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THE AMINO ACID SE· .�UENCE OF TRYPriC PEPTIDEo

OF SHEEP HEAHT PHOSPHOFRUC1rOKINA3E.

A thesis presented in partiRl fulfillment

of the requirements for the

degree of J>octor of Philosophy

in Biochemistry at Massey University.

Stephen Oliver Brennan

July 1974

ACI\NO\,;LEDGEf1L'Wr.S

I wish to thank my su�ervisor Dr G. G_l"1idwinter for his

encouragement and sug5estions throughout this investigation.

I am also indebted to Dr C. H,Noore for his advice. I wish

to express my gratitude to Dr E. N. Baker for performing the

x-ray crystallography and to Professor R.Hodges for

performing the mass spectrometry. I '""ould like to thank

my wife Susan for help in the preparation of this thesis.

ii

i1 f3.BHJ�VJ: A'ri ONS

l'Fl� l·'lTC l'rH l"rc Dll00

l_r'r I I'� JJif� Ep (6.5) Ep ( 2.1 )

Phosphofructokinase

Phenylisothiocyanate

3-phenyl-2-thiohydantoin

}ncnylthiocrtrbamyl

Dimethyl sulphoxide

Trifluoroncetic acid

?r:1ction

f'iolc:cular <·Teight Dansyl Electrophor�tic mobility nt pH 6.5

Electrophoretic mobility at pH 2.1

iii

iv

J·ho��pho.fructokin::tr;e "'·e1s purified frum sheep hcr1rt. The

sr>dimente.t:i.on pattern of the purific(1 tm��ym0. \·l<·\S investicatcd

over n protein cone entr::d;ion rc:mr,c 0.? to 1'+. 5 mc;/ml. Two

rljr-;tinct 7 and 30 S l)(mJlrl��ries \H'T'r; oll:::·.erve<l at all conccntra-

t:;ions. A minor amount of 19 S ma.t cri<:J.l was also prcs ent.

The 30 S boundary was asymmetric and its concentration

dependence was characteristic of a pol;ymerisine; s ys tem in

rapid reversible e quilibrium . The dissolved crystalline enzyme usually sediment ed as a

single trailing 30 S boundary; the molecular vreie;ht of this

component was estimated at 1.5 x 106• This value was

consistent with x-ray data, which indicated unit cell

dimensions of 600 x 250 x 220 �' implying a protein weight of

greater than 106

daltons per asymmetric unit. In one

experiment the 30 S component appeared to be undergoing a

trimerisation to a 53 S form.

Sodium dodecylsulphate gel electrophoresis indicated a

protomer molecular wei�1t of 80,000 to 85,000, which was

consistent with a corrected sedimentation coefficient of

3.8 S and a molecular weight of 90,000 for maleyl­

phosphofructokinase, and with a corrected sedimentation

coefficient of 3.9 S for the urea-dissociated enzyme.

When maleylation was carried out on carboxymethyl­

phosphofructokinase in 7.5 M urea, the enzyme was further

dissociated to a 40,000 molecular. weight ,;subuni t. Peptide

mapping of tryptic peptides( �- � :· ·� l¥b.ic� : : ·crtginine-, histidine-,

tryptophan- and tyrosine-containing peptides were located;

was consistent with'an 85,000 form composed of two

identical subunits.

The enzyme was digested with trypsin. :B'orty-three different

peptides were isolated using a combination of: gel

filtration, ion exchange chromatography (on Dowex 50 and

DE 32 cellulose), paper electrophoresis, and paper chromato­

graphy. · The complete amino acid sequence was established

for 38 of these peptides. The amino acid compositions (and

l'Prtinl se·quences ) w e re cntablishc<l for the other five tryptic peptides. A summary of the amino flcid sequence

dot a obtain ed for the t rypti c peptid e1:; is shovm in

'ruble V .

S even carboxyme t hylcysteine�containing pep t ides were isolat ed in this inv es t iga t i on, while ei(:':;ht hElve been i8olated from ra bb i t muscle ( Coffee et al. 197)). Six of

these peJ>tirles hnd ver,y oimilo.r compo::.;i tions bcbveon the

tvJO FJpccies. '.rhe rabbit enzyme contr-1inecl two carboxy-

m e t hylcyst ein e-con taininc; p eptid es v1hich w e r e not found in

r:>heep h eart and the sh e ep enzym e contained a 20 residue pept ide not found in rabbit muscl e. This probably r efl ects

gen e tic variation betw e en the two species.

