Amino-Acid Sequence of NADP-Specific Glutamate Dehydrogenase of Neurospora crassa

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Proc. Nat. Acad. Sci. USA Vol. 71, No. 11, pp. 4361-4365, November 1974

Amino-Acid Sequence of NADP-Specific Glutamate Dehydrogenase of Neurospora crassa (homology with vertebrate enzymes)

JOHN C. WOOTTON*, GEOFFREY K. CHAMBERS*, ANTHONY A. HOLDER*, ANDREW J. BARON*, JOHN G. TAYLOR*, JOHN R. S. FINCHAM*, KENNETH M. BLUMENTHALt, KENNETH MOONfI:, AND EMIL L. SMITHI * Department of Genetics, University of Leeds, Leeds LS2 9JT, U.K.; t Department of Biological Chemistry, UCLA School of Medicine

and Molecular Biology

Institute, University of California, Los Angeles, Calif. L. Contributed by Emil Smith, August £8, 1974

A tentative primary structure of the ABSTRACT NADP-specific glutamate dehydrogenase [L-glutamate: NADP oxidoreductase (deaminating), EC 1.4.1.41 from Neurospora crassa has been determined. The proposed sequence contains 452 amino-acid residues in each of the identical subunits of the hexameric enzyme. Comparison of the sequence with that of the bovine liver enzyme reveals considerable homology in the amino-terminal portion of the chain, including the vicinity of the reactive lysine, with only shorter stretches of homology within the carboxyl-terminal regions. The significance of this distribution of homologous regions is discussed.

Investigation of structure-function relationships in enzymes has progressed to the state where assignment of a specific function to certain amino-acid residues in a protein is possible. Information on the function of individual residues in the sequence can be obtained from specific chemical modifications, from amino-acid replacements during the course of evolution, or from artificially induced mutations within a species. Investigations of the sequence of the NADP-specific glutamate dehydrogenase (GDH) from Neurospora crassa were undertaken with a view to attacking all phases of this problem. Thus, comparison of the sequence proposed herein with that obtained earlier for the vertebrate enzymes should afford some general information on the importance of groups of residues for catalysis, for regulation of activity, and for conformation. Moreover, determination of the sites of substitution in the mutants described by Fincham and coworkers (1, 2) should provide specific information on the role of individual residues. In this communication a summary of our findings will be presented; the laboratories involved will report separately upon the experimental details elsewhere. RESULTS AND DISCUSSION

Like the vertebrate enzymes, GDH (NADP) of Neurospora is composed of six identical polypeptide chains each having a molecular weight of about 48,800 (3, 4), somewhat less than the subunit weight of 55,393 calculated from the sequence of the bovine enzyme (5). The proposed sequence of the Neurospora enzyme, shown in Fig. 1, was derived by studies in Leeds of the peptides obtained by hydrolysis of the intact

90024

protein with trypsin§, chymotrypsin , pepsin. , and the neutral protease from Staphylococcus aureus (6)11, and in Los Angeles by chemical cleavage with cyanogen bromide, followed, in most cases, by secondary hydrolysis of the purified CNBr peptides with the aforementioned enzymes.** Overlapping of tryptic peptides was facilitated by limitation of the sites of cleavage to arginine residues by maleylation (7), or to lysine by treatment with cyclohexanedione (8). Detailed sequence work was performed either by manual Edman degradation (9) or by the dansyl-Edman technique (10). Currently, we have established the NHrterminal sequence of 49 residues, the COOH-terminal sequence of 361 residues, and two interior segments of 31 and 11 residues. Thus, the tentative sequence shown in Fig. 1 depends upon single residue overlaps at residues 49-50, 81-82, and 92-93; however, it seems unlikely that any peptides have been omitted in this part of the sequence. As shown in Table 1, the composition of peptide CN I (residues 1-109), the fragment comprising all of the single residue overlaps, is in good agreement with that calculated from the proposed sequence. It has not been possible, as yet, to purify larger overlapping peptides containing Arg-49, Arg-81, or Lys-92. The remaining overlaps in the molecule are firmly established, and most of the sequence has been determined at least twice on different types of peptides. Various aspects of the sequence work are worthy of special mention, either because of unexpected properties or difficulties encountered. The amino terminus of the protein is N-acetylserine, in agreement with the finding that the intact molecule lacks an a-amino group (3). The NHr2terminal staphylococcal protease and chymotryptic peptides were at first thought to have pyrrolidone carboxylic acid at their NH2 termini. Treatment with methanolic HCl (11), intended to open the pyrrolidone ring, did render the peptides susceptible to Edman degradation but revealed N-terminal serine. A sample of the staphylococcal protease peptide was kindly analyzed for us by Dr. Howard Morris at the Medical § Wootton, J. C., Jackson, A. A., Chambers, G. K., Taylor, J. G. & Fincham, J. R. S., in preparation. ¶ Holder, A. A., Chambers, G. K., Wootton, J. C. & Fincham, J. R. S., in preparation. 1I Wootton, J. C., Baron, A. & Fincham, J. R. S., in preparation. ** Blumenthal, K. M., Moon, K. & Smith, E. L., in preparation.

