materials  recombinant human aldh2 was expressed in and 
  purified from escherichia coli 
  ( 16 ) . gtn ( nitropohl 
  ampoules ; g. pohl - boskamp gmbh & co , hohenlockstedt germany ) was obtained 
  from a local pharmacy as a 1 mg / ml aqueous isotonic solution , containing 49 
  mg / ml glucose monohydrate , and diluted in 50 mm triethanolamine / hcl 
  ( ph 7.4 ) .
[ 2-c]gtn ( 50 mci / mmol ) , manufactured by american 
  radiolabeled chemicals , was supplied locally by humos diagnostica ( maria 
  enzersdorf , austria ) .
1,2-gdn and 1,3-gdn , used as standards in radio - thin 
  layer chromatography , were obtained from lgc promochem ( wesel , germany ) . 
 
naphthoic 
  acid was purchased from abcr gmbh & co kg ( karlsruhe , germany ) .
determination of the inactivation kinetics  to determine the 
  rate of inactivation of aldh2 by gtn , the optical absorption of a 1-cm quartz 
  cuvette ( type 105.200-qs , hellma , mllheim , germany ) containing substrate 
  ( acetaldehyde or p - npa ) and additional compounds like nad 
  or dtt as indicated in 50 mm potassium pi ( ph 7.4 ) , was 
  monitored at 340 ( dehydrogenase ) or 400 nm ( esterase ) in a hewlett - packard 
  8452a diode array spectrophotometer .
after 12 min aldh2 was added 
  and the reaction was allowed to proceed for 23 min .
subsequently , 
  gtn ( or in some instances nad ) was added to start the 
  inactivation .
usually , when inactivation was complete , dtt , substrate , or both 
  were added to reactivate the enzyme .
the total volume of the samples after gtn 
  addition was 250 l . the total time over which the reaction was monitored 
  was 2030 min . although new conditions were always explored with a 
  single sample for optimal time resolution , concentration dependences or 
  variations of cofactor or reductant ( see  results  ) were usually 
  studied with up to 6 samples per experiment with the 89073a multicell 
  transport accessory .
the temperature of the samples was kept at 20 c by a 
  refrigerating circulating water bath ( lauda rm6 , bartelt gmbh , austria ) .
the 
  various segments of the time traces ( initial activity , restored activities ) 
  were fitted linearly , except for the inactivation , which was fitted to a 
  combination of a single - exponential and a straight line .
the linear component 
  yielded the residual activity ; the exponential component yielded the apparent 
  inactivation rate constant , as can be derived as follows : if the uninhibited 
  activity is ve = kee , 
  with ke and e representing the catalytic rate 
  constant and the enzyme concentration , respectively , inactivation will lower 
  the activity by decreasing the catalytically competent enzyme fraction in a 
  mono - exponential fashion with et = 
  e0e , 
  in which e0 is the total enzyme concentration and 
  ki is the apparent inactivation rate constant .
the 
  activity will therefore change in time according to the equation 
  ve(t ) = ke  
 
consequently , the absorbance will display simple monoexponential behavior 
  during inactivation according , 
  ( eq .
1)\documentclass[10pt]{article } 
 \usepackage{amsmath } 
 \usepackage{wasysym } 
 \usepackage{amsfonts } 
 \usepackage{amssymb } 
 \usepackage{amsbsy } 
 \usepackage{mathrsfs } 
 \usepackage{pmc } 
 \usepackage[euler]{upgreek } 
 \pagestyle{empty } 

 \oddsidemargin -1.0 in 

 \begin{document } 
 \begin{equation*}\;\;a(t)=a_{0}+{\delta}{\epsilon}\frac{v_{0}}{k_{i}}(1-e^{-k_{i}t})\;\;\end{equation*}\end{document } 
  in which a0 and  correspond to the initial 
  absorbance and the absorbance difference coefficient between product and 
  substrate , respectively .
inactivation of aldh2 by preincubation with gtn (  gtn 
  preconditioning)purified aldh2 ( 0.27 mg / ml ) was 
  preincubated with 1 m gtn in 50 mm napi 
  ( ph 7.5 ) containing 0.2 mm mgcl2 and 2 mm 
  nad at 37 c for 10 min .
dehydrogenase activity was determined 
  as the formation of nadh from nad in the presence of acetaldehyde 
  by monitoring the increase in absorbance at 340 nm ( 340 = 
  6.22 mm cm ) .
esterase activity was 
  measured by monitoring the formation of p - nitrophenolate from 
  p - nitrophenylacetate ( p - npa , 400 = 16 
  mm cm ) .
the reaction mixture contained 
  substrate ( 0.2 mm acetaldehyde or 0.1 mm 
 p - npa ) , 2 mm nad , 10 mm 
  mgcl2 , and 1.5 g / ml aldh2 in 50 mm napi 
  ( ph 7.5 ) at 25 c . in a second series of experiments , preincubation with 1 or 10 
  m gtn was performed in 0.1 m mes ( ph 6.5 ) containing 
  0.2 mm mgcl2 at 37 c for 10 min , dehydrogenation 
  was measured at ph 9.0 ( 50 mm napi ) , and esterase 
  activity was determined in the absence of nad 
  ( 17 ) .
determination of 1,2-gdn formation by radio - thin layer 
  chromatography  the rate of the conversion of gtn to 1,2- and 
  1,3-gdn was determined according to a described protocol 
  ( 12 ) .
purified aldh2 ( 4 g ) 
  was incubated with c - labeled gtn ( 2 m ; 50,000 
  dpm ) at 37 c for 10 min in a final volume of 100 l of 50 
  mm potassium pi ( ph 7.4 ) , containing 3 mm 
  mgcl2 , 1 mm nad , 1 mm edta , 1 
  mm egta , and 2 mm gsh with 2 mm dtt as 
  indicated . after incubation , reaction mixtures were filtered over ym-10 
  columns ( millipore gmbh , vienna , austria ) and washed twice with 60 l of 50 
  mm potassium pi ( ph 7.4 ) to yield 200-l filtrate and 
  20-l residues containing the enzyme .
the recovered protein was incubated 
  again with c - labeled gtn ( 2 m , 50,000 dpm ) at 
  37 c for 10 min in a final volume of 200 l of potassium pi 
  buffer ( 50 mm , ph 7.4 ) , containing 3 mm 
  mgcl2 , 1 mm nad , 1 mm edta , 1 
  mm egta , and 2 mm gsh with 2 mm dtt as 
  indicated .
reaction products from the filtrate of the first incubation 
  ( pre - column ) and from the reaction mixture of the second incubation 
  ( post - column ) were extracted twice with 1 ml of diethyl ether , separated by 
  thin layer chromatography , and quantified by liquid scintillation counting . 
 
