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UNIVERSITI PUTRA MALAYSIA
EFFECT OF STORAGE ON THE CHANGES IN CATHEPSIN D ACT I VITY, NUCLEOTIDE CONTENTS , PEPTIDE PROFILES AND MUSCLE ULTRASTRUCTURE OF ARISTICHTHYS NOBILIS, R.
JAMILAH BAKAR
FSMB 1993 1
EFFECT OF STORAGE ON THE CHANGES IN CATHEPS I N D ACTIVITY, NUCLEOTIDE CONTENTS , PEPTIDE PROFILES AND MUSCLE
ULTRASTRUCTURE OF ARISTICHTHrS NOBILIS, R.
JAMILAH BAKAR
DOCTOR OF PHILOSOPHY
UNIVERSITI PERTANIAN MALAYSIA
1993
EFFECT OF STORAGE ON THE CHANGES IN CATHEPSIN D ACTIVITY , -�CLEOTIDE CONTENTS , PEPTIDE PROFI LES AND MUSCLE
ULTRASTRUCTURE OF ARISTICBT.HTS NOBILIS,R .
BY
JAMILAH BAKAR
The s i s submitted in f u l f i lment o f the requ i r ement s f o r t h e D e g r e e o f D o c t o r o f P h i l o s o p h y i n t h e
Faculty of Food Sc ience and Biotechnology
Universiti Pertanian Malays ia
May , 1993
Dedicated to my husband and
a ll m y c hil dren
last but not least my mu m
and in memory of
my late father.
ACKNOWLEDGEMENT
PE'RPUSTAKAAN .JNIVERSITI PERTANIAN MALAV8I1
The author expre s se s her s i ncere appreci at ion t o her
supervisor, Assoc . Pro f . Dr . Yu Swee Yean , of the Department of
Food Technology , Facu lty of Food Science and Biotechnology for
her gu idance and encouragement throughout the course of her
study . She wou ld a l s o l ike to extend her thanks to her co-
supervisor, Dr . Abdul lah Abu Bakar , of the Department of Food
Technology, Facu lty of Food Science and Biotechnology .
Many thanks and appreciat ion are also due to Profes sor Dr .
Mohamed Mahyudd in Dahan, Dean , Facu lty o f Food Science and
B iotechnology for his support .
The author also wishes to acknowledge her appreciation to
a l l the Heads of Department and the technical s t a f f in the
Faculty of Food Science and Biotechnology for their cooperat ion .
Many thanks is a l so due to Pro f . Dr . Syed Jalaluddin Syed
Salim and staff at the Rumen Microbiology Laboratory , Facu lty
of Veterinary Medicine and Animal Science , Universiti Pertanian
Ma l ay s i a, for a l l owing her to u s e t he i r f ac i l i t ie s at the
beginning o f the pro j ect .
i i i
L a s t ly, s h e wou l d a l s o l i ke t o t h ank t h e Ma l ay s i a n
Government and t h e Un ivers iti Pertanian Malaysia f o r making
it pos s ible for her to undergo her PhD programme .
Ac know l edgement i s a l s o due t o t h e A s i a n F i s h e r i e s
S o c iety and the I nternat ional Deve lopment Re search Centre
( IDRC ) of Canada for their financial assistance ( Grant no: 89-
003-J) .
iv
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i i i.
LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
L I ST OF FIGURES x i
LIST OF PLATES xv
ABSTRACT xviii
ABSTRAK
CHAPTER
1
2
xx
INTRODUCTION 1
LITERATURE REVIEW 6
Changes in Fish Muscle during Spoilage 6
Common Parameters Invest igated 8
Adenos ine-5 '-triphosphate ( ATP ) Catabol ites . 16
E lectrophoretic Pattern of Pept ides
Ultrastructural Changes
Role of cathepsin D in Muscle Deteriorat ion
Activity Animal s
of Catheps in D in Terrestrial
Activity of Cathepsin D in Fish Muscle
Extract ion , cathepsin D
Pur if icat ion and Character ist ics o f
p H Optimum and Stability
Temperature Opt imum and stability
I sozymes
Spec ificity
v
20
2 3
27
28
3 2
34
3 6
3 8
3 9
4 0
3
Page
Molecu lar Weight . . . . . . . . • • . . • • • • • • • • • • • • • • 42
PRELIMINARY INVESTIGATIONS ON THE EXTRACTION AND PURIFICATION OF CATHEPSIN D • • • • . • • • • • • • • • • • • • •
Introduct ion
Materials and Methods
Materials
Methods
Results and Discuss ion
44
44
45
45
46
5 1
Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . • . . 5 6
4 EXTRACTION , PURIFICATION , CHARACTERISTICS AND ACTIVITY OF CATHEPS IN D FROM BIGHEAD CARP ( A .
nobilis , R . )
I ntroduction
Materials and Methods
Mater ials
Methods
Results and Discuss ion
Sephadex GlOO - 120 Elution Prof ile
Carboxymethyl Ce llu lose ( CMC ) E lut ion
59
59
60
60
60
66
70
Prof ile . • • • • • . . . . • • • . • . . • • . • • • • • • • • . • • . • • • 72
pH Activity Profile 77
Temperature Activity Prof ile and Stabil ity • . 7 7
Pepstatin Inhibit ion 83
Molecu lar Weight 8 3
Summary 84
vi
5 DETERMINATION OF I SOCRATI C HPLC AND
I ntroduction
Materials and Methods
Materials
Methods
ADENOS INE NUCLEOTI DES BY ESTIMATION OF K1-VALUE
Results and Discussion
Page
87
87
89
89
90
92
Summary • • . . . . • • . . . . . . . . . • • . • . . • • • • . • • • • • • • • • • • 9 9
6 EFFECT OF STORAGE TEMPERATURE ON NUCLEOTI DE CONCENTRATIONS AND CATHEPTIC ACTIVITY OF BIG-HEAD CARP ( A . nobilis , R . ) MUSCLES 106
I ntroduction 106
Materials and Methods 108
Materials 108
Methods 109
Results and Discuss ion 1 1 1
Summary .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 1 3 3
7 EFFECT O F STORAGE TEMPERATURE ON SDS-PAGE OF MUSCLE PROTEINS AND ULTRASTRUCTURAL CHARACTERI S-TICS OF BIGHEAD CARP ( A . nobilis,R . ) MUSCLES 1 3 8
I ntroduct ion 1 3 8
Mater ials and Methods 140
Materials 140
Methods 140
Results and Discuss ion 142
Summary 167
vii
8 CONCLUSION AND RECOMMENDATION
BIBLIOGRAPHY
APPENDICES
BIOGRAPHICAL SKETCH
viii
Page
170
176
194
201
Table
1
2
3
4
S
6
7
LIST OF TABLES
pH Optima for Aquat ic and Terrestrial Animals with Hemoglobin as the Substrate
Temperature optima of Cathepsin 0
The Molecu lar Weight of Catheps in D Isolated from Various Animal s • • . . . • • • • .
Partial Puri f icat ion of Cathepsin D from B ighead Carp According to the Procedure of Makinodan et al e ( 1982 ) . . . • . . . • . • . •
Part ial Pur i f icat ion of Cathepsin D from B ighead Carp According to the Procedure of Barrett ( 1973) . . . . . . . . . . . • • • . . . . • • •
E ffect of Short-term Frozen storage ( -2 0 °C ) on the Extraction o f Cathepsin 0
The Percentage of White, Red and Belly Muscle of A. nobil is,R . . . . . . . . • . . . . . •
8 Part ial Purif icat ion of Cathepsin D from White, Red and Bel ly Muscle of A .
9
10
11
12
13
nobilis,R.
Extract ion and Purif ication of cathepsin D from A. nobilis,R. . . . . . . . . . . • . . . . . •
Pepstat in Act ivity
Inhibit ion of Cathepsin 0
The Relat ive Migration D istance of Protein Molecular Weight Markers and Cathepsin D . . • . . . . • . . . . . . . . . . . . . . . . . . .