Abbreviation: GDH, glutamate dehydrogenase. t Present address: School of Biochemistry, University of New South Wales, Kensington 2033, Sydney, Australia. 4361

Biochemistry: Wootton et al.

4362

Proc. Nat. Acad. Sci. USA 71 (1974)

20 1 Ac -Ser-Asn- Leu- Pro- Ser-Glu- Pro-Glu-Ph~e-Glu-Gln-Ala-Tyr-ILys -Glu-Leu-Ala-Tyr-Thr-Leu-Glu-Asn-Ser-Ser-Leu- Phe-Gln- Lys-Hi s -Pro-Glu-Tyr- Arg-Thr-Ala'T TT s SP

40

36

sP SP'

-

'

60

Leu-Thr-Val-Ala-Ser- Ile-Pro-Glu-Arg-Val-Ile-GlnT 'T

Asn-Val-Gln-Val-Asn-Arg-Gly-Tyr-Arg-Val-Gln- Phe-AsnPhe-ArgHVal-Val-Trp-Glu-Asp-Asp-Asp-GlyT ' ' ''T P_

P

C

-C

CHD-T+ SP

SP

CNI

80

71

Ser-Ala-Leu-Gly- Pro-Tyr-Lys T

100

s-Pro-Ser-Val-Asn-Leu-Ser- Ile-Leu-LysqPhe- Leu-Gly- Phe-Glu-Gln- Ile-Phe-Lys-Asn-Ala-Leu-Thr-Gly-Glyr-Leu-ArgHLeu-Hi T ' T --'-T

