MORITA, RICHARD Y. (University of Nebraska, Lincoln) AND ROGER D. HAIGHT. Malic dehydrogenase activitv at 101 C under hydrostatic pressure. J. Bacteriol. 83:1341-1346. 1962.-No malic dehydrogenase activity was found to occur at 101 C at various hydrostatic pressures from 1 to 700 atm. However, activity was demonstrated with hydrostatic pressures above 700 atm, with optimal activity at 1,300 atm at the same temperature. Explanation of the data is based upon thermal denaturation of the enzyme, which involves a molecular volume increase of the enzyme. The molecular volume increase is counteracted by hydrostatic pressure. Pressures above 700 atm to 1,500 atm (highest employed) were sufficient to offset the denaturation by 101 C which probably resulted in an incomplete denaturation of malic dehydrogenase

Haight, Roger D.

Morita, Richard Y.

English

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Papers in Microbiology Papers in the Biological Sciences 
January 1962 
MALIC DEHYDROGENASE ACTIVITY AT 101 C UNDER 
HYDROSTATIC PRESSURE 
Richard Y. Morita 
University of Nebraska-Lincoln 
Roger D. Haight 
University of Nebraska-Lincoln 
Follow this and additional works at: https://digitalcommons.unl.edu/bioscimicro 
 Part of the Microbiology Commons 
Morita, Richard Y. and Haight, Roger D., "MALIC DEHYDROGENASE ACTIVITY AT 101 C UNDER 
HYDROSTATIC PRESSURE" (1962). Papers in Microbiology. 2. 
https://digitalcommons.unl.edu/bioscimicro/2 
This Article is brought to you for free and open access by the Papers in the Biological Sciences at 
DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Papers in Microbiology by an 
authorized administrator of DigitalCommons@University of Nebraska - Lincoln. 
MALIC DEHYDROGENASE ACTIVITY AT 101 C UNDER
HYDROSTATIC PRESSURE
RICHARD Y. MORITA' AND ROGER D. HAIGHT
Department of Microbiology, University of Nebraska, Lincoln, Nebraska
Received for publication January 5, 1962
ABSTRACT
MORITA, RICHARD Y. (University of Nebraska,
Lincoln) AND ROGER D. HAIGHT. Malic dehydro-
genase activitv at 101 C under hydrostatic
pressure. J. Bacteriol. 83:1341-1346. 1962.-No
malic dehydrogenase activity was found to occur
at 101 C at various hydrostatic pressures from 1
to 700 atm. However, activity was demonstrated
with hydrostatic pressures above 700 atm, with
optimal activity at 1,300 atm at the same
temperature. Explanation of the data is based
upon thermal denaturation of the enzyme, which
involves a molecular volume increase of the
enzyme. The molecular volume increase is
counteracted by hydrostatic pressure. Pressures
above 700 atm to 1,500 atm (highest employed)
were sufficient to offset the denaturation by 101 C
which probably resulted in an incomplete
denaturation of malic dehydrogenase.
It is well known that high temperature will
denature enzymes and that the denaturation
process involves a molecular volume increase
(Johnson, Eyring, and Polissar, 1954). Molecular
volume increases are opposed by pressure. Brown,
Johnson, and Marsland (1942) presented evidence
that, above optimal temperature for luminescent
bacteria, pressure increased the luminescence by
reactivating the thermally inactive luciferase. At
temperatures causing a progressive destruction of
luminescence at favorable pH, pressure retarded
the irreversible denaturation (Johnson et al.,
1945). Johnson and Campbell (1945) demon-
strated that hydrostatic pressure retarded the
precipitation of highly purified human globulin
and egg albumen at 65 C. Pressure also retarded
the thermal denaturation of tobacco mosaic
virus (Johnson, Baylor, and Frazer, 1948).
Evidence was presented that substrate must be
l Present address: Department of Microbiology,
Oregon State University, Corvallis.
present to demonstrate the protective effect of
pressure on the enzymes, phenylglycosidase
(Berger, 1958) and aspartase (Haight and Morita,
1962), against thermal inactivation.
MATERIALS AND METHODS
Source and preparation of enzyme. The malic
dehydrogenase preparation was obtained from
R. J. Downey of this laboratory. The enzyme
was prepared by treating a thick cell suspension
of Bacillus stearothermophilus NCA 2184 with a
10-kc Raytheon sonic oscillator for 30 min and
centrifuging it for 60 min at 23,500 X g at 0 C in
a Servall RC-2 refrigerated centrifuge. The
supernatant liquid was decanted and centrifuged
at 144,000 X g for 120 min in a Spinco Model L
preparative ultracentrifuge. The clear straw-
colored supernatant liquid, which contained the
enzyme, malic dehydrogenase, was decanted and
frozen. When needed, the enzyme preparation
was thawed and diluted 1:10 (v/v) with tris
(hydroxymethyl)aminomethane-HCl (pH 8.5,
0.2 M). This enzyme preparation contained 153
jig protein nitrogen per 0.5 ml.