blank values , determined in the absence of protein under identical conditions , 
  were subtracted .
mean values  s.e . were calculated from reaction rates 
  measured as duplicates in three independent experiments . to evaluate the effect of the procedure on enzyme activity , dehydrogenase 
  and esterase assays
the filtered enzyme was washed twice with 60 l of 50 
  mm napi ( ph 9.0 or ph 7.5 for the dehydrogenase and 
  esterase determination , respectively ) .
assay conditions were : 50 mm 
  napi ( ph 9.0 ) , 0.2 mm acetaldehyde , 2 mm 
  nad , 10 mm mgcl2 , and 1.5 g / ml aldh2 for 
  the dehydrogenase activity ; 50 mm napi ( ph 7.5 ) , 0.1 
  mm p - npa , 10 mm mgcl2 , and 1.5 
  g / ml aldh2 for the esterase activity . inactivation by gtn and reactivation by thiols of aldh in 
  mitochondria  rat liver mitochondria were prepared according to a 
  previously published method 
  ( 18 ) .
rat livers were 
  homogenized in tris buffer ( 250 mm sucrose , 10 mm tris , 
  5 mm egta , 2 mm mgcl2 , ph 7.4 ) and 
  centrifuged at 600  g ( 10 min at 4 c ) .
the 
  pellet was resuspended in tris buffer and centrifuged at 15,000  
  g for 5 min .
the mitochondrial fraction 
  ( 2030 mg / ml protein ) was frozen at -70 c until use .
mitochondria ( 0.8 mg / ml ) 
  were preincubated with gtn ( 100 m ) in tris buffer for 10 min at 
  37 c .
after preincubation , the mixture was centrifuged ( 15,000  
  g , 5 min at 4 c ) and washed three times with tris buffer to 
  remove gtn .
the pellet was resuspended in tris buffer at a final concentration 
  of 8 g / ml and mixed with 0.1 mm nad in a cuvette at 
  37 c . after 3 min ,
the dehydrogenase substrate monal 62 ( 3 
  m ) was added and fluorescence was monitored 
  ( ex 310 nm , em 360 nm ) in a ls50b 
  luminescence spectrometer ( perkinelmer life sciences ) .
after another 3 min , 
  thiol ( dtt , -mercaptoethanol , gsh , cys , or lpa - h2 ) was added 
  at the indicated concentrations and fluorescence was measured for 8 more min . 
  at the end of the experiment ,
signal intensity was calibrated by comparison 
  with the fluorescence of known concentrations of naphthoic acid .
inhibition of dehydrogenase activity by gtn  to study the 
  inactivation of aldh2 by gtn in real time we monitored the formation of nadh 
  from nad spectrophotometrically at 340 nm and determined how the 
  activity of aldh2 was affected by the addition of gtn . 
 