The Retent ion Time of Various Nucleotides Using D i f ferent Mobi le Phases on a Reverse-phase HPLC Column . . • • . . • . . . . • •
Gel Compos it ion for Sodium Dodecyl Sul fate Polyacrylamide Gel E lectropho-res i s ( 50S-PAGE ) . . . . . . • . . . • . • . • • • . • • • •
ix
Page
37
39
43
S 2
54
57
67
68
69
84
8 5
93
1 9 5
14
1 5
1 6
The Nucleotide Changes in White , Red and Belly Muscle of A . nobilis ,R. Stored at Ambient Temperature • • . • • • • • • • • • • • • • • • •
The Nucleot ide Changes in White , Red and Belly Muscle of A . nobilis,R. Kept at 00 C • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •
The Nucleot ide Changes in White , Red and Belly Mu scle of A . nobilis,R. Kept at -20 ° C • • • • • • • . • • . • . • . . • • • • • • • • • • • • • • • • •
1 7 The K1-values ( % ) of White , Red and Belly Muscles of A. nobil is,R. Kept at Ambient
18
Temperature , O ° C and -2 0 ° C
The Cathept ic Act iv it ies of White , Red and Bel ly Muscles of A . nobilis,R. Kept at Ambient Temperature , O ° C and -2 0 ° C
x
Page
196
197
198
199
200
F igure
1
2
LIST OF FIGURES
TMAO Degradat ion React ions . • . . . . • . . . . .
General Scheme o f Autoxidation o f Lipids and Further Breakdown o f Unsaturated Fatty Acid . . . . . . . . . . . . . . . • . . . • . . . . . • • •
3 Breakdown o f ATP to it s Catabo l ites by E ndogenous Fish Mu scle Enzymes
4
5
6
7
8
9
10
1 1
12
13
The Format ion o f Uric Ac id Hypoxanthine by Xanthine Oxidase
from
The Oxidazed B-chain o f Insu l in Showing Maj or and Minor Cleaving Points by Catheps in D . . . . . . . . . . . . . . . . . . . . . . . . • . .
Summary o f the Extract ion tion steps of Cathepsin nobilis , R.
and D
Puri f icafrom A .
The Carboxymethyl Ce llulose E lution Pro f i le of Acetone Fract ion ( pH 4 . 2 ) o f Catheps in D Extract . . . . . . . . . . . . . . • • • . .
E lution Prof ile of catheps in D Extract on Sephadex G100 - 120 Column . . . . • • . . • . • . •
E lut ion Profile of Sephadex Fract ion No . 9 on I on-Exchange Cel lulose Column
E lution Pro f i le of Sephadex Fract ion No . 19 on Ion-exchange Cel lulose Column
pH Activity Pro f i le of Crude and Acetone Fract ions of cathepsin D . . . . . . • . . . . . . . .
Temperature Act ivity Prof ile o f Crude and Acetone Fract ions of Cathepsin D . • • . • . .
Temperature Stabi l ity Curve D in the Acetone Fraction
of Catheps in
14 Myof ibr i l Digest ion by Cathepsin D in the Acetone Fraction
xi
Page
9
13
1 7
19
4 1
47
58
7 1
7 3
7 4
7 8
7 9
8 1
8 2
1 5
16
17
18
19
2 0
2 1
2 2
2 3
2 4
2 5
26
Separat ion o f Nuc leot ide Standards Relevant to ATP Catabo l ism in Fish Muscle with 0 . 04 M P04 Buf fer , pH 4 . 5 • . • . • • . • .
Separat ion Relevant to with 20% 0 . 04 M P04
o f Nuc leot ide Standards ATP Catabol ism in Fish Muscle
Acetonitrile , 70% H20 and 1% Buf fer , pH 4 . 5 . . . . . . . . . . . • •
Separat ion o f Nucleot ide Standards Relevant to ATP Catabo l i sm in Fish Muscle with 20% Acetonitr ile , 70% H20 and 10% 0 . 04 M P04 Bu f fer , pH 4 . 5 . . . . . . . . . . . . •
Separat ion of Nuc leot ide Standards Relevant to ATP Catabol ism in Fish Muscle with 10% Acetonitrile , 90% 0 . 04 M P04 Buf fer , pH 4 . 5 . . . . . . . . . . . . . . . . . . • • . • • • .
