Studies on the primary structure of rat liver phenylalanine hydroxylase

Vol.

126,

No. 2, 1985

January

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STUDIES ON THE PRIMARY RAT LIVER PHENYLALANINE Masayoshi Iwakil, 1 Department

922-932

STRUCTURE OF HYDROXYLASE

Michael A. Parniak2 and

Kaufman3

Seymour

of Biochemistry, Shiga University of Medical Science Ohtsu, Shiga, 520-21, Japan

2 Lady Davis Institute for Medical Research Sir Mortimer B. Davis - Jewish General Hospital Montreal, Canada H3T lE2 3 Laboratory Received

December

of Neurochemistry, National Institute Bethesda, Maryland 20205

of Mental Health

6, 1984

The primary structure of phenylalanine hydroxylase purified from rat liver was investigated with high speedgel filtration chromatography, cyanogen bromide cleavage and end group analyses of polypeptides derived from the enzyme. On gel filtration in the presence of 6M guanidine hydrochloride, the enzyme gave a single peak corresponding to a molecular weight of 52,000. In the same system the enzyme that had been cleaved with cyanogen bromide gave two peptides (Cal, M,= 32,800 and CB2, M,= 20,400). Sequence studies showed that the alignment of these two peptides was CBI - CB2. Furthermore, in experiments using 32P phosphorylated enzyme, the site of phosphorylation by CAMP-dependent protein kinase was found to be located on the CBl peptide. The NH2-terminus of this enzyme, which was found to be blocked, was shown to be N-acetylalanine. By both carboxypeptidase A digestion and hydrazinolysis, the carboxyl terminus was identified as serine. These data indicate that the phenylalanine hydroxylase molecule from rat liver is composed of subunits which are homogenousor, at least, very similar in their primary structure.

Phenylalanine hydroxylase

(EC1.14.16.1), the enzyme that

conversion of phenylalanine to tryrosine, was first obtained from pure

rat

catalyzes

liver in essentially

form by the procedure of Kaufman and Fisher (1). These workers reported

polyacrylamide-gel electrophoresis

under

the

that

on

non-denaturing conditions, the hydroxylase

migrates as two closely-spaced bands, both with hydroxylase activity (1). During SDS-stacking

gel electrophoresis, two bandsM,=

49,000

and

50,000, accounting for at least

95% of the protein, were detected (2). Subsequently, the enzyme purified by other procedures

has also been shown to migrate as two bandsin the presence (3) or absence

Abbreviations used:

SDS, sodium

dodecylsulfate;

chromatography.

0006-291X/85

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HPLC, high performance liquid

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Vol. 126, No. 2, 1985

There have also been reports,

of SDS (4). hydroxylase

is detectable

during electrophoresis

however,

that only a single band of

under non-denaturing

(5,6) as well as

under denaturing conditions (7). The molecular clarified.

basis for these different

The possibility

forms of the hydroxylase

has been considered that they represent

different

the enzyme (2). It has also been suggested that the smaller molecular proteolytic

has not been subunits of

weight form is a

degradation product derived from the larger form (3). In addition, there is

evidence that --in vitro phosphorylation

of the enzyme from rat liver (8) and from human

and monkey liver (9) can convert one of these species into the other. In view of the variety of explanations that have been presented to account for the occurrence