Assay of malic dehydrogenase activity. Malic
dehydrogenase activity was measured by the
procedure of Marsh (1956), which involves the
reduction of triphenyltetrazolium chloride
(TPTZ) to formazan (reduced TPTZ) in the
presence of NaCN, diphosphopyridine nucleotide
(DPN), tris buffer, L-malic acid, and malic
dehydrogenase.
The reaction mixture (4.0 ml total volume)
contained: tris-HCl buffer, 1.3 ml (pH 8.5, 0.2
M); L-malic acid, 1.0 ml (pH 8.5, 400 ,umoles/ml);
TPTZ, 0.5 ml (0.02 M); NaCN, 0.2 ml (0.05 M);
DPN, 0.5 ml (1 mg/ml); and the diluted malic
dehydrogenase preparation (0.5 ml). When
total volumes larger than 4.0 ml were needed, the
respective reagent volumes were scaled accord-
ingly.
The reaction mixture was placed in test tubes
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MORITA AND HAIGHT
(12 by 16.5 mm) and stoppered with no. 00
white rubber stoppers. No air phase remained in
the stoppered test tubes. The test tubes were then
placed in the pressure cylinder for treatment.
Certain experiments called for the pretreatment
of either the enzyme or DPN before addition to
the rest of the reaction mixture.
: .:.*.
*.*::.:
* :.
.. :.:...:.....;......~~~~~000 . 2000t.....
........ .... :; . ..0.
* :: .::.: |l ,.Fo30
After incubation at the desired temperature
and pressure, 4 ml of the reaction mixture were
pipetted immediately into screw-cap test tubes
containing 1.0 ml of chilled trichloroacetic acid
(50%) to stop the reaction. All tubes were kept
cold in an ice bath and in the dark until analyzed.
The formazan was extracted with 5.0 ml of ethyl
acetate. A sharp interface between the ethyl
acetate and the reaction mixture was brought
about by centrifugation for 5 min at 1,230 X g.
A known amount of ethyl acetate containing
formazan was diluted with a known amount of
ethyl acetate to obtain a reading within the
linear range of the standard curve. The standard
curve was obtained by diluting known amounts of
formazan with ethyl acetate, and the optical
densities of the solutions were read at 495 m,u on
a spectrophotometer (Bausch and Lomb Spec-
tronic 20). The activity of the malic dehydro-
genase preparation was expressed as mg formazan
produced per ml of reaction mixture. No formazan
was produced under the conditions of our
1500
0
-
1400
0
. 1300
4c
z 12000
4 1100m
0 1000z
z 900
i-
0D 800
w 700
600
a500
Q 400
t 300
(I)
, 200
a.
-* 100
z
c
FIG. 1. Pressure cylinder used for studies at
101 C at various hydrostatic pressures.
0
0
0
AP= Pf- P;
I 200 400 600 800 1000
INITIAL PRESSURE, Pi, AT 57 C (ATM)
FIG. 2. Initial pressure-final pressure relation-
ship for the pressure cylinder illustrated in Fig. 1.
r, 0 0
I I I I I I
0
-
I
I
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MALIC DEHYDROGENASE ACTIVITY AT 101 C
experiments in the absence of the enzyme
preparation.
Pressurization technique. The pressure appara-
tus and technique described by ZoBell and
Oppenheimer (1950) was employed with one
exception. The pressure-cylinder cap was fitted
with a three-way valve so that a pressure gauge
could be attached (Fig. 1). Rupturing of the
neoprene "O" ring seals at 101 C under pressure
caused difficulties. For this reason, constant
observation of the pressure gauge was necessary
to insure correct incubation times.
The pressure cylinder was filled with water and
equilibrated to 57 C. After the stoppered test
tubes filled with reaction mixture were placed in
the pressure cylinder and the pressure-cylinder
cap secured, the pressure was elevated to the
desired initial pressure at 57 C (Pi in Fig. 2). If
the pressure cylinder was equilibrated to 101 C
before pressure was applied, the enzyme would
undergo thermal denaturation before the hydro-
static pressure could be elevated. A total of 6
min was required for the initial pressure in the
cylinder to reach the final pressure (Pf in Fig. 2).
The data in Fig. 2 were obtained by use of the
pressure cylinder shown in Fig. 1. In all cases
where the temperature of 101 C was used, the
pressure cylinder was cooled with cold water
before the pressure was released and the pressure-
cylinder cap removed to obtain the reaction
mixture. This process took approximately 1 min.
For experiments at 1 atm and 101 C, the
pressure-cylinder cap was removed and the
cylinder filled with ethylene glycol and equili-
brated at 57 C. The reaction mixture was added
to a long test tube fitted with a vapor stop and
was then placed in the pressure cylinder. The
entire assembly was immersed in the 101 C
ethylene glycol bath as shown in Fig. 3. This
procedure was necessary since the increase in
temperature from 57 to 101 C would increase the
pressure if the pressure cylinder was capped.
Also, approximately the same time element was
required for it to obtain thermal equilibrium as
the entire pressure cylinder.
An Aminco constant-temperature bath, type R,
which can maintain a liquid temperature of up to
200 C (i 0.2 C), was utilized for this investiga-
tion. The temperature was checked with a
National Bureau of Standards Certified thermo-
meter.
