E nergy-rich compou nds
3
3.1 Theoretical part
Cell as a complicated astern requires the large amount of free encr for all important activities:
the performance of mechanical work in the muscle contraction, and other cellular movemcnts, the
active transport of molecules and ions, and the synthesis of macromoleculcs and other biomoleculcs.
Free enCf tised iR these processes maintains an org:Inism in a state that is far from the equilibrium,
All these processes mentioned above are energetically very demanding and cells use encry u’hich
is special for all living systcms — chemical energy.
This form of energy is puts in some chemical compounds and is liberated in hydrolysis of some of
group which is bounded io this compound by the high energy bonds (- ).
There is nothing special about the bonds themselves. There are high-crier bo rids in the sense the i
much free ener is released when they are hydrolyscd for the reason gi›cn above.
lf âi•h energy bonds are hy0rolysed, prod ecu of reaction go to the energetically lo«’cr siaic hat
is expressed as marked change of Gibbs encr ' of system (g .
Most of chemical bonds in compounds of orq•anism as ester, pcpiidc and glycosi‹lc bonds arc not
high energy and in hydrolqis of these bonds libefaicd energy is about HIS iJ›mo1 of compound. If
h's h energy compound (bond) is hy‹lro1;sed much more free energy is liberated (30 —60 kJ,'mol of huh
ener compound).
Energy-rich molecules are formed by the oxidation of substrates which cull obtains from the
environmenc
The crealion of enefgy-fiCh compoun‹is in cells is carried on by ihree main ways:
t. During oxidation of substrates (for instance glucose) are formed iniermediates ’iih high-energy
phosphate group.
2. ATP - the most important high-encry phosphate compound and its phosphoanhydri‹le bonds
are referred to as high-energy bonds and is created in the process of oxidative phosphorylation in
mitochondria.
5. Some energy-rich compounds are produced so that phosphate is transferred from ATP to anoihef
molecule in the reaction which is catalysed by kinase anal high-criers bond is preserved.
3.1.1 Kinds of high-energy bonds
Energy-rich compounds in cells compr:se fi e kinds of high-eaer bonds: phosphoanhydride, acvl
phosphate, enolphosphaie, guanidine pbospha:e anal ihioesier bonds (Fig. 3.1).
Phosphoanhydride bond is formed be: ecn I 'o molecules of pbosphoriC 0Cid (FisPO‹). In hydrolysis of I mol of this bond is liberated approximatelly 30,5 kJ’mol bond. These bonds we can find in
nucleotides. Typical representative of high-enery compound with phosphoanhydride bond (diphosphase bond) is ATP (adenosine triphosphaie). In this compound are two high-energy diphosphaie
bonds (phosphoanhydride bonds). The ihird phosphate bond bci ecn phosphate and ribose is not
enerp’-rich, ii is phosphate ester bond.
Similar diphosphate bonds afe in all Si- and iripâosphates of purine and pyrimidinc nucleosi‹lcs.
Energy of diphosphate bonds has a ¡;rmi imporNlnce in the metabolism of a cell. ATP sexes as ihe
principal immediate donor of free ener in biological systems in most ender*onic reactions of iâc
cell, in the active transport of molecules across membranes, muscle contraction, iransmlssion of neo’c
impulse, and the oiher processes which requise energy.
Despite that fact that ATP is the principal donor of enerp (source of encre’), for some meiabolic
paihwajs can be used energy of diphosphate bonds of another nucleosides as GTP (tuanosine
triphosyhate) which is donor of energy in the proicosvnthcsis and also in the gluconeogcncsis. L/TP
(uridinc iriphosphate) is imporiam nucleoiide in ihe meiabolism of saccharidcs and CTP (q’iid inc
triphosphatc) in the mctabolism o( lipiüs.
19
Enolphosphate l›onsd is: formed
whcn: phosphate gro,up is a ttached to
the hydrox group. «hich is böunüćd
to cs rbon with double bond.
:CÕHP.õ.UN0
æ,s
ș PHQŞPHOAHHYORIOE’!’’ ,AfĘ
: ğ ’" ğ
1ïs cncrgy-rich bonü can be transform ATP (this process is: c«I:lüd yhos-
:EKBL...:
y.tt,PfłbSPHATE
t PH ŁI.S^H0E"0L’üY,RU V’ATč’
ö1
ghorvlaiion o/ the substrate).
Acy,lph sphate bond iS formed by
ICC ÊCa,Cțloțl Of carbo 3’lJc acid n ith
phosphate troops. ,In , the hydrolysis is
liberated approximately 4 k3im 1 I
energy. This type of bond is in Ip-ñis-
phosphöglş-ceraië:and is föimëd in the
is
c*ÎÌ-l'„O
GCÄFIO'l8E
PHß:ŠPŁIAT”Ë
ATP.
