Planta (Berl.) 78, 213--225 (1968)

The Effect of Heat Hardening on the Heat Resistance of Some Enzymes from Plant Leaves N. L. FELl)MAN Institute of Cytology and Komarov Botanical Institute, Academy of Sciences of the USSR, Leningrad Received September 28, 1967

Summary. Heat hardening of leaves which leads to an increase in the heat resistance of their cells, also increases the heat resistance of their enzymes (urease, acid phosphatase, ATPase). As judged by the temperature reducing enzyme activity by 50%, the heat resistance increased by about 6~ and 4 ~ respectively, for urease and acid phosphatase of cucumber, about 7~ for acid phosphatase of wheat, and 1,5~ for ATPase of Caragana. Increased heat resistance of acid phosphatase and ATPase caused by heat hardening was accompanied by a decrease in the activity of these enzymes. The activity of urease was not affected by heat hardening. It is assumed that the cause of this increase in thermal resistance of enzymes is a stabilization of protein macromolecules during heat hardening of leaves. Introduction

Some e n v i r o n m e n t a l factors cause changes of resistance in p l a n t cells ; these changes can be considered as a p r o t e c t i v e r e a c t i o n in response to u n f a v o u r a b l e conditions. A n e x a m p l e is h e a t hardening, i.e. increase of t h e t h e r m o r e s i s t a n e e of the cells in response to t h e action of sufficiently high t e m p e r a t u r e s on t h e whole p l a n t or its p a r t s (AL~xAND~OV, 1956). H e a t h a r d e n i n g is a p h e n o m e n o n easily reproducible u n d e r e x p e r i m e n t a l conditions (ALExANDROV, 1963, 1964, 1965a; etc.) a n d is also o b s e r v e d u n d e r n a t u r a l condition (AL~xAND~OV a n d YASKULIEV, 1961; LANCe, 1961 ; u 1964; S~KHTINA, 1965). A n e l u c i d a t i o n of t h e m e c h a n i s m u n d e r l y i n g t h e reversible resistance increase after h e a t h a r d e n i n g would be of considerable interest. One of t h e m o s t p r o b a b l e causes of increased resistance of h a r d e n e d p l a n t s is s t a b i l i z a t i o n of t h e p r o t o p l a s m i c p r o t e i n s (ALExA~cD~OV, 1956, 1964). This is in t h e first place i n d i c a t e d b y t h e fact t h a t h e a t h a r d e n i n g increases t h e resistance of t h e p l a n t to intensive s h o r t t e r m periods of heating, t h e injurious effect of which is k n o w n to d e p e n d on h e a t denat u r a t i o n of cellular p r o t e i n s (NAssoNov a n d ALEXANDROV, 1940; AL~XA~D~OV, 1963, 1964; USnAKOV, 1965). This view is also s u p p o r t e d b y the fact t h a t resistance is increased n o t only to h e a t i n g b u t also to o t h e r injurious a g e n t s which are able to d e n a t u r e t h e proteins, such as high h y d r o s t a t i c pressure, alcohol, p o t a s s i u m t h i o c y a n a t e a n d c a d m i u m chloride (ALExANDROV,1963, 1964). 15a Planta (Berl.), Bd. 78

214

N.L. FELDI'~AlV:

The increase of resistance b y h e a t h a r d e n i n g affects the m a i n structures a n d f u n c t i o n s of cells: selective p e r m e a b i l i t y , photosynthesis, respiration, protoplasmic streaming (ALExA~D~OV, 1964, 1965a) a n d the a b i l i t y of the chloroplasts for phototactic m o v e m e n t s (LOMAGINet M., 1966). C o n s e q u e n t l y the changes leading to heat h a r d e n i n g should be ]ocMized i n c o m p o n e n t s c o m m o n to all cellular structures. F i r s t of all we m a y consider the proteins. A significant evidence i n f a v o u r of the stabilizing role of proteins i n heat h a r d e n i n g is the s t r e n g t h e n i n g of the b o n d s b e t w e e n chlorophyll a n d the lipoprotcin complexes of the chloroplasts after heat h a r d e n i n g in relation to the s u b s e q u e n t h e a t i n g (LYvTOVA, 1963). However, a direct comparison of heat resistance of proteins from h a r d e n e d a n d from n o n - h a r d e n e d p l a n t s should d e m o n s t r a t e more conclusively t h a t heat h a r d e n i n g results in ~ stabilization of p r o t e i n molecules. W e decided to s t u d y this question, using e n z y m e s extracted from leaves, because the level of their heat resistance can be measured easily b y changes i n their activity. The e n z y m e chosen - - n a m e l y , acid phosphatase, adenosinetriphosp h a t a s e (ATPase) a n d u r e a s e - - are f o u n d i n tissues of m a n y higher plants. T h e y differ i n their i n t r a c e l i n l a r ]ocMization and, hence, are represent a t i v e of different s t r u c t u r a l c o m p o n e n t s of the cell.