Separat ion of Nucleot ide Standards Relevant to ATP Catabol ism in Fish Muscle with 2% Acetonitrile and 98% 0 . 04 M P04 Bu ffer , pH 4 . 5 . . . . . . . . . . . . . . . . . . . . • . • • .
Separat ion of Nuc leot ide Standards Relevant to ATP Catabo l i sm in Fish Muscle with 2% Acetonitrile and 98% 0 . 04 M P04 Bu f fer , pH 5 . 5 . . . . . . . . . . . . . . . . • . . . . . . • .
Separation of Nucleot ide Standards Relevant to ATP Catabol ism in F ish Muscle with 2% Acetonitrile , 98% 0 . 04 M P04 Buf fer and PIC A , pH 5 . 5 . . . . • . . • . . . . • •
Hypoxanthine Standard Curve
Inos ine Standard Curve
Inosine Monophosphate Standard Curve
Separation of Nucleotides from White Muscle of A . nobilis,R. us ing LiChrosorb RP-18 . . . . . . . . . . . . . . . . . . . . . . . . . • . • . . . . . •
Separat ion of Nuc leotides from Red Muscle o f A. nobil is,R. us ing LiChrosorb RP-18
xii
Page
94
9 5
9 5
9 6
96
9 7
9 7
101
102
103
104
104
2 7
2 8
2 9
3 0
3 1
3 2
3 3
3 4
3 5
3 6
3 7
3 8
3 9
separation of Nucleotidea from Belly Muscle of A . nobilis ,R. us ing LiChrosorb RP- 18 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Nucleot ide Changes in White Mu scle of A . nobilis ,R. Kept at Ambient Temperature
Nuc leot ide changes in Red Muscle of A . nobilis,R. Kept at Amb ient Temperature
Nucleot ide Changes in Belly Muscle of A . nobilis ,R. Kept at Ambient Temperature
Nucleot ide Changes in White Muscle o f A . nobil is ,R. Kept at O ° C . . . • . . . . . . . • • .
Nucleot ide Changes in A . n obil is ,R. Kept at ooC
Red Muscle of
Nuc leot ide Changes in Belly Mu scle of A . nobilis ,R. Kept at ooC . . . . • . . . . . . • . .
Nuc leot ide Changes in White A . nobil is , R . Kept at -2 0 o C
Nucleot ide Changes in Red A . nobilis ,R. Kept at - 2 0 o C
Nucleot ide Changes i n Belly A. n obil is ,R. Kept at -20 ° C
Muscle o f
Muscle o f
Muscle o f
K1-value of White , Red and Bel ly Muscles Ambient of A . nobilis ,R. Kept at
Temperature
K1-value of White , Red and Bel ly Muscles of A. nobil is,R. Kept at ooC . . . . . • • . . • .
K1-value of White , Red and Bel ly of A. nobilis ,R. Kept at -2 0 o C
Muscles
40 cathept ic Act ivity of White , Red and Belly Muscles of A . nobil is,R. Kept at Ambient
4 1
Temperature
cathept ic Act ivity of White , Red and Bel ly Muscles of A . nobil is ,R. Kept at O °C
xiii
Page
105
112
113
1 14
117
118
119
120
122
123
125
1 2 6
1 2 7
130
132
42
43
Catheptic Activity of White , Red and Belly Muscles of A . nobil is ,R. Kept at -20 ° C • • •
The Standard Curve o f Tyrosine
x iv
Page
134
194
P late
1
2
3
4
5
6
7a
7b
8
9
1 0
1 1
LIST OF PLATES
Electrophoretic Pattern ( PAGE ) o f the Various Fractions of Cathepsin 0 Extract
Electrophoret ic Pattern ( SOS-PAGE ) o f the Various Fract ions of cathepsin 0
Extracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
E lectron Micrograph o f Fresh White Muscle of A . nobilis ,R . . . . . . . . . . . • . . . . •
Electron Micrograph of White Muscle o f A . n obilis ,R. After 8 h r of storage at Ambient Temperature . . . . . . . . . . . . . . . . . . • . .