uC

-

~~~~~~~P

P

C

z

MT * 120 106 140 Gly-.Leu-Ser-Met-Gly-Gly-Gly-Lys -Gly-Gly-Ala-Asp- Phe-Asp- Pro-Lys-Gly-Lys-Ser-Asp-Ala-Glu- Ile-Arg-Arg- Phe -Cys -Cys -Ala- Phe-Met-Ala-Glu- Leu-His T

T

T

T

C

SP CN n

160 141 Lys -Hi s -Ile -Gly-Ala-Asp-Thr-Asp-Val-Pro-Ala-Gly-Asp- Ile-Gly-Val-Gly-Gly-Arg-Glu- Ile-Gly-Tyr-Met-Phe-Gly-Ala-Tyr-Arg-Lys -Ala-Ala-Asn-Arg- Phe-

T

C

__'

T

Z~~C ~-

C

SP

-

CNN 200 180 Glu-Gly-Val-Leu-Thr-Gly-ILys -Gly-Leu-Ser-Trp-Gly-Gly-Ser-Leu- Ile-Arg- Pro-Glu-Ala-Thr-Gly-Tyr-Gly- Leu-Val-Tyr-Tyr-Val-Gly-Hi s-Met- Leu-Glu-Tyr-

176

T

C

P

''C

C

*'

C

-'

CN X+ T

CN =

211 220 240 S er-Gly-Ala-Gly-Ser-Tyr-Ala-Gly-Lys -Arg-Val-Ala-Leu-Ser-Gly-Ser-Gly-Asn-Val-Ala-Gln-Tyr-Ala-Ala- Leu-Lys -Leu- Ile-Glu- Leu-Gly-Ala-Thr-Val-ValT T

P

|C

-

-

.-

C

246

C

260

280

Ser- Leu-Ser-Asp-Ser-Lys -Gly-Ala- Leu-Val-Ala-Thr-Gly-Glu-Ser-Gly- Ile-Thr-Val-Glu-Asx- Ile-Asx-Ala-Val-Met-Ala-Ile -Lys -Glu-Ala-Arg-Gln-Ser- Leu-

T

T C

SP

CNY + CNM[ 300 Thr- Ser- Phe-Gln-Hi s-Ala-Gly-Hi s-Leu-Lys -Trp- Ile-Glu-Gly-Ala-Arg- Pro-Trp- Leu-Hi s-Val-Gly-Lys-Val-Asp- Ile -Ala- Leu-Pro-Cys -Ala-Thr-Glu-Asp-Glu281

T

T

T

C

C-

SP

340 316 320 Val-Ser-Lys -Glu-Glu-Ala-Glu-Gly-Leu-Leu-Ala-Ala-Gly-Cys -Lys -Phe-Val-Ala-Glu-Gly-Ser-Asn-Met-Gly-Cys-Thr-Leu-Glu-Ala- Ile-Glu-Val- Phe-Glu-AsnT sP

380 351 360 Asn-Arg-Lys -Glu-Lys -Lys -Gly-Glu-Ala-Val-Trp-Tyr-Ala-Pro-Gly-Lys -Ala-Ala-Asn-Cys -Gly-Gly-Val-Ala-Val-Ser-Gly- Leu-Glu-Met-Ala-Gln-Asn-Ser-GlnT

--T- Z

T

C

C

C N ME 400 420 386 Arg-Leu-Asn-Trp-Thr-Gln-Ala-Glu-Val-Asp-Glu-Lys -Leu-Lys -Asp- Ile-Met-Lys -Asn-Ala-Phe-Phle-Asn-Gly-Leu-Asn-Thr-Ala-Lys -Thr-Tyr-Val-Glu-Ala-AlaT T T C

-s P

SP

SP

'

'

'_C

CNI

CN

421

440

-

452

Glu-Gly-Gln-Leu- Pro-Ser-Leu-Val-Ala-Gly-Ser-Asn-Ile-Ala-Gly-Fne-Val- Lys -Val-Ala-Gln-Ala-Met-Hi s-Asp-Gln-Gly-Asp-Trp-Ser-Lys -Asn-COOH T

P

T

CN X

FIG. 1. Tentative amino-acid sequence of Neurospora glutamate dehydrogenase (NADP). Lysine-113, reactive with pyridoxal phosphate and NV-ethylmaleimide, is indicated by the asterisk. Single residue overlaps at residues 49-50, 81-82, and 92-93 are shown by double vertical lines. In general, only those peptides essential for providing overlaps are shown and none of the peptides derived by enzyme digestion or CNBr peptides are shown, unless they provided unique overlaps. Peptides are designated as either tryptic (T), chymotryptic (C), peptic (P), staphylococcal protease (SP), or cyanogen bromide (CN). Some peptides derived from tryptic digests of either maleylated (MT) or cyclohexanedione treated (CHDT) cyanogen bromide fragments are also shown. Peptide CHD-T+SP was isolated after tryptic hydrolysis of cyclohexanedione-treated peptide CN-I, and by further cleavage of the large peptides derived therefrom with staphylococcal protease.

Neurospora NADP-Specific Glutamnate Dehydrogenase

Proc. Nat. Acad. Sci. USA 71 (1974)

14

10

20

Ala-Asp-Arg-Glu-Asp-Asp-Pro-Asn-Phe-Phe-Lys-Met-Val-Glu-Gly-Phe-Phe-Asp-Arg-Gly-Ala-Ser-Ile-Val-Glu-Asp

(B)

Acetyl

(N)

4363

Ser-Asp-Leu-Pro-Ser-Glu-Pro-Glu-Phe-Glu-Gln-Ala-Tyr.y

t

10

40 -ProLeu-Val-Glu-As-ILeu- Lys -Thr-Arg-Gln-Thr-lGln-Glui Gln-LyssArg-Asn-Arg-ValAgGly- Ile Arj- Ile- Ile- Lys Ser- IleLyfHi s -Pro-Glu-TyrtAr fThr-Alai LuThr-Val-AlaLeuSer-SerGlu-Leu-Ala-Tyr-Thr- Leu-Glu-Asn140 20 50

