NOTES

Two Forms of a Mitschondrial Endonuclease in Neurospora crassn CHARLES E. MARTINAND ROBERT P. WAGNER Can. J. Biochem. Downloaded from www.nrcresearchpress.com by Depository Services Program on 11/11/14 For personal use only.

iJepartmet~tof Zoology, Ut~iversityof Texas, Austin, Texas 75712

Received January 2, 1975 Martin, C. E. & Wagner, R. P. (1975) Two Forms of a Mitochondria1 Endonuclease in Neurosporcz crassa. Can. J. Biochem. 53, 823-825 Mitochondrial nuclease activity in Neurospora crassa occurs in membrane-bound and soluble forms in approximately equal proportions. These activities apparently are due to the same enzyme, which has an approximate molecular weight of 120 000. A portion of the insoluble enzyme appears to be associated with the inner mitochondrial membrane and is resistant to solubilization by detergent treatment as well as by physical disruption methods. Martin, C . E. & Wagner, R. P. (1975) Two Forms of a Mitochondria1 Endonuclease in ~Venrosporncrnssa. Cart. J. Biocitem. 53, 823-825 Chez Neurosprn cmssa, I'activitC nucl6asique mitochondriale se partage en proportions approximativement Cgales entre une forme liCe i la membrane et une forme soluble. Ces deux formes seraient dues i la meme enzyme dont Be poids molCculaire est d'environ 120 Bo. Une partie de 19enzymeinsoluble serait associCe ii la membrane mitochondriale interne et elle rdsiste i la solubilisation par traitement avec un detergent de mCme qu'aux techniques physiques [Traduit par le journal] de degradation.

Gilford recording spectrophstometer. Protein was estiIntroduction mated by the method of Lowry et nl. (7). previously Linn and Lehman (I) have Routine nuclease assays were run with native DNA the existence of an endonuclease acting on single- (Sigma, type I) using the hyperchrornicity method of stranded and double-stranded DNA, as well as Kunitz (8) with the following modifications: assays were conducted at 34 "C in a Gilford recording spectrophoRNA, to produce 5~~p~osp~omonoester~termitometer with a reaction mixture of 100 gg DNA, 2 mM in Neuros~ora MgSO,, and 50 m M Tris buffer (pH 7.7) in a total volume mitochondria. Other mitochondria1 nuclease ac- of 1 ml. tivities have been found also in yeast (2), rat Assays involving comparison of activity upon native liver (31, and mammalian cells (4); however, their and denatured DNA substrates were performed by substrate specificities appear to vary from those adding enzyme to 1 ml reaction mixtures containing 500 gg DNA, 2 m M MgSQ4,and 50 m M Tris (pH 7.5) or of the Neurospora enzyme. 0.1 M phosphate buffer (pH 6.5). The reaction was In the course of studies to elucidate the func- terminated by the addition of 1 ml of lo:& tricl~loroacetic tion of this nuclease activity and its localization acid (TCA) or 0.57; uranyl acetate in 1 OC8; TCA. Portions within the organelle it became apparent that it were mixed and allowed to stand in an ice bath for 10 min by centrifugation at 3000 r.p.m. for 20 min. occurs in membrane-bound and soluble forms. followed Samples of the supernatant were diluted then wit11 water, Furthermore, these forms appear to represent the to determine the absorbance at 260 nm. Denatured DNA same enzyme. was prepared by immersing stock solutions of DNA in a

Materials and Methods

Mitochondria from wild type Ne~~rosporu C I ~ U S S Ustrain LSDT (1969) were prepared by the sand-grind method previously described (5). The crude mitochondria1 fraction used for digitonin fractionation experiments was purified by the sucrose gradient method of Cassady et a1. (5, 6). All other experiments were performed with tnitochondria washed three times by differential centrifugation with 50 m M Tris-HC1 buffer (pH 7.7) containing 0.15%; bovine serum albumin and 0.25 A4 sucrose. Bigitonin fractionation and marker enzyme assays were also performed according to the previously published methods (6) with the exception that succinate cytochreme c reductase (complex 11-1 11) was assayed at 35 "C in a

boiling water bath for 10 min, following by quick-cooling in an ice water slurry. Both nuclease assay methods gave results which were linear with respect to protein concentration and time. Solubilization of nuclease activity from mitochoildrial membranes was achieved by treating purified mitochondria with 4 mg sodium deoxycholate per milligram of mitochondrial protein at 4 "C, followed by centrifugation at 140 000 g for 30 min to remove a clear pellet which contained no nuclease activity. Triton X- 100 (20';G) was added to the supernatant to a final concentration of 2(,; and the mixture was fractionated on a G-50 column at room temperature with 0.05 M Tris (pH 7.8) as the eluting buffer. The active fractions from the column were pooled and concentrated by ultrafiltration in a Diaflo apparatus with a PM-30 membrane filter. No detectable