Electron Micrograph o f White Muscle o f A . n obilis ,R. After 24 h r o f storage at Ambient Temperature . . . . . . . . . . . . . . • . . . • • •
E lectron Micrograph of Fresh Red Muscle of A. n obil is R. • • • • • • • • • • • • • • • • • • • • • • • •
E lectron Micrographs of Red Muscle o f A . n obilis ,R. After 8 h r of storage at Ambient Temperature . . . . . . . . . . . . . . • . . . • . .
E lectron Micrographs of Red Muscle o f A . n obil is ,R. After 8 hr of storage at Ambient Temperature . . . . . . . . . . . . . . . . . . • • •
E lectron Micrograph A . n obil is ,R. After Ambient Temperature
of Red Muscle o f 24 h r o f storage at
Electron Micrograph of Fresh Belly Muscle o f A. n obilis ,R. . . . . . . . . . • . • • . . • . . . . . • • .
Electron Micrograph o f Bel ly Mu scle o f A . n obilis ,R. After 8 h r o f storage at Ambient Temperature . . . . . . . . . . . . . . . • . . . . •
E lectron Micrograph o f Belly Muscle o f A . n obilis ,R. After 24 h r of Storage at Ambient Temperature . . . . . . . . . . . . • • • • • • • • •
xv
Page
7 5
7 6
143
143
145
145
146
146
148
148
149
149
1 2 Electron Micrograph of White Muscle of A . nobil is ,R. After 24 hr of storage at Ambient Temperature . • . . . . . . . . . . . . . . . . • • •
1 3 a E lectron Micrograph of White Muscle o f A . nobilis ,R. After 9 D ays of storage at
Page
1 5 1
o oe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 5 1
13b Electron Micrograph of White Muscle of A . nobil is , R. After 9 Days o f storage at o oe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
14a Electron Micrograph A . nobil is ,R. After 3
of Red Muscle o f Days of storage at
0 0 c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14b E lectron Micrograph of Red Muscle o f A . nobil is ,R. After 3 Days o f storage at 0 0 c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
15 E lectron Micrograph of Red Muscle o f A . nobil is ,R. After 9 Days of storage at
16a
0 ° C • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •
Electron Micrograph of A. nobil is,R. After 3
Belly Muscle o f Days of storage at
152
154
1 5 4
OOC • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 1 5 6
1 6b E lectron Micrograph of Belly Muscle o f A . n obil is,R. After 9 Days of storage at o oe . • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 5 6
1 7 a E lectron Micrograph of Be lly Muscle o f A . nobilis ,R. After 9 Days of Storage at
17b
1 8
19
o °C . • . . . . . . . . . . . . . . . . . . . . . . • . . . • • . • • • . .
Electron Micrograph of Belly Muscle o f A . nobilis , R. after 9 Days of Storage at o °C . • . . • . . . . . • . . . . . . . . . . . . . . • . . . . . . • . . •
The Electron Micrograph of White Muscle of A. nobil is ,R. After 1 Month o f Frozen Storage at -2 0°C . . . . . . . . . . . • . . • • . . • . • . .
E lectron Micrograph of White Muscle o f A . nobil is ,R. After 4 Months o f Frozen Storage at -2 0°C . . . . . . . . . . . • . . . • • • . . • • •
xvi
1 5 7
1 5 7
1 5 9
1 5 9
20
2 1
22
23
E lectron Micrograph A . nobilis ,R. after
of Red Muscle of 1 Month of Frozen
storage at -20°C • • . . . • • . • • . • • • • • • • • • • • •
E lectron Micrograph of Red Muscle o f A . nobili s , After 4 Months o f Frozen storage at -20oe ....................... .