FIG. 3. Apparatus used for 1 atm studies at 101 C
RESULTS
Effect of pH. Preliminary studies on the malic
dehydrogenase activity at 60 C demonstrated
that substrate saturation level was 50 MAmoles/ml
of reaction mixture. In order that the substrate
would not be a limiting factor, 100 ,umoles of
L-malic acid/ml of reaction mixture were used.
This level showed no substrate inhibition. The
rate of enzyme activity during a 6-min period
did not decrease. By use of a substrate level of 100
,umoles/ml of reaction mixture, the reaction
mixtures were made up with varying pH values
by using 0.2 M tris(hydroxymethyl) a mino me-
thane (tris) buffer and incubated for 35 min at
60 C. The data in Fig. 4 show an optimal pH of
approximately 8.5 and also show that the activity
curve in relation to pH is not sharp. For all
subsequent studies, a pH of 8.5 was selected.
Stability of DPN. Treatment of DPN at
various pressures and temperatures for a period
of 44 min before addition to the rest of the
reaction mixture revealed that activity was lost
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MORITA AND HAIGHT
at 100 C (Table 1). DPN, in a concentration of 1
mg/ml, was employed in the reaction mixture
since 60% activity was lost when DPN was
treated at 100 C and 1 atm for 44 min. Destruc-
tion of DPN occurred more when treated at 100
C and 1 atm for 44 min than when treated at
100 C and 1,500 atm for 44 min.
Activity of malic dehydrogenase at various
-I
a w
l- cc3
N x 25
c z 200 0
co - I-
4 W 10
C 5
A. 5
TEMPERATURE * 60 C.
7.7 7.9 8.1 8.3 85
pH OF REACTION
w
x
-2
0i:
4)
Id
a:
2
4
N
0
IL.
0)
I
9 4
9
a:
0L
8.7 8.1
FIG. 4. Effect of pH on malic dehydrogenase
activity at 60 C. The reaction mixture contained
tris-HCl buffer, 1.3 ml (various pH levels, 0.2 M);
L-malic acid, 1.0 ml (various pH levels, 400 ,umoles/
ml); TPTZ, 2.5 ml (0.02 M); NaCN, 0.2 ml (0.05
M); DPN, 0.5 ml (1 mg/ml); and 0.5 ml of diluted
malic dehydrogenase preparation (153 ,ug protein
N/0.5 ml). Values are corrected for controls. In-
cubation time was 35 min.
TABLE 1. Stability of DPN at various hydrostatic
pressures and temperatures as measured by the
amount of formazan produceda
Treatment of DPNb Fonnazan ActivityTreatmentDPNproducedc loStd
zg/ml %
28 C, 1 atm, 44 min........... 190
60 C, 1 atm, 44 min........... 190 0
100 C, 1 atm, 44 min........... 79 60
100 C, 1,500 atm, 44 min....... 108 40
No DPN.................... 0
a The reaction mixture contained tris-HCl
buffer, 1.3 ml (pH 8.5, 0.2 M); L-malic acid, 1.0
ml (pH 8.5, 400 ,umoles/ml); TPTZ, 0.5 ml (0.02
M); NaCN, 0.2 ml (0.05 M); 0.5 ml DPN (1 mg/ml);
and 0.5 ml of the diluted malic dehydrogenase
preparation (153 ug per protein N per 0.5 ml).
I Before addition to the malic dehydrogenase
reaction mixture.
c At 60 C for 35 min; tests were run in duplicate
and corrected for controls.
d Relative to treatment at 28 C and 1 atm for
44 min.
55 60 65 70 7
TEMPERATURE (C)
FIG. 5. Effect- of temperature on malic dehy-
drogenase at 1 atm. The reaction mixture was
identical to that in Fig. 4 except that the pH level
was 8.5. Incubation time was 35 min. Values are
corrected for controls.
temperatures at 1 atm. When the enzyme reaction
mixture was subjected to various temperatures,
an optimum of 55 C was noted (Fig. 5). No
activity of the enzyme could be detected at 80 C,
which indicated that thermal inactivation oc-
curred just below 80 C.
Activity of malic dehydrogenase at 101 C at
various pressures. When the malic dehydrogenase
reaction mixtures were subjected to a temperature
of 101 C at various pressures, it was noted that
no activity could be demonstrated at 1 to 700
atm (Fig. 6). However, activity of the malic
dehydrogenase was demonstrated when hydro-
static pressure was elevated above 700 atm. Since
it took 6 min for the initial pressure to reach the
final pressure in the system, the 6-min points
served as the base line for these studies. At 1,300
atm and 101 C, the amount of formazan pro-
duced by the reaction mixture in 41 min was
approximately 350% greater than the amount
produced in 6 min. The net activity at 101 C at
various pressures, represented in Fig. 6 as delta
(A), is the difference between the amount of
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MALIC DEHYDROGENASE ACTIVITY AT 101 C
gX 350.2 M
x 35
Z 300
I-~~~~~~~C.)