PhetGn

80

6o

Cys-Asn-His Val Leu-Ser-Leu-Ser-XPhe-Pro-Ile-Arg-Arg Asp-Asp-Gly Ser-Trp-Glu4 Ile-Glu4Gly-Tyr-Arg Al-a+G Asp-Gly Asn-Val-Gln4Val Asn-Arg4G1y-rTyr-Arg Val4Glnr Val Ile-Gln- Phe-Arg-Val-Val-Trp-Glu-Asp Pro-G-u-Arg 6 50 100 Ala4r4 His-Ser-His- Gln- Arg-Thr ly-Gly Ie 4ATyr-Ser-Thr-Asp 4VaI4 Ser-Va1-Asp-Glu-Valf sTAlaf

Phe-Asn-Ser-Ala-Leu-Gly 70

ErhLs-Gly-Gly

Leu4Argj Leu-His-Pro-Ser4ValJAsn-Leu-Ser-Ile-Leufj' Phe Leu3- Gly80 90

126 120 Val-Lys-Ile-AsnPro-Lys AsnSer-Leu.-Met-Thr-ffl+Lys Cys Ala Val-Val-Asp-Val-Pro-Phe4Gly-Gly Phe-Glu-Gln-Ile-Phet 4Is Asn 4~ejLeu-Thr-Gly-Leu-Ser-Met :La-Asp-Phe-Asp Pro-Lys Gly4Gly-Gly4Gly0AlyGly120 110 113 100 16o 140 Pro-Gly-ValLys-Gly-Phe + I Tyr-Thr Asp Glu-Asp- Leu-Glu-Lys-Ile-Thr-Arg-Arg+ Phe Thr-Met Glu-Leu Ile- Gly+Ala-Asp-ThrLys-Ser As Ala-Glu-Ie-Arg-Arg-Phe-Cys-Cys-Alaj+ fMet-Ala4Glu-LeuHisLys His143 142 140 130

AlafLy-sAla-Gly AlafLyE

Asp-Val- Pro-Ala

ufArg-Glu Pro-Asn-Met-Ser-Thrf~lyGly4~g-Glu+

Asp-Val-Pro-AlaaGly-Asp-Ile-Gly-Valt qly

180 Ala-Ser-Thr-Ile-Gly-Hi sMet-Ser-Trp-Ile-Ala-Asp-Thr+ Ile-Gly-Tyr-Met-Phe-G1y-Ala TyrsArg-Lys-Ala-Ala-Asn-Arg-

170 160 200 Tyr-Astp-Ile-Asn-Ala-His-Ala-Cys-Val Thr Lys Pro[GlyJ le f fGinf(1y-Gly Ie-His,-GlyfXPg Ile-Ser fliir-l1 Leu Thr Gly-Lys 4Gl+ Ser Trp Giv-GJyJ Ser-Leu-Ile Arg Pro-Glu Ala-eThr-Gly Phe-Glu-Gly-Vali717 0 178 240 220 Phe-Gly-Hi s -Ile-Glu-Asn- Phe -Ile GuAsn-Ala- Ser-Tyr- Met -Ser- Ile-Leu-Gly-Met -Thr- Pro-Gly- Phe -GlyTyr-Ser-Gly-Ala- Gly-Ser-Tyr-AlaLeu-Val-Tyr-Tyr-Val-Gly-Hi s-Met-Leu Glu 210 209 200 150

ArgfGly+-yValTyr4Glyf

260

His-Arg-Phe fl-Alab LyS-CysPhe 4Gly-Asn-ValiGGy-Leu-His-Ser-Met-Arg-Tyr Asp 4 Thr-'Phela Val-Gln Se=rGly-Asn-Val.Ala-Gln-Tyr-Ala-Ala-Leu-Lys .Leu Ile-Glu-Leu G3-AJa Thr-ValGly Lys Arg-Val Ala Leu-Ser.4GlyT 220 2140 230

280

|ValAla-Val-Gly-Glu

AspGyS er- Ile -Trp -Asn-Pro-Asp-Gly-Ile-Asp-Pro-Lys-Glu-Leu-Glu-Asp-Phe-Lys-Leu-Gln-

Ser-Leu-Ser-Asp j~erLys 4Gly Ala-Leu-Val-Ala-Thr-Gly-Glu-Ser-Gly-Ile-Thr-Val-Glu-Asx-Ile-Asx-Ala-Val-Met270 260 250 320 300 Ie Leu Glu-Val-Asp-Cys-Asp-Ile-Leu-Ile-ProHi s -Gly-Thr-le-Leu-Gly-Phe-Pro-s-Ala- Lys-Ile-Tyr-Glu-Gly-S Ala- Ile- Lys -Glu-Ala-Arg-Gln- Ser- Leu-Thr-S er- Phe -Gln-Hi s-Ala-Gly- His *Leu Lys -Trp- Ile -Glu-Gly-Ala-Arg- Pro -Trp290 280

|

340 Ala-Ala-Ser-Glui Gln-Leu-Thr-Lys-Ser-Asn-Ala-Pro-Arg-Val-LysLeu-Hi s-Val-Gly Ly Val-Asp. Ile-Ala-Leu-Pro-Cys-Ala-Thr-.Glu,'4Asp-Glu-Val- Ser-Lys -Glu-Glu-Ala-Glu-Gly- Leu-Leu320 310 300

JA60

3141

AlaX LyeIle-Ile Ala-Glu-GiyAla Asn Gly-Pro-Thr Thr Pro-Gin Ala sp-Lys-Ile T Leu-Glu-Arg-AsnGlu-Asn-Asn-ArgAla-Ala-Gly-Cys {~}sPhe-VaiAlGlu-Gly.Ser+Asn Met-Gly-Cys jfLeu-Glu AlaIle-Glu-Val 350 340 330 380 Ile-Met-Val-Ile-Pro-As-Leu-Tyr-Leu-Asn Ala Giy Gly Val-Thr-Val-Ser-Tyr-Phe-Glx-Leu-Lys-Asn-Leu-Asn-Hi s-ValIvs-Glu-Lys-Lys-Gly-Glu-Ala-Val-Trp-Tyr Ala Pro Gl Lys-Ala-Ala-Asn-Cys-Gly-Gly-Val-Ala-Val-Ser-Gly-Leu-Glu370 360 1400 Ser-Tyr-Gly-iArg -Leu-Thr-Phe-Lys -Tyr-Glu-Arg-Astp-Ser-Asn-Tyr-Hi s -Leu- Leu-Met -S er-Val-Gln-Glu- Ser-Leu-Glu-;Arg-

Met-Ala-Gln-Asn-Ser-Gln-Arg-Leu-Asn-Trp-Tr-Gln-Ala-Glu-Val-Asp-Glu-Lys-Leu-Lys-Ap- Ile-Met-Lys-Asn-Ala-Phe380