824

CAN. 3. BIBCHEM. VQL. 53, 1975

TABLEI . Distribution of nucleaae activity in e~itochondrialsubfractions following treatment with 0.6 mg digitonin

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per milligram of mitochondria1 protein@

Fraction S-1 B- l $2 B-2

Kynurenine 3-hydroxylase

-..-.

.-

-

Tf.Act,

(,'cC.

T. Act.

22 7.7 4.3 3.9

965 33

82 2.8

12 0

L .O 0.0

1.59 2.30 2.03 1.76

38.1

1010

44$ 4

1180

,

.

Eildonuclease

SCCR

Proteiu (mg)

-

C.

T. Act.

10,S 15,3

1160 77.5 65 41.3

o,oz

13.5 Il,7

"b

c.

52.6 22.5 21.3 13.5

Amount

recovered Control

85.6 100

7.67 15

51 180

344

133

394

1 0

OMitochondria were incubat& with digltonjn and sucrose step-gradients were prepared aceardlng te previously described procedures (6). Control activities are from whole mitachorP$rls,Triton X-100 (1 O/o) was added to the nuclease contr~lto measure totat activity. Enzyme activities are i n the following units: kynurenine 3-hydroxylam, micromoles 3-hydroxykynurenine produced per hour: ~uccinufecytochrom c reductase (SCCR),micromoles cytochrome c redw& per minula; endonuclease, O.D.:ao units per 8 min, Suoroae gradient fractions are listed from the top sf the centrifuge tube as follows: S-1,O.l M s w ~ o m B-B, ; interface of 0.1 and 1.0 IMsucrose; SJ, 1,U M ~ P ~ C P Q J BB-2, ; interface between 1.0 M sucxose and 1.9 bf sucrose cushion, Abbreviations: T. Act., total ilCtlvlty; % C., pergentage of control activity.

loss of total activity was observed with this procedure. This cancentrated fraction is unstable in the absence of deoxychofate and has a tendency to form a precipitate with time. In Jater experiments sodium desxycholate was readded to these preparations to prevent this. Sucrose gradients (5-28(:1,) were centrifuged 17 h at 5 "C in an S W 41 rotor at 35 000 r.p.m. Beef heart catdase, Neuruspra malate dehydrogenase and dihyciroxy;t~~id &hydrataw (EC 4.2.1.9) were used as molecular weight markers. Catalase activity was determined by the methad sf Beers and Sizer (9).

estimated by sucrose gradient centrifugation utilizing flanking enzymatic molecular weight markers. Treatment of mit~shondriawith 0.6 mlg digitonin per milligram ~f mitochondria1 protein, and separation of mita~hondrialsubfractions on sucrose step gradients (Table I) yields a fraction (B-2) which is devaid of kynurenine 3-hydroxylase activity and yet coatwins both SCGR and nuclease activity, giving evidence of intrinsic attachment of a partion af the insoluble nuclease Results to the inner membrane. The S-l fraction contains Initial experiments showed that digitonin only approximately oneshalf sf the total nuclease partially releases nuclease activity from Neuro- qctivity, which apparerltly corresponds to the spur4 mitoch~ndriawhen compared with enzy- "soluble" fraction, while the remainder of the matic markers of the various organelle subfrac: nuclease activity is distributed through the tians, Low concentrations of digitonin released gradient in a pattern similar to that observed nuclease in quantities paralleling those of rnalslfp for SCCR. dehydcogenase, a soluble matrix enzyme, ap@ Although these data suggest the presence of kynurenine 3-hydroxylase (EC 1.14.13.9), p R: two forms of nuclease activity in the mitosumed outer membrane marker (5). ~ i g $fkR- chondrion, several fines of evidence indicate that centrations (0.4 mg digitonin per milligfp-? gf this may represent two forms of the same enmitochondria1 protein) indicated a &~hggge zyme, rather than two different proteins. activity which is as insoluble as the jager'ineffl: brane marker, succinate cytochrome F ~ Q u c t q g g Results-2 Both fractions are primarily endonucleolytic, (SCCR). Approximately equal a m o i ~ ~f t~ uble" and "insofuble" nuclease acfjyjgjes ,.. . y g ~using ~ a test described by Mills and Fraser (10). The rate of increase in absorbance of assays found under these conditions. Triton X-IOOand physical metJtq& g p d ~2g terminated by trichloroacetic acid (TCA) is osmotic shock, sonication and fre~tg.-thagi~sabout two times greater than those terminated also produce membrane-bound and sql~blef q f ~ $ with uranyl-acetate - TCA. Furthermore, both in similar proportions. The soluble q g c FEE~ ~fractions ~ ~ are ~ capable of hydrolyzing native and kased by these methods, as well as by digit@^&? single-stranded DNA as well as RNA at similar has a molecular weight of approximaf~ly120 Ratf, rates.