'< 25w
_j20
z
N 1-5
L^ IIo
CD
1 500 700 900 1100 13001500
FINAL PRESSURE (ATM)
FIG. 6. Activity of malic dehydrogenase at 101
C under various hydrostatic pressures. The reaction
mixture was identical to that in Fig. 4 except that
the pH level was 8.5. Delta (A) represents the differ-
ence between the 6 min and 41 min incubation times.
Values are corrected for controls.
formazan produced in 41 min minus the amount
formed in 6 min.
Malic dehydrogenase displayed activity with
time at 101 C and 1,300 atm, whereas the curve
at 101 C and 1 atm curve showed no activity
(Fig. 7). The curve at 101 C and 1,300 atm is
definitely not as pronounced as the curve at 58 C
and 1 atm.
DISCUSSION
Since it is extremely difficult to measure pH
shifts with increased hydrostatic pressure and
temperature, tris-HCl buffer was employed.
According to Kauzmann (personal communica-
tion), the organic amine types of buffers possess a
small A volume (v) (Av = +2 to +4 ml at 25 C
and 1 atm at infinite dilution) as compared to
phosphate buffer (Av = -29 ml at 25 C and 1
w z C 0
:
, 210
X 195
IS180
z
o 165
o ISO
4c
c 135
I 120
a 105
z 9 0
49
N4 754
0
coIL 45
a 30
49
0 '5
0
pH - 8.5, 0.2 M TRIS*HCI BUFFER
I C- I ATM
101 C-1300ATM
M 0 0 101 C-1 ATM
I I I I
0 6 12 18 24 30 36
INCUBATION TIME (MIN) AT STATED CONDITIONS
FIG. 7. Rate of malic dehydrogenase activity.
The reaction mixture was identical to that in Fig. 4
except that the pH level was 8.5. The curve of 101 C
and 1,300 atm is corrected for the 6-min period
required for the initial pressure to reach the final
pressure. Values are corrected for controls.
atm at infinite dilution). The pH shifts with
pressure are minimized with systems that
possess small Av values. Since the pH curve at
60 C and 1 atm (Fig. 4) does not display a sharp
peak, a slight shift in pH would not alter the
main point of this investigation, which is the
activity of malic dehydrogenase at 101 C under
hydrostatic pressure.
Malic dehydrogenase activity at 1 atm was
destroyed at approximately 78 C (Fig. 5), but,
when the pressure was increased beyond 700 atm
(Fig. 6), the molecular volume increase brought
about by the increase in temperature was coun-
teracted by the applied hydrostatic pressure.
As a result of the applied pressure, the enzyme
does not undergo complete thermal denaturation.
The extent of denaturation of the enzyme under
the pressure applied at 101 C awaits further
investigation. Since a pressure cylinder has not
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MORITA AND HAIGHT
been developed to compensate for the increase
in pressure brought about by increased tem-
perature, there is undoubtedly some denaturation
of the enzyme during the time the pressure
cylinder goes from its initial pressure to the
final pressure (Fig. 2). Denaturation would
probably take place more at the lower pressures
applied in our studies.
We also realize that the rate of activity at 101 C
and 1,300 atm is quite low compared to the rate
at 58 C and 1 atm. However, it should be remem-
bered that we are probably not operating under
the optimal temperature for the malic dehydro-
genase system. The data obtained, however,
show clearly that malic dehydrogenase can
operate at 101 C when hydrostatic pressures of
700 to 1,500 atm are applied.
ACKNOWLEDGMENTS
The authors wish to acknowledge W. Kauz-
mann of Princeton University for information
concerning the effect of pressure on pH.
This investigation was supported by research
grant A-3784 from the National Institute of
Arthritis and Metabolic Diseases, U.S. Public
Health Service.
LITERATURE CITED
BERGER, L. R. 1958. Some effects of pressure on
a phenylglycosidase. Biochim. et Biophys.
Acta 30:522-528.
BROWN, D. E., F. H. JOHNSON, AND D. A. MARS-
LAND. 1942. The pressure-temperature rela-
tions of bacterial luminescence. J. Cellular
Comp. Physiol. 20:151-168.
HAIGHT, R. D., AND R. Y. MORITA. 1962. The
interaction between the parameters of hy-
drostatic pressure and temperature on as-
partase of Escherichia coli. J. Bacteriol.
83:112-120.
JOHNSON, F. H., M. B. BAYLOR, AND D. FRASER.
1948. The thermal denaturation of tobacco
mosaic virus in relation to hydrostatic pres-
sure. Arch. Biochem. 19:237-245.
JOHNSON, F. H., H. EYRING, AND M. J. POLISSAR.
1954. The kinetic basis of molecular biology.
John Wiley & Sons, Inc., New York.
JOHNSON, F. H., AND D. H. CAMPBELL. 1945.
The retardation of protein denaturation by
hydrostatic pressure. J. Cellular Comp.
Physiol. 26: 43-46.
JOHNSON, F. H., H. EYRING, R. STEBLAY, H.
CHAPLIN, C. HUBER, AND G. GHERARDI.
1945. The nature and control of reactions in
bioluminescence. With special reference to
the mechanism of reversible and irreversible
inhibitions by hydrogen and hydroxyl ions,
temperature, pressure, alcohol, urethane, and
sulfanilamide in bacteria. J. Gen. Physiol.
