Biochem. J. (1977) 163, 449453
`Printed in Great Britain
`
`449
`
`Presence of a High-Molecular-Weight Form of Catalase in Enzyme
`Purified from Mouse Liver
`
`By MALCOLM B. BAIRD, HAROLD R. MASSIE and LINDA S. BIRNBAUM
`Masonic Medical Research Laboratory, 2150 Bleecker Street, Utica, NY 13503, U.S.A.
`
`(Received 16 November 1976)
`
`Ultracentrifugation studies of purified mouse hepatic catalase revealed that 5-7% of
`the total material consists of a form with a higher molecular weight than the bulk of the
`catalase. The two components were separated by sucrose-gradient centrifugation.
`Polyacrylamide-gel
`electrophoresis
`buffer)
`high-
`(in
`demonstrated
`that
`borate
`molecular-weight catalase is enriched in a more slowly migrating component, and
`sodium dodecyl sulphate/polyacrylamide gel-electrophoresis demonstrated that the
`molecular weight of the subunits of the high-molecular-weight material is identical with
`that of the subunits of the major form. These results suggest that high-molecular-weight
`catalase consists of subunits that are not markedly distinct from those present in the
`normal catalase tetramer.
`
`Catalase (EC 1.11.1.6) is a ubiquitous enzyme
`found in most tissues of aerobic organisms (de Duve
`& Baudhuin, 1966; Deisseroth & Dounce, 1970).
`The exact role of catalase within intact organisms
`remains obscure, although recent results strongly
`support the argument that a relationship exists
`between hepatic catalase activity and lipid meta-
`bolism (Reddy & Krishnakantha, 1975).
`Much is known about both the physical and chemi-
`cal properties of catalases from a variety of sources
`(Deisseroth & Dounce, 1970). The native enzyme is
`a slightly ellipsoidal protein, consisting of four
`polypeptide chains (Schroeder etal., 1969), each chain
`binding one molecule of protohaematin (Greenfield
`& Price, 1956). The molecular weights of several
`mammalian catalases have been tabulated, and range
`from 230000 to 250000 (Deisseroth & Dounce,
`1970).
`We have been interested in the possible role of
`catalase, as well as that of its substrate, H202, in
`senescence (Baird & Samis, 1971; Nicolosi et al.,
`1972; Samis et al., 1972; Baird et al., 1974, 1976;
`Massie & Baird, 1976). During the course of these
`studies hepatic catalase was purified from mouse
`liver, and examined by a variety of methods. In
`the present paper we give evidence that an active
`catalase molecule of higher than normal molecular
`weight exists in enzyme preparations purified from
`mouse liver.
`Vol. 163
`
`Experimental
`Materials
`C57B1/6J male mice (2 months old) were used
`throughout the study, and were maintained as
`described previously (Baird & Samis, 1971). SDS*
`(electrophoresis purity) was purchased from Bio-
`Rad Laboratories, Richmond, CA, U.S.A. Benzidine
`and Coomassie Blue were purchased from Sigma,
`St. Louis, MO, U.S.A. All other reagents and
`materials were of the best grade available.
`Methods
`Animals were killed at 09:00h EST. Livers were
`excised and pooled and catalase was purified by the
`method of Price et al. (1962). Catalase activity was
`assayed by a spectrophotometric method essentially
`as previously described (Baird & Samis, 1971).
`Sedimentation-velocity determinations were per-
`formed with a Beckman model E ultracentrifuge.
`Purified catalase was applied to 5-15% (w/v)
`sucrose (in water) gradients and centrifuged in a
`Beckman L5-40 ultracentrifuge. Pertinent details
`of these and other methods are given in the appro-
`priate Figure legends.
`electrophoresis of catalase
`Polyacrylamide-gel
`was performed in borate:buffer as described by
`Baird et al. (1976), or in 0.1 % SDS as described in
`detail by Welton & Aust (1974). Peroxidatic activity
`* Abbreviation: SDS, sodium dodecyl sulphate.
`
`Dexcom Inc. v. WaveForm Technologies, Inc.
`IPR2017-01051
`Exhibit 1019
`
`