Material and Methods

1. Plant Material and Heat Hardening The experiments were carried out with cucumbers, Cucumis sativus L., ev. "Vyaznikovski" (leaves from 3~4~ week old plants); wheat, Triticum aestivum L., cv. "Diamant" (10-day-old seedlings); and Caragana arborescens L. The former two plants were grown in a greenhouse at a temperature of 23~ and a light intensity of 5,000 lux. Material of Caragana, consisting of fully expanded leaves, was obtained from bushes growing under natural conditions in the Botanical Garden in Leningrad. The activity of urease was determined in extracts from cucumber leaves, that of acid phosphatase in extracts from cucumber leaves and wheat seedlings, and that of ATPase in extracts from Caragana leaves. Heat hardening of the leaves was performed by placing the leaves for 18 hours in a moist chamber at 36--40 ~ for cucumbers and wheat, and at 42~ for Caragana. Control leaves were kept the same time in a moist chamber at room temperature. The leaves were then dried with filter paper and weighed. The extracts were prepared in the cold room. The leaves were cut into small pieces and ground for 15--20 rain in a mortar of Plexiglass in the appropriate extracting solutions.

2. Determination o] Enzyme Activity Acid Phosphatase was extracted with 1.5 volumes of a 5% solution of NaC1 ( S A ~ w ~ and KRIS~A~, 1961). After standing for 60 rain at 0% the extract was filtered through nylon. The enzymatic activity was determined in the crude homogenate, as acid phosphotase is localized preferentially in the large-particale frac-

Heat Hardening and the I-Ieat I~esistance of Enzymes from Leaves

215

tions. In part of the tests the homogenate was first dialyzed in the cold for 20 hours against a 50 fold volume of 0.01 M citrate buffer, pH 5.9. During this time the buffer was renewed twice. The activity of the enzyme in the extract was determined as the amount of inorganic phosphorus (Pi) liberated from sodium/~-glyeerophosphate after I0 min (wheat) or 60 rain (cucumbers), at a temperature of 30 ~ The incubation mixture consisted of 0.5 ml of extract, 0.5 ml of 0.I M citrate buffer, pH 5.6, and 0.i Inl of a 20 % solution of sodium fl-glycerophosphate. The final pK of the mixture was 5.6. The samples with the incubation mixture were held for i0 rain at 30 ~ before introducing the substrate. Enzyme activity was stopped by heating the incubation mixture for 7 rain in a boiling water bath. The Pi liberated was determined by the LowI~Y and LoPEz method as modified by WAYGOOl) (194:8). ATPase was extracted from the leaves with a threefold volume of 0.5 M KCI solution (PoGI~AZOV, 1956). After i hour in the refrigerator the crude extract was pressed through nylon and dialysed against neutralized distilled water, conditions and time of dialysis otherwise being similar to those used for acid phosphatase. After dialysis, the extract was centrifuged at 12,000 • g for 20 rain. The supernatant was diluted with i0--25 volumes of 0.5 M KCl solution and the ATPase activity determined as the amount of Pi liberated from ATP during 5 min of incubation at 30 ~ The incubation mixture consisted of ].0 ml of extract, 1.0 ml of 0.I M borate buffer, pI-I 8.0, 0.I ml of 0.5 M MgCI 2 solution and 0.i ml 10% ATP solution (final pl-I 8.0). Prior to introducing the substrate, the test tube with the incubation mixture was held at 300 for i0 rain. After the end of the reaction the enzyme was inactivated for 7 mill in a boiling water bath, similarly to the procedure with acid phosphatase, and Pi was determined after W),YGoo?0 (1948). Urease was extracted from leaves with 1.5 volume of I/3 3/[ phosphate buffer, pH 7.5. Cystein and ethylenediaminetetraacetate were added to the buffer directly before starting the experiment, to make the final concentration 3 • 10 -5 M. The mixture was pressed through nylon and centrifuged at 12,000 • g for 20 min. The supernatant was used for further experiments either immediately, or after dialysing it against 1/30 M phosphate buffer at pH 7.5 (FEL~)MAN, 1966). The activity of the urease was determined using CosJwAY's microdiffusion method (DILLEu and WALKER, 1961). The incubation mixture was composed of 1.0 m] extract and l.O ml of 10% urea solution. The reaction was stopped after 60 rain by mixing the incubation mixture with 2.0 ml saturated K2CO a solution. Isothermic ammonia distillation was carried out for 24 hours at room temperature. In all cases, enzyme activity was related to protein content. Protein was determined by the micro-Kjeldal method. All enzymes were subjected to preliminary investigations in order to determine the relationship between enzyme activity and incubation time, and between substrate concentration and amount of extract. Based on the results of these studies such proportions of components and times of incubation were chosen which gave a sufficiently high level of enzyme activity and a direct relationship between enzyme activity and amount of extract in the presence of excess substrate.