1a shows a 
  typical trace recorded for the inhibition of dehydrogenase activity with 
  acetaldehyde as the substrate .
it illustrates how the linear increase of the 
  nadh concentration is gradually reduced to a very low residual level .
fitting 
  the curve corresponding to this process to a single exponential yielded a 
  first - order rate constant that can be equated to the apparent inactivation 
  rate constant under this particular set of conditions ( see  experimental 
  procedures  ) .
the magnitude of reactivation was not affected 
  by the concentrations of dtt ( between 0.2 and 2 mm ) and 
  acetaldehyde ( up to 2 mm ) .
for instance , at a fixed concentration of 0.43 mm acetaldehyde the 
  inactivation rate constant increased from 2.96  0.08  
  10 s at 0.05 mm gtn to 21.1  0.5 
   10 s at 0.6 mm gtn .
by contrast , 
  inactivation slowed down at higher acetaldehyde concentrations : at a fixed 
  concentration of 0.5 mm gtn the rate constant decreased from 23 
   5  10 s at 0.1 mm 
  acetaldehyde to 2.9  0.3  10 s at 20 
  mm acetaldehyde .
a detailed description of the 
  effects of substrate and inhibitor concentrations on the inactivation rate is 
  given under the supplemental materials ( fig .
panel a shows a time trace for the formation of 
  nadh from nad , monitored at 340 nm . at t
= 0 the cuvette 
  contained 0.2 mm acetaldehyde and 0.2 mm nad 
  in 50 mm potassium pi ( ph 7.4 ) . at t = 80
s , 
  catalysis was initiated by the addition of 33 g / ml aldh2 .
inactivation 
  started at t = 480 s by the addition of 0.05 mm gtn .
after 
  inactivation of the enzyme , at t = 1160 s , an attempt was made to 
  restore activity by the addition of 1 mm dtt .
the dots 
  represent the data points , whereas the continuous lines are best fits 
  to the data .
linear fits were applied to the phases before ( no catalysis 
  ( -0.10  0.06 )  10 absorbance units 
  ( a.u.)/s ) , and after aldh2 addition ( initial activity , ( 8.03  
  0.05 )  10 a.u./s ) , and to the activity after dtt admission 
  ( restored activity ( 1.378  0.006 )  10 a.u./s ) ; for 
  the inactivation after gtn addition a combination of a single exponential and 
  a linear fit was applied ( apparent inactivation rate constant ( 7.77  
  0.06 )  10 s ; residual activity ( 0.133 
   0.007 )  10 a.u./s ) .
panel b compares the 
  residual and restored rates of acetaldehyde dehydrogenation after addition of 
  gtn and dtt , respectively .
experimental conditions : 33 g / ml aldh2 , 0.43 
  mm acetaldehyde , 0.4 mm nad , 0.4 
  mm dtt , and concentrations of gtn as indicated in 50 mm 
  potassium pi ( ph 7.4 ) .
initial dehydrogenase activities under the 
  conditions applied here amounted to 289  13 nmol min 
  mg , which corresponds to a turnover number of 69  3 
  min .
panel a shows a time trace for the formation of 
  nadh from nad , monitored at 340 nm . at t
= 0 the cuvette 
  contained 0.2 mm acetaldehyde and 0.2 mm nad 
  in 50 mm potassium pi ( ph 7.4 ) . at t = 80
s , 
  catalysis was initiated by the addition of 33 g / ml aldh2 .
inactivation 
  started at t = 480 s by the addition of 0.05 mm gtn .
after 
  inactivation of the enzyme , at t = 1160 s , an attempt was made to 
  restore activity by the addition of 1 mm dtt .
the dots 
  represent the data points , whereas the continuous lines are best fits 
  to the data .
linear fits were applied to the phases before ( no catalysis 
  ( -0.10  0.06 )  10 absorbance units 
  ( a.u.)/s ) , and after aldh2 addition ( initial activity , ( 8.03  
  0.05 )  10 a.u./s ) , and to the activity after dtt admission 
  ( restored activity ( 1.378  0.006 )  10 a.u./s ) ; for 
  the inactivation after gtn addition a combination of a single exponential and 
  a linear fit was applied ( apparent inactivation rate constant ( 7.77  
  0.06 )  10 s ; residual activity ( 0.133 
   0.007 )  10 a.u./s ) .
panel b compares the 
  residual and restored rates of acetaldehyde dehydrogenation after addition of 
  gtn and dtt , respectively .
experimental conditions : 33 g / ml aldh2 , 0.43 
  mm acetaldehyde , 0.4 mm nad , 0.4 
  mm dtt , and concentrations of gtn as indicated in 50 mm 
  potassium pi ( ph 7.4 ) .
initial dehydrogenase activities under the 
  conditions applied here amounted to 289  13 nmol min 
  mg , which corresponds to a turnover number of 69  3 
  min .
the residual activity and the extent of restoration in the presence of dtt 
  diminished when the concentration of gtn was increased 
  ( fig .
reactivation was not appreciably improved by extra dtt : at 0.032 mm 
  acetaldehyde and 0.1 mm gtn , reactivation amounted to 8  2 
  and 10  1% with 0.2 and 1.0 mm dtt , respectively . 
  similarly , the substrate concentration had little effect on reactivation 
  ( supplementary fig .
s1c ) and addition of extra substrate after 
  inactivation did not affect the restored activity either .
panel a , restoration by different thiols of aldh2-catalyzed 
  dehydrogenation after inactivation by gtn .
plotted are the relative residual 
  activities ( vresidual / vinitial ) after 
  inactivation by 0.1 mm gtn and the relative restored activities 
  ( vrestored / vinitial ) after addition of 
  1 mm dtt , lpa - h2 , or gsh . initial conditions : 25 
  g / ml aldh2 , 0.45 mm acetaldehyde , and 0.4 mm 
  nad in 0.2 m potassium pi ( ph 7.4 ) . 
 
panel b , protection by dtt of dehydrogenase activity against 
  gtn - mediated inactivation .
the figure shows representative time traces of the 
  aldh2-catalyzed formation of nadh , as monitored at 340 nm before and after 
  addition of gtn in the presence or absence of dtt .
experimental conditions : 33 
  g / ml aldh2 , 0.45 mm acetaldehyde , 0.4 mm 
  nad , and 0.1 mm gtn with or without 1 mm dtt 
  in 50 mm potassium pi ( ph 7.4 ) .
panel a , restoration by different thiols of aldh2-catalyzed 
  dehydrogenation after inactivation by gtn .
plotted are the relative residual 
  activities ( vresidual / vinitial ) after 
  inactivation by 0.1 mm gtn and the relative restored activities 
  ( vrestored / vinitial ) after addition of 
  1 mm dtt , lpa - h2 , or gsh . initial conditions : 25 
  g / ml aldh2 , 0.45 mm acetaldehyde , and 0.4 mm 
  nad in 0.2 m potassium pi ( ph 7.4 ) . 
 
panel b , protection by dtt of dehydrogenase activity against 
  gtn - mediated inactivation .
the figure shows representative time traces of the 
  aldh2-catalyzed formation of nadh , as monitored at 340 nm before and after 
  addition of gtn in the presence or absence of dtt .
experimental conditions : 33 
  g / ml aldh2 , 0.45 mm acetaldehyde , 0.4 mm 
  nad , and 0.1 mm gtn with or without 1 mm dtt 
  in 50 mm potassium pi ( ph 7.4 ) . as illustrated by fig . 
 
2a , lpa - h2 restored gtn - inactivated 
  dehydrogenation to about the same level as dtt ( from 6  1 to 20 
   3% of the initial activity with lpa - h2 compared with 26 
   1% with dtt ) .
gsh , on the other hand , had no effect ( 7  2% ) . 
 
1b and 
  2a ) , it largely 
  prevented inactivation if added before gtn 
  ( fig .
dtt slowed 
  down inactivation by 0.2 mm gtn ( 7.4  
  0.6)-fold3 with an 
  ec50 of 10  1 m ( supplementary fig .
the 
  results indicate that dtt strongly protects against inactivation , but can not 
  completely abolish it . whereas gsh , cys , and ascorbate did not prevent 
  inactivation
, lpa - h2 lowered the inactivation rate constant to a 
  similar extent as dtt ( results not shown ) .
inhibition of the esterase activity of aldh2 by gtn  in 
  addition to the nad - dependent dehydrogenation of aldehydes , aldh 
  is known to catalyze the hydrolysis of certain esters in a reaction that does 
  not require nad 
  ( 20 ) .
when the effect of gtn 
  on this activity was examined under standard conditions , i.e. in the 
  absence of nad , gtn hardly affected the rate of hydrolysis of 
  p - npa ; however , subsequent addition of nad did cause 
  inactivation ( fig . 
 
