Biochem. J. (1983) 215, 447-455 447Printed in Great Britain

Regulation of activity of purified guanylate cyclase from liver that isunresponsive to nitric oxide

Su-Chen TSAI, Ronald ADAMIK, Vincent C. MANGANIELLO and Martha VAUGHANLaboratory ofCellular Metabolism, National Heart, Lung, and Blood Institute, National Institutes ofHealth,

Bethesda, MD 20205, U.S.A.

(Received 7 April 1983/Accepted 21 July 1983)

Guanylate cyclase was purified from rat liver supernatant. Electrophoresis underdenaturing conditions revealed one major peptide of Mr approx. 69 OQO. On the basis ofthe Stokes radius (4.7nm) and s20,w (6.4S), the calculated Mr value of the nativeenzyme was 133000, i.e. it is apparently a homodimer. Kinetics of inactivation bydiamide (which was reversible with djthiothreitol) suggested that oxidation of a singleclass of thiol sites was involved. In the absence of other additions, cyclase activityassayed with Mn2+ was over 7 times that assayed with Mg2+; maximal effects wereobserved with approx. 5mM of each (with 1mM-GTP). The purified enzyme wasmarkedly activated by nitrosylhaermoglobin. Relative activation was much greater inassays with- Mg2 than with Mn2t, although maximal activities were similar. Whenassayed with Mg2+, the enzyme exhibited a single Km (0.35 mM) for GTP; with Mn2+,plots of 1/v versus 1/[SI were nofi-linear. Activator or nitrosylhaemoglobin increasedVmax but did not alter Km in the presence of either Mg2+ or Mn2+. The enzyme wasinhibited by Na3VO4, Na2WO4 and Na2B407. Reduction from vv to VIv abolished theinhibitory effect of vanadate. Na2B407 (2 mM) inhibited activity with Mn2 , but not with-Mg2+. In assays with Mg2+, but not with Mn2 , FMN, NAD+ and NADH (each0.5 mM) inhibited activation by protoporphyrin IX and nitrosylhaemoglobin. Rotenone(0.6mM) inhibited activity with.protoporphyrin IX to a greater extent than with nitrosyl-haemoglobin. Methylene Blue (1 mM) inhibited activation by nitrosylhaemoglobin,protoporphyrin IX and actiyator. It appears that this enzyme purified from rat liverlacks haem (and perhaps other components) required for activation by NO, and itshould be particularly useful for elucidating the mechanism of action of NO, proto-porphyrin IX and other activators.

NO and compounds from which it can arise (e.g.nitroprusside, azide) activate soluble guanylatecyclase from virtually all mammalian tissues (forreview see Mittal & Murad, 1977). Craven &DeRubertis (1978) first demonstrated the import-ance of haem and haemoproteins in NO activation.Although it has been suggested that the purifiedenzyme can be directly activated by NO (Braughleret al., 1 979a, b), mnore recent reports indicate the pre-seAce of haem in purified preparations of guanylate,cyclase that respond to NO, nitroprusside orprotoporphyrin IX (Gerzer et al., 198 1a,b; Ignarroetal., 1982a,b).As a step towards elucidating the mechanisms of

action of NO and other activators, we have purifiedsoluble guanylate cyclase from liver in a form that is

* Abbreviations used: Hb, haemoglobin: SDS, sodiumdodecyl sulphate.

activated little, if at all, by NO and apparently lackshaem. We report here some physical and kineticcharacteristics of, and effects of nitrosylhaemo-globin and a heat-stable activator on, the purifiedenzyme. We have also compared effects of Hb andactivator on NO activation as well as effects of anumber of inhibitors on enzyme activity.

Materials and methodsMaterials

Nitroprusside, haematin, haemoglobin, proto-porphyrin IX, phenylmethanesulphonyl fluoride,GTP-agarose, GTP, cyclic GMP, cytochrome c,ferredoxin, FMN, NAD+, NADH, Methylene Blueand reduced glutathione were from Sigma ChemicalCo.; rotenone was from Aldrich Chemical Co.;diamide was from Calbiochem; DEAE-cellulose wasfrom Whatman; phenyl-Sepharose and Affi Blue

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S.-C. Tsai, R. Adamik, V. C. Manganiello and M. Vaughan

Sepharose were from Pharmacia; [a-32P]GTP andcyclic [3H]GMP (purified on t.l.c.) were from NewEngland Nuclear; Ultrogel AcA-34 was from LKB;DEAE-Bio-Gel was from BioRad; NO (99%) wasfrom Matheson Gas; Na2WO4, Na2B407 andNa3VO4 (ortho) were from Fisher Scientific;Na3VO4 (VV) was reduced to the Vlv level byaddition of 10-fold concentrated ascorbate for30min (Cantley et al., 1977).