Electron Micrograph o f Bel ly Muscle o f A . nobilis ,R. After 1 Month of Frozen storage at -20 ° C . . . . . . . . . • • • • • . • . • • • • • • •
E lectron Micrograph of Bel ly Muscle o f A . nobil is, R. After 4 Months of Frozen storage at -20oe . . . . . . . . . . . . . . . . . . . . . . .
24 SOS-PAGE Prof i les of White Muscle o f A . nobil is ,R. for Varying storage Time
Page
160
160
1 6 1
1 6 1
( hr ) a t Ambient Temperature . . . . . . . . . . . . 163
2 5
2 6
2 7
2 8
SOS-PAGE Profiles o f Wh ite Muscle of A . nobilis, R. for Varying Storage T ime ( Days ) at oDe • • • • • • • • • • • • • • • • • • • • • • • • • •
SOS-PAGE Prof iles of Red Muscle o f A . nobilis ,R. for Varying storage Time ( hr ) at Ambient Temperature . . . • • . . . . . . .
SOS-PAGE Pro files o f Red Muscle o f A . nobil is ,R. for Varying Storage Time ( hr ) at Ambient Temperature . . . . . . . . . • . . •
SOS-PAGE Profiles o f Bel ly Muscle of A . nobilis ,R. for Varying Storage Time ( hr ) at Ambient Temperature . . . . • . . • . . . • .
xvii
1 6 5
1 6 6
166
168
Abs tract o f the The s i s Presented to the senate o f Univers it i Pert ani an Malays ia in Fu lfilment of the Requirements
for Degree of Doctor of Philosophy .
EFFECT OF STORAGE ON THE CHANGES IN CATHEPS IN D ACTIVITY ,
NUCLEOTIDE CONTENTS , PEPTIDE PROFI LES AND MUSCLE ULTRASTRUCTURE OF ARISTICHTBrS NOBILIS, R .
by
JAMILAH BAKAR
MAY , 1993
Supervisor Yu Swee Yean , PhD
Faculty Food Sc ience and B iotechnology
Cathepsin D from the muscle of bighead carp (A ristichthys
n o b i l i s , R . ) w a s e x t r a c t e d , p u r i f i e d a n d p a r t i a l l y
characterized . The extract ion and puri f ication of the enzyme
w a s a c h i e v e d b y a u t o l y s i s o f t h e mu s c l e , a c e t o n e
prec ipitat ion , gel filtrat ion on Sephadex G100-120 and on ion-
exchange carboxymethyl cel lulose ( CMC ) column chromatography .
It had a molecu lar weight ( m . w ) of 37 , 500 - 38 , 000 dalton ( D )
w i t h a pH opt imum at 3 . 2 and temper ature opt imum o f 5 0 ° C .
Myof ibril was also optima lly digested at pH 3 . 2 . The pur i f ied
enzyme had a s i ngle ma j or pept ide band on sod ium dodecyl
sulfate polyacrylamide gel electrophoresis ( SDS-PAGE ) and was
completely inhibited by pepstatin .
xviii
S t o r ag e stud i e s at 2 8 ° ( ambient ) , 0 ° and - 2 0 ° C we re
carr ied out t o qu a l i f y and qu ant i fy changes i n c at hept i c
activity, nuc leotide concentrations from ATP catabo l i sm, K1-
value, SDS-PAGE pro f i le and the ultrastructure o f white, red
and be l ly muscles of bighead carp ( A . nobilis , R ) . Red muscle
had the highest init ial cathept ic act ivity and K1-value ( 34 . 0 1
± 1 . 0% ) a s compared to wh ite and be l ly mu s c l e s . The rate o f
change o f K1-value was temperature dependent - being faster at
higher temperature . Dif ferent patterns and rate of change o f
K 1 - v a l u e w e r e o b s e r v e d a m o n g t h e mu s c l e . I n o s i n e
- S ' -monophosphate ( IMP ) was the mo st abundant nuc leot ide i n
a l l fresh mu sc l e s . It decrea sed rapidly dur ing storage and
had an inverse relationship with time ( r = - . 9 1 ) for red ;
r = - . 83 for be l ly and r = -.72 for white ) . Inos ine ( HxR )
a c c ummu l a t e d i n a l l mu s c l e s d u r i n g t h e t h r e e s t o r a g e
temperatures studied .