`

`430
`
`M. 13. BAIRD, HI. R. MASSIE AND L. S. 7BIRNBAUMI
`
`of material on the gels was detected by staining with
`benzidine (Welton & Aust, 1974). Benzidine was
`handled with extreme caution as it is known to be
`carcinogenic in mammalian systems. The peroxidase-
`stained gels were scanned at 615nm in a linear gel
`transport with a Gilford 2400S spectrophotometer.
`We have found that such stains are stable when the
`gels are rinsed in water immediately after the
`appearance of the blue colour. Duplicate gels were
`stained for protein with Coomassie Blue (Fairbanks
`et al., 1971) and scanned at 550nm. Reference
`standards (with molecular weights in parentheses)
`were as follows: phosphorylase a (94000); bovine
`serum albumin (66500); pyruvate kinase (57000);
`aldolase (40000); lactate dehydrogenase (36000);
`lysozyme (14300); cytochrome c (11700).
`
`Results and Discussion
`The u.v.-visible spectrum of purified mouse liver
`catalase is presented in Fig. 1. We estimated that this
`material was greater than 99% pure (A407/A276 1.14),
`assuming that the criteria for purity of rat liver
`
`0.6r
`0.5
`0.4
`0.3-
`0.2h
`0 I0
`
`'1
`
`50k
`
`1000
`
`I0
`104x Molecular weight
`Fig. 3. Relationship between s2O,W and molecular weightfor
`purified mammalian catalases and subunits of catalase
`generated by various treatments
`Data were taken from Deisseroth & Dounce (1970).
`The stars (*) represent the s value for high-molecular-
`weight catalase and normal mouse liver catalase.
`
`I.0
`
`> 0
`
`.4.I
`cd
`
`.6
`
`1. 4
`
`0
`
`I .c
`
`0.8
`
`0.6
`
`0.41
`
`0.21
`
`0
`
`0
`
`200
`
`300
`500
`600
`400
`Wavelength (nm)
`Fig. 1. U.v.-visible scan ofpurified nmuse hepatic catalase
`Protein concentration was 300,ug/ml in 0.02M-sodium
`phosphate buffer, pH7.0.
`
`700
`
`800
`
`(a)
`
`(b)
`
`(c)
`
`g
`Fig. 2. Tracings of representative scans ofpurified mouse
`hepatic catalase after (a) 16, (b) 26 and (c) 36min of
`centrifugation in a Beckman model E ultracentrifuge
`Samples were centrifuged at 56000rev./min in 0.02M-
`sodium phosphate buffer, pH6.8, and scanned at
`407nm. Protein concentration was 520pg/ml. Direc-
`tion of force is represented by g.
`
`Fraction no.
`Fig. 4. Sucrose-density-gradient centrifugation of mouse
`hepatic catalase
`Purified catalase (O.1 ml, 4.55mg/ml) was applied to
`a 5-15%/ (w/v) sucrose gradient. The material was
`centrifuged for 7.5h, at 40000rev./min, in a 50.1
`rotor in a Beckman L540 ultracentrifuge. Fractions
`(10 drops) were collected and 0.1 ml of 0.02M-sodium
`phosphate buffer, pH6.8, was added to each
`fraction, and the
`fractions were analysed for
`A407 (m) and total units of catalatic activity (A).
`
`catalase established by Price et al. (1962) are
`applicable to mouse catalase.
`Centrifugation of this material in the model E
`ultracentrifuge (0.8A unit) revealed the presence of
`1977
`
`Dexcom Inc. v. WaveForm Technologies, Inc.
`IPR2017-01051
`Exhibit 1019
`
`