Purification ofguanylate cyclaseGuanylate cyclase was purified from rat liver by a

modification of our published procedure (Tsai et al.,1978, 1980). Results for a representative prepara-tion are shown in Table 1. Liver (880g) washomogenized in 1800 ml of 0.25 M-sucrose in 10mM-Tris/HCl buffer, pH 7.4, containing 2 mM-dithio-threitol and 1mM-EDTA (buffer A), with 0.5mM-phenylmethanesulphonyl fluoride. The homogenatewas centrifuged (7000g, 60min), and the super-natant was applied to DE-52 DEAE-cellulose (1.5litres of gel), which was washed successively with 2litres of buffer A, 2 litres of buffer A containing75mM-NaCl, and 2 litres of buffer A containing175 mM-NaCl. Guanylate cyclase was eluted with 2litres of buffer A containing 200mM-NaCl anddirectly applied to phenyl-Sepharose (700ml). Thephenyl-Sepharose was washed with 2 litres of bufferA, then with 1.5 litres of 300mM-Tris/HCl buffer,pH 8.8, containing 0.25 m-sucrose, 2 mM-dithio-threitol, 1 mM-EDTA and 7.5% (v/v) propyleneglycol. Guanylate cyclase was eluted with buffer Acontaining 20% (v/v) dimethylformamide andimmediately transferred to a Sephadex G-25 column(4cm x 38cm) to remove dimethylformamide. Theguanylate cyclase was then applied to a column(4cm x 40cm) of DEAE-Bio-Gel and eluted with alinear gradient of NaCl (50-350mM). The enzymefractions were pooled, concentrated with Aquacide(Calbiochem) to 30ml, dialysed against buffer A,and applied to a column (2.5 cm x 80 cm) of UltrogelAcA-34 equilibrated and eluted with the samebuffer. Active fractions were pooled and dialysedagainst a solution containing 10% (v/v) glycerol,

0.5 M-sucrose, 2 mM-dithiothreitol in 10 mM-Tris/HClbuffer, pH 7.4 (buffer B), and then stored at -65 0C.At this stage (step 5, Table 1), activity was stable formonths at -6-5 0C.The enzyme from step 5 was applied to 45 ml of

GTP-agarose in a fritted-glass filter, which wasequilibrated with 50mM-sucrose in 20mM-Tris/HClbuffer, pH 7.6, containing 2 mM-dithiothreitol and1 mM-NaN3 (buffer C). The pass-through fraction,containing most of the guanylate cyclase, was col-lected and the gel was washed with 68 ml of buffer C.The pooled fraction plus wash (70% of appliedactivity) was made 3 mm in MgCI2 and transferred to54 ml of GTP-agarose. The gel was washed with5 vol. of buffer C containing 3 mM-MgCl2 and 4 vol.of buffer C containing 25 mM-NaCl. Guanylatecyclase was then eluted with 3 vol. of buffer C con-taining 100mM-NaCl, immediately dialysed againstbuffer B (45min at 2°C) and stored at -65°C.(Recovery of this step was unusually low for thepreparation indicated in Table 1.)

The enzyme was further purified by using pre-parative polyacrylamide-gel electrophoresis (Tsaiet al., 1978). Specific activities of peak fractionsfrom electrophoresis (corresponding to 106-110 inFig. la) were 1.5-2.0,umol/min per mg of protein.Polyacrylamide-slab-gel electrophoresis in the pre-sence of SDS of samples from fractions indicated thepresence of a major protein band of M, approx.69000 (Fig. lb) that correlated with enzymeactivity. The purified guanylate cyclase prepara-tions contained small amounts of one or two otherpeptides that migrated more slowly than the enzymeon electrophoresis in the presence of SDS and haveproved to be very difficult to remove from rat liver(Fig. lb) or calf liver (results not shown) prepara-tions. The purified enzyme exhibited a Stokesradius of 4.7nm and an s20, wof 6.4 S, from which wehave calculated an Mr value of 133 000 for the nativeenzyme. When stored in buffer B at -650C, thepurified enzyme from preparative electrophoresis(10-30ug of protein/ml) was stable for severalmonths. It was however, extremely unstable at 0°C.Therefore, for most of the experiments reported in

Table 1. Purification ofguanylate cyclasefrom rat liverPreparation from 880g of rat liver was as described in the Materials and methods section. Guanylate cyclaseactivity was assayed with 5 mM-MnCl2 and activator.