3. Determination el the Heat Resistance 0] Enzymes To determine the heat resistance of the enzyme, the extract was poured into test tubes of equal diameter and heated in a water bath at different temperatures, for 15 rain with urease and 30 min with acid phosphatase and ATPase. After cooling in water at room temperature enzyme activity was determined using the methods described above. The level of heat resistance was judged by the level of residual activity, the activity of non-heated extract being taken as 100 %. 15"

216

N.L. FELDMAN: Results 1. Heat Resistance o/Cells

Prior to investigating the effect of heat hardening on heat resistance of extracted proteins, we had to prove that the heat treatment used did, in fact, increase the heat resistance of the plants investigated. Different methods of determination were used for the different species. With cucumber we determined the temeperatures at which cytoplasmic streaming ceased in t h e epidermal cells of hardened and non-hardened leaves. The maximum temperature, when applied for 5 min, after which movement of spherosomes could still be observed in the cells was 46.7 ~ in the hardened and 45.0 ~ in the control leaves. Thus, the heat resistance of the leaf cells of cucumber was increased by 1.7 ~ after heat hardening. In wheat seedlings and Caragana leaves movement of spherosomes cannot be observed in the leaf cells; in these cases we had to use other criteria for judging the heat resistance. In the case of wheat we made use of the experience of KISLYVK (1962) who found that after heat hardening the seedlings became more thermoresistant. This was indicated by lesser injury to leaves and meristems after exposure to high temperatures. Repeating these experiments, hardened seedlings and controls were heated for 30 rain at 63 ~ then placed into a cabinet at 20 ~ and about 5,000 lux. In 4 or 5 days the surviving plants were counted. 100 % of the control plants died while among the hardened plants the death rate was only 21%. For judging heat resistance in Caragana leaves the temperature sensitivity of photosynthesis was used. First, the initial value of photosynthesis in hardened and non-hardened leaves was measured; then, the suppression of photosynthesis after a heat treatment was determined, in either case by radiometric methods using C140e. In full agreement with results obtained with other plants (L~uTOVA, 1962) the rate of photosynthesis in the leaves of Caragana was substantially reduced as a result of hardening (Table 1), namely, to 14% of the controls. Despite of this the hardened plants showed quite a measurable rate of photosynthesis. Table 1. Photosynthesis rate and heat resistance o/photosynthesis in Caragana leaves a]ter heat hardening at 40--42 ~ for 18 hours

Control Hardened specimen

No. of experiments

Initial rate (rag C02/g dry wt./h)

l~te after heating (5 rain at 44~

4 4

50 -t- 5 7 ~- 1

15 • 2 5 -5 1

P

mg COe/g % of inidry wt./h tim value 30 72

< 0.001 < 0.05

Heat Hardening and the Heat Resistance of Enzymes from Leaves Dark absorption of CO~ in the hardened plants was 0.4 mg/g dry weight/hour, i.e. 5% of the assimilation of CO~ in the light. Both control and hardened leaves were heated for 5 rain at 44 ~ and the rate of photosynthesis measured again. The ratio of the value thus obtained to the initial vahie of photosynthesis is an indication of the heat resistance of photosynthesis. The data in Table 1 show t h a t in the hardened leaves the heat treatment had very little effect on photosynthesis, while in the control leaves photosynthesis was reduced by70%. Consequently, the heat resistance of photosynthesis had been increased b y heat hardening. Thus, the hardening temperatures used in our experiments clearly resulted in an increase in the heat resistance of the cells of cucumber and Caragana leaves and of wheat seedlings.