of multiple molecular

study of the primary liver. primary

structure

weight forms of this enzyme, we have undertaken of the subunits of phenylalanine

Our results show that the subunits are either identical structure,

and make unlikely

enzyme contain significant

the possibility

hydroxylase

a

from rat

or very similar in their

that these preparations

amounts of any species that is proteolytically

of the

derived from

a larger one. MATERIALS

AND METHODS

Materials: 6-Methyltetrahydropterin was obtained from Calbiochem-Behring. LPhenylalanine, cyanogen bromide, iodoacetic acid, CAMP, ATP, carboxypeptidase A (treated with diisopropylfluorophosphate) and partially purified CAMP-dependent protein kinase were purchased from Sigma. Iodoacetic acid was twice crystallized from toluene. Guanidine hydrochloride was from Bethesda Research Laboratories. CLChymotrypsin and pepsin were from Worthington Biochemical. All the reagents for Edman degradation, hydrazine anhydrous and carboxypeptidase Y were purchased from Pierce. N-Acetylalanylalanine was synthesized from alanylalanine and acetic anhydride by Dr. Robert S. Phillips. The purity and the structure was confirmed on reverse phase HPLC and on mass spectrometry. y -[. 32P I-ATP (approximately 5000 Ci/mmol) was obtained from New England Nuclear. Preparation of Phenylalanine Hydroxylase: Purified phenylalanine hydroxylase was prepared from frozen livers from male Sprague Dawley rats (Rockland Farms) by a combination of published methods (1,2). Homogenization of rat livers, preparation of crude extracts, ethanol precipitation and ammonium sulfate precipitation were carried out as described by Kaufman and Fisher (I). The ammonium sulfate precipitate was subjected to phenyl-Sepharose CL-4B (Pharmacia) chromatography (4). The recovery of hydroxylase activity from crude extracts was 30-40%; these preparations were homogenous on SDS polyacrylamide gel electrophoresis. The concentration of pure hydroxylase was determined by measurement of the absorbance at 277 nm, taking 11.1 as the absorbance of a 1% solution of the pure enzyme in water. This value was determined by measurements of absorbance and dry weight (IO). In vitro phosphorylated hydroxylase was prepared as described (I 1). Carboxymethylation and CNBr Cleavage: Purified phenylalanine hydrox lase was reduced and carboxymethylated according to the method of Crestfield et al. r 12) in the presence of 6M guanidine hydrochloride. Cyanogen bromide cleavage of 923

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carboxymethylated phenyialanine hydroxylase was carried and Porter (13) in 70% (V/V) formic acid for 24 h at OoC. Amino Acid Analysis: Purified enzyme protein or extensively and were hydrolyzed with constant boiling h. The amino acid composition of the hydrolysate amino acid analyzer, Beckman model 119C, with the (14).

out by the method of PrahI

derived peptides were dialyzed HCl --in vacua at 105oC for 24-72 was analyzed with an automatic use of the citrate buffer system