esterase inactivation also occurred when gtn was 
  added in the presence of nad 
  ( fig .
the rate of 
  inactivation was independent of the order in which gtn and nad 
  were added .
the rate of inactivation increased when the gtn concentration was raised , 
  and decreased at higher concentrations of p - npa . at a fixed 
  concentration of 0.5 mm p - npa
the inactivation rate 
  constant increased from 2.8  0.3  10 
  s at 0.05 mm gtn to 11.7  0.8  
  10 s at 1.0 mm gtn ; at a concentration of 
  0.1 mm gtn the inactivation rate constant decreased from 12.6 
   0.9  10 s to 1.5  0.5 
   10 s , when the p - npa concentration 
  was raised from 0.05 to 1.0 mm .
the residual esterase activity after inactivation by gtn was affected by 
  the concentration of p - npa : the relative activity 
  ( vresidual / vinitial ) increased from 
  0.024  0.002 to 0.12  0.03 when the substrate concentration 
  was raised from 0.2 to 1.0 mm ( supplementary fig .
s4a ) . on 
  the other hand , the activity was not affected by the gtn concentration 
  ( supplementary fig .
thiol - induced reactivation of 
  p - npa hydrolysis could not be determined , because of the ability of 
  thiols ( not just dtt but also gsh ) to hydrolyze p - npa in the absence 
  of aldh ( results not shown ) . in line with previous reports 
  ( 21 ) , nad enhanced 
  esterase activity , and an intermediate stimulation was observed in the 
  presence of nadh ( supplementary fig .
the 
  apparent rate constant for gtn - induced inactivation increased by an order of 
  magnitude in the presence of nad , and again the effect of nadh was 
  intermediate ( fig .
although 
  the presence of nad(h ) is no prerequisite for the reduction of gtn by aldh2 , 
  it is conceivable that the enzyme uses nadh as a facultative electron donor . 
 
however , we observed no absorbance changes at 340 nm upon incubating aldh2 
  with gtn and nad or nadh , which rules out the participation of 
  either nucleotide as a redox agent in the decomposition of gtn .
the figure shows the formation of 
  p - nitrophenol from p - npa , as monitored at 400 nm .
panel 
  a shows that , in the absence of nad , gtn did not 
  significantly affect esterase activity . however , inactivation set in 
  immediately after the addition of nad .
experimental conditions 
  were : p - npa ( 0.2 mm ) in 50 mm potassium 
  pi ( ph 7.4 ) was present at the start of the reaction ; aldh2 ( 33 
  g / ml ) , gtn ( 0.1 mm ) , dtt ( 0.2 mm ) , and 
  nad ( 0.2 mm ) were added as indicated .
1 yielded ( 1.0  0.5 )  10 absorbance 
  units ( a.u.)/s for the uncatalyzed reaction , ( 1.07  0.02 ) 
   10 a.u./s for the initial rate , ( 1.083  0.008 ) 
   10 a.u./s and ( 1.15  0.01 )  
  10 a.u./s after addition of gtn and ddt , respectively , ( 1.051 
   0.008 )  10 s for the apparent 
  inactivation rate constant after addition of nad , and ( 7.2 
   0.1 )  10 a.u./s for the residual activity . 
 
experimental conditions : p - npa ( 0.2 
  mm ) and nad ( 0.2 mm ) in 50 mm 
  potassium pi ( ph 7.4 ) were present at the start of the reaction ; 
  aldh2 ( 33 g / ml ) and gtn ( 0.1 mm ) were added as indicated .
curve 
  fitting yielded the following results : ( 0.048  0.009 )  
  10 a.u./s for the uncatalyzed reaction , ( 3.65  0.08 ) 
   10 a.u./s for the initial activity , ( 2.6  0.1 ) 
   10 s for the apparent inactivation rate 
  constant , and ( 0.0567  0.0008 )  10 a.u./s for the 
  residual activity .
the figure shows the formation of 
  p - nitrophenol from p - npa , as monitored at 400 nm .
panel 
  a shows that , in the absence of nad , gtn did not 
  significantly affect esterase activity . however , inactivation set in 
  immediately after the addition of nad .
experimental conditions 
  were : p - npa ( 0.2 mm ) in 50 mm potassium 
  pi ( ph 7.4 ) was present at the start of the reaction ; aldh2 ( 33 
  g / ml ) , gtn ( 0.1 mm ) , dtt ( 0.2 mm ) , and 
  nad ( 0.2 mm ) were added as indicated .
1 yielded ( 1.0  0.5 )  10 absorbance 
  units ( a.u.)/s for the uncatalyzed reaction , ( 1.07  0.02 ) 
   10 a.u./s for the initial rate , ( 1.083  0.008 ) 
   10 a.u./s and ( 1.15  0.01 )  
  10 a.u./s after addition of gtn and ddt , respectively , ( 1.051 
   0.008 )  10 s for the apparent 
  inactivation rate constant after addition of nad , and ( 7.2 
   0.1 )  10 a.u./s for the residual activity . 
 