r ~)o

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L§247

2. Enzyme Activity alter Hardening The absolute activity of the enzymes under investigation varied widely from experiment to experiment, particularly in the case of acid phosphatase and ATPase. The causes of this variation are not completely understood. Only in a few cases has it been possible to establish a relation with the developmental phase of the plant or with the season of the year. Thus, the activity of ATPase in extracts from Caragana leaves dropped at the end of the growing season. LUBIMOVA et al. (1966) made similar observations for Mn++-activated ATPase extracted from the leaves of a number of higher plants. Irrespective of the level of initial activity of acid phosphatase and ATPase, heat hardening of leaves always leads to an inhibition of these two enzymes (Table 2). This agrees with the depression of a number of cellular functions, for example photosynthesis, rate of protoplasmic streaming, 15b

Planta (Berl,), Bd. 78

217

r

~o ~0 ~0 o0 ~ o0

r r

O 0 0

218

N.L. F~LDMA~:

and growth, t h a t was observed after heat hardening (ALwxA~I)~ov, 1964, 1965a). I n contrast, the activity of urease was not reduced after heat hardening and in undialysed extracts was in fact somewhat higher t h a n in the control (Table 3). After dialysis this difference was reduced due to an increase of activity in the control. Table 3. Initial level o/ urease activity in extracts/tom hardened and non-hardened cucumber leaves Temperature and time of hardening (o C/hrs) 36~ 36~

No. of Dialysis experiments

12 5

--]-

Activity in ~g NH3/mg protein-N/60 rain/22 ~ Control

Hardened

Activity in hardened extract as percent of control

26~:2 30 4- 4

344-4 32 :j::2

1334-8 107 ~ 5

Some rise of activity in the control extract after dialysis was also observed with acid phosphatase (Table 2). This phenomenon is perhaps explained b y the presence of some low-molecular substance(s) suppressing the activity of acid phosphatase and of urease. I t is known, for example, t h a t urease is suppressed b y K + and Na + ions (FAs~Azr and NI~MAzr 1951). I t is conceivable t h a t substances with an inhibitory action are absent, or are present in lower concentrations, in extracts from hardened leaves. 3. Heat Resistance o / E n z y m e s after Heat Hardening a) Urease. D a t a for the temperature inactivation of enzymes in extracts from hardened and non-hardened leaves are given in Figs. 1--3. I n the case of urease, a statistically significant decrease of activity in the control, t h a t is in extracts from non hardened leaves, was observed after 15 rain of heating at 50 ~ Further temperature increase led to a progressive drop in activity (Fig. 1). I n contrast, heating of extracts from hardened leaves at 400--50 ~ resulted in some stimulation of the activity of the enzyme (Fig. 1) ; a reduction of activity was observed only after heating at higher temperatures (70~ The slope of the temperature-inactivation curve for urease was less steep for the control than for the extract from hardened leaves (Fig. 1). b) Acid Phosphatase a~,d A T P a s e . As mentioned before, heat hardening of the leaves resulted in a reduction in the activity of acid phosphatase and ATPase (Table 2). Concerning temperature inactivation of these enzymes, 30 min of heating of the extracts caused a reduction of enzyme activity in the control at temperatures at which the hardened

Heat Hardening and the Heat Resistance of Enzymes from Leaves

219

extract still retained its initial level of activity. Thus, in the case of acid phosphatase of wheat and cucumbers, the lowest temperatures of heating used in our experiments were 40 and 45 ~, respectively. A t such temperatures the control showed a statistically significant decrease in activity (Figs. 2 a and b), b u t in extracts from hardened leaves such a decrease took place only at 55 ~ for wheat and 52.5 ~for c u c u m b e r (Figs. 2 a and b).

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\

7

9 --'Cootro

o "6

ext=

>,

~o W

\\1

o Extract from hardened \ leaves

3tO Z;'O 5tO iO Temperature

r

7tO~ 80

Fig. 1. Changes in urease activity in dialyzed extracts from hardened and nonhardened cucumber leaves after 15 rain of heating at different temperatures Similar d a t a were obtained for A T P a s e (Fig. 3). E n z y m e inhibition in the control was observed after heating at 44 ~, b u t in the extract from hardened leaves only after heating at 47.5 ~. The slopes of the temperature-inactivation curves of acid phosphatase and A T P a s e in extracts from the control and hardened material were different; the activity decrease in the control was steeper. Therefore after heating at relatively high temperatures the curves came closer to (Fig. 2b) or even crossed one another (Figs. 2a and 3). The comparison of temperature inactivation for all three enzymes shows clearly t h a t heat hardening changes the sensitivity of the enzyme to high temperature. These differences are most pronounced when we compare the relation between residual activity (that is, activity after thermal injury, expressed as percentage of the initial level) and the temperatures of heating (FELDMA~, 1966; FV,LD~[A~" et al., 1966). As can be seen from Figs. 4 6, the degree of inhibition of activity in an extract from hardened leaves was lower at all temperatures t h a n in the control.

