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Complete hydrolysis of glucagon by leucine aminopeptidase
VOL. 31 (I959)
SHORT CO~tMONICATIOI~S
257
Complete hydrolysisof glucagon by leucine aminopeptidase" Previous studies have shown that leucine aminopeptidase (LAP) catalyzes the stepwise degradation of the amino-terminal portion of several polypeptides and proteins 1,2. Complete hydrolysis of polypeptides has been achieved with this enzyme in the case of the oxidized A-chain of insulin which contains 2i residues 1, and the naturally occurring octapeptide hormone, hypertensin a. We would now like to report the complete enzymic hydrolysis of crystalline glucagon, the 29-residue, polypeptide hormone of porcine pancreas 4. 7 mg (2.0 ~moles) of crystalline glucagon** (Lot No. 258-234B-54-2, Eli Lilly and Co.) was incubated at 4 °0 and pH 8.5 with 4.35 mg of Mg++-activated LAP with C1 = 55"**. Hydrolysis was estimated by quantitative ninhydrin determinations on aliquots removed at intervals after starting the reaction. After 3o min, the initially insoluble glucagon had completely dissolved. After 2 h, 77 % hydrolysis was found while complete hydrolysis occurred after 7-h incubation. At this time, LAP was inactivated by adjusting the reaction mixture to pH 4 with dilute HC1, and the amino acids separated by exhaustive dialysis. The dialysates were concentrated to dryness in vacuo, dissolved in o.15 M sodium citrate, pH 2.1, and analysed by a modification of the chromatographic procedure of SPACKMAN,MOORE AND STEIN5. The results of a representative experiment are shown in Table I. It is evident that there is stoichiometric recovery of each amino acid found in glucagon with the exception of glutaminee which is not detected because of conversion to pyrrolidonecarboxylic acid. Under the conditions of the analysis asparagine is not resolved from serine. TABLE I AMINO ACIDS RECOVERED AFTER HYDROLYSIS OF GLUCAGON BY LEUCINE AMINOPEPTIDASE Amino acid
Moles~mole Composition glucagon o~ glucagon Amino acid
Moles/mole Composition gltwagon o/glucagon A mino acid
Mole.s/mole Composi*ion glucagon o/gluragott
Asp
3 .00
3
Ala
I .oo
x
Phe
1.84
2
Thr
3. o I
3
Val
I. 19
I
Try
I.OO
i
Set + Asp NH~
4.85
5
Met
0.93
i
Lys
i.I 3
I
Glu
o.o
o
Leu
2.02
2
His
o.93
i
Gly
1.03
i
Tyr
1.86
2
A rg
1.85
2
In order to estimate the liberated glutamine and asparagine, a sample of the amino acid mixture was heated in 2 N HC1 in a sealed tube at IiO ° for z h. This causes deamidation of asparagine to aspartic acid and conversion of pyrollidonecarboxylic acid to glutamic acid. Since 4.i8 residues of aspartic acid were recovered whereas only 3.0 residues were found after enzymic hydrolysis (Table I), only one asparagine residue * T h i s i n v e s t i g a t i o n w a s s u p p o r t e d in p a r t b y a g r a n t f r o m t h e N a t i o n a l I n s t i t u t e s of H e a l t h , U.S. P u b l i c H e a l t h Service. ** C r y s t a l l i n e g l u c a g o n w a s k i n d l y s u p p l i e d b y Dr. WILLIAM W. BROMER of t h e L i l l y R e ~ a r c h Laboratories. *** T h e p r o c e d u r e s w h i c h were r o u t i n e l y used for d e g r a d a t i o n of p o l y p e p t i d e s a n d p r o t e i n s w i t h L A P are d e s c r i b e d in ref. x.