experimental conditions : p - npa ( 0.2 
  mm ) and nad ( 0.2 mm ) in 50 mm 
  potassium pi ( ph 7.4 ) were present at the start of the reaction ; 
  aldh2 ( 33 g / ml ) and gtn ( 0.1 mm ) were added as indicated .
curve 
  fitting yielded the following results : ( 0.048  0.009 )  
  10 a.u./s for the uncatalyzed reaction , ( 3.65  0.08 ) 
   10 a.u./s for the initial activity , ( 2.6  0.1 ) 
   10 s for the apparent inactivation rate 
  constant , and ( 0.0567  0.0008 )  10 a.u./s for the 
  residual activity .
activities of aldh2 after gtn preconditioning  in the 
  experiments described so far , we studied the inactivation of aldh in real time 
  by monitoring the dehydrogenase and esterase activities in the presence of 
  gtn .
although this procedure enables the determination of the inactivation 
  kinetics , the presence of gtn after inactivation may compromise subsequent 
  attempts to restore enzyme activity .
moreover , because of the inevitable 
  presence of the substrate during preincubation , fairly high gtn concentrations 
  were required .
therefore , in a different procedure , referred to as gtn 
  preconditioning , we preincubated the enzyme in the presence of gtn followed by 
  determination of the activity ( dehydrogenase or esterase ) after 200-fold 
  dilution of the preincubation mixture . in this way
inactivation takes place in 
  the absence of substrate , allowing application of much lower gtn 
  concentrations and determination of activities in the virtual absence of 
  gtn .
figure 4.effect of nad and nadh on the rate of inactivation by gtn of 
  ester hydrolysis .
the figure shows the effects of nad and nadh 
  ( 0.2 mm ) on the apparent inactivation rate constant in the absence 
  and presence of 0.4 mm dtt .
( n = 3 ) . further conditions : 33 g / ml aldh2 , 0.2 
  mm p - npa , and 0.1 mm gtn in 50 mm 
  potassium pi ( ph 7.4 ) .
effect of nad and nadh on the rate of inactivation by gtn of 
  ester hydrolysis .
the figure shows the effects of nad and nadh 
  ( 0.2 mm ) on the apparent inactivation rate constant in the absence 
  and presence of 0.4 mm dtt .
( n = 3 ) . further conditions : 33 g / ml aldh2 , 0.2 
  mm p - npa , and 0.1 mm gtn in 50 mm 
  potassium pi ( ph 7.4 ) . as is shown in fig . 5 , 
  preincubation with 1 m gtn at ph 7.5 resulted in complete 
  inactivation of acetaldehyde dehydrogenation , whereas the enzyme remained 
  active after preincubation in the absence of gtn .
part of the dehydrogenase 
  activity that was lost after preincubation with gtn was recovered in the 
  presence of dtt .
partial reactivation occurred in the presence of 
  lpa - h2 as well , although to a smaller degree .
comparable results 
  were obtained when preincubation took place at ph 6.5 and when 
   standard  assay conditions were applied for both reactions ( ph 
  9.0 with nad for the dehydrogenase reaction , and ph 7.5 without 
  nad for the esterase reaction ) ( see supplemental fig .
time dependence of irreversible aldh2 inactivation  to 
  further elucidate the mechanism of irreversible inactivation , we performed a 
  series of experiments of the type shown in 
  fig .
1a and varied the 
  time of dtt addition , leaving all other conditions equal . the results , which 
  are described in greater detail under supplemental materials ( fig .
s7 and 
  accompanying text ) , showed that most of the inactivation took place within the 
  same time span in which nad reduction was inhibited by gtn in the 
  absence of dtt , although a slow additional inactivation was also apparent .
the 
   rapid  irreversible inactivation was exacerbated when the gtn 
  concentration was increased , whereas the slower process was not affected .
the figure shows the effects of preincubation of aldh2 at 
  ph 7.5 with 1 m gtn on the subsequent determination of 
  dehydrogenase activity in the presence of nad at ph 7.4 in the 
  absence and presence of 2 mm dtt and lpa - h2 . 
 
experimental conditions for preincubation were : 0.27 mg / ml aldh2 , 1 
  m gtn , 0.2 mm mgcl2 , 2 mm 
  nad , 50 mm napi ( ph 7.5 ) for 10 min at 37 
  c .
assay conditions were : 1.5 g / ml aldh2 , 2 mm 
  nad , 10 mm mgcl2 , and 0.2 mm 
  acetaldehyde in 50 mm napi ( ph 7.5 ) at 25 c .
the figure shows the effects of preincubation of aldh2 at 
  ph 7.5 with 1 m gtn on the subsequent determination of 
  dehydrogenase activity in the presence of nad at ph 7.4 in the 
  absence and presence of 2 mm dtt and lpa - h2 . 
 
experimental conditions for preincubation were : 0.27 mg / ml aldh2 , 1 
  m gtn , 0.2 mm mgcl2 , 2 mm 
  nad , 50 mm napi ( ph 7.5 ) for 10 min at 37 
  c .
assay conditions were : 1.5 g / ml aldh2 , 2 mm 
  nad , 10 mm mgcl2 , and 0.2 mm 
  acetaldehyde in 50 mm napi ( ph 7.5 ) at 25 c .
we also determined the effect of incubation time in experiments of the type 
  described in fig . 5 , in which 
  we preincubated the enzyme in the presence and absence of gtn ( 0.18 
  mm ) and dtt ( 2 mm ) for different lengths of time , and 
  then measured the dehydrogenase activity after 15-fold dilution . the results , 
  presented in fig .
6 , show that 
  in the absence of dtt the enzyme tends to lose activity over time ( 34  
  2% in 1 h ) .
the same trend was observed in the presence of gtn , but in that 
  case approximately half of the activity was additionally lost within the first 
  15 min . as a result , 13  4% of the activity remained after 1 h of 
  preincubation .
dtt offered moderate protection against the gradual 
  inactivation in the absence of gtn ( 20  3% activity loss after 1 h ) . 
  in the combined presence of dtt and gtn ,
inactivation and reactivation of aldh2-catalyzed gtn 
  reduction  to assess how gtn affects its own reduction by 
  mitochondrial aldh we measured gtn reductase activity , removed low molecular 
  weight ingredients from the assay mixture by gel filtration , and repeated the 
  activity assay with the recovered enzyme . before gel filtration , 1,2-gdn 
  formation
was always observed , but omission of dtt from the reaction mixture 
  reduced pre - column activity by 85  2% 
  ( fig .
the activity in the 
  absence of dtt corresponded to approximately one turnover ( 280 nm 
  1,2-gdn were formed by 332 nm aldh ) , in line with the proposal that 
  reduction of gtn results in oxidation of active site cysteine residues .
after 
  gel filtration , the recovered enzyme showed no activity in the absence of dtt , 
  but produced a greater than stoichiometric amount of 1,2-gdn in its presence , 
  in agreement with the proposed role of dtt in regeneration of active site 
  thiols .
fig . 7 also shows that 
  the recovered enzyme was significantly less active than before treatment , 
  suggesting partially irreversible inactivation . in parallel experiments , the 
  same procedure ( enzymatic assay , enzyme recovery , repeated assay ) did not 
  affect dehydrogenase and esterase activities ( dehydrogenation : 0.39  
  0.05 and 0.37  0.07 mol / mg / min before and after enzyme recovery , 
  respectively ; ester hydrolysis : 0.219  0.003 and 0.223
 0.004 
  nmol / mg / min before and after enzyme recovery , respectively ) , which rules out 
  that partial inactivation of gtn reduction was an artifact .
the enzyme was preincubated for various times , after which dehydrogenase 
  activities were determined by monitoring the linear absorbance increase at 340 
  nm over a period of 5 min .
preincubation was performed in the absence 
  ( open symbols ) and presence ( filled symbols ) of dtt , and in 
  the absence ( circles ) or presence ( squares ) of gtn . 
 
