Studies on hexose mono-phosphate oxidation

65 Pages·02.915 MB·English

by  Moldave, Kivie

STUDIES OK HBXOSB MOMO-PHOSPHATE
OXIPATXOK
A Thesis
Presented to
the Faculty o f the Department o f Biochem istry
U n iversity o f Southern C alifornia
In P artial F ulfillm ent
o f the Requirements for the Degree
M aster o f Science
by
K ivie Moldave
February 1950
UMI Number: EP41308
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This thesis, written by
Kivio_Mo_ldave___-__..................................
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A '
and approved by all its members, has been
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i i
TABLE OF CONTENTS
CHAPTER PAGE
I* INTRODUCTION.............................................................. 1
U nitarfan .Theory — , . . . . “ , 1
Re syntlies is Theory of Meyerhof 1
© xidation-R eduotion Theory o f Lipmann. • • , 2
Shunt Theory o f Sngelhardt and Barkash . . » 2
II, THEORETICAL . , ..........................., ............................. . . 3
Meyerhof Scheme. . . . . . . . . . . . . . . 3
The Krebs Tri-C arboxylic Acid C ycle. . . . . 5
The Hydrogen Transport System • 3
The Kexose Monophosphate Shunt . . . . . . . 9
I I I . EXPERIMENTAL .................................... 16
C alibration o f the Manometers • • • . • • , 16
The Preparation o f the Homogenate . . . . . 17
Chemical Preparations Employed . . . . . . . 18
Incubation Techniques* .............................. 23
A n alytical Methods 26
IT. RESULTS AND CONCLUSIONS . . . . . . . . . . . . 28
Manometer C alibrations 28
The Development o f the System 28
An E valuation o f the System......................... * • • 34
A G lycolytic Pathway Which Does not
Involve Hexose Diphosphate as an
Interm ediate .................................... . . . . . . . 36
i i i
CHAPTER PAGE
The T issue S u rv ey ...........................................................................39
V. SUMMARY ............................... « . 34
BIBLIOGRAPHY . . . ................ . . . . . . . . . . . . . 56
iv
LI S3? OP TABLES
TABLE PAGE
I , Comparison of Substances Involved in tlie
Breakdown of one Mole o f Glucose to Pyruvic
Acid by the Meyerhof and the Hexose Mono-
Phosphate Shunt Schemes . . . . . . . . . . . 15
I I , Plask C o n te n ts ......................................................................... E4
I I I , C alibration Bata ........................... 39
IT, Oxygen Consumption and Pyruvate form ation o f
Adrenal Gland Homogenates • 40
V. Oxygen Consumption and Pyruvate Formation o f
Brain Homogenates ........................................ 42
VJ. Oxygen Consumption and Pyruvate Formation o f
Heart Homogenates 44
¥11. Oxygen Consumption and Pyruvate Formation o f
Liver Homogenates 46
V III, Oxygen Consumption and Pyruvate Formation of
(
Kidney Homogenates ................................... 47
IX, Hexose Diphosphate Disappearance, Oxygen
Consumption and Pyruvate Formation o f
Adrenal Gland Homogenates . . . . . 49
X. Summary of R esults 51
V
LIST OF FIGURES
FIGURE PAGE
1 , Meyerho f-Bmbd an-Parnas- Go r i -Warburg Cycle
(M odified from Dorfman) . . • . . .......................... 4
E. Tli© T ricarboxylic Acid Cycle (M odified from
Krebs} . ......................... 6
3 . The Hydrogen Transports System • 10
4* The Hexose Monophosphate Shunt (M odified
From Diekens) ..................... . . . . . . . . . . . 14
5. The E ffeot o f Homogenization Time on Oxygen
Consumption . . . . . . . . . . . . 31
CHAPTER I
INTRODUCTION
In 1876, Louis P asteur made the observation th a t the
quantity of alcohol formed by yeast ferm entation under
anaerobic conditions is g reater than th at formed in the
presence of oxygen. Decrease in ferm entation products
formed due to aerobic conditions was called "the Pasteur
Reaction" by Otto Warburg in 1926. This reactio n has been
proved to be general for organisms and tissu e s which have an
aerobic and an anaerobic system.
The Pasteur Reaction has been in v estig ated by many
workers, and numerous th eo ries explaining i t have been put
fo rth . G enerally, three explanations have received wide
acceptance.
U nitarian Theory. This explanation is based on the
assumption th a t an interm ediate is formed under anaerobic
conditions and th a t th is interm ediate is oxidized in the
presence of oxygen. This would mean th a t, anaerobically,
animal tis s u e s m etabolize glucose to la c ta te which is oxi­
dized to carbon dioxide plus w ater in the presence of oxygen.
Resynthesis Theory of Meyerhof.' This theory sta te s
th a t oxidation of p a rt of the anaerobically-form ed interm ed­
ia te supplies energy for the resyn th esis of carbohydrate
2
from th at interm ediate. Thus anaerobically, glucose would
be m etabolized to la c ta te , and aero b ically a p a rt of th is
would be oxidized to carbon dioxide y ield in g energy which
could be used for the resy n th esis o f glucose from la c ta te .
Q xidation-Reduction Theory of Lipmann. According to
th is theory the high oxidation-reduction p o te n tia l estab­
lish ed by oxygen in h ib its anaerobic steps of glucose break­
down and thus interm ediates do not form so rap id ly in the
presence of oxygen as in i t s absence.
Ihunt Theory of Bngelhardt and Barkash. A fo u rth
hypothesis holds th at an a lte rn a te aerobic pathway of carbo­
hydrate degradation e x ists, i .e . oxidation of glucose through
the so -called Hexose Monophosphate Shunt.
I f oxygen is presumed to in h ib it a step of anaerobic
glucose degradation (compare Lipmann theory) and there is
a su b stitu te oxidation of glucose through an a lte rn a te path?
way, the reduction of la c tic acid production under aerobic
conditions would be explicable.
This th e sis is concerned w ith the experim ental evalu­
a tio n of the Hexose Monophosphate Shunt.
CHAPTER I I
THEORETICAL
Meyerhof Scheme. G lycolysis, the degradation of
glucose has been widely studied in animal tis s u e s . I t is
only through the combined e ffo rts of many in v e stig a to rs th at
the gross aspects of th is breakdown scheme have been c la r i­
fie d . The f i r s t stages involved in the breakdown o f carbo­
hydrate have been shown to proceed through a se rie s of phos-
phorylated compounds leading to the form ation of la c ta te or
pyruvate from glucose or glycogen depending on the condi­
tio n s used. Figure 1 shows the known interm ediates and
some of the cofactors for the reactio n s as modified from
Dorfman (1 0 ).
Though th is o v erall pictu re of the reactions involved
serves to acquaint one w ith the g ly co ly tic pathway, i t is
oversim plified. A great number of fac to rs complicate the
p ictu re i f an in v itro study of th is scheme is made.
A d etailed and complete discussion of the reactio n s
involved in the g ly co ly tic degradation, of the necessary
enzymes, and the electro n tra n sfe r agents involved in the
oxidative processes is beyond the scope of th is treatm ent.
The scheme in Figure 1, however w ill be used in developing
fu rth e r discussions in th is th e sis.