This tjpe of hi,h-energy bon‹ls is
0
Ć
glycolysis ::,and ,can be also transferred
from this! compo.und. to ADD° to form
PH.0SPH0CREÅ„TI NE
xa
-»e-o
41,
formed also, in the activation of fatty
acids and am.ino acids when these react
with ATP. Product is acyladenylate
resp. aminoacyladenylate.
:Gu:aai:d ine ph!osphate bonÒ is .förmed if phösphaie group is attached to
guanidine g:roup. Energy of the hydï ,: U‹1 ‹*’ 43 /N
*:
JP tant cömpouñd with this bond is phosphocieati,ne. êhösphöcrNilfie. is first
of all in: muscle celts here is a reserve
of energy for this” tïssue.
0g
In cells rms type of bond is formed
which is one of the.princïyat er›črçctical substr3‹cs oí thc ccll.
3.1.2 Energy-rich compounds in muscle cells
The muscle contraction is mechanical work and muscle cell is actua.lly „machine“ where chemical
energy of hìghmnergy compounds is changing to mechanical work.
As the,principal,söurce ofenefgy,.ìn ihe muscle coRtraciioascwes ATP. In resting mmclethe mmcle
cell creates definite level of ATP an4 ratio of ATP:ADP is about 10: 1. Duri•s the muscle work the
great amount of ATP is use4: The muscle cell has the eng matic equipment for producing ATP in the
glycolysis and also in the oxidative pho,sphorylaiion.
la active muscle level of ATP decreases. The reduced enemy charge uf active muscle stimulates the
glycogen„breakdown,,glycolysis, citric acid„cycle, and the oxidative phtisphorylaiion. These prticusses
are,the relative Contributions to the generation of ATP.
In the siäte when the müscle is acii e (vigorously wording) appears the anaerobic condiiioni and
so Uhe generation of ATP by the oxidation of substrates is considerably limited.
So in the resting state muscle cell uses phosphocreaiine hich contains phosphoguanido group to
store high-potential phosphoryl group in the muscle. The concentration of phosphocreatine in the
muscle in the resting state is five more times higher than the: level of ATP. R’urliing musclu is able to
use ener of phosphoguanido group of phosphocreaiine for the re cncrution uf ATP.
Ăhosp1oceatine is /ormad by the reaction:
ATP + creatinc
phO5pho,crcatine. + ADP.
kinase
And in the muscle contraction the reaction is rmerse:
phöiphocreatinc + ADP
—- ——
ł:inase.
ATP + creatine
The reaction is catalysed by kinase. High activities of this ename:are first of all in muscle cells and
aho in-,:myomrd.
3.2 The .aiæ of practical exercise
Thcaim of laboratory practice is to determine activity of creative kinase in the muscle, thc mjucard
and the liver of a rabbit. We compare acti›i in homogenates of these tissues. First of all in the muscle,
but also in the myoœrd this enzyme plays important rote ia the #nergetics of cell and its activity in
thae tissues ú high. On the contrary the İiver cell does not w•e phosphocreatiae as reserve of ener
an4 so aciiviy of weatinc kinase in ib‹e liver tissue is low.
The acti iș of creative is also determined in the clinical laboratory. In injury of myocard this
cngee is liberated into blood an4 so the determination of ac:i› iij’ of crcz line kinase is one of the basic
parameters in diagnosis of the infarct of myocard.
3.3 Practical part
33.1 The determination of phosphocreati.ne Ńnase in tissues of muscle, mș ocard
liver of rabbit
Creatine ki:nase forms ATP from phosphocreatine and ADP:
CK
2"
CODH
’H-ûH2" Cß0H
in
0,5
0,5
Incubatio:n 15 minutes
0,5
0,0
0;5
0,5
0,5
0o
0,5
0,5
0,5
0,5
Samples let siand. for i0 minutes
Physiologica1›alam ofcreatime kinase in serious tissues are very different. For insJance,in the liver
activity o( creatine kinase is only 12 nka,t/g fresh tissue, in the myocard is activity about 6ykai/g tiss,ue
an,d!:in the muscle 33 ¿i kai/g1issue. If we compare activity in the liver 8 ñd i.n the muscle, hero is activity
21
3.4.2 The ev.aIuation
From obtained absorbance values of ’arious tissues we read the amount of created crmiine in the
react ion from the analytical cun’e.
Then from these values we calculate the amount pcr 1 C of fresh iissue. Actin ity of crcaiine kinase
c express in nkat or kat per gram of fresh tissue.
Illustrate we acti âiy of enp’mc in column graphs and determinc how many time is the acti icy of
creai ine kinase higher in the muscle tissue than in the liver and the m ocurd.