SDS Polyacryamide Gel Electrophoresis and High Performance Liquid Chromatography: SDS polyacryamide gel electrophoresis was carried out according to the method of Weber and Osborn 05) with the use of phosphorylase b (Mr.= 94,000), bovine serum albumin (M,= 66,300), ovalbumin (M,= 43,000), carbonic anhydrase (Mr.= 30,000), soybean trypsin inhibitor (M,= 20,100) and a-lactalbumin (M,= 14,400) as standards. High performance liquid chromatography (HPLC) was performed with the use of a Gilson Gradient System 42 equipped with the HM Holochrome Variable Wavelength Detector or the Gilson Model 302 system equipped with a 111B ultraviolet detector (280 nm). High pressure gel filtration in the presence of 6M guanidine hydrochloride was carried out essentially according to Ui (16) with the use of a TSK G3000 SW column (7.5~600 mm, Altex). Reverse phase HPLC was performed with an Ultrasphere ODS 5 urn column (4.6~250 mm, Altex) at room temperature with an isocratic or linear gradient solvent systems. Conditions are specified in the legends to the figures. Terminal Analyses of Peptides: For sequential NH2-terminal analysis, Edman degradation of peptides was carried out as described (17). Liberated anilinothiazolinone derivatives- of amino acids were converted to phenylthiohydantoin amino acids and analyzed on HPLC as described (18). Confirmatory data were obtained by thin layer chromatography on silica gel plates (Si-HPF, Baker) carried out according to the method of Jeppsson and Sjbquist (19). The COOH-terminal residues of peptides were analyzed both by carboxypeptidase A digestion (20) and hydrazinolysis; the latter procedure was carried out essentially according to the method of Braun and Shroeder (21). After S-carboxymethylation, the sample (0.2 Amol) was dialyzed against water, lyophilized and dried over phosphorous pentoxide. Hydrazinolysis was performed b vacua in 2 ml of hydrazine anhydrous with 50 mg of Amberlite CC-50 (100-200 mesh, Rohm and Haas) at 8OoC for 30 h. The COOH-terminal amino acid was separated from amino acid hydrazides with the use of P-cellulose chromatography in 0.4 M pyridineformic acid buffer (pH 3.2) and was identified by comparison of its elution pattern with appropriate standards on the amino acid analyzer. The analysis of blocked NH2-terminus of the peptides was carried out essentially according to the method described by Tsuanasawa and Narita (22). Lyophilized hydroxylase protein (20 mg) was suspended in 2 ml of 1 mM HCl. After adjusting the pH to 2.0 with 1 M HCI, 1 mg of pepsin was added to the suspension. Incubation was carried out at 370C for 6 h with gentle stirring. The digest was lyophilized, applied to a Dowex Ag50-x2 column (H+ form, 1x5 cm) equilibrated with water and washed with 20 ml of water. This water eluate (acidic fraction) was lyophilized, the residue dissolved in 200 u-11of 0.1% trifluoroacetic acid and the solution applied to the reverse phase HPLC for the further purification. The conditions used were as follows: column, Ultrasphere ODS 5 ,um (4.6~250 mm); solvent system, linear gradient of 0 to 50% CH3CN in 0.1% trifluoroacetic acid over 34 min; flow rate, 1 ml/min; sample size, acidic fraction (100 ul) produced from 192 nmol of carboxymethylated hydroxylase. Rate of the release of amino acids from carboxymethylated phenylalanine hydroxylase by digestion with carboxypeptidase A was carried out in a mixture that contained carboxymethylated hydroxylase (2.5 mg, 48 nmol), carboxypeptidase A (2 nmol), 50 nM SDS and 50 mM Tris-HCI buffer (pH 7.0) in a total volume of 3 ml. Aliquots (0.5 ml) were taken into 0.5 ml of 10% trichloroacetic acid, centrifuged and subjected to amino acid analysis. 924

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RESULTS Weight

Molecular

purified

phenylalanine

polyacrylamide guanidine methods

Determination hydroxylase

gel electrophoresis

hydrochloride

(Fig. ZA).

Group Analyses

End

of Edman

of the

degradation,

amino acids appeared

of the Subunit:

appeared

as

(Fig.

and on high

IA)

The relative

was 50,000 (SDS gel electrophoresis) The NH2-terminus

cycles

and

no

both

on

speed gel filtration weight

determined

by both

subunit

detectable

was

phenylthiohydantoin

analyzed

by

derivative

at any of these three cycles.

II-

Ill-

(+) B

A

SDS gel electrophoresis of purified phenylalanine derivative (B). CNBr digest of its carboxymethyl A. B.

About

10 ug of protein

was applied

About 65 ug of lyophilized

SDS

in 6M

(-)

Fig. 1:

of

and 52,000 (gel filtration).

S-carboxymethylated but

species

a homogenous

molecular

The subunit

hydroxylase (A) and the

to a gel.

CNBr digest was used. 925

three

of

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

I

0

0.016Amm

4

8

12

16

TIME (min) Fig. 2:

High speed gel filtrations of phenylalanine hydroxylase and its CNBr digest in the presence of 6M guanidine hydrochloride. Column, TSK G-3000 SW (7.5~600 mm); mobile phase, 10 mM potassium phosphate buffer (pH 6.8) containing 1 mM EDTA and 6M guanidine hydrochloride; flow rate, I ml/min. (A) Phenylalanine hydroxylase (110 Augj was preincubated in the above elution buffer containing 0.1 M dithiothreitol at 370C for I h and then analyzed by gel filtration. (B) The CNBr digest of the carboxymethylated enzyme (170 up) was preincubated in the elution buffer at 370C for I h and then analyzed by gel filtration. (Cj 32P-phosphorylated phenylalanine hydroxylase (40 ug, 1.17~103 DPM) was subjected to CNBr cleavage as described in Methods and subjected to gel filtration after it had been treated as in ‘(A),ractions (250 ~1) were collected and radioactivity (25~1) was measured.