FractionSuperantantDE-52 DEAE-cellulosePhenyl-SepharoseDEAE-Bio-GelUltrogel AcA-34GTP-agaroseElectrophoresis

Protein(mg)

107001020510140

0.9

Activity Specific activity(nmol/min) (nmol/min per mg)

0.26400 0.66100 6.08500 175190720

378001700

Step1234567

Recovery(%)

1009513380116

1983

448

Guanylate cyclase

6(

4

. >

c)

4 ._o E

E,C: a

>C): 2(

io (a)0

0-~~ ~ ~

88 96 104 112 120 128 13692 100 108 116 124 132_ X - _- ........~~~. . . ..:_::.:.

.. ..: ..::..::_~_~

STD 70 88 96 104 110 114 120 128 STD80 92 100 108 112 116 124 132

Fraction no.

Fig. 1. (a) Preparative polyacrylamide-gel electro-phoresis of guanylate cyclase and (b) polyacrylamide-slab-gel electrophoresis in the presence of SDS of

fractionsfrom the preparative electrophoresis(a) Enzyme (0.2mg) from step 6 was subjected toelectrophoresis on 8% polyacrylamide gel (Tsaiet al., 1978). Samples of fractions (50-200ng ofprotein) were assayed with 5 mM-MnCl2 and acti-vator (10ug). Total activity per fraction is recordedin nmol/l0min. (b) Proteins in samples of fractionfrom the preparative electrophoresis shown in (a)were precipitated with 10% (w/v) trichloroaceticacid, dissolved in 1% (w/v) SDS/5% (v/v) 2-mercaptoethanol (3min, 100°C), transferred to an8% polyacrylamide slab gel, and subjected toelectrophoresis in the presence of SDS (Laemmli,1970). Standard proteins (STD) are, from the top,phosphorylase b, bovine serum albumin andovalbumin.

the present paper, guanylate cyclase purified up tostep 6 (chromatography on GTP-agarose) was

used. These preparations usually had specific ac-tivities of 0.6-1.2,umol/min per mg of protein andexhibited one or two contaminating protein bands onelectrophoresis in the presence of SDS.

Preparation ofdeoxyHb andNOHbStock solutions of deoxyHb (0.5 mM) were pre-

pared as described by Craven & DeRubertis (1978).Na2S204 (final conc. 2mg/ml) was added to asolution of Hb (75% methaemoglobin) in 50mM-Tris/HCl buffer, pH7.6 for 5min on ice under N2.The solution was applied to a column of SephadexPG-10 equilibrated and eluted with the Tris buffer.The fractions containing deoxyHb were imme-diately flushed with N2 and stored at 40C until used

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(within 1 week). NOHb was prepared immediatelybefore use by flushing a solution of deoxyHb(0.5mM) with NO under atmosphere of N2 for 2-3min at 0°C. The tube was then capped and after2-3 min was flushed with N2.

Guanylate cyclase was assayed in a total volumeof 100,ul containing 5mM-MnCI2 or 6mM-MgCl2.1 mM-[a-32P]GTP (3 x 106 c.p.m.), 1-2 mM-dithio-threitol and 50mM-Tris/HCl buffer, pH 7.6 (Tsaiet al., 1978, 1980). Theophylline (6mM) and cyclicGMP (1 mM) were included in assays of enzymefrom steps 1 to 5 of purification. After incubation at37°C for 10min, assays were terminated by ad-dition of 5% (w/v) trichloroacetic acid containing1 mM-GTP and 1 mM-cyclic GMP. Cyclic GMP wasisolated for radioassay by using Dowex 1 (Cl- form),and alumina (Nesbitt et al., 1976). To assess effectsof NO and NOHb on guanylate cyclase activity,enzyme samples (100-200,u1) were incubated at0°C with NO under N2 or with NOHb for 2min inbuffer containing 2 mM-dithiothreitol before addi-tion of other assay components. Although freshsolutions of NOHb were prepared for each ex-periment, considerable variation in their effective-ness was noted. For the experiment illustrated in Fig.2, deoxyHb at the indicated concentration wasadded to the enzyme sample at 0°C; NO (twobubbles) was introduced and assays were initiatedimmediately with addition of substrate. With thisprocedure, activation was more reproducible andgreater than that observed with the same nominalconcentration of preformed NOHb (see, for exam-ple, Table 2). The guanylate cyclase activator wasprepared as described previously (Tsai et al., 1980,1981).