`

`HIGH-MOLECULAR-WEIGHT CATALASE
`
`451
`
`0.4
`
`(a)
`
`0.3
`
`0.2
`
`0. l
`
`o
`
`(b)
`
`0.5 _
`
`l.
`
`}
`
`I
`
`\
`
`0.4
`
`0.3
`
`0.2
`
`0.1
`
`o
`
`0.2 (c-
`
`0.
`
`0
`
`Vol. 163
`
`IT
`
`I
`
`1
`
`3
`2
`Distance (cm)
`
`4
`
`5
`
`6
`
`two components (Fig. 2). The major component
`had s20,w 11.1S, and the minor component had
`520ow 14.8S. The minor component accounted for
`5-7% of the total material.
`We have plotted (Fig. 3) the data collected by
`Deisseroth & Dounce (1970) relating corrected s
`values and molecular weights of native catalases as
`well as for subunits of catalase. We estimated by
`extrapolation that the molecular weight of the major
`component of mouse hepatic catalase was 235000,
`and that of the high-molecular-weight catalase was
`345000. This value, of course, assumes certain
`similarities between the two catalase molecules, and
`may be in error.
`The two components were readily separated on a
`sucrose gradient (Fig. 4), in terms of both A407 and
`catalatic activity. Since the ratio between the absorb-
`ance of the Soret band (407nm) and that of the pro-
`tein band (276nm) in purified mouse hepatic catalase
`is nearly unity (Fig. 1), the A407 is a useful estimate
`content. We determined from the
`of protein
`results of numerous sucrose gradients that
`the
`specificenzyme activity (enzyme activity/A407) of both
`the major form ofcatalase and high-molecular-weight
`catalase is identical. Therefore we suggest that the
`haematin content of both forms is similar, although
`we have not directly measured the haematin content
`of high-molecular-weight catalase.
`Purified enzyme, as well as the material under the
`l ltwo peaks resolved by centrifugation (Fig. 4), were
`subjected to both SDS/polyacrylamide-gel electro-
`phoresis and gel electrophoresis in borate buffer.
`Separation of the purified material on the basis of
`size and charge (borate buffer) revealed the presence
`of two components with peroxidatic activity (Fig. 5a).
`Electrophoresis of the material under the two peaks
`resolved on a sucrose gradient demonstrated that the
`material under the major peak (Fig. 5b) corresponds
`to the more rapidly migrating material in the purified
`catalase, and the material in the minor peak is en-
`riched in more slowly migrating material (Fig. 5c).
`Separation of the material in the purified catalase,
`as well as the material in both peaks in the presence of
`0.1% SDS, reveals the presence of two components
`(Fig. 6). The more rapidly migrating component
`has mol.wt. 60000 and the more slowly migrating
`minor component has mol.wt. 120000. We suggest
`that the 120000-mol.wt. component is a contaminant,
`
`Fig. 5. Non-dissociating polyacrylamide-gel electrophoresis
`ofpurified mouse hepatic catalase activity before and after
`fractionation on a sucrose gradient
`Origin of the run was at the cathode (0). Gels were
`stained for peroxidatic activity with benzidine.
`(b) major component (see
`(a) Purified catalase;
`Fig. 3); (c) high-molecular-weight catalase (more
`rapidly sedimenting component, see Fig. 3).
`
`Dexcom Inc. v. WaveForm Technologies, Inc.
`IPR2017-01051
`Exhibit 1019
`
`