220

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N . L . F~LDHAN:

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I "~~ extracts o ~ fromhardened teQves 20O

%

E 50

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160 140 120 100

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_

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~

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5'0 ~

&

20

0 Temper~ture

3'0

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i

50 ~

b

Figs. 2a and b. Changes in acid phosphatase activity in dialyzed extracts from hardened and non-hardened cucumber leaves (a) and wheat seedlings (b) after 30 rain of heating at different temperatures

~o ?'

f

~s

4 F ~3

:~2

=ii Zes •

LU

3qO

4TO 510 oC Temperature

60

:Fig. 3. Changes in ATPase activity in dialyzed extracts from hardened and nonhardened leaves of Caraganaafter 30 rain of heating at different temperatures

Heat Hardening and the Heat Resistance of Enzymes from Leaves F o r instance, after heating at 30 ~ the residual activity of acid phosphatase in the extract from hardened material was 30 % higher t h a n t h a t in the control (Figs. 5 a and b). If judged b y the temperature causing an activity decrease of 50 %, the heat resistance of enzymes from hardened leaves was higher b y a b o u t 6 ~ for urease, b y 4 ~ and 7 ~ for acid phosphatase of c u c u m b e r and wheat, respectively, and 1.5 ~ for ATPase. B o t h dialysed and non dialyzed extracts showed higher heat resistance of enzymes after heat hardening (Table 4). We shall consider this fact later, when discussing the causes of the increase in heat resistance after hardening.

221

120 -~ -~ 100 !~ ~ so :~_ 60 ~ 40 ~ 20

leaves

"U

10

4

I

I

f

50 60 Temperature

7~

0 ~

80

Fig. 4. Residual activity of nrease in dialyzed extracts from hardened and non hardened cucumber leaves after 15 rain of heating of extracts at different temperatures

Table 4. Heat resistance o[ acid phosphatase and urease in dialyzed and non-dialyzed extracts/tom cucumber leaves

Enzyme

Tempera- Diature and lysis time of hardening (o C/hrs)

No. of experiments

Acid phosphatase

40~

-~-

Urease

36~

-+

Temperature of 50% inactivation (~C)

Difference between hardened and control extracts (~C)

control

hardened

4 3

50.5 49.0

53.0 53.0

2.5 4.0

5 5

63.0 67.0

76.0 75.0

13.0 8.0

Discussion At the beginning of our paper we suggested t h a t the change in the sensitivity of cells to heating after heat hardening m a y be related to an increase of the heat resistance of protein. Our experiments have in fact shown t h a t heat hardening increased the heat resistance of urease, acid phosphatase and A T P a s e in homogenates of leaves of several plants. I t is difficult to say something about the biological meaning of heat hardening - - whether it is in the increase of protein heat resistance per se or in the change of level of conformational flexibility of protein molecules

222 ~100

N.L. FELDMAN: 9- - " Control ]:

'>--~

from hardened leaves

.~ 80 "s o~ .._~ 6o

imp_.

8

>.N,

l.fi

20

b o

'

s'o

a

4tO 6o Temperature

,

T

T 5'0

I

~ i 60

b

Figs. 5a and b. Residual activity of acid phosphatase in dialyzed extracts from hardened and non-hardened cucumber leaves (a) and wheat seedlings (b) after 30 rain of heating at different temperatures ~120