Results

At all stages of purification, activity was greater inassays with Mn2+ rather than Mg2+, as has beenobserved previously (Craven & DeRubertis, 1978;Tsai et al., 1978, 1980; Braughler et al., 1979a,b;Gerzer et al., 1981a,b; Ignarro et al., 1982a,b). Atseveral steps in purification, the enzyme exhibitedthe same Stokes radius (4.7nm) and s20,w (6.4 S),indicating that the apparent size of the nativeenzyme (Mr 133000) was not affected by thepresence of other proteins or the procedures em-ployed (results not shown).NO did not enhance activity of the enzyme

purified through preparative electrophoresis (step 7)(Table 2), and at higher concentrations it wasinhibitory, more so in assays with Mn2+ than withMg2+. NO plus haem did not alter activity (resultsnot shown), but activity was markedly enhanced byNOHb; the percentage activation was greater withMg2+ than with Mn2+ (Table 2). Hb alone increasedactivity relatively little. Activator produced the same

449

S.-C. Tsai, R. Adamik, V. C. Manganiello and M. Vaughan

Table 2. Activation ofpurified guanylate cyclase by NO,NOHb, deoxyHb or activator

Samples of purified guanylate cyclase from prepara-tive electrophoresis (0.12,ug of protein) were assayedwith 5 mM-MnCl2 or 6 mM-MgCl2 and other additionsas indicated. For full details see the text.

Guanylate cyclase activity(nmol/min per mg of protein)r

AdditionsNoneNO (100pl)NO (200u1)DeoxyHb (25,UM)NOHb (10puM)NOHb (25 pM)Activator (10,ug)Activator plus NO (100pl)

MgCl249403899

15002600240200

MnCI2390380210620

2000

1500980

percentage increase with Mg2+ and Mn2+; it did notsupport NO activation (Table 2).

After elution from DE-52 DEAE-cellulose (step2), cyclase activity was markedly enhanced by NO;responsiveness to NO decreased with succeedingsteps of purification, and was minimal or absentafter step 6 and step 7 (Table 2). As reported byothers (Gerzer et al., 1981b; Ignarro et al., 1982b),the cyclase from bovine lung remained responsive toNO after binding to Affi Blue Sepharose and elutionwith GTP. As with the rat liver enzyme (Table 2),the bovine lung cyclase was activated little, if at all,by NO after chromatography on GTP-agarose(results not shown).

With the liver enzyme purified through chromato-graphy on GTP-agarose (step 6), 2.5-25 pM-deoxyHb increased activity by 200% in assays withMn2+ or Mg2+ (Fig. 2). NOHb (1 ,UM) increasedactivity with Mg2+ 120-fold and only about one-tenth that with Mn2+. Higher concentrations ofNOHb were less effective (Fig. 2), as were lowerconcentrations (results not shown). Another proteinthat contains haem, cytochrome c, and an iron-containing protein, ferredoxin, also increased basalguanylate cyclase activity; unlike Hb, however, theydid not support NO activation (results not shown).

The concentration of Mn2+ required to produce ahalf-maximal activity (1.2mM) was consistently lessthan the concentration of Mg2+ that supportedhalf-maximal activity (3pM). Neither NOHb (Fig. 3)nor activator (results not shown) altered the de-pendence on Mg2+ or Mn2+. Maximal activity (with1 mM-GTP) was observed with 5mm of either (Fig.3).

In assays with Mn2+ with or without activator,activity was maximal at pH7.2-7.6 and declinedwith increasing pH (results not shown), as weobserved with less-purified preparations of guanylate

0

0

0.5

E

0

0.05l_

0 10 20Coyncn. of Hb or NOHb (#M)

Fig. 2. Effects of NOHb and Hb on guanylate cyclaseactivity

Samples of guanylate cyclase (0.13,pg of protein)from step 6 were incubated at 0°C with theindicated concentrations of NOHb (A and A) ordeoxyHb (0 and 0) for 2 min before assay with5 mM-MnCl2 (0 and A) or 6mM-MgCl2 (O and A).For full details see the text.

cyclase (Tsai et al., 1978). A similar pH optimumwas observed in assays with Mn2+ or Mg2+ andNOHb; basal activity with Mg2+, however, did notdecrease with increasing pH from 7.6 to 8.5 in Trisbuffer (results not shown) as noted also byBraughler (1980).

