Download 34r PROCEEDINGS OF THE BIOCHEMICAL SOCIETY Centre

34r
PROCEEDINGS OF THE BIOCHEMICAL SOCIETY
Spranger, J. W., Koch, F., MoKusick, V. A. Matzschka,
J., Wiedemann, H. R. & Zellweger, H. (1970). Helv.
paediat. Acta, 25, 337.
Van Hoof, F. & Hers, H. G. (1968). Eur. J. Biochem. 7, 34.
enzyme (B) has a molecular weight of approx.
340000. In concentrated solution (10-40mg of
protein/ml) this B form can aggregate to a larger,
less active, form (A). On dilution or on detergent
treatment the B form dissociates into probably
two similar subunits.
Properties of Phosphoenolpyruvate Carboxyl- Lowe, J. & Slack, C. R. (1971). Biochim. biophy8. Acta,
235, 207.
ase Isolated from Maize Leaves
By M. W. KERR and A. ROBERTSON. ('Shell'
Re8earch Ltd., Wooddtock Agricultural Research
Centre, Sittingbourne, Kent, U.K.)
Phosphoenolpyruvate carboxylase (EC 4.1.1.31) Carbonic Anhydrase: Multiple Forms in
has been purified 100-fold from maize leaves in Plants
25% yield. The purification method differs from
that of Lowe & Slack (1971) and involves By B. D. PATTERSON,* C. A. ATKINS and D.
isoelectric focusing as the final step (pI5.2-5.3). GRAHAM. (Plant Physiology Unit, Commonwealth
Specific activities of better than 40U/mg of protein Scientific and Indu8trial Re8earch Organization
Divi8ion of Food Re8earch, Sydney, N.S.W.
were obtained.
The enzyme was most stable in 1 mM-dithio- 2113, Au8tralia)
threitol at 150C at pH7.0. When glycerol was
Carbonic anhydrase (EC 4.2.1.1) catalyses the
added to the enzyme solution to a final concentra- hydration
of carbon dioxide to give HC03- and
activity
its
specific
it
increased
tion of 50% (v/v)
H+
ions:
and helped to maintain its activity.
C02 + H20 = HC03-+H+
If purified enzyme was fractionated on an agarose
column (Bio-Gel A1.5), two protein bands emerged: It is present in the green parts of plants, and
first a small band of low-activity protein (A) appears to be important for the efficient fixation
followed by a large band of lighter high-activity of carbon dioxide (Graham & Reed, 1971). Isoprotein (B). If samples of band B material were enzymes of carbonic anhydrase from animals can
re-run on the same column after 24h storage at often be located after starch-gel electrophoresis by
15°C, two band were eluted at the same positions using their relatively weak esterase activity
as bands A and B; after 5 days' storage the pro- (Tashian, 1965). We could not detect any esterase
portion of band A material had increased at the activity associated with the carbonic anhydrase
expense of band B material.
from the leaves of the angiosperm Trade8cantia
Band B material sedimented as an homogeneous albiflora Kunth. We therefore devised a method to
protein in the analytical ultracentrifuge with an locate the enzyme after electrophoresis on polyestimated 8 0 W value of 13.5±0.3 S (possible vari- acrylamide gels. The gels are surface-stained with
ation in the slope of the extrapolation to zero con- Bromocresol Purple and exposed to carbon dioxide.
centration). The diffusion coefficient was estimated H+ ions produced by the enzyme-catalysed
to be 3.77 x 10 7cm2 .s- and the partial specific hydration of carbon dioxide change the colour of
volume as 0.74cm3 g-1. From these data a the indicator. The differential staining of the
molecular weight of 340 000 ± 8000 was calculated. enzyme bands is a transient phenomenon, but
When a solution of band B material was diluted further hydration is stopped by freezing the gels on
from 7mg of protein/ml to 5.5mg/ml, a second com- solid carbon dioxide. At low temperatures solid
ponent was seen with a sedimentation rate cor- solutions of Bromocresol Purple fluoresce brightly
responding to approximately half the molecular under u.v. light, the acid form yellow and the base
weight of the main component.
form pink, and the position of carbonic anhydrase
Treatment of band A material and band B is therefore shown on the frozen gels as yellow bands
material with sodium dodecyl sulphate followed against a pink background.
by polyacrylamide-gel electrophoresis in the preExtracts of leaves from higher plants were separsence of this detergent showed a single component ated by electrophoresis through gradients of- pQlywith similar mobility in each case. Comparison acrylamide (Margolis & Kenrick, 1968). All the
with the mobilities of protein standards indicated extracts examined gave at least two major bands
a molecular weight of approx. 160000 for the of carbonic anhydrase activity. Although it is
detergent-treated enzyme.
* Present address: East Malling Research Station,
Phosphoenolpyruvate carboxylase from maize
leaves appears to exist in several forms. The active Maidstone, Kent, U.K.