`

`452
`
`M. B. BAIRD, H. R. MASSIE AND L. S. BIRNBAUM
`
`(a)
`
`3.6
`
`3.2
`
`2.8
`
`2.4
`
`2.0
`
`1.6
`
`1.2
`
`0.8
`
`0.40c
`
`I_
`
`I
`
`I
`
`I
`
`I
`
`I
`
`° 3.2 -
`
`(b)
`
`2.8-
`
`2.4-
`
`-2.0-
`
`1.6
`
`1.2-
`
`0.8
`
`0.4
`
`0.4
`
`(c
`
`0
`
`2
`
`6
`4
`Distance (cm)
`
`8
`
`10
`
`inasmuch as it does not possess any detectable
`peroxidatic activity. Therefore high-molecular-weight
`catalase appears to consist of subunits that are
`identical with those present in normal tetrameric
`catalase.
`We have not determined whether high-molecular-
`weight catalase possesses some function in vivo, or
`is generated during the purification process. There are
`conditions under which ox liver catalase aggregates
`into higher-molecular-weight species (Itoh et al.,
`1962). However, spontaneous aggregation into high-
`molecular-weight catalase most likely does not occur,
`as identical electrophoretic results are obtained
`whether performed in the presence or absence of di-
`material,
`dilution of the
`thiothreitol.
`Further,
`followed by ultracentrifugation, does not alter the
`percentage of the total material accounted for by
`high-molecular-weight catalase. Finally, high-mole-
`cular-weight catalase is not present in catalase pre-
`parations purified from CFN rat liver.
`Other studies from our laboratory (Baird et al.,
`1976) indicate that the response of mouse liver
`catalase activity to the pharmacological agent allyl-
`isopropylacetamide is different from that observed
`in rats. High-molecular-weight catalase appears to
`be another anomalous feature ofmouse liver catalase.
`
`This research was supported by the Masonic Founda-
`tion for Medical Research.
`
`References
`Baird, M. B. & Samis, H. V., Jr. (1971) Gerontologia 17,
`105-115
`Baird, M. B., Zimmerman, J. A., Massie, H. R. & Samis,
`H. V. (1974) Gerontologia 20, 169-178
`Baird, M. B., Samis, H. V., Massie, H. R., Zimmerman,
`J. A. & Sfeir, G. A. (1976) Biochem. Pharmacol. 25,
`1101-1105
`de Duve, C. & Baudhuin, P. (1966) Physiol. Rev. 46,
`323-357
`Deisseroth, A. & Dounce, A. L. (1970) Physiol. Rev. 50,
`319-375
`Fairbanks, G., Steck, T. L. & Wallach, D. F. H. (1971)
`Biochemistry 10, 2606-2616
`Greenfield, R. E. & Price, V. E. (1956) J. Biol. Chem.
`220, 607-617
`Itoh, M., Nakamura, Y. & Shibata, K. (1962) Can. J.
`Biochem. Physiol. 40, 1327-1334
`
`Fig. 6. SDS/polyacrylamide-gel electrophoresis ofpurified
`mouse hepatic catalase before and after fractionation on
`a sucrose gradient
`Origin of the run was at the cathode (0). Gels were
`stained with Coomassie Blue. (a) Purified catalase;
`(b) major component (see Fig. 3); (c) high-molecular-
`weight catalase (more rapidly sedimenting com-
`ponent, see Fig. 3).
`
`1977
`
`Dexcom Inc. v. WaveForm Technologies, Inc.
`IPR2017-01051
`Exhibit 1019
`
`

`

`HIGH-MOLECULAR-WEIGHT CATALASE
`
`453
`
`Massie, H. R. & Baird, M. B. (1976) Mech. Ageing Dev. 5,
`39-43
`Nicolosi, R. J., Baird, M. B., Massie, H. R. & Samis,
`H. V. (1972) Exp. Gerontol. 8, 101-108
`Price, V. E., Sterling, W. R., Tarantola, V. A., Hartley,
`R. W., Jr. & Rechcigl, M., Jr. (1962) J. Biol. Chem.
`237, 3468-3478
`Reddy, J. K. & Krishnakantha, T. P. (1975) Science 190,
`787-789
`
`Samis, H. V., Baird, M. B. & Massie, H. R. (1972) in
`Molecular Genetic Mechanisms in Development and
`Aging (Rockstein, M. & Baker, G., eds.), pp. 133-143,
`Academic Press, New York
`Schroeder, W. A., Shelton, J. P., Shelton, J. B. & Olsen,
`B. M. (1969) Arch. Biochem. Biophys. 131, 653-655
`Welton, A. F. & Aust, S. D. (1974) Biochem. Biophys. Res.
`Commun. 56, 898-906
`
`Vol. 163
`
`Dexcom Inc. v. WaveForm Technologies, Inc.
`IPR2017-01051
`Exhibit 1019
`
`