In assays with Mg2+ (4.8mm in excess of GTP),the enzyme exhibited a single Km for GTP(0.35mM), which was not consistently altered byNOHb or activator (Fig. 4). Variations in pHbetween 6.8 and 8.3 altered Vmax but not the Km forGTP (results not shown). Data from assays withMn2+ (2mm in excess of GTP) yielded plots of 1/vversus 1/[SI that were non-linear (Fig. 5), withapparent Km values of 50-60pM and 1.5mm for

1983

450

Guanylate cyclase

0~~~~~~0 04A

0

~40

20-

0 1 2 3 4 5 10

Concn. of MnCI2 orMgCl2 (mM)Fig. 3. Effects ofMgCl2 and MnCl2 on guanylate cyclase

activitySamples of guanylate cyclase (step 6, 0.27 pg ofprotein) were assayed with 1 mM-GTP and theindicated concentration of MgCl2 (O and A) orMnCI2 (0 and A) without (O and 0) or with (Aand A) 3,uM-NOHb. For full details see the text.Maximal activities (100%) with MgCl2 without andwith NOHb were 26 and 300nmol/min per mg ofprotein respectively, and with MnCI2 were 50 and700nmol/min per mg of protein respectively.

0* 0.3

0.--

0.0

i.n

0 0.2

E-. 0.1

-

i/

12 16 20-4 0 4 81/[SI (mM-)

0.01 'C

00.30-o

0.02 v.0boE1).0.

0._

E0O-e

I X

Fig. 4. Effect of substrate concentration on guanylatecyclase activity with MgC12

Samples of guanylate cyclase from step 6 (0.13,ug ofprotein) were assayed with the indicatedconcentrations of MgGTP plus 4.8mM-MgCl2 with7pg of activator (0), 0.7,uM-NOHb (A) or noadditions (-). For full details see the text.

GTP, which were not significantly altered by NOHbor activator (Fig. 5) or by higher concentrations ofexcess Mn2+ (results not shown). Double-reciprocal

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U.3

000

sE 0.03

0.0

.0.01 A

0 4 8 12 16 201/[SI (mw1)

Fig. 5. Effect of substrate concentration on guanylatecyclase activity with MnCl2

Samples of guanylate cyclase from step 6 (0.26 pg ofprotein) were assayed with the indicatedconcentrations of MnGTP plus 2mM-MnCl2 withlO,g of activator (0), 5pM-NOHb (A) or noadditions (0). For full details see the text.

Table 3. Inhibition of guanylate cyclase activity byvanadate, tungstate and borate

Samples of guanylate cyclase (step 6, 0.27pg ofprotein) were assayed with MgCl2 or MnCl2,1.8,uM-NOHb, activator (lOpg), 50,uM-Na3VO4,1 mM-Na2WO4 and/or 2mM-Na2B407 as indicated.For full details see the text. Activities relative tocontrol values (=100) are given in parentheses.

Assay withMgCl2 (6 mM)Plus NOHbPlus activatorMnCl2 (5 mM)Plus NOHbPlus activator

Guanylate cyclase activity(nmol/min per mg of protein)

Control Na3VO4 Na2WO4 Na2B40714 (100) 6 (43) 2 (14) 12 (86)

270 (100) 130 (48) 15 (6) 220 (82)110 (100) 56 (51) 19 (17) 97 (88)120 (100) 52 (43) 6 (5) 67 (56)510 (100) 310 (61) 27 (5) 190 (37)820 (100) 540 (66) 570 (69) 410 (50)

plots were also non-linear when assays were per-formed at pH 6.5, 7.3 or 8.5 (results not shown).

The purified guanylate cyclase was inhibited 50%by 50,uM-Na3VO4 in assays with Mg2+ or Mn2+ withor without NOHb or activator (Table 3). Reductionof vanadate from vv to Viv abolished the inhibitoryeffect (results not shown). Na2WO4 (1 mM) causedvirtually complete inhibition under all conditionsexcept in the presence of Mn2+ and activator (Table3). In assays with Mn2 , with or without NOHb oractivator, 2mM-Na2B407 was inhibitory, but it hadrelatively little effect in assays with Mg2+ (Table 3).

