, revealed that T2b resulted from a non-specific cleavage of leucinecarboxymethylcysteine bond by trypsin. Here again, the NH,-terminal peptide, containing hydrophobic residues, was not recovered. Bovine B T, peptide was cleaved with chymotrypsin. The chymotrypsin digest was subjected to gel filtration on Sephadex 6-15 (Fig.8). Peptides T3Cd and T,C, (Table 4) were obtained in pure form in the second and third peak, respectively. Two peptides T,C, and T3Cb were isolated from the first peak by paper electrophoresis, T,Cb exhibiting the highest mobility towards the cathode. Composition and amino acid sequence of bovine B T, peptide and Eur. J. Biochem. 39 (1973)
241
G. Maghuin-Rogister and G. Hennen
Table 4. Amino-mid sequence of bovine ,!? T3 peptide Amino acid compositions are expressed as molar ratios of the various amino acids. The numbers in parentheses are theoretical values Peptide
Procedure
TS
Complete
Amino acid sequence and composition
Glu-Ala-CmCys-Pro-Val-CmCys-Ile-Thr-Phe-Thr-Thr-Ser-Ile-CmCys-Ala-Gl~T~TsCa TaCb T3Cc +.
.(
.(
CmCys-Pro-Ser-Met-Lys-OH.
-
T&b TsCd
,
------+
Glx-Ala-CmCys-Pro-Val-CmCys-Ile-Thr
Dansyl-Edman Carboxypeptidases A
+B
Chymotrypsin peptides TSCa
---+
--f----f
--+
--f
---+
15 min: Ser, 0.2; Met, 0.3; Lys, 1.0 1 h: Ser, 0.5; Met, 0.6; Lys, 1.0 residues/mole.
Glu, 1.0 (1); Ala, 1.0 (1); CmCys, 1.6 (2); Pro, 1.2 (1); Val, 1.0 (1); Ile, 1.0 (1); Thr, 1.0 (1); Phe, 0.7 (1) Glx-Ala-CmCys
Dansyl-Edman
-+
----*----+
Leucine aminopeptidase TSCb
4 h: Glu, 1.0; Ah, 1.0 residues/mole.
Thr, 1.9 (2); Ser, 1.9 (2); Ile, 0.9 (1); CmCys, 2.2 (2); Ala, 1.0 (1); Gly, 1.2 (1); Tyr, 0.6 (1); Pro, 1.1 (1); Met, 1.1(1); Lys, 1.1 (1)
Thr-Thr-Ser-Ile-CmCys-Ala
Dansyl-Edman
---T4--+-+
--3-
Thr, 1.9 (2); Ser, 1.0 (1); Ile, 0.9 (1); CmCys, 1.1 (1); Ala, 1.0 (1); Gly, 1.0 (1);
TF, 0.7 (1) Dansyl-Edman
-
Thr-Thr-Ser
Carboxypeptidases A
+B
TsCd
--%
--+
30 min: T a , 0.8
residues/mole.
CmCys, 1.1(1); Pro, 1.1 (1); Ser, 0.7 (1); Met, 0.8 (1); Lys, 0.9 (1) CmCys-Pro-Ser
Dansyl-Edman
__t
-+ --+
1. O r 2.0
1
I
I
I
I
0
20
40
.60
80
F r a c t i o n no.
Fig.7. Gel filtration 012 S e p M e x G-25 (superfine) of the thermolytic digest of bovine ,!? T , ( 3 mg). Column, 1.5 x 88 cm; developing buffer, 0.05 M ammonium bicarbonate; flow rate, 10 ml/h; sample volume, 0.5 ml; fraction size, 2 ml; temperature, 25 "C Eur. J. Biochem. 39 (1973)
1
10
20 ~
30
40
50
60
F r a c t i o n no.