28:463-536.
MARSH, J. M. 1956. A genetic study of thermo-
phily. Thesis, University of Nebraska, Lin-
coln.
ZOBELL, C. E., AND C. H. OPPENHEIMER. 1950.
Some effects of hydrostatic pressure on the
multiplication and morphology of marine
bacteria. J. Bacteriol. 60:771-781.
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MALIC DEHYDROGENASE ACTIVITY AT 101 C UNDER HYDROSTATIC PRESSURE

DigitalCommons@University of Nebraska

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Papers in Microbiology Papers in the Biological Sciences January 1962 MALIC DEHYDROGENASE ACTIVITY AT 101 C UNDER HYDROSTATIC PRESSURE Richard Y. Morita University of Nebraska-Lincoln Roger D. Haight University of Nebraska-Lincoln Follow this and additional works at: https://digitalcommons.unl.edu/bioscimicro  Part of the Microbiology Commons Morita, Richard Y. and Haight, Roger D., "MALIC DEHYDROGENASE ACTIVITY AT 101 C UNDER HYDROSTATIC PRESSURE" (1962). Papers in Microbiology. 2. https://digitalcommons.unl.edu/bioscimicro/2 This Article is brought to you for free and open access by the Papers in the Biological Sciences at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Papers in Microbiology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. MALIC DEHYDROGENASE ACTIVITY AT 101 C UNDERHYDROSTATIC PRESSURERICHARD Y. MORITA' AND ROGER D. HAIGHTDepartment of Microbiology, University of Nebraska, Lincoln, NebraskaReceived for publication January 5, 1962ABSTRACTMORITA, RICHARD Y. (University of Nebraska,Lincoln) AND ROGER D. HAIGHT. Malic dehydro-genase activitv at 101 C under hydrostaticpressure. J. Bacteriol. 83:1341-1346. 1962.-Nomalic dehydrogenase activity was found to occurat 101 C at various hydrostatic pressures from 1to 700 atm. However, activity was demonstratedwith hydrostatic pressures above 700 atm, withoptimal activity at 1,300 atm at the sametemperature. Explanation of the data is basedupon thermal denaturation of the enzyme, whichinvolves a molecular volume increase of theenzyme. The molecular volume increase iscounteracted by hydrostatic pressure. Pressuresabove 700 atm to 1,500 atm (highest employed)were sufficient to offset the denaturation by 101 Cwhich probably resulted in an incompletedenaturation of malic dehydrogenase.It is well known that high temperature willdenature enzymes and that the denaturationprocess involves a molecular volume increase(Johnson, Eyring, and Polissar, 1954). Molecularvolume increases are opposed by pressure. Brown,Johnson, and Marsland (1942) presented evidencethat, above optimal temperature for luminescentbacteria, pressure increased the luminescence byreactivating the thermally inactive luciferase. Attemperatures causing a progressive destruction ofluminescence at favorable pH, pressure retardedthe irreversible denaturation (Johnson et al.,1945). Johnson and Campbell (1945) demon-strated that hydrostatic pressure retarded theprecipitation of highly purified human globulinand egg albumen at 65 C. Pressure also retardedthe thermal denaturation of tobacco mosaicvirus (Johnson, Baylor, and Frazer, 1948).Evidence was presented that substrate must bel Present address: Department of Microbiology,Oregon State University, Corvallis.present to demonstrate the protective effect ofpressure on the enzymes, phenylglycosidase(Berger, 1958) and aspartase (Haight and Morita,1962), against thermal inactivation.MATERIALS AND METHODSSource and preparation of enzyme. The malicdehydrogenase preparation was obtained fromR. J. Downey of this laboratory. The enzymewas prepared by treating a thick cell suspensionof Bacillus stearothermophilus NCA 2184 with a10-kc Raytheon sonic oscillator for 30 min andcentrifuging it for 60 min at 23,500 X g at 0 C ina Servall RC-2 refrigerated centrifuge. Thesupernatant liquid was decanted and centrifugedat 144,000 X g for 120 min in a Spinco Model Lpreparative ultracentrifuge. The clear straw-colored supernatant liquid, which contained theenzyme, malic dehydrogenase, was decanted andfrozen. When needed, the enzyme preparationwas thawed and diluted 1:10 (v/v) with tris(hydroxymethyl)aminomethane-HCl (pH 8.5,0.2 M). This enzyme preparation contained 153jig protein nitrogen per 0.5 ml.Assay of malic dehydrogenase activity. Malicdehydrogenase activity was measured by theprocedure of Marsh (1956), which involves thereduction of triphenyltetrazolium chloride(TPTZ) to formazan (reduced TPTZ) in thepresence of NaCN, diphosphopyridine nucleotide(DPN), tris buffer, L-malic acid, and malicdehydrogenase.The reaction mixture (4.0 ml total volume)contained: tris-HCl buffer, 1.3 ml (pH 8.5, 0.2M); L-malic acid, 1.0 ml (pH 8.5, 400 ,umoles/ml);TPTZ, 0.5 ml (0.02 M); NaCN, 0.2 ml (0.05 M);DPN, 0.5 ml (1 mg/ml); and the diluted malicdehydrogenase preparation (0.5 ml). Whentotal volumes larger than 4.0 ml were needed, therespective reagent volumes were scaled accord-ingly.The reaction mixture was placed in test tubes1341 at UNIV OF NEBRASKA-LINCOLN on August 30, 2007 jb.asm.orgDownloaded from MORITA AND HAIGHT(12 by 16.5 mm) and stoppered with no. 00white rubber stoppers. No air phase remained inthe stoppered test tubes. The test tubes were thenplaced in the pressure cylinder for treatment.Certain experiments called for the pretreatmentof either the enzyme or DPN before addition tothe rest of the reaction mixture.: .:.