451

IF

S.-C. Tsai, R. Adamik, V. C. Manganiello and M. Vaughan

Table 4. Effects ofinhibitors on guanylate cyclase activitySamples of guanylate cyclase (step 6, Expt. I, 0.1,ug of protein; Expt. II, 0.12,ug of protein) were assayed withMgCl2 or MnCl2, luM-protoporphyrin IX, luM-NOHb, activator (3,ug) and the addition indicated in Expt. I,0.5mM-FMN, 0.5mM-NAD+ or 0.5mM-NADH; in Expt. II, 1mM-NaCN, 1mM-K3Fe(CN)6, 0.6mM-rotenone orI mM-Methylene Blue.

Guanylate cyclase activity (nmol/min per mg of protein)

MgCl2 MnCl2

AdditionExpt. INoneFMNNAD+NADH

Expt. 2NoneNaCNK3Fe(CN)6RotenoneMethylene Blue

Control + Protoporphyrin IX

17252619

140103111

104

25237

1125

3703701501733

200

O 150

0

0

>1 100

50

0 0.5 1.0 1.5

Concn. of Methylene Blue (mm)

Fig. 6. Inhibition of guanm/7ate cvclase activity with

Methylene Blue

Samples of guanylate (step 6, 0.12pug of protein)were assayed with the indicated concentrations of

Methylene Blue and mm-GTP in the presence of

6 mm-MgCl2 or 5 mm-MnCl2. For full details see the

text. Control activities (100%) with M9Cl2 with no

addition (U), 1lpm-protoporphyrin (A) and 1,pm-NOHb (EJ) were 27, 128 and 545 nmol/min per mg

of protein respectively, and with MnCI2 without (0)and with lOpg of activator (@) were 78 and

726 nmol/min per mg of protein respectively.

1-v

ct.-

C._._

8.->1

O

Fi

0 3 6 9 12

Time (min)

ig. 7. Inactivation of rat liver guanylate cyclase bydiamide

Guanylate cyclase from step 6 (180ug/ml) wasincubated with 0.24 mM- (0), 0.3 mM- (A), 0.36 mM-(*), 0.42 mM- (0) or 0.48 mM- (0) diamide at220 C. At the indicated times, samples (5,ul, 0.9,ug ofprotein) were assayed. For full details see the text.

Concentrations of inhibitors used in Table 3 wereselected on the basis of experiments in which manyconcentrations of each were tested with enzymefrom step 5 (results not shown).

In assays with Mg2+ or Mn2+ or with Mn2+ plusactivator, activity was unaffected by 0.5mM-FMN,-NAD+ or -NADH. In assays with Mg2+, these

1983

+ NOHb

750490104340

1150133052060067

Control

59626871

220180150150220

+ Activator

320310340320

720740510510320

452

Guanylate cyclase

compounds inhibited activation by NOHb byapprox. 50% and that by protoporphyrin IX byapprox. 20% (Table 4). In assays with Mg2+, with orwithout NOHb and protoporphyrin IX, and withMn2 , with or without activator, NaCN (1 mM) hadlittle effect on activity (Table 4). K3Fe(CN)6 (1 mM)inhibited activity in all assays with Mg2+ to a greaterextent than with Mn2+ (Table 4). Rotenone (0.6 mM)inhibited activity under all conditions and virtuallyabolished activation by protoporphyrin IX. Methyl-ene Blue (1 mM) had little effect on basal activity(Table 4) or increased it somewhat in the presence ofMn2+ (Fig. 6), but it markedly inhibited activationby either NOHb or protoporphyrin IX in assayswith Mg2+ as well as effects of activator in assayswith Mn2+ (Table 4). The concentration-dependenceof the effects of Methylene Blue is shown in Fig. 6.Methylene Blue also inhibited the effects of cyto-chrome c and ferredoxin on cyclase activity (resultsnot shown).

Activity of the enzyme purified throughchromatography on GTP-agarose was inhibited bydiamide or oxidized glutathione in a reversiblefashion, as reported previously (Tsai et al., 1981).Inactivation with diamide at 22°C obeyed pseudo-first-order kinetics (Fig. 7). A plot of log (1000/t0.5)versus log (diamide concentration) yielded a straightline with a slope of 1.1. Similar results were obtainedwith oxidized glutathione. Treatment with diamideor p-chloromercuribenzoate inhibited activation byNOHb (S.-C. Tsai, R. Adamik, V. C. Manganiello& M. Vaughan, unpublished work).