Fig. 8. Gel filtration on S e p M e x G-15 of a chymotryptic digest of bovine j3 Ts.Column, 1.6 x 100 cm; developing buffer, 0.05 M ammonium bicarbonate; flow rate, 11ml/h; sample volume, 2 ml; fraction size, 2.3 ml; temperature, 25 "C
242
Primary Structure of the Bovine and Porcine Luteinizing-Hormone p-Subunit
Table 5. Amino-acid sequence of bovine B T10 peptide Amino acid compositions are expressed as molar ratios of the various amino acids. The numbers in parentheses are theoretical values. n.d., not determined Peptide
Procedure
Amino acid sequence and composition
TlO
Complete
Thr-Glna-Pro-Leu-Ala-CmCys-Asp-His-Pro-Pro-Leu-Pro -Asp- (Ile,Leu)- OH. -Ti&,TioCb --f
Dansyl-Edman
Thr-Glx-Pro-Leu-Ala-CmCys-Asx -3-+
Carboxypeptidase A
++
-+
--+
Hydrazinolysis
15min Ile 0.158 Leu 0.074 Asp, traces of Ile
Dansyl
Thr, 0.9 (1); Glu, 1.0 (1); Pro, 1.0 (1); Leu, 1.0 (1) Thr
TloCa
2h 4h 0.161 0.170 0.095 0.098 residues/mole. and Leu
d
Ala, 1.0 (1); CmCys, 1.0 (1); Asp, 1.9 (2); His, 0.9 (1); Pro, 3.0 (3); Leu, 1.1; Ile, 0.2 Ala-CmCys-Asx-(His) b-Pro-Pro-Leu-Pro-Asx
TioCb
Dansyl-Edman
--+ -+
-+ -+
--+
3
--+ -.-+
--+
28 h: Ala, 1.0; CmCys, 1.0; Asp, 0.7; His, n.d.
Leucine aminopeptidase
residueslmole.
* Electrophoretic mobility at pH 6.5 of the tetrapeptide bovine /3 Tl0Caindicates it is a neutral peptide migrating as glycine b
No dansyl amino acid was identified.
1.5
2 .o
:
Ln
c7 N
+ m
g 1.o
0,
LD
5 1 .o n
m
N
L
0 vl
c
m
n
6
a, S
m
f (I)
n
Q
0.5
0 1
I
10
20
I
I
30 40 Fraction no.
I 50
Fig.9. Gel filtration o n Sephadex G-15 of a chymotryptic digest of bovine Ti,,. The experimental conditions were those used in Fig.8
0
-
80
-- --L CNErI C N B r l P CNArIU CNBrlI.
100
120
140
160
180
ZOO
F r a c t i o n no
of its chymotryptic fragments are given in Table 4. The chymotryptic fragments could easily be ordered as T3Ca -+ T, Cc -+ T,Cd (Table 4). The complete structure of bovine /? T,,, subsequently identified as the carboxy-terminal peptide (vide infra), was established from composition and sequence data of its chymotryptic fragments TloCa and T,,Cb (Table 5). These chymotryptic peptides were obtained in pure form by gel filtration on Sephadex G-15 (Fig.9). The leucine and isoleucine
Fig. 10. Gel filtration on Sephadex G-50 (fine) of the cyanogen bromide cleavage products of the reduced and carboxymethylated bovine p-subunit. Column, 2 x 200 cm; developing buffer, 0.05 M ammonium bicarbonate; flow rate, 18.4 ml/h; sample 80 mg in 5 ml buffer; fraction size, 3.2 ml; temperature 25 "C
contents of bovine T,, as well as data of carboxypeptidase digestion could be due to il variability in the chain elongation. Eur. J. Biochem. 39 (1973)
G. Maghuin-Rogister and G. Hennen
243
Table 6. Compositions of bovine p cyanogen bromide peptides (CNBr I-CNBr I V ) and of the tryptic glycopeptide (MT,)of maleylated, reduced and curboqmethylated bovine p-subunit Data expressed as molar ratios of the various amino acids. The numbers in parentheses are theoretical values. Methionine was determined as homoserine MT, composition
Cyanogen bromide peptide compositions
Amino acid
CNBr I
CNBr I1
0.9 (1)
0.8 (1)
Lysine Histidine Arginine Carboxymethylcysteine Aspartic acid Threon ine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
2.1 (2) 3.6 (5) 1.6 (1) 3.4 (4) 2.8 (3) 3.2 (3) 5.0 (5) 2.0 (2) 4.5 (5) 1.3 (1) 0.8 (1) 2.4 (3) 3.4 (3) 0.9 (1) 0.9 (1)
Total residues Sugars
41 Present
Table I. Partial sequences of bovine Fragment
CNBr 111
CNBr IV
0.4 (1) 1.4 (3) 1.8 (2) 1.2 (1) 0.6 (1) 1.3 (1) 1.8 (2) 5.0 (5) 2.1 (2) 0.7 (1) 2.7 (3) 0.9 (1)
1.2 (2) 1.9 (2) 4.7 (5) 3.2 (3) 2.3 (2) 3.4 (4) 1.3 (1) 5.7 (7) 3.1 (3) 2.2 (2) 2.2 (2)
2.0
1.4 (1)
2.9 (3) 1.9 (2) 1.0 (1) 0.6 (1) 2.1 (2)
0.4 (0-1) 4.1 (4-5)
2.0 (2) 0.4 (1) 0.5 (1)
0.9 (1)
27
11
38-40
41 Present
B cyamqen bromide fragments Procedure
Sequence
Dansyl
Trace of Ser
Dansyl-Edman
Lys-Arg-Val-Leu-
CNBr I
. ..