The COOH-terminal peptidase with

residue

A and hydrazinolysis.

carboxypeptidase

of the same sample During

A, serine

16 h), and was followed

by leucine

digestion

and glutamine

are not separable

the citrate

buffer

(141, analysis

amino

acids

system

liberated

amino

acid liberated

66%) and alanine

asparagine

on the amino

was also performed

by carboxypeptidase.

Following 926

with

both carboxy-

of the carboxymethylated

was the fastest (yield

was analyzed

(yield

acid

(yield

12%).

acid analyzer after

hydroxylase 88% after

Since

serine,

with the use of

acid hydrolysis

hydrolysis,

however,

of the the

Vol. 126, No. 2, 1985

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amount of serine that could be detected

after

carboxypeptidase

digestion

was not

changed. By hydrazinolysis, the analyzer.

only serine (yield 62%) was detected as a free amino acid on

According

to the results of these two different

that serine is the single COOH-terminal

residue of the hydroxylase

Cyanogen Bromide Cleavape of the Subunit: covalent structure phenylalanine pattern

methods, we conclude subunit(s).

To obtain more information

about the

of the subunit, cyanogen bromide cleavage of S-carboxymethylated

hydroxylase

was carried out. Fig.

of the whole cyanogen bromide digest.

III, 19,000) could be detected.

1B shows the SDS gel electrophoresis Three bands (I, M,= 50,000; II, 32,000;

Since Band I coincided in mobility with the subunit itself,

Band I was assumed to be the uncleaved subunit (which often occurs because of some oxidation

of methionine

residues).

To determine more precisely the molecular

weights

of the cyanogen bromide peptides, as well as to isolate them, high speed gel filtration the presence of 6M guanidine hydrochloride

was performed.

Fig. 2B shows the elution

corresponding

to Band I to III on SDS gel

pattern of the CNBr digest.

Three fractions

electrophoresis

The two CNBr peptides indicated

were

were seen.

pooled separately;

comparison

their

relative

in

molecular

weights

by bars (CBl and CB2) were

determined

by

of their elution positions with those of standard proteins during high speed

gel filtration

in 6M guanidine hydrochloride

found for CBl and CB2, respectively.

(16).

Values of 32,800 and 20,400 were

The amino acid composition of the CNBr peptides

and the native subunit were analyzed for comparison (Table 1). In vitro 32P phosphorylated

phenylalanine hydroxylase

the products applied to high speed gel filtration above (Fig. 2C).

Radioactivity

was cleaved with CNBr and

essentially under the same conditions as

was found to be associated

exclusively

with

the

uncleaved subunit and with CBI. N-Terminal

Analyses

Blocked NH2-Terminal

of CNBr Residue:

Peptides

and Isolation

of the

Two steps of Edman degradation were carried out on

the whole CNBr digest, and on CBl and CB2.

The sequence Tyr-Thr

the whole CNBr digest and in CB2 with reasonable yields. for CBl.

and Identification

The blocked NH2-terminal

appeared both in

No NH2-terminus

was found

peptide was isolated from the hydroxylase

pepsin digestion as described in Methods.