*.*.*::.:* :... :.:...:.....;......~~~~~000 . 2000t............. .... :; . ..0.* :: .::.: |l ,.Fo30After incubation at the desired temperatureand pressure, 4 ml of the reaction mixture werepipetted immediately into screw-cap test tubescontaining 1.0 ml of chilled trichloroacetic acid(50%) to stop the reaction. All tubes were keptcold in an ice bath and in the dark until analyzed.The formazan was extracted with 5.0 ml of ethylacetate. A sharp interface between the ethylacetate and the reaction mixture was broughtabout by centrifugation for 5 min at 1,230 X g.A known amount of ethyl acetate containingformazan was diluted with a known amount ofethyl acetate to obtain a reading within thelinear range of the standard curve. The standardcurve was obtained by diluting known amounts offormazan with ethyl acetate, and the opticaldensities of the solutions were read at 495 m,u ona spectrophotometer (Bausch and Lomb Spec-tronic 20). The activity of the malic dehydro-genase preparation was expressed as mg formazanproduced per ml of reaction mixture. No formazanwas produced under the conditions of our15000-14000. 13004cz 120004 1100m0 1000zz 900i-0D 800w 700600a500Q 400t 300(I), 200a.-* 100zcFIG. 1. Pressure cylinder used for studies at101 C at various hydrostatic pressures.000AP= Pf- P;I 200 400 600 800 1000INITIAL PRESSURE, Pi, AT 57 C (ATM)FIG. 2. Initial pressure-final pressure relation-ship for the pressure cylinder illustrated in Fig. 1.r, 0 0I I I I I I0-II1342 [voL. 83-- at UNIV OF NEBRASKA-LINCOLN on August 30, 2007 jb.asm.orgDownloaded from MALIC DEHYDROGENASE ACTIVITY AT 101 Cexperiments in the absence of the enzymepreparation.Pressurization technique. The pressure appara-tus and technique described by ZoBell andOppenheimer (1950) was employed with oneexception. The pressure-cylinder cap was fittedwith a three-way valve so that a pressure gaugecould be attached (Fig. 1). Rupturing of theneoprene "O" ring seals at 101 C under pressurecaused difficulties. For this reason, constantobservation of the pressure gauge was necessaryto insure correct incubation times.The pressure cylinder was filled with water andequilibrated to 57 C. After the stoppered testtubes filled with reaction mixture were placed inthe pressure cylinder and the pressure-cylindercap secured, the pressure was elevated to thedesired initial pressure at 57 C (Pi in Fig. 2). Ifthe pressure cylinder was equilibrated to 101 Cbefore pressure was applied, the enzyme wouldundergo thermal denaturation before the hydro-static pressure could be elevated. A total of 6min was required for the initial pressure in thecylinder to reach the final pressure (Pf in Fig. 2).The data in Fig. 2 were obtained by use of thepressure cylinder shown in Fig. 1. In all caseswhere the temperature of 101 C was used, thepressure cylinder was cooled with cold waterbefore the pressure was released and the pressure-cylinder cap removed to obtain the reactionmixture. This process took approximately 1 min.For experiments at 1 atm and 101 C, thepressure-cylinder cap was removed and thecylinder filled with ethylene glycol and equili-brated at 57 C. The reaction mixture was addedto a long test tube fitted with a vapor stop andwas then placed in the pressure cylinder. Theentire assembly was immersed in the 101 Cethylene glycol bath as shown in Fig. 3. Thisprocedure was necessary since the increase intemperature from 57 to 101 C would increase thepressure if the pressure cylinder was capped.Also, approximately the same time element wasrequired for it to obtain thermal equilibrium asthe entire pressure cylinder.An Aminco constant-temperature bath, type R,which can maintain a liquid temperature of up to200 C (i 0.2 C), was utilized for this investiga-tion. The temperature was checked with aNational Bureau of Standards Certified thermo-meter.FIG. 3. Apparatus used for 1 atm studies at 101 CRESULTSEffect of pH. Preliminary studies on the malicdehydrogenase activity at 60 C demonstratedthat substrate