390

400ko

440 £ ~ -Hi ~ s -Gly-Gly-Thr ~ ~ ~ -nIe-Pro ~ ~ -le er71Gu Il-Val- Pro-Thr-Ala-Glu- Phe-Gln;-Asp-Arg- Ile- Ser- Gly-A~lal e Gl-yvs LysLye-The Lys Phe-Asntg yLeu-Asn-Thr-Ala-Lys -Thr-Tyr-Val-Glu-Ala-Ala-Glu-Gly-Gln- Leu- Pro- Ser- Leu-Val-Ala-Gl-y *Sr Asn- Ile-

420

-Phef

-

460 450 Asp-nIe-Val-Hi s-Ser-Gly-Leu-Ala-Tyr-Thr-Met -Glu-Arg- Ser-Ala-Arg-Gln- Ile-Met-Arg-Thr- Ala-Met -Lys-Tyr-Asn- Leu-

Ala-Gly-Phe-Val-Lys-Val-Ala-Gln-Ala-Met-His-Asp-Gln-Gly-Asp-Trp-Ser-I~s--Asn-COOH

440 450 480 Gly-Leu-Asp-Leu-Arg-Thr-Ala-Ala-Tyr-Va.1-Asn-Ala- Ile-Glu-Lys-VP1-Phe-Arg-Val-Tyr-Asn-Glu-Ala-Gly-Val-Thr-Phe500 Thr-COOH

FIG. 2. Comparison of the amino-acid sequences of bovine (B) and Neurospora (N) glutamate dehydrogenases. Residues that occupy identical positions in the two proteins appear in boxes and residues related by single base changes in the respective codons are underlined. The numbers above the sequences refer to the bovine enzyme, below, to the Neurospora enzyme. Gaps, which have been introduced to maximize homology, are indicated by empty spaces.

Proc. Nat. Acad. Sci. USA 71

Biochemistry: Wootton et al.

4364

TABLE 1. Comparison of the amino-acid composition of CN I with that calculated for the proposed sequence Residues Amino acid Tryptophan Lysine Histidine Arginine Aspartic acid

Threonine Serine Glutamic acid Proline Glycine Alanine Valine Homoserine + lactone Isoleucine Leucine Tyrosine Phenylalanine

Total residues

Analysis

0.8(1) 5.2(5) 2.0(2) 5.7(6) 9.9(10) 3.8(4) 6.7(7) 14.0(14) 5.8(6) 7.6(8) 7. 4(7) 6.8(7) 1.0(1) 3.7(4) 12.3(12) 4.2(5) 5.3(6) (105)

Sequence (Residues 1-109) 1 5 2 6 10 4 9 15 6 7 6 8 1 4 13 5 7 109

The peptide was hydrolyzed for 24 and 70 hr. Values for threonine, serine, and tyrosine were obtained by extrapolation to zero time, and the 70-hr hydrolysate was used to obtain values for isoleucine and valine. The value for tryptophan was obtained by hydrolysis with p-toluenesulfonic acid (18).

Research Council Laboratory for Molecular Biology, Cambridge, using mass spectrometry, and N-acetylserine was found as the terminal residue (Morris, personal communication). As reported elsewhere (12), the Asp-Pro bond at position 119-120 was found to be highly labile to acidic conditions. More surprising was the finding that cleavage occurred carboxyl to six of the tryptophanyl residues in the molecule during cleavage with CNBr, although all of the tryptophan containing CNBr peptides were also isolated in intact form, with the exception of Peptide CN VI; this part of the molecule was isolated in two fragments-residues 272-298 and 299-338 of the linear sequence. Such cleavage was not ob served at Trp-52, located in a highly acidic region of the chain. Although the yield of the cleavage could not be unequivocally determined, it was at least 20% in some cases. Homology relationships of the bovine and Neurospora enzymes