leading to an increase of their heat resistance (AL~xANDI~OV, 1965b). Nevertheless it is interesting to 100 discuss the question, what causes "5 the increase of thermal stability of protein molecules? This becomes even more interesting as a similar reaction to high temperature is observed not only in higher plants ~ 40 but also in different microorganisms (LA~cGI~IDG~, 1963). I The increase in heat resistance 40 ' 5'0 ~ might be caused b y "de-nova" Temperature synthesis of protein with different Fig. 6. Residual activity of ATPase in dialyzed extracts from hardened and primary structure and with higher non hardened leaves of Carctganaafter stability. This appears possible in cases of long incubations of an 30 rain of heating at different temperatures organism at high temperature. Such data m a y be found for bacteria (CAMPBWLL, 1955; B~OWN et al., 1957), yeast (CHRISTOFHERSENand PR~CHT, 1950,1951), and infusoria ( S ~ A w N et al., 1965). I n heat hardening of plant leaves the synthesis of molecules with new properties is, however, much less probable as heat hardening with all its characteristic properties can be accomplished b y heat t r e a t m e n t of very short duration, namely 1--15 sec (Lo~AGI~r 1961; YARWOOD, 1961, 1962; ZAVADSKAYA, 1963).

~

"o

%

Heat Hardening and the Heat Resistance of Enzymes from Leaves

223

Another possible cause for the increase in protein stability after heat hardening is a selection of stable molecules during the action of hardening temperature. Such a selection would result in a decrease of the initial level of enzyme activity. This, however, was not observed in our experiment with urease (Fig. 1) and, consequently, for this enzyme selection of more stable enzyme molecules cannot be the cause of increased thermal resistance. The cases of acid phosphatase and ATPase are more complex. Hardening did decrease the activity of these enzymes. Yet an examination of Figs. 2 a and 3 shows that at intense heating the curves of temperature inactivation of the acid phosphatase from cucumber and the ATPase of Caragana cross one another; that is, although the activity of the control extract was initially higher, it becomes lower than that of the "hardened" extract. This would not be possible if selection of more stable enzyme molecules, present in the cells, was the only cause of the increase in heat stability. I n the case of acid phosphatase of wheat no crossing of the temperature inactivation curves was observed. But if we examine Fig. 2b we find that the slope of the curves in the control and hardened samples is different, being less steep in the latter so that the curves come nearer to each other at higher temperatures. I t is thus quite probable that at still higher temperatures of heating of the extracts the curves would intercross similarly to those of the acid phosphatase of cucumbers and the ATPase of Caragana. Thus, there is little probability that the increase of heat stability in both acid phosphatase and ATPase is caused by the selection of pre-existing, more heat-stable enzyme molecules. However, in order to exclude this possibility completely, a detailed study of the enzyme characteristics after heat hardening would have to be made, to show that their isozymic composition has not changed. I t m a y be assumed that the higher heat resistance of enzymes after heat hardening is related to the accumulation of some low molecular protective substances stabilizing the protein macromolecules. Such results have been described from in-vitro experiments (for example, PVT~A~, 1957). I n our case, however, this possibility should be rejected, since the differences in temperature sensitivity of enzymes in extracts from hardened and non-hardened leaves were observed also after dialysis, during which the concentration of low-molecular protective substances should have been greatly decreased. Thus, the most likely remaining assumption is that heat hardening causes an increase in the resistance of existing protein molecules. I n this connection it is interesting to cite some data of STeWArT and HALVo~so~ (1954) and C~u~c~ and HALVOi~SO~ (1959), showing that the enzymes in the spores of some bacteria are more heat resistant than enzymes from

224

N . L . FELD~IAN:

v e g e t a t i v e cells, t h e r e a s o n b e i n g t h a t t h e e n z y m e s in t h e s p o r e s ~re in a c o m p l e x w i t h d i p i c o l i n i c a c i d w h i c h s t a b i l i z e s t h e s e e n z y m e s . I t is p r o b a b l e t h a t a s i m i l a r p r o c e s s o c c u r s also a t h e a t h a r d e n i n g . A n inc r e a s e i n t h e p r o t e i n s t a b i l i t y m i g h t be a c h i e v e d as a r e s u l t of f o r m a t i o n of c o m p l e x e s w i t h s o m e s u b s t a n c e s of t h e c y t o p l a s m . The author is greatly indebted to I. E. KAMENTSEVAfor technical assistance. Thanks are also due to M. I. LYUTOVAfor the determinations of photosynthesis.

Note added in proo]: After our paper was sent to publisher we got to know the paper of E. J. Ki31maAe~tmr e t a l . and CH. J. SVLLIVAX et al. [Crop Science 7, 148, 241 (1967)] which seems to be in good agreement with our results. References

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H e a t Hardening and the Heat Resistance of Enzymes from Leaves

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The effect of heat hardening on the heat resistance of some enzymes from plant leaves.

Heat hardening of leaves which leads to an increase in the heat resistance of their cells, also increases the heat resistance of their enzymes (urease...
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