During reverse 921

after

phase HPLC analysis of the

Vol. 126, No. 2, 1985

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 1: Amino Acid Compositionof PhenylalanineHydroxylase andits CNBr-Peptides CBI Asp Thra Sera ClU

Pro GlY

21.5 13.5 19.1 34.4

(22) (14) (19) (34)

1;.-4 (15)

Leu Tyr Phe

:t*3 t::; 13:s (14)

Ar g cysd

Hsee Total

E 24:2 25.4 28.9 22.9 0.9 24.5 47.7 21.9

18.2 4.6 25.5 4.1 0.6

(18) (5) (26) (4) (1)

(275)

6.4 (6) 18.5 (19) Yi.

Native 39.9 (40) 22.8 (23)

17.4 (17) 7.8 (8) 13.7 (14)

ti-: I::,’ 9:9 (IO) 19.7 (20)

Ala Valb Met Ileb

g His

CBI + CB2

CB2

Ii;

I::; (24) (25) (29) (23) (1) (25) (48) (22)

‘3 13’ 29.0 (29) 10.7 (11)

10.7 (11) 4.1 (4) 6.4 (6) 2.1 (2) 0 (0)

. I? 21*:

(183)

(458)

(457)

Unlessotherwiseindicated,the resultsof the aminoacid analysesare averagesof two separateanalysesafter hydrolysisfor 24 and 48 h; residueswere calculatedbased on assumedmolecular weightsof 32,000(CBl), 20,000(CBZ) and 52,000(the native subunit). The numbersin parentheses are the nearestintegers. E Valuesextrapolated to zero-time hydrolysis. Valuesat 72 h hydrolysis. : Determinedby the spectrophotometricmethodof BeavanandHoliday (29). Determinedascarboxymethylcysteine. e Homoserineplushomoserine lactone.

acidic fraction derived from the AG50-x2 chromatography, a major peak (P-l) was detected. The properties of P-l are shownin Table 2. Direct injection of P-l into the amino acid analyzer did not give any ninhydrin-positive peaks, but after acid hydrolysis, a single amino acid, alanine, was detected (yield, about 130%, i.e., 0.50ymol from 0.38 ).rmol (20 mg) of the subunit). After digestion of P-l with carboxypeptidase Y, alanine was again the only amino acid detected (yield, 43% of the alanine content of P-1). Hydrazinolysis of this compound also gave alanine (39% recovery).

Analyses of the

carboxypeptidase Y digest of P-l on reversed phaseHPLC yielded a product, designated P-l-CP, which had a different retention time from P-l. Amino acid analysis of the acid hydrolysate of P-I-CP, however, also gave alanine (Table 2). The previous results indicated that P-l might be N-acetyalanylalanine and P-l-CP might be N-acetylalanine.

To test this possibility, standard N-acetylalanylalanine and 928

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126,

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No. 2, 1985

TABLE

Characterization

2:

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of the Acidic Peptides Derived from Pepsin Digestion of Phenylalanine Hydroxylase P-l

Peptides

P-I-CP

Pepsin digestion of phenylalanine hydroxylase

Origin

Carboxypeptidase Y digestion of P-l

Amino acid analysis HCI hydrolysate recovery (%I

Ala (134)a

Ala (42F

Carboxy ptidase digest IFrecovery (%)

Ala (431,

N.D.e

Ala

N.D.

Hydrazine digestd recovery (%I

(39F Percent recovery was calculated as the micromoles of alanine micromole of subunit (Mr.= 52,000) that was used for the analyses.

recovered

Per

P-l (200 nmoll was digested with carboxypeptidase Y (8 nmoll in 50 mM ammonium bicarbonate solution at 250C for 2 h and then lyophilized. Percent recovery was calculated as the micromoles of alanine recovered from the micromoles of alanine in the HCI hydrolysate of P-l that was used for the analysis. P-l (20 nmol) was hydrazinolysed with 0.2 ml of hydrazine anhydrous at 8OoC for 18 h. Excess hydra&e was removed by lyophilization and applied to amino acid analysis. N.D. = not determined.

standard

N-acetylalanine

solvent

The

systems.

acetylalanylalanine CNBr

peptide

sequence

were data

compared shown

and P-I-CP (CB2)

when the analysis

in Table

out

on 280 nmol

several

with

the

Edman

the use of various

conclusion

degradation

of the

that

P-l

is

of the isolated

material.