258
SHORT COMMUNICATIONS
VOL. 3 1
(1959)
was present originally. 4.o6 residues of serine were now recovered in accord with the composition of glucagon (Table I). Similarly, 3.05 residues of glutamic acid were found corresponding to the glutamine content of glucagon. It is apparent that complete hydrolysis of many polypeptides by LAP should be feasible, thus permitting estimation of asparagine, glutamine and tryptophan, all of which are destroyed by conventional acidic hydrolysis. It may be noted that LAP has been used to determine the amide distribution in peptides obtained after tryptic digestion of ribonuclease 7. Furthermore, the differential analysis of amides, as described above, should not be necessary if a chromatographic system is used which separates glutamine and asparagine from other amino acids. It is noteworthy that a relatively low concentration of LAP is required for complete hydrolysis of glucagon. Complete hydrolysis of the oxidized A-chain of insulin is found in 24 h at a molar ratio of substrate to enzyme of 3201, whereas complete hydrolysis of glucagon occurs in 24 h at a molar ratio of 148o. Although this rate difference reflects the absence of residues in glucagon which would be expected to limit the rate of hydrolysis by LAP, e.g., proline and cysteic acid, secondary structural features and disulfide bridging also can limit the susceptibility of polypeptides and proteins to hydrolysis by LAP 1. The susceptibility of glucagon to LAP suggests that this enzyme may play a role in controlling levels of glucagon at the cellular level. Indeed, it has been reported that the supernatant fraction from rat-or rabbit-liver homogenates can hydrolyse glucagon to free amino acidss. The suggestion that this might be due to hydrolysis by LAP is supported by the present experiments as well as by observations that LAP activity is readily detected in the submicroscopic particles of rat-liver homogenates9. Although it is tempting to suggest that LAP may regulate the levels of other polypeptide hormones, it is important to note that oxytocin is only slowly hydrolysed, even at high LAP concentration1.
Laboratory/or Study o/Hereditary and Metabolic Disorders, University o/Utah College o~ Medicine, Salt Lake City, Utah (U.S.A.)
ROBERT L. HILL EMIL L. SMITH
1 R. L. HILL AND E. L. SMITH, J. Biol. Chem., 228 (1957) 577. 2 R. L. HILL AND E. L. SMITH, J. Biol. Chem., 231 (1958) 117. 3 D. F. ELLIOTT AND W. S. PEART, Biochem. J., 65 (1957) 246. 4 A. STAUB, L. SINN AND O. K. BEHRENS, J. Biol. Chem., 214 (1954) 619. 5 D. H. SPACKMAN, W. H. STEIN AND S. MOORE, Anal. Chem., 30 (1958) 119o. e W. W. BROMER, A. STAUB, E. R. DILLER, H. L. BIRD, L. G, SINN AND O. K. BEHRENS, J . Am. Chem. Soc., 79 (1957) 2794. 7 C. H. W. HIRS, W. H. STEIN AND S. MOORE, in Symposium on Protein Structure, J. Wiley and Sons, New York, in the press. s A. J. KENNEY, Proc. Biochemical Sot., Gt. Britain, April 18-19, 1958. 9 R. L. HILL AND E. L. SMITH, u n p u b l i s h e d observations.
Received June I3th, 1958
SHORT CO~tMONICATIOI~S
257
Complete hydrolysisof glucagon by leucine aminopeptidase" Previous studies have shown that leucine aminopeptidase (LAP) catalyzes the stepwise degradation of the amino-terminal portion of several polypeptides and proteins 1,2. Complete hydrolysis of polypeptides has been achieved with this enzyme in the case of the oxidized A-chain of insulin which contains 2i residues 1, and the naturally occurring octapeptide hormone, hypertensin a. We would now like to report the complete enzymic hydrolysis of crystalline glucagon, the 29-residue, polypeptide hormone of porcine pancreas 4. 7 mg (2.0 ~moles) of crystalline glucagon** (Lot No. 258-234B-54-2, Eli Lilly and Co.) was incubated at 4 °0 and pH 8.5 with 4.35 mg of Mg++-activated LAP with C1 = 55"**. Hydrolysis was estimated by quantitative ninhydrin determinations on aliquots removed at intervals after starting the reaction. After 3o min, the initially insoluble glucagon had completely dissolved. After 2 h, 77 % hydrolysis was found while complete hydrolysis occurred after 7-h incubation. At this time, LAP was inactivated by adjusting the reaction mixture to pH 4 with dilute HC1, and the amino acids separated by exhaustive dialysis. The dialysates were concentrated to dryness in vacuo, dissolved in o.15 M sodium citrate, pH 2.1, and analysed by a modification of the chromatographic procedure of SPACKMAN,MOORE AND STEIN5. The results of a representative experiment are shown in Table I. It is evident that there is stoichiometric recovery of each amino acid found in glucagon with the exception of glutaminee which is not detected because of conversion to pyrrolidonecarboxylic acid. Under the conditions of the analysis asparagine is not resolved from serine. TABLE I AMINO ACIDS RECOVERED AFTER HYDROLYSIS OF GLUCAGON BY LEUCINE AMINOPEPTIDASE Amino acid
Moles~mole Composition glucagon o~ glucagon Amino acid
Moles/mole Composition gltwagon o/glucagon A mino acid
Mole.s/mole Composi*ion glucagon o/gluragott
Asp
3 .00
3
Ala
I .oo
x
Phe
1.84
2
Thr
3. o I
3
Val
I. 19
I
Try
I.OO
i
Set + Asp NH~
4.85
5
Met
0.93
i
Lys
i.I 3
I
Glu
o.o
o
Leu
2.02
2
His
o.93
i
Gly
1.03
i
Tyr
1.86
2
A rg
1.85
2
In order to estimate the liberated glutamine and asparagine, a sample of the amino acid mixture was heated in 2 N HC1 in a sealed tube at IiO ° for z h. This causes deamidation of asparagine to aspartic acid and conversion of pyrollidonecarboxylic acid to glutamic acid. Since 4.i8 residues of aspartic acid were recovered whereas only 3.0 residues were found after enzymic hydrolysis (Table I), only one asparagine residue * T h i s i n v e s t i g a t i o n w a s s u p p o r t e d in p a r t b y a g r a n t f r o m t h e N a t i o n a l I n s t i t u t e s of H e a l t h , U.S. P u b l i c H e a l t h Service. ** C r y s t a l l i n e g l u c a g o n w a s k i n d l y s u p p l i e d b y Dr. WILLIAM W. BROMER of t h e L i l l y R e ~ a r c h Laboratories. *** T h e p r o c e d u r e s w h i c h were r o u t i n e l y used for d e g r a d a t i o n of p o l y p e p t i d e s a n d p r o t e i n s w i t h L A P are d e s c r i b e d in ref. x.