Degradat ion of connect ive tissues ( per imys ium, endomys ium/
p l a s m a l emma ) w a s t he mo s t c o n s p i cu o u s c h ange i n ambi e n t
temperature storage . However , progress ive detachment o f the
mus c le f iber ends from the myocommata were observed in samples
stored at O O C . Minima l degrad ation o f u l tra structure o f
mus c les was observed for frozen stored muscle kept less than 4
mont h s . Change s in pept ide patterns o f mu s c l e s were o n l y
observed after pro longed storage a t ambient ( > 1 6 hr ) and at
OGC ( > 5 day s ) . No o bv i o u s change o f pept ide bands w a s
observed during frozen storage up t o 5 months .
xix
Abstrak tesis yang dikemukakan kepada Senat univers it i Pertan ian Malaysia sebaga i memenuhi syarat
keper luan untuk I j azah Doktor Fal safah .
EFFECT OF STORAGE ON THE CHANGES IN CATHEPSIN D ACTIVITY , NUCLEOTIDE CONTENTS, PEPTIDE PROFILES AND MUSCLE
ULTRASTRUCTURE OF ARISTICHXUYS NOBILIS,R .
Oleh
JAMlLAH BAKAR
MEl , 1993
Penyel ia Yu Swee Yean , Ph . D
Fakulti Sains Makanan dan B ioteknologi.
Catheps in D dari otot ikan kap kepa la besar ( A ristich thys
n obilis , R . ) telah diekstrak , ditul inkan dan disepara cirikan .
Pengek s t r a kan dan penu l in a n e n z im t e l ah berjaya d i l akukan
dengan autol i s i s otot , pemendakan aset on , penurasan ge l di
atas Sephadex GIOO-120 dan kromatograpi penukaran ion-lajur di
a t a s c a r bo xyme t h y l c e l l u l o s e ( CMC ) . la mempu n y a i be r a t
mo l iku l sebanyak 3 7 , 5 0 0 - 3 8 , 000 dalton dengan p H opt ima d i
3 . 2 dan suhu optima pada 50°C. Myof ibr i l j uga dihadamkan pada
pH opt ima 3 . 2 . Enzim yang ditul inkan mempunyai satu jalur
utama pept id di atas e lektropore s i s natr ium dodecyl s u l f at e
( SDS-PAGE ) dan dihalang sepenuhnya oleh pepstatin .
xx
Kaj ian peny impanan di suhu 2 8 ° C ( b i l i k ) , 0 ° dan - 2 0 ° C
j uga d i j a lankan untuk meni lai jumlah dan mutu perubahan di
dalam aktiviti katept ik , kepekatan nukleot id dari katabolisma
ATP , n i lai K1 , profil 50S -PAGE dan u ltrastruktur otot put ih ,
merah dan perut ikan kap kepala besar ( A . n obi li s , R ) . otot
merah mempunyai nilai awalan akt ivit i katept ik dan K1 ( 34 . 0 1 ±
1 . 0% ) yang tertinggi berbanding dengan otot putih atau perut .
Kadar perubahan nilai K1 bergantung kepada suhu - lebih cepat
di suhu yang t inggi . Corak dan kadar perubahan nilai K1 yang
ber1ainan didapat i didalam otot-otot . Inos ine -5 ' -monofos fat
( IMP ) adalah nukleot id yang terbanyak s ekal i di da1am s emua
otot s egar . I a menurun dengan cepat semasa pengstoran dan
menunj ukkan kaitan yang song sang dengan masa ( r = - . 9 1 bag i
otot merah; r = - . 8 3 bag i otot perut dan r = - . 7 2 bagi otot
put ih ) . Inos ine ( HxR ) didapat i terkumpu l di dalam semua otot
semasa penyimpan di ketiga-tiga suhu pengstoran yang dikaj i .