In Fig. 2, the proposed sequence of Neurospora GDH (NADP) is compared to the sequence of the bovine enzyme. The bovine GDH contains 48 more residues than does the Neurosopra enzyme. The two proteins have been aligned to exhibit maximal homology, with the bovine enzyme containing 13 more residues at the NH2 terminus and 36 more at the carboxyl end. In addition, three small gaps have been introduced in the Neurospora sequence, corresponding to residues 156-157, 194-197, and 229-235 of the bovine enzyme, and a single gap has been introduced in the bovine enzyme, corresponding to residues 315-328 of the Neurospora GDH. As aligned, the two sequences reveal identity at 81 residues; 182 residues differ by single base changes in the respective amino acid

codons.

(1974)

Bovine, chicken, and Neurospora glutamate dehydrogenases are all inactivated by reaction of a single lysine residue of unusually low pKa With pyridoxal 5'-phosphate (3, 13, 14). As indicated in Fig. 2, this reactive lysine (113 in the Neurospora sequence) is situated in the region exhibiting greatest homology, where the sequences of the two proteins are almost identical. Furthermore, in the 115 residue sequence of residues

55-169 of the Neurospora enzyme, 38 are identical and 45 (mostly conservative changes) are related by single base changes in the codons of the respective amino acids. The homology in this region has been noted in an earlier communication (2). Almost as interesting is the relative paucity of homology in the 100-residues near the COOH terminus. That the vertebrate enzymes are subject to strong allosteric regulation by purine nucleotides is well known (15), and it has been shown that nitration of Tyr-406 in bovine GDH results in allosteric desensitization to GTP (16). The Neurospora enzyme does not respond allosterically to GTP and ADP, although its activity may be influenced by a number of ligands that are without effect upon the activity of the bovine enzyme (17). Neurospora GDH apparently lacks a counterpart to the bovine Tyr-406, and the only short region of homology that is apparent in the COOH-terminal half of the chain includes residues 330-337, corresponding to residues 342-349 of bovine GDH. The proposed sequence of Neurospora GDH (NADP) now ,makes possible study of both mutant forms and chemically modified derivatives of the enzyme. This study was independently undertaken in both Leeds and Los Angeles. On learning of each other's activities we decided to pool our information and efforts. It is a pleasure to acknowledge the effective collaboration of the two laboratories. The Leeds group thanks A. A. Jackson, Mollie Crawford, and Margaret Burnley for expert technical assistance and the Science and Medical Research Councils for financial support. The Los Angeles group thanks Ingeborg Kolbe for skilled technical assistance and Dorothy McNall for amino-acid analyses. The research was supported by Grant GM-11061 from the National Institutes of Health, U.S. Public Health Service.

1. Fincham,

J. R. S. & Stadler, D. R. (1965) "Complementation relationships of Neurospora am mutants in relation to their formation of abnormal varieties of glutamate dehydrogenase," Genet. Res. 6, 121-129. 2. Wootton, J. C., Chambers, G. K., Taylor, J. G. & Fincham, J. R. S. (1973) "Amino-acid sequence homologies between the NADP-dependent glutamate dehydrogenase of Neurospora and the bovine enzyme," Nature New Biol. 241, 42-43. 3. Blumenthal, K. M. & Smith, E. L. (1973) "Nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase of Neurospora. I. Isolation, subunits, amino acid composition, sulfhydryl groups, and identification of a lysine residue reactive with pyridoxal phosphate and Nethylmaleimide," J. Biol. Chem. 248, 6002-6008. 4. Wootton, J. C., Taylor, J. G. & Fincham, J. R. S. (1972) "The subunit structure of the nicotinamide adenine di-