preincubation conditions were : 0.48 mg / ml aldh2 , 1 mm 
  nad , and 0.18 mm gtn and 0.2 mm dtt as 
  indicated in 300 mm potassium pi ( ph 7.4 ) .
assay 
  conditions were : 19 g / ml aldh2 , 1 mm acetaldehyde , 1 
  mm nad , and 2 mm dtt in 50 mm 
  potassium pi ( ph 7.4 ) .
the enzyme was preincubated for various times , after which dehydrogenase 
  activities were determined by monitoring the linear absorbance increase at 340 
  nm over a period of 5 min .
preincubation was performed in the absence 
  ( open symbols ) and presence ( filled symbols ) of dtt , and in 
  the absence ( circles ) or presence ( squares ) of gtn . 
 
preincubation conditions were : 0.48 mg / ml aldh2 , 1 mm 
  nad , and 0.18 mm gtn and 0.2 mm dtt as 
  indicated in 300 mm potassium pi ( ph 7.4 ) .
assay 
  conditions were : 19 g / ml aldh2 , 1 mm acetaldehyde , 1 
  mm nad , and 2 mm dtt in 50 mm 
  potassium pi ( ph 7.4 ) .
figure 7.effect of preincubation of aldh2 with gtn on the aldh2-catalyzed 
  reduction of gtn .
gtn reduction was determined in the absence or presence 
  of dtt ( pre - column activity ) .
subsequently , the enzyme was rapidly recovered 
  from the assay mixture by ym-10 chromatography , and the gtn reductase activity 
  was measured again in the absence or presence of thiols ( post - column 
  activity ) .
experimental conditions were : 4 g of aldh2 , 2 m 
  [ c]gtn ( 50 000 dpm ) , 3 mm mgcl2 , 1 
  mm nad , 1 mm edta , 1 mm egta , and 
  2 mm gsh with 2 mm dtt as indicated in 50 mm 
  potassium pi ( ph 7.4 ) for 10 min at 37 c .
gtn reduction was determined in the absence or presence 
  of dtt ( pre - column activity ) .
subsequently , the enzyme was rapidly recovered 
  from the assay mixture by ym-10 chromatography , and the gtn reductase activity 
  was measured again in the absence or presence of thiols ( post - column 
  activity ) .
experimental conditions were : 4 g of aldh2 , 2 m 
  [ c]gtn ( 50 000 dpm ) , 3 mm mgcl2 , 1 
  mm nad , 1 mm edta , 1 mm egta , and 
  2 mm gsh with 2 mm dtt as indicated in 50 mm 
  potassium pi ( ph 7.4 ) for 10 min at 37 c .
the figure depicts the effect of preincubation with gtn ( 0.1 
  mm , 10 min , 37 c ) on the dehydrogenation of monal 62 in the 
  absence and presence of various thiols .
experimental conditions were : 8.1 
  g / ml rat liver mitochondria , 3 m monal 62 , 0.1 
  mm nad , and dtt ( 2 mm ) , -me ( 50 
  mm ) , or lpa - h2 ( 0.1 mm ) as indicated , in 10 
  mm tris ( ph 7.4 ) , 250 mm sucrose , 5 mm egta , 
  and 2 mm mgcl2 . inhibition by gtn and reactivation by thiols of aldh in intact 
  mitochondria .
the figure depicts the effect of preincubation with gtn ( 0.1 
  mm , 10 min , 37 c ) on the dehydrogenation of monal 62 in the 
  absence and presence of various thiols .
experimental conditions were : 8.1 
  g / ml rat liver mitochondria , 3 m monal 62 , 0.1 
  mm nad , and dtt ( 2 mm ) , -me ( 50 
  mm ) , or lpa - h2 ( 0.1 mm ) as indicated , in 10 
  mm tris ( ph 7.4 ) , 250 mm sucrose , 5 mm egta , 
  and 2 mm mgcl2 .
inactivation by gtn and reactivation by thiols of aldh in intact 
  mitochondria  to probe the inactivation by gtn and reactivation by 
  thiols in a physiologically more relevant setting , we investigated the 
  inhibition of aldh by gtn and its reactivation by thiols in isolated rat liver 
  mitochondria .
dehydrogenase activity was determined as the conversion of the 
  aldehyde monal 62 to naphthoic acid by fluorescence spectroscopy in the 
  presence of intact mitochondria preincubated with 100 m gtn ( 37 
  c , 10 min ) . as illustrated in fig . 
 
8 , preincubation with gtn caused a loss of activity of 84  
  2% that was partly ( 51  5% ) restored by 2 mm dtt . 
 
lpa - h2 ( 2 mm ) and -mercaptoethanol ( -me ) at 
  the very high concentration of 50 mm reactivated the enzyme to 
  comparable levels . at a concentration of 2 mm ,
-me , cys , and 
  gsh caused no or marginal reactivation ; at a physiologically relevant 
  concentration of 0.1 mm , lpa - h2 was ineffective as well 
  ( supplemental fig .
s8a ) . these results are in good agreement with the 
  observations for the purified enzyme and with a previous report on aldh 
  inactivation in intact mitochondria 
  ( 30 ) . raising the gtn 
  concentration
gradually increased the extent of inactivation from 20  
  10% at 1 m to 81  4% at 100 m gtn with 
  an ic50 of 2.7  0.5 m ( supplemental fig . 
 