Oegradas i ti su penyambung ( perimysium , endomys ium/plasma
lemma) didapat i sang at nyata berlaku ket ika pengstoran di suhu
b i l ik . Perkembangan kearah pencabutan hu j ung gent ian otot
dar ipada myocommata dil ihat di dalam sampe l yang d i s impan di
suhu a o c . Oegradasi ultrastruktur yang minimum didapat i bagi
otot yang d i s impan s e j ukbeku yang t idak me leb ihi 4 bulan .
Perubahan j alur pept id otot hanya ketara selepas pengstoran
yang l ama ( > 1 6 j ) pada suhu b i l ik dan >5 har i pada a o c .
Tiada perubahan yang nyata diperolehi bag i j alur pept id otot
yang disej ukbekukan sepanj ang masa kaj ian .
xxi
CHAPTER 1
INTRODUCTION
I n the year 2 000 , an estimated 104 mil l ion tons of fish
wi l l be needed for human consumpt ion of which 9 0 % w i l l be
consumed in deve loping countries ( Pedrosa - Menabr ito and
Regenstein, 1 9 8 8 ) . To meet this demand , they suggested
that the postharvest los ses of fish be reduced by increased
ut i l i zat ion of available resources and underut i l ized fish,
increased act iv ity in the aquacu lture sector and final ly to
divers ify fishing effort s .
F i sh i s a very p e r i s hab l e commodity and i t has b e e n
e s t imated that 2 5 percent of the cat ch for human consumpt ion
is lost during postharvest ( Santos , 199 1 ) . A better knowledge
of s poilage mechan i sm w i l l prov ide pos s ib i l it ie s in dev i s ing
techniques to reduce this postharvest los s .
Spoilage of fish can b e defined a s unacceptable changes
occurr ing in f i s h mu s c l e pos tmort em whi ch inherent ly w i l l
affect the qual ity ( Makundan e t al . , 1982 ) . Fish spoilage is
t r iggered by the ac t i v i ty of endoen zyme s of f i s h mu s c l e
( proteas e s , catheps ins , pept idas e s , et c . ) on pept ides and
proteins ; hence , estab l i shing a favourab le environment for the
propagation of spoi lage microogan i sms ( Pedrosa-Menabrito and
Regenstein, 1988 ) .
2
The process ing and stor age characterist i c s of cold and
temperate water f i s h are well e s tabl i s hed but r elat ive ly
l ittle i s known about tropical fi s h . Pou lter e t a l . ( 1 9 8 2 )
also found that proteins of some tropical fish are more stable
during process ing than proteins of species from co lder waters .
Very l itt le i s known about en zymes which part ic ipate in
the deterioration of f i s h . I nvest igat ions o n cathep s i n s
indicate that fundamental differences exist between mammalian
and fish enzyme spec ificities which in turn are completely
d ifferent from those occu rring in meat or any other protein
r ich food ( S iebert and Schmitt , 1980 ) .
Most of the informat ion ava ilable on cathept ic en zymes
were obta i ned from work done on s p l ee n , kidney and l iver
terrestr ial animal wh ile l ittle informat ion is ava i l able on
muscle which cou ld be due to the relat ive ly low act ivity of the
en zyme in it ( Fruto n , 1 9 60 ) . Siebert ( 1 9 5 8 ) fou nd that the
c athept ic act ivity of fish mu scle is ten t ime s greater than
mammal ian muscle and also observed that fish muscle cathep s in s
p l ayed a r o l e in t h e s po i l age o f f i s h p r i o r to p ro c e s s ing
( S iebert , 1 9 62 ) . The work of s i ebert t r iggered research
activities in the sc ient ific commun ity whose main interest is
i n f i s h deteriorat ion . Gron inger ( 19 6 4 ) pub l i shed work on
proteinase from albacore foll owed by Ting e t al . ( 1 9 6 8 ) who
found that the crude cat hept ic act ivi ty in Ch inook s a lmon
( Onco rhynchus tshawytscha) mu scle was forty times higher than