nucleotide phosphate-dependent glutamate dehydrogenase of Neurospora crassa," Biochem. J. 127, 31P-32P. 5. Moon, K. & Smith, E. L. (1973) "Sequence of bovine liver glutamate dehydrogenase. VIII. Peptides produced by specific chemical 6leavage. The complete sequence of the protein," J. Biol. Chem. 248, 3082-3088. 6. Drapeau, G. R., Boily, Y. & Houmard, J. (1972) "Purification and properties of an extracellular protease of Staphylococcus aureus," J. Biol. Chem. 247, B.6720-6726. S. & Leberman, 7. Butler, P. J. G., Harris, J. I., Hartley, R. (1969) "The use of maleic anhydride for the reversible

Proc. Nat. Acad. Sci. USA 71 (1974) blocking of amino groups in polypeptide chains," Biochem. J. 112, 679-689.

8. Patthy, L. & Smith, E. L. (1974) "Reversible modification of arginine residues. Application to sequence studies by restriction of tryptic hydrolysis to lysine residues," J. Biol. Chem., in press. 9. Peterson, J. D. & Steiner, D. F. (1972) "Determination of the amino acid sequence of the monkey, sheep, and dog proinsulin C-peptides by a semi-micro Edman degradation procedure," J. Biol. Chem. 247, 4866-4871. 10. Gray, W. R. (1967) "Sequential degradation plus dansylation," in Methods in Enzymology, ed. Hirs, C. H. W. (Academic Press, New York), Vol. 11, pp. 469-475. 11. Kawasaki, I. & Itano, H. A. (1972) "Methanolysis of the pyrrolidone ring of amino-terminal pyroglutamic acid in model peptides," Anal. Biochem. 48, 546-556. 12. Piszkiewicz, D., Landon, M. & Smith, E. L. (1970) "Anomalous cleavage of aspartyl-proline peptide bonds during amino-acid sequence determinations," Biochem. Biophys. Res. Commun. 40, 1173-1178.

Neurompora NADP-Specific Glutamate Dehydrogenase

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13. Moon, K., Piszkiewicz, D. & Smith, E. L. (1972) "Glutamate dehydrogenase: amino-acid sequence of the bovine enzyme and comparison with that from chicken liver," Proc. Nat. Acad. Sci. USA 69, 1380-1383. 14. Piszkiewicz, D., Landon, M. & Smith, E. L. (1970) "Bovine liver glutamate dehydrogenase: sequence of a hexadecapeptide containing a lysine residue reactive with pyridoxal-5'phosphate," J. Biol. Chem. 245, 2622-2626. 15. Frieden, C. (1965) "Glutamate dehydrogenase. VI. Survey of purine nucleotide and other effects on the enzyme from various sources," J. BIol. Chem. 240, 2028-2035. 16. Piszkiewicz, D., Landen, M. & Smith, E. L. (1971) "Bovine glutamate dehydrogenase. Loss of allosteric inhibition by guanosine triphosphate and nitration of tyrosine-412," J. Biol. Chem. 246, 1324-1329. 17. West, D. J., Tuveson, R. W., Barratt, R. W. & Fincham, J. R. S. (1967) "Allosteric effects in nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase of Neurospora," J. Biol. Chem. 242, 2134-2138. 18. Liu, T.-Y. & Chang, Y. J. (1971) "Hydrolysis of proteins with p-toluenesulfonic acid," J. Biol. Chem. 246, 2842-2848.

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