dtt ( 2 mm ) and lpa - h2 ( 10 mm ) 
  restored activity to 80 and 60% of the original activity with 1 and 100 
  m gtn , respectively .
these results suggest that partially 
  irreversible aldh inactivation occurs in intact mitochondria as well .
inactivation of mitochondrial aldh by gtn  in agreement with 
  previous reports ( 10 , 
  22 , 
  23 ) , we found that gtn caused 
  time - dependent inactivation of both the dehydrogenase and esterase activities 
  of aldh2 .
the inactivation rates 
  exhibited a tendency toward saturation at high gtn and low substrate 
  concentrations , and we could fit the curves of kobs 
 versus [ gtn ] and 1/[acald ] or 1/[p - npa ] to hyperbolic 
  functions . the data also show that any contribution from reversible 
  competitive inhibition can be neglected ( see supplemental materials for a 
  discussion of this issue ) .
rapid - equilibrium binding of substrate and inhibitor , followed by a slow 
  inactivation step , is described by an equation of the form , 
  ( eq
. 2)\documentclass[10pt]{article } 
 \usepackage{amsmath } 
 \usepackage{wasysym } 
 \usepackage{amsfonts } 
 \usepackage{amssymb } 
 \usepackage{amsbsy } 
 \usepackage{mathrsfs } 
 \usepackage{pmc } 
 \usepackage[euler]{upgreek } 
 \pagestyle{empty } 

 \oddsidemargin -1.0 in 

 \begin{document } 
 \begin{equation*}\;\;k_{{\mathrm{obs}}}=\frac{k_{i}{\times}[i]}{[i]+k_{i}{\times}(1+[{\mathrm{s}}]/k_{s})}\end{equation*}\end{document } 
  in which [ i ] and [ s ] are the concentrations of inhibitor and 
  substrate , ki and ks are the 
  respective dissociation constants , and ki is the 
  inactivation rate constant .
this model predicts the observed hyperbolic 
  dependence of kobs on the gtn concentration and the 
  reciprocal substrate concentration .
however , our results suggest an apparent 
  dissociation constant ( ks ) for acetaldehyde some 23 
  orders of magnitude above the published km . in fact , 
  analysis of the data suggests values for the apparent dissociation constants 
  for acetaldehyde , p - npa , and gtn in the order of 10 
  m ( see supplementary materials ) .
similar discrepancies have been 
  reported for the mutual inhibition of esterase and dehydrogenase activities 
  ( 21 , 
  24 ) .
interestingly , inhibition 
  of gtn reduction by aldh2 also required more than 1000-fold higher 
  concentrations of acetaldehyde than might have been expected 
  ( 9 ) .
this was ascribed to the 
  absence of nad , which has been reported to increase the substrate 
  affinity of mitochondrial aldh 
  ( 20 ) .
because our results 
  suggest that the phenomenon persists in the presence of nad , an 
  alternative explanation seems to be required .
it has been debated whether the 
  dehydrogenase and esterase reactions take place at the same site .
this matter 
  appears settled now , with convincing evidence in favor of a single active 
  site , although some of the earlier data that suggested a 2-site model remain 
  unexplained ( 21 , 
  2428 ) . 
  to accommodate the disparate observations , we propose that all substrates 
  react in the same active site pocket , but that the unnatural esterase 
  substrates and gtn can occupy any of a number of poorly defined low - affinity 
  positions , whereas the dehydrogenase substrates can and must be bound to a 
  strictly defined high - affinity site to enable hydride transfer ( supplementary 
  materials scheme s1 ) .
somewhat surprisingly , nad profoundly affected the rate of 
  inactivation of the esterase reaction by gtn . in the presence of dtt , 
 
a 
  substantial decrease of the dissociation constant for gtn in the presence of 
  nad seems to be an implausible 
  explanation.4 it is 
  more likely that nad binding exposes a target to gtn that is 
  poorly accessible in the absence of the cofactor .
protection and restoration of the dehydrogenase reaction by 
  thiols  the inhibition of mitochondrial aldh by isosorbide dinitrate 
  is completely reversed by -me 
  ( 23 ) .
inhibition of yeast aldh 
  by no was almost completely prevented and largely reversed by dtt , whereas 
  -me and gsh were less efficient 
  ( 29 ) .
mitochondrial aldh was 
  partially protected against gtn - induced inactivation by dtt and 
  lpa - h2 , but not by gsh 
  ( 30 ) .
it has been demonstrated 
  that the conversion of gtn to 1,2-gdn by mitochondrial aldh requires the 
  presence of a reducing agent 
  ( 3 ) .
the highest activity was 
  observed in the presence of dtt , whereas lpa - h2 was about half as 
  effective ; activity was low in the presence of cys and -me and virtually 
  absent in the presence of gsh . in line with these reports , we found that dtt 
  and lpa - h2 , but not cys or gsh , protected dehydrogenation against 
  gtn - induced inhibition .
accordingly , inactivation of aldh2 seems to be due to 
  the oxidation and subsequent disulfide formation of an essential cysteine 
  residue .
the poorer performance of gsh compared with dtt has been ascribed to 
  a combination of the lower redox potential of dtt and the presence of two 
  thiol groups per molecule , which gives dtt a kinetic advantage over gsh ( and 
  -me ) in achieving complete reduction of the disulfide bridge 
  ( 29 ) .
when dtt was added to the enzyme after complete inactivation by gtn , the 
  thiol could only rescue a minor fraction of dehydrogenase activity , suggesting 
  that at that time most of the enzyme was no longer present as a disulfide . 
  both residual and thiol - restored activities diminished when the gtn 
  concentration was increased .
this might suggest that the magnitude of residual 
  and restored activity is determined by competition between gtn and substrate ; 
  however , the acetaldehyde concentration hardly affected either activity . 
 