NOTES

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Complete solubilization of the nuclease can be obtained with virtually no loss of activity by treatment of mitochondria with a mixture of deoxycholate and Triton X-100, followed by fractionation on Sephadex to remove sucrose and most of the detergent mixture. Sedimentation of this solubilized enzyme in a sucrose gradient with 1 mg of deoxycholate per millilitre reveals a single, homogeneous peak of activity with a sedimentation value consistant with that found for the soluble enzyme. Thermal denaturation of the solubilized enzyme at 38 "C is linear, also indicating the existence of a single enzyme. Kinetic data from the two fractions reveal no significant differences with regard to either substrate specificity or pH dependence. The ratio of activity of these fractions on native DNA (pH 6.5/7.5) is 0.97 and 0.92 for the soluble and membrane-bound forms, respectively. Similarly, the native/denatured substrate activities at pH 7.5 are 0.97 and 0.92. These data point out a marked difference between the Neursspora enzyme and a yeast enzyme described by other workers (2). The small differences in hydrolytic rates between native and denatured substrates contrast strongly with the high denatured/native activity ratio of the yeast enzyme. In addition, the optimal pH for hydrolysis of native DNA by the yeast enzyme is in the range of pH 5.2, while the Neursspsra enzyme exhibits a pH optimum in the range of pH 7.0-7.8 (1) and has no detectable activity below pH 6.0.

Discussion Dual localization of rat liver mitochondria1 nuclease has been reported previously (3), however, that enzyme differs from the Neurospom enzyme both with regard to substrate specificity and localization of the membrane-bound form. The function of these nucleases still is not clear, although localization of a portion of the Neurospora enzyme on the inner membrane raises the possibility of its involven~entin mitochondrial nucleic acid metabolism. The authors would like to thank Ms. Deborah Lin for her expert technical assistance and Dr. Guy Thompson for his helpful criticisms. This research was supported by National Institutes of Wealth grants GM-52877, GM12323, NIH career award SM-18383 and a grant from the Robert A. Welch Foundation. I . Linn, S . & Lehman. I. R. (1966) J , Biol. Cl~em.241, 2694-2699 2. Paoletti, C., Couder, H. & Guerineau, hl. (1972) Blochem. Biopla)~~. Res. Commulr. 48, 950-958 3. Morais, R. (1969) Biochetn. Biopllys. Ast6t 189, 38-45 4. Durphy, M . , Manly, P. N. & Friedberg, E. C. (1974) J . Cell Biol. 62, 695-706 5. Cassady , W. E. & Wagner, R. P. (1 97 1) J. Cell Biol. 49, 536-541 6 Cassady, W. E., Leiter, E. H., Bergquist. A. & Wagner, R. P. (1972) J. CeN Biol. 53, 66-72 7. bowry, 0. H., Rosebrough, J. H., Farr, A. L. & Randall, R. J. (1951) J. Biol. Clrem. 193, 265-275 8. Kunitz, M. (1950) J. Getl. Pl~ysiol.33, 349-362 9. Beers, R. F., Jr. & Sizer, I. W. (1952) J. Riol. Claem. 195, 133-140 10. Mills, C. & Fraser, M. J. ( 1973) Catr. J. Bioclrem. 51, 888-895

Erratum: Lysolecithin-Cholesterol Interaction. A Spin-Resonance and Electron Micrographic Study A. D. PURDON, J. e. HSIA,L. PINTERIC, AND D. 0. TINKER Departments of Biochemistry arrd Pknrmacology, Universitj~of Toronto, Toronto, Ontario 1w5S IAN AND

R. P. RAND Department of Biological Sciences, Broock U~tiversity,St. C~itharirres,Ontario LZS JA1

Received July 8, 1974 (Ref. : Can. J. Biochem. 53, 196-286 (1975))

The legends for Figs. 4 and 5 should be interchanged.