V

INDEX

1

2

3

INTRODUCTION

2.1

2.2

2.3

2.4

2.6

2.7

2.8 2.9 2.10

2.11

2.12

2.13

2.14

2.15

l'Iaterials

Enzyme purification and crystRllisation

Polyacrylamide gel electrophoresis

Ultracentrifugation

2 .4.1

2.4.? 2.4.3 2.4.'+

Sedimentation analysis

Diffusion analysis Corrected coefficients

I�l�cul�r weight determinations

Carboxymethylation

l'1aleylation

Tryptic diG·�sti on of ent.�ym�: Feptide r:1RppJn(3 Preparative electrophoresis

c14 Detection

Amino acid analysrs

N-terminal analysis ( peptides)

N-terminal sequ ence analysis

N-terminal analysis ( protein)

Amino acid sequence determination

2.15.1

2.15.2

Dansyl-Edman technique

Mass spectrometry

2.16 Digestion of peptides

RESULTS (1)

Crystals

Gel electrophoresis

Sedimentation studies on the purified

enzyme

Sedimentation studies on the dissolved

�rystalline enzyme

3.5 Molecular weight determinations under

dissociating conditions

vi

Page Ho.

1

8

8

8

11

12

12

13

13

14

15

15

16

16

17

18

18

18

20

20

21

21

22

22

24

24

24

25

27

28·

5

6

3-7

Ll.1

L�. 2 Lj . • 3 '+ . 4

L�. 5

N-terminal an�lysis J'eptide m.:�ps

Teptidc separRtion, chPractorisation and

amino acid sesuence detcrmin�tion Fraction A Fraction B Irraction C

Hraction D

ii..CID-INSOLU.3Ll; 'l1HY1-''11IC P.EPTllJES

5-3

li'raction Y Fraction X

Fraction \.J

DISCUSSION

Polymerisation Subunit structure

Primary structure

APPENDIX I:

1Th'FEREN C ES :

31

31 32 '+9 59 63 65 65 71 74 75 75 77 79

90 91

1 .... and B 2

3

4

5

6

7

8A

8B

9

1 hosphofructoL i nase cr:n-: tn.l�; 0ediment::o.tion r jttern of rurified

�1osphofructokinase

Cone entro.tion dependenc t:.' of tiH' sed imen­tRtion coefficient of purified lFK 0edimentation pattern of dissolved

cryst;.1lline 1-FK Sedimentation pattern of dissolved

crystalline PFI\. ( L�th prepc1r.1.tion) Concentration dependence of the

,fter

25

26

27

27

seclimentn.tion. coefficiC'nt of rnnleyl-PFK 2B

Graph for the rteterminHtion of

molecular vreic;ht of PFK by e;el

electrophoresis

Peptide map of acidic EJn(t b:�.sic tryptic

peptides of carboxymethyl-} JiTK �optide map of neutral tryptic peptides

of carboxymethyl-PTI(

El,ltion profile of soluble tryptic

peptides on Sephadex G- 50

29

30

30

31

1 0A and B Elution profile of fraction A peptides

11

12

13

1 4

1 5

16

17

on Dowex 50

:B'ragments observed on mass spectrometry

of peptide A(G0-63).46

Feptide map of fraction B peptides

Feptide fraction C on Jm 3� CPllulose

1-eptide fraction D on lll; 7·;) r.P.1lulose

Elution profile of insoluble peptides on

Sephadex G 50

Peptide fraction Y on DE 32 cellulose

Peptide fraction X on DE 32 cellulose

32

40

49

59

63

64

65

71

I II

Enzyme purification

Data for determination of the sedimentation

rate of the purified enzyme

Ill Sedimentation and diffusion coefficients of

IV maleyl-PFK

Amino acid analyses of chyrnotryptic (CH) and

thermolytic (TH) peptides of peptide X (86-84)

V Summary of amino acid sequence data obtained

Page I'o. 10

26

28

73

for sheep heart FFL 83

VI Comparison of amino acid compositions of

CM -Cys - containinG peptides of rabbit muscle

(H) and sheep-heart (S) PFK 86

VII Amino acid composition of peptides in

relation to composition of the original

enzyme 87

ix