saturation level was 50 MAmoles/mlof reaction mixture. In order that the substratewould not be a limiting factor, 100 ,umoles ofL-malic acid/ml of reaction mixture were used.This level showed no substrate inhibition. Therate of enzyme activity during a 6-min perioddid not decrease. By use of a substrate level of 100,umoles/ml of reaction mixture, the reactionmixtures were made up with varying pH valuesby using 0.2 M tris(hydroxymethyl) a mino me-thane (tris) buffer and incubated for 35 min at60 C. The data in Fig. 4 show an optimal pH ofapproximately 8.5 and also show that the activitycurve in relation to pH is not sharp. For allsubsequent studies, a pH of 8.5 was selected.Stability of DPN. Treatment of DPN atvarious pressures and temperatures for a periodof 44 min before addition to the rest of thereaction mixture revealed that activity was lost134319621 at UNIV OF NEBRASKA-LINCOLN on August 30, 2007 jb.asm.orgDownloaded from MORITA AND HAIGHTat 100 C (Table 1). DPN, in a concentration of 1mg/ml, was employed in the reaction mixturesince 60% activity was lost when DPN wastreated at 100 C and 1 atm for 44 min. Destruc-tion of DPN occurred more when treated at 100C and 1 atm for 44 min than when treated at100 C and 1,500 atm for 44 min.Activity of malic dehydrogenase at various-Ia wl- cc3N x 25c z 200 0co - I-4 W 10C 5A. 5TEMPERATURE * 60 C.7.7 7.9 8.1 8.3 85pH OF REACTIONwx-20i:4)Ida:24N0IL.0)I9 49a:0L8.7 8.1FIG. 4. Effect of pH on malic dehydrogenaseactivity at 60 C. The reaction mixture containedtris-HCl buffer, 1.3 ml (various pH levels, 0.2 M);L-malic acid, 1.0 ml (various pH levels, 400 ,umoles/ml); TPTZ, 2.5 ml (0.02 M); NaCN, 0.2 ml (0.05M); DPN, 0.5 ml (1 mg/ml); and 0.5 ml of dilutedmalic dehydrogenase preparation (153 ,ug proteinN/0.5 ml). Values are corrected for controls. In-cubation time was 35 min.TABLE 1. Stability of DPN at various hydrostaticpressures and temperatures as measured by theamount of formazan producedaTreatment of DPNb Fonnazan ActivityTreatmentDPNproducedc loStdzg/ml %28 C, 1 atm, 44 min........... 19060 C, 1 atm, 44 min........... 190 0100 C, 1 atm, 44 min........... 79 60100 C, 1,500 atm, 44 min....... 108 40No DPN.................... 0a The reaction mixture contained tris-HClbuffer, 1.3 ml (pH 8.5, 0.2 M); L-malic acid, 1.0ml (pH 8.5, 400 ,umoles/ml); TPTZ, 0.5 ml (0.02M); NaCN, 0.2 ml (0.05 M); 0.5 ml DPN (1 mg/ml);and 0.5 ml of the diluted malic dehydrogenasepreparation (153 ug per protein N per 0.5 ml).I Before addition to the malic dehydrogenasereaction mixture.c At 60 C for 35 min; tests were run in duplicateand corrected for controls.d Relative to treatment at 28 C and 1 atm for44 min.55 60 65 70 7TEMPERATURE (C)FIG. 5. Effect- of temperature on malic dehy-drogenase at 1 atm. The reaction mixture wasidentical to that in Fig. 4 except that the pH levelwas 8.5. Incubation time was 35 min. Values arecorrected for controls.temperatures at 1 atm. When the enzyme reactionmixture was subjected to various temperatures,an optimum of 55 C was noted (Fig. 5). Noactivity of the enzyme could be detected at 80 C,which indicated that thermal inactivation oc-curred just below 80 C.Activity of malic dehydrogenase at 101 C atvarious pressures. When the malic dehydrogenasereaction mixtures were subjected to a temperatureof 101 C at various pressures, it was noted thatno activity could be demonstrated at 1 to 700atm (Fig. 6). However, activity of the malicdehydrogenase was demonstrated when hydro-static pressure was elevated above 700 atm. Sinceit took 6 min for the initial pressure to reach thefinal pressure in the system, the 6-min pointsserved as the base line for these studies. At 1,300atm and 101 C, the amount of formazan pro-duced by the reaction mixture in 41 min wasapproximately 350% greater than the amountproduced in 6 min. The net activity at 101 C atvarious pressures, represented in Fig. 6 as delta(A), is the difference between the amount of1344 [VOL. 830 at UNIV OF NEBRASKA-LINCOLN on August 30, 2007 jb.asm.orgDownloaded from MALIC DEHYDROGENASE ACTIVITY AT 101 CgX 350.2 Mx 35Z 300I-~~~~~~~C.)'