Discussion

From estimates of the Stokes radius (4.7 nm) andS20 w (6.4 S) at several stages during purification, thecalculated Mr value of the native enzyme is 133 000.Electrophoresis of the purified enzyme underdenaturing conditions indicates that the enzyme is ahomodimer with a subunit Mr of 69 000. Theseestimates of apparent molecular size and subunitcomposition are in basic agreement with thosepublished for the enzymes purified from rat liver andbovine lung (Braughler et al., 1979a,b; Ignarroet al., 1982a,b). No previous workers, however, haveattempted to correlate peptide patterns (electro-phoresis in the presence of SDS) with activity inindividual and successive fractions isolated duringthose final step(s) that yield the purified enzymes.Such analyses might be important in understandingdifferences in responsiveness to NO, protopor-phyrin IX and NOHb in preparations from differentsources.

Gerzer et al. (1981a,b) reported that purifiedpreparations of guanylate cyclase from bovine lungcontained 1 mol of haem (ferroprotoporphyrin IX)and 1 mol of copper per mol of native enzyme. The

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lung enzyme purified by Ignarro et al. (1982a,b)apparently also contained haem, and it seemsprobable that haem was present in the purified liverguanylate cyclase described by Braughler et al.(1979a). These preparations were markedly acti-vated by nitroprusside or NO, whereas the purifiedenzyme described in the present paper was acti-vated little, if at all, by NO. The precise mechanismsresponsible for loss in responsiveness in our pre-parations and those of others (Gerzer et al., 198 la;Ignarro et al., 1982a,b) remain to be defined. Byusing procedures similar to those of Gerzer et al.,(1981a) and Ignarro et al. (1982a,b), NO-respon-sive preparations were isolated from bovine lung.Chromatography of the lung preparations as well asrat liver preparations described in the present paperon agarose-GTP was sufficient to decrease re-sponsiveness to NO markedly. When Hb was addedto the purified liver cyclase, NO increased activity tovalues (2-8pmol/min per mg of protein) similar tothose reported for the lung enzyme (Gerzer et al.,198 lb; Ignarro et al., 1982b). Craven & DeRubertis(1978) first reported that haem or haemoproteinscould restore responsiveness of liver guanylatecyclase to NO, nitroprusside and related com-pounds. Although the specific activities of ourguanylate cyclase preparations were more than10-fold higher, there were many similarities in theresponses to Hb, NO and NOHb as well as theeffects of Mg'+ versus Mn2+. Not all haem-con-taining proteins, however, are capable of supportingactivation of guanylate cyclase by NO; for example,cytochrome c did not restore responsiveness to NO.Essentially all of the observations on enzymepreparations such as those described in the presentpaper that presumably lacked haem (Ignarro et al.,1982a,b) or contained haem (Gerzer et al., 198 la,b;Ignarro et al., 1982a,b) are consistent with theconclusion that haem is required for activation byNO and related compounds. The proximate acti-vating molecule is in all probability NO-haem.

In the presence of NOHb, cyclase activity was atleast as great with Mg2+ as it was with Mn2+; similarfindings have been reported by Craven &DeRubertis (1978) with partially purified liverenzyme and by Ignarro et al. (1982a) with a purifiedlung guanylate cyclase. In the absence of activator,guanylate cyclase activity was higher with Mn2+than with Mg2+, as has been observed repeatedlywith the enzymes at all stages of purification(Craven & DeRubertis, 1978; Tsai et al., 1978-1980; Braughler et al., 1979a,b; Gerzer et al.,1981a,b; Ignarro et al., 1982a,b). In assays withMg2+, with or without NOHb or activator, theenzyme described in the present paper exhibited aKm for GTP of approx. 0.35 mm, whereas with Mn2+Lineweaver-Burk plots were non-linear (apparentKm values approx. 60pM and 1.5mM). Similar

453

454 S.-C. Tsai, R. Adamik, V. C. Manganiello and M. Vaughan

differences between assays with Mn2+ and Mg2+were also noted for the lung enzyme (Ignarro et al.,1982a,b). Neither activator nor NOHb alteredmetal-ion- or pH-dependence of enzyme activity orKm values for GTP (in assays with Mn2+ or Mg2+).There seems to be increasing agreement that Mg2+ isprobably the physiologically relevant bivalent cationand that Mn2+, in addition to substitution for Mg2+in the catalytic process, can activate the guanylatecyclase (Gerzer et al., 198 la; Ignarro et al., 1982a;Braughler, 1980).