The

The same sequence

Tyr-Thr-Pro-Gln-Pro-Asx.

was repeated

analysis

3 support

is acetylalanine.

was carried

was obtained:

by HPLC

following

was obtained

times.

DISCUSSION Our

liver

provide

phenylalanine

of which results

results

hydroxylase

is derived support

homogeneous

convincing

from

peptide.

is composed

the other

the conclusion

evidence

that

The following

against

of a mixture

by proteolysis. the subunit results 929

the proposal

of two different

In a more

general

of our preparation

are in accord

(3) that

with

purified

rat

species,

one

sense, the present of the enzyme

this conclusion:

is a (1) on

Vol.

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The Retention Times of P-l and P-I-CP on HPLC Compared to Authentic Acetylalanine and Acetylalanylalanine

3:

Retention

Time (min) P-I-CP

Acetylalanylalanine

P-l

Sytem

BIOPHYSICAL

Acetylalanine

Ia

6.85

6.84

5.88

5.89

2b

5.84

5.86

--

--

3c

7.39

7.40

5.80

5.80

4d

--

--

4.21

4.21

HPLC was performed at room temperature using Ultrasphere ODS 5 pm (4.6~250 mm). The flow rate was I ml/min. Retention times were measured by the Data Master data analysis system (Model 620, Cilson) using an injection detector. About 2 nmol of each sampIe was injected. a Linear gradient of 0 to 50% CH3CN in 0.1% trifluoroacetic b Isocratic system of 2% CH3CN in 0.1% trifluoroacetic c

Linear min.

d

Isocratic

gradient

system of 0 to 50% methanol

acid over 34 min.

acid.

in 0.1% trifluoroacetic

system of 3.5% CH3CN in 0.1% trifluoroacetic

acid over 36

acid.

both high speed gel filtration in 6M guanidine hydrochloride and SDS polyacrylamide gel electrophoresis, the hydroxylase gives a single sharp peak (M,= 52,000); procedure, the CNBr digest of the S-carboxymethylated peptides (CBl

and

CB2).

Both

the

sum

of their

molecular

subunit

degradation of the whole CNBr digest, the NH2-terminal

yields

weights

acid compositions correspond very well with those of the intact sequence

(2) by the same CNBr-

of their amino

and

subunit; was

two

(3) shown

by

Edman

to be Tyr-

Thr, which corresponds to the NH2-terminal sequenceof the peptide, CB2 (see below); (4) N-acetylalanine was identified as the single NH2-terminal residue (see below) and serine was the only COOH-terminal residue. Our results in

rat

have also clarified the question of the number of methionine residues

liver phenylalanine hydroxylase.

Values

ranging

from one to four residues per

subunit have been reported for this amino acid (7,23,24,25). Not only do the results of our amino acid analysis (see (4,7),

but

our

Table

I) confirm the findings of one residue

per subunit

observation that the enzyme is cleaved by CNBr into only two peptides

provides strong independent evidence that there is only one methionine residue per subunit

of

the

rat

liver

hydroxylase.

930

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 126, No. 2, 1985 The present results covalent

modification

have also provided structural

of the hydroxylase.

residue that is phosphorylated

CNBr peptide (CBl).

another type of covalent modification amino acid.

physiological

Secondly, we have discovered

Just as with many other blocked NH2-termini

importance

subunits

of this modification

is relevant

protein kinase

of the enzyme, namely acetylation

of proteins,

that rat liver hydroxylase

to the interpretation

of recent

results

[Mercer et al. (27). Based on results obtained from the --in vitro translation messenger

RNA,

these workers

forms of rat liver phenylalanine are encoded by different separate hydroxylase characteristic (28).

the pattern (28).

of the two different

the

is composed reported

by

of rat liver

molecular

weight

that have been observed in some studies

species of messenger RNA which

might be products

of two

strains of rats have

molecuIar weight forms of the hydroxylase

for the strain of rats used in the present study, Sprague Dawley,

was variable,

some rats having one of the two forms, others having both

It would be of interest

molecular

different

genes. They have also reported that different

patterns

Unfortunately,

concluded that the two hydroxylase

of the NH2-

is unclear.