< 25w_j20zN 1-5L^ IIoCD1 500 700 900 1100 13001500FINAL PRESSURE (ATM)FIG. 6. Activity of malic dehydrogenase at 101C under various hydrostatic pressures. The reactionmixture was identical to that in Fig. 4 except thatthe pH level was 8.5. Delta (A) represents the differ-ence between the 6 min and 41 min incubation times.Values are corrected for controls.formazan produced in 41 min minus the amountformed in 6 min.Malic dehydrogenase displayed activity withtime at 101 C and 1,300 atm, whereas the curveat 101 C and 1 atm curve showed no activity(Fig. 7). The curve at 101 C and 1,300 atm isdefinitely not as pronounced as the curve at 58 Cand 1 atm.DISCUSSIONSince it is extremely difficult to measure pHshifts with increased hydrostatic pressure andtemperature, tris-HCl buffer was employed.According to Kauzmann (personal communica-tion), the organic amine types of buffers possess asmall A volume (v) (Av = +2 to +4 ml at 25 Cand 1 atm at infinite dilution) as compared tophosphate buffer (Av = -29 ml at 25 C and 1w z C 0:, 210X 195IS180zo 165o ISO4cc 135I 120a 105z 9 049N4 7540coIL 45a 30490 '50pH - 8.5, 0.2 M TRIS*HCI BUFFERI C- I ATM101 C-1300ATMM 0 0 101 C-1 ATMI I I I0 6 12 18 24 30 36INCUBATION TIME (MIN) AT STATED CONDITIONSFIG. 7. Rate of malic dehydrogenase activity.The reaction mixture was identical to that in Fig. 4except that the pH level was 8.5. The curve of 101 Cand 1,300 atm is corrected for the 6-min periodrequired for the initial pressure to reach the finalpressure. Values are corrected for controls.atm at infinite dilution). The pH shifts withpressure are minimized with systems thatpossess small Av values. Since the pH curve at60 C and 1 atm (Fig. 4) does not display a sharppeak, a slight shift in pH would not alter themain point of this investigation, which is theactivity of malic dehydrogenase at 101 C underhydrostatic pressure.Malic dehydrogenase activity at 1 atm wasdestroyed at approximately 78 C (Fig. 5), but,when the pressure was increased beyond 700 atm(Fig. 6), the molecular volume increase broughtabout by the increase in temperature was coun-teracted by the applied hydrostatic pressure.As a result of the applied pressure, the enzymedoes not undergo complete thermal denaturation.The extent of denaturation of the enzyme underthe pressure applied at 101 C awaits furtherinvestigation. Since a pressure cylinder has not19621 1345 at UNIV OF NEBRASKA-LINCOLN on August 30, 2007 jb.asm.orgDownloaded from MORITA AND HAIGHTbeen developed to compensate for the increasein pressure brought about by increased tem-perature, there is undoubtedly some denaturationof the enzyme during the time the pressurecylinder goes from its initial pressure to thefinal pressure (Fig. 2). Denaturation wouldprobably take place more at the lower pressuresapplied in our studies.We also realize that the rate of activity at 101 Cand 1,300 atm is quite low compared to the rateat 58 C and 1 atm. However, it should be remem-bered that we are probably not operating underthe optimal temperature for the malic dehydro-genase system. The data obtained, however,show clearly that malic dehydrogenase canoperate at 101 C when hydrostatic pressures of700 to 1,500 atm are applied.ACKNOWLEDGMENTSThe authors wish to acknowledge W. Kauz-mann of Princeton University for informationconcerning the effect of pressure on pH.This investigation was supported by researchgrant A-3784 from the National Institute ofArthritis and Metabolic Diseases, U.S. PublicHealth Service.LITERATURE CITEDBERGER, L. R. 1958. Some effects of pressure ona phenylglycosidase. Biochim. et Biophys.Acta 30:522-528.BROWN, D. E., F. H. JOHNSON, AND D. A. MARS-LAND. 1942. The pressure-temperature rela-tions of bacterial luminescence. J. CellularComp. Physiol. 20:151-168.HAIGHT, R. D., AND R. Y. MORITA. 1962. Theinteraction between the parameters of hy-drostatic pressure and temperature on as-partase of Escherichia coli. J. Bacteriol.83:112-120.JOHNSON, F. H., M. B. BAYLOR, AND D. FRASER.1948. The thermal denaturation of tobaccomosaic virus in relation to hydrostatic pres-sure. Arch. Biochem. 19:237-245.JOHNSON, F. H., H. EYRING, AND M. J. POLISSAR.1954. The kinetic basis of molecular biology.John Wiley & Sons, Inc., New York.JOHNSON, F. H., AND D. H. CAMPBELL. 1945.The retardation of protein denaturation byhydrostatic pressure. J. Cellular Comp.Physiol. 26: 43-46.JOHNSON, F. H., H. EYRING, R. STEBLAY, H.CHAPLIN, C. HUBER, AND G. GHERARDI.1945. The nature and control of reactions inbioluminescence. With special reference tothe mechanism of reversible and irreversibleinhibitions by hydrogen and hydroxyl ions,temperature, pressure, alcohol, urethane, andsulfanilamide in bacteria. J. Gen. Physiol.28:463-536.MARSH, J. M. 1956. A genetic study of thermo-phily. Thesis, University of Nebraska, Lin-coln.ZOBELL, C. E., AND C. H. OPPENHEIMER. 1950.Some effects of hydrostatic pressure on themultiplication and morphology of marinebacteria. J. Bacteriol. 60:771-781.1346 [VOL. 83 at UNIV OF NEBRASKA-LINCOLN on August 30, 2007 jb.asm.orgDownloaded from 

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MALIC DEHYDROGENASE ACTIVITY AT 101 C UNDER HYDROSTATIC PRESSURE

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