Ignarro et al. (1 982a) found that haem-con-taining lung guanylate cyclase was activated 30-40-fold by protoporphyrin IX (to the same extent asby NO or NO-haem in assays with Mg2+). Morerecently, they reported activation by protopor-phyrin IX of lung guanylate cyclase preparationsdeficient in haem (Ignarro et al., 1982b). Proto-porphyrin IX, which increased activity of theenzyme described in the present paper, did notsupport activation by NO. Whether the strikingactivation by protoporphyrin IX observed byIgnarro et al. (1982a,b) was due to the presence ofsome component lacking in our guanylate cyclasepreparation remains to be determined.We (Tsai et al., 1980) and others (Brandwein

et al., 1981) have reported that the soluble liverguanylate cyclase can be reversibly inactivated bycertain thiol-reactive reagents and disulphide com-pounds. Inactivation of the enzyme by diamide, asshown in the present work, apparently results fromthe oxidation of a single class of thiol groups. Thepurified guanylate cyclase was also inhibited byapprox. 50% by 50pM-Na3VO4 in assays with Mg2+or Mn2+ with and without NOHb or activator. Inview of the proposed role of redox reactions in theregulation of guanylate cyclase, it is of interest thatreduction of vv to VIV abolished the inhibitory effectof vanadate. The concentration of V043- required toinhibit guanylate cyclase is, however, much greaterthan that required to inhibit the Na+ + K+-de-pendent ATPase (Cantley et al., 1977) and, in lightof the reported vanadate content of tissues, the effectmay not be of physiological significance. Na2WO4,at considerably higher concentrations, was alsoinhibitory under all assay conditions; however,2mM-Na2B407, which caused approx. 50% inhibi-tion in assays with Mn2+, had relatively little effect inassays with Mg2+. It is possible that B4072- inhibitsthe postulated activating effect of Mn2+ more than itdoes the catalytic activity of the guanylate cyclase.FMN, NAD+ and NADH selectively inhibited

activation by NOHb and protoporphyrin IX inassays with Mg2+ without altering effects of acti-vator in assays with Mn2 . Ohlstein et al. (1982)have also reported that FMN inhibited activation byNO-haem and protoporphyrin IX without alteringbasal activity. Rotenone inhibited activation by

protoporphyrin IX to a greater extent than that byNOHb or activator. Whether these compoundsinhibited activity solely by virtue of their redoxproperties is unknown. Methylene Blue markedlydecreased activation by protoporphyrin IX, NOHbor activator as well as by the proteins Hb,cytochrome c and ferredoxin without inhibiting basalactivity. In some experiments, Methylene Blueactually increased basal activity. In increasing basalactivity, Methylene Blue may mimic (perhaps byinteraction with hydrophobic domains in the cyc-lase) some of the effects of haemoglobin, cyto-chrome c and activator. Inhibition of activation byprotoporphyrin IX and especially by NOHb may berelated to the redox properties of Methylene Blue.

Though apparently deficient in haem, the enzymepreparation described in the present paper re-sponds dramatically to NOHb (50-100-fold acti-vation). Its activity can also be effectively regulated(with or without added haem) by the bivalent cationsMn2+ and Mg2+, by several kinds of molecules (i.e.,activator; proteins such as Hb, cytochrome c andferredoxin; compounds such as Methylene Blue) anda number of compounds that show some selectivityin apparently inhibiting the effects of differentactivators. In utilizing Hb for activation by NO, thecyclase may be analogous to prostaglandin endo-peroxide synthase, which requires haem for activitybut when isolated in the haem-deficient state can beactivated by certain haem-containing proteins (Uenoet al., 1982). The earlier work of Craven &DeRubertis (1978) and the more recent observa-tions by Ignarro et al. (1982b) suggest thathaem-deficient preparations of guanylate cyclase canbind haem and be activated by NO. A purifiedguanylate cyclase, such as that described in thepresent paper, should be particularly useful fordefining the nature of the interaction with haemand elucidating the mechanism of haem-dependentprocesses (i.e., NO activation) as well as haem-independent processes (i.e., activation by proto-porphyrin IX and other effectors).

We thank Mrs. D. Marie Sherwood for expertsecretarial assistance.

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