258
SHORT COMMUNICATIONS
VOL. 3 1
(1959)
was present originally. 4.o6 residues of serine were now recovered in accord with the composition of glucagon (Table I). Similarly, 3.05 residues of glutamic acid were found corresponding to the glutamine content of glucagon. It is apparent that complete hydrolysis of many polypeptides by LAP should be feasible, thus permitting estimation of asparagine, glutamine and tryptophan, all of which are destroyed by conventional acidic hydrolysis. It may be noted that LAP has been used to determine the amide distribution in peptides obtained after tryptic digestion of ribonuclease 7. Furthermore, the differential analysis of amides, as described above, should not be necessary if a chromatographic system is used which separates glutamine and asparagine from other amino acids. It is noteworthy that a relatively low concentration of LAP is required for complete hydrolysis of glucagon. Complete hydrolysis of the oxidized A-chain of insulin is found in 24 h at a molar ratio of substrate to enzyme of 3201, whereas complete hydrolysis of glucagon occurs in 24 h at a molar ratio of 148o. Although this rate difference reflects the absence of residues in glucagon which would be expected to limit the rate of hydrolysis by LAP, e.g., proline and cysteic acid, secondary structural features and disulfide bridging also can limit the susceptibility of polypeptides and proteins to hydrolysis by LAP 1. The susceptibility of glucagon to LAP suggests that this enzyme may play a role in controlling levels of glucagon at the cellular level. Indeed, it has been reported that the supernatant fraction from rat-or rabbit-liver homogenates can hydrolyse glucagon to free amino acidss. The suggestion that this might be due to hydrolysis by LAP is supported by the present experiments as well as by observations that LAP activity is readily detected in the submicroscopic particles of rat-liver homogenates9. Although it is tempting to suggest that LAP may regulate the levels of other polypeptide hormones, it is important to note that oxytocin is only slowly hydrolysed, even at high LAP concentration1.
Laboratory/or Study o/Hereditary and Metabolic Disorders, University o/Utah College o~ Medicine, Salt Lake City, Utah (U.S.A.)
ROBERT L. HILL EMIL L. SMITH
1 R. L. HILL AND E. L. SMITH, J. Biol. Chem., 228 (1957) 577. 2 R. L. HILL AND E. L. SMITH, J. Biol. Chem., 231 (1958) 117. 3 D. F. ELLIOTT AND W. S. PEART, Biochem. J., 65 (1957) 246. 4 A. STAUB, L. SINN AND O. K. BEHRENS, J. Biol. Chem., 214 (1954) 619. 5 D. H. SPACKMAN, W. H. STEIN AND S. MOORE, Anal. Chem., 30 (1958) 119o. e W. W. BROMER, A. STAUB, E. R. DILLER, H. L. BIRD, L. G, SINN AND O. K. BEHRENS, J . Am. Chem. Soc., 79 (1957) 2794. 7 C. H. W. HIRS, W. H. STEIN AND S. MOORE, in Symposium on Protein Structure, J. Wiley and Sons, New York, in the press. s A. J. KENNEY, Proc. Biochemical Sot., Gt. Britain, April 18-19, 1958. 9 R. L. HILL AND E. L. SMITH, u n p u b l i s h e d observations.
Received June I3th, 1958