Our evidence in support of the conclusion of identical

we have shown that the site of the

by the action of cyclic AMP-dependent

(26) is located on the NH2-terminal

terminal

First,

details about several types of

to carry out comparative

structural

studies on the two

weight forms reported by Mercer et al. (28) with the methods that we have

employed in the present study. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Kaufman, S., and Fisher, D. B. (1970) 3. Biol. Chem. 245, 4745-4750. Kaufman, S., and Fisher, D. B. (1974) Molecular Mechanisms of Oxygen Activation, pp. 285-369, Academic Press, New York. Choo, K. H., Cotton, R. G. H., Danks, D. M., and Jennings, I. C. (1979) Biochem. J. 181, 285-295. Shiman, R., Gray, D. W., and Pater, A. (1979) J. Biol. Chem. 254, 11300-11306. Al-Janabi, J. M. (1980) Arch. Biochem. Biophys. 200, 603-608. Webber, S., Harzer, G., and Whiteley, J. M. (1980) Anal. Biochem. 106, 63-72. Nakata, H., and Fujisawa, H. (1980) Biochim. Biophys. Acta. 614, 313-327. Donlon, J., and Kaufman, S. (1980) J. Biol. Chem. 255, 2146-2152. Smith, S. C., Kemp, B. E., McAdam, W. J., Mercer, 3. F. B., and Cotton, R. G. H. (1984) J. Biol. Chem. 259, 11284-11289. Kupke, D. W., and Dorrier, T. E. (1978) Methods Enzymol. 48, 155-162. Phillips, R. S., and Kaufman, S. (1984) J. Biol. Chem. 259, 2474-2479. Crestfield, A. M., Moore, S., and Stein, W. H. (1963) J. Biol. Chem. 238, 622-627. Prahl, J. W., and Porter, R. R. (1968) Biochem. J. 107, 753-763. Spackman, D. H. (1967) Methods. Enzymol. 11, 3-11. Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4414. Ui, N. (1979) Anal. Biochem. 97, 65-71. 931

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Bll)mback, B., Blt)mback, M., Edman, P., and Hassel, B. (1966) Biochim. Biophys. Acta 115, 371-396. Zimmermann, C. L., Pisano, J. J., and Appella, E. (1977) Anal. Biochem. 77, 569-573. Jeppsson, J. O., and Sjbquist, J. (1967) Anal. Biochem. 18, 264-269. Ambler, R. P. (1967) Methods Enzymol. 11, 436-445. Braun, V., and Schroeder, W. A. (1967) Arch. Biochem. Biophys. 118, 241-252. Tsuanasawa, S., and Narita, K. (1982) J. Biochem. 92, 607-613. Fisher, D. B., Kirkwood, R., and Kaufman, S. (1972) J. Biol. Chem. 247, 51615267. Dbskeland, A., Ljones, T., Skotland, T., and Flatmark, T. (1982) Neurochem. Res. 7, 407-421. Shiman, R. (1980) 3. Biol. Chem. 255, 10029-10032. Abita, J. P., Milstein, S., Chang, N., and Kaufman, S. (1976) J. Biol. Chem. 251, 5310-5314. Mercer, J. F. B., Hunt, S. M., and Cotton, R. G. H. (1983) J. Biol. Chem. 258, 5854-5857. Mercer, J. F. B., Grimes, A., Jennings, I., and Cotton, R. G. H. (1984) Biochem. 3. 219, 891-898. Beavan, G. H., and Holiday, E. R. (1952) Advan. Prot. Chem. 7, 319-386.

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