CNBr I1 +
+
A
4
. ..
CNBr I11 Dansyl-Edman CNBr I V Dansyl-Edman
Indeed, carboxy-terminal heterogeneity of bovine luteinizing hormone /I-subunit was previously demonstrated by Rasco [26], whose data suggest a Asp-IleLeu-OH carboxy-terminal sequence in the case of the most elongated peptide chain beside a shorter sequence ending with a carboxy-terminal aspartic residue.
Cyanogen-Bromide Fragments of the Reduced and Carboxymethylated Bovine-Luteinixing-Hormone /3-Subunit As expected from the methionine content of bovine luteinizing hormone /3-subunit [12], four peptides were isolated by gel filtration of the cyanogen bromide hydrolysate (Fig. 10). Information conEur. J. Biochem. 39 (1973)
1.3 5.1 1.o 3.8 1.9 3.1 4.7 1.5 4.9 1.2 0.9 2.6 2.6 0.6 1.o
.. .
Pro-Glx-Arg-Val-CmCys-
++++
--$
Val-Ser-Phe-Pro-Val-Ala-Leu-Ser-CmCys..
+-++-++++++
cerning their amino acid composition and partial sequence are given in Tables 6 and 7. Bovine /3 CNBr I was only a glycopeptide, CNBr IV contained no homoserine. The determination of its NH,-terminal sequence permitted partial filling of a gap not elucidated during the study of the tryptic peptides.
Tryptic Digestion of the Maleylated, Reduced and Carboxymethylated Bovine /3-Subunit of Luteinixing Hormone The maleylated, reduced and carboxymethylated protein was digested with trypsin and the resulting digest was fractionated on Sephadex 6-50 under conditions similar t o those described in Fig.10. The glycopeptide, bovine /3 MT,, emerged in the first
Primary Structure of the Bovine and Porcine Luteinizing-Hormone /?-Subunit
244
Table 8. The amino-acid sequence of bovine b-subunit 5 10 CHO 15 20 1 E - S e r - A r g - G l y - P r o - L - ~ g - ~ oI - ~ ~ - ~ s - ~ l n -I P r o - I l e - A s ~ - A ~ ~ - T ~ - ~ ~ - A l a - ~ l a - G l u - ~ y s - G l u - A l a - ~ s + -
(TI)
(T,)
-+
>-
(T2b)
(MTA (CNBr I)
4 4
35 40 45 I I Pro-Val-Cys-Ile-Thr-Phe-Thr-Thr-Ser-Ile-Cys-Ala-Gly-T~-~s-Pro-Ser-Met-Lys-~g-Val-Leu-Pro(T,) (T,) + (MT,) + < (CNBr I) 25
30
I
+
55
50
-
65
60
I Val-Ile-Leu-Pro-Pro-Met-Pro-Glu-Arg-Val-Cys-T~-T~His-Glu-Leu-~g-Phe-Ala-Ser-Val-Arg-Leuc--(T,) --+ c (T,) + : (TJ c
-(CNBr 11) -+ 70
(CNBr 111)
4
75
80
90
85
Pro-Gly-Cys-Pro-Pro-Gly-Val-As~-Pro-~~et-Val-Ser-~he-Pro-~al-Ala-~u-Ser-C~s-(His,Cys,Cly,Pro)I I (Tsa)
-+
(Tab)
(CNBr 111)
(CNBr IV)
95 100 105 110 115 Cys-Arg-Leu-Ser-Ser-Thr-*s*-C~s-Gly-Pro-Gl~Arg-Thr-Cln-Pro-Leu-Ala-Cys-Asp-IIis-P~o-~~-ProI I
(T,)
-----t