conceivably , the gtn concentration could also modulate restored activity by 
  competition with dtt , because the gtn to dtt ratio might affect the 
  steady - state levels of the inactive and active aldh2 species in a reaction 
  cycle involving oxidation / inactivation by gtn and reduction / reactivation by 
  dtt . however , the dtt concentration also did not affect restored activity .
we 
  propose that , in addition to the dtt - reversible oxidation of the active site 
  cysteine by gtn , the oxidized cysteinyl and/or some other active site residues 
  may engage in a secondary reaction with gtn to yield an enzyme species that 
  does not support dehydrogenation and is not reduced by dtt .
scheme 1 shows a simple 
  model for gtn - induced inactivation that accommodates most of the observations , 
  particularly the observations that ( i ) irreversible inhibition in the absence 
  of dtt is only partial and ( ii ) the extent of irreversible inhibition 
  increases with the gtn concentration .
accordingly , partitioning between 
  reversible and irreversible inhibition in the absence of a reductant will 
  depend on the ratio of the rates of disulfide formation 
  ( krev ) and irreversible inactivation 
  ( kirr[gtn ] ) , the latter of which depends on the 
  gtn concentration . in the presence of a reductant ( e.g. dtt )
, 
  reversible inactivation will be abolished by regeneration of the active enzyme 
  state ( esh ) from the disulfide species 
  ( es - s ) , and irreversible inhibition will be slowed down by 
  the dtt - induced reduction of the oxidized thiol species 
  ( es ) .
the model correctly predicts that the 
  fraction of aldh2 that is irreversibly inactivated by gtn in the absence of 
  dtt will depend on the gtn concentration but not on the concentrations of dtt 
  and acetaldehyde applied to reactivate the enzyme .
scheme 1.irreversible inactivation of aldh - catalyzed gtn reduction . under low 
  turnover conditions ( [ gtn ] , [ dtt ] < < km , 
  km ) ,
the enzyme cycles between the reduced 
  thiol / thiolate , oxidized thiyl , and oxidized disulfide states . at higher 
  concentrations , gtn and the thiol reductant ( here represented by dtt )
compete 
  for the oxidized thiyl state , resulting in direct regeneration of the reduced 
  enzyme with dtt , or in irreversible inactivation with gtn 
  ( esh , es- , es - s , 
  and ei represent the reduced ( thiol / thiolate ) , oxidized 
  thiyl , oxidized disulfide , and irreversibly inactivated enzyme states ; 
  kox , krev , kirr , 
  kred , and kred stand for the 
  rate constants for gtn - induced thiolate oxidation , dtt - reversible disulfide 
  formation , gtn - induced dtt - irreversible inactivation , and dtt - exacted 
  reduction of the es - s and es 
  enzyme states , respectively ) .
note that because of the branching of the 
  reaction after thiol oxidation , irreversible inactivation in the absence of 
  dtt will only be partial , with the extent depending on the concentration of 
  gtn .
inactivation will be slower in the presence of dtt but , on account of the 
  continuous recycling of the disulfide to the active reduced thiol , all of the 
  enzyme should eventually be inactivated irreversibly .
irreversible inactivation of aldh - catalyzed gtn reduction . under low 
  turnover conditions ( [ gtn ] , [ dtt ] < < km , 
  km ) ,
the enzyme cycles between the reduced 
  thiol / thiolate , oxidized thiyl , and oxidized disulfide states . at higher 
  concentrations , gtn and the thiol reductant ( here represented by dtt )
compete 
  for the oxidized thiyl state , resulting in direct regeneration of the reduced 
  enzyme with dtt , or in irreversible inactivation with gtn 
  ( esh , es- , es - s , 
  and ei represent the reduced ( thiol / thiolate ) , oxidized 
  thiyl , oxidized disulfide , and irreversibly inactivated enzyme states ; 
  kox , krev , kirr , 
  kred , and kred stand for the 
  rate constants for gtn - induced thiolate oxidation , dtt - reversible disulfide 
  formation , gtn - induced dtt - irreversible inactivation , and dtt - exacted 
  reduction of the es - s and es 
  enzyme states , respectively ) . note that because of the branching of the 
  reaction after thiol oxidation , irreversible inactivation in the absence of 
  dtt will only be partial , with the extent depending on the concentration of 
  gtn .
inactivation will be slower in the presence of dtt but , on account of the 
  continuous recycling of the disulfide to the active reduced thiol , all of the 
  enzyme should eventually be inactivated irreversibly . as a rather trivial alternative explanation for the dependence of the 
  extent of irreversible inactivation on the concentration of gtn , one could 
  assume that gtn or one of its metabolites is inactivating the enzyme in a 
  totally unrelated reaction .
although such a reaction would be expected to 
  affect the rate rather than the extent of irreversible inactivation , it might 
  explain the observations of fig . 
  1 ,
provided the reaction is slow and the time of gtn and dtt 
  additions did not vary much between experiments . to investigate that 
  possibility we performed the experiments illustrated by figs . 
  6 and supplemental s7 , in which 
  we varied the time between gtn and dtt addition .
the results indicated that 
  most of the activity was lost within the same time frame in which gtn 
  inhibited the enzyme ( in the absence of dtt ) , in agreement with the 
  mechanism - based model of inactivation presented in 
  scheme 1 .
although a slower reaction was also apparent , it did not depend on the gtn 
  concentration and also occurred in the absence of gtn , suggesting that it 
  represents unspecific deterioration of the enzyme upon prolonged incubation . 
  in the preincubation experiments ( fig . 
 
6 ) we made the observation that , although dtt protected the enzyme 
  against inactivation in the absence of gtn , it exacerbated the inactivation in 
  its presence . although paradoxical at first sight , this observation is in line 
  with expectations for the mechanism - based model of irreversible inactivation 
  proposed by us . in the absence of dtt , the enzyme undergoes only one turnover 
  in the presence of gtn , and mechanism - based inactivation will be halted after 
  that . in the presence of dtt , multiple turnovers with gtn
will occur , which 
  will result in complete inactivation , even if dtt diminishes the fraction of 
  irreversibly inactivated enzyme per turnover .
taken together , these results 
  provide strong support for mechanism - based inactivation as the origin of 
  irreversible inactivation .
the strictly kinetic nature of the present study precludes conclusions 
  about the site or identity of the structural changes underlying the 
  inactivation processes , although the literature may offer some clues .
a 
  secondary reaction , in which gtn or one of its metabolites reacts with an 
  oxidized or nitrated thiol before generation of the disulfide can occur , might 
  result in irreversible formation of thiol sulfinate and sulfonate products 
  ( 6 , 
  14 ) .
one candidate 
  inactivating agent is nitroxyl ( no ) , which has been proposed to be 
  a product of gtn metabolism 
  ( 31 ) , and which was reported 
  to cause partially irreversible inhibition of aldh , presumably by the 
  formation of sulfinamides 
  ( 32 ) .
gtn can 
  stimulate sgc in the presence of purified aldh2 , and this stimulation is 
  mediated by no even though the proposed mechanism for aldh - supported 
  decomposition of gtn predicts nitrite as the only product 
  ( 12 ) .
formation of no in the 
  secondary reaction occurring at higher gtn concentrations might explain those 
  observations .
we are currently carrying out mass spectrometric studies with 
  wild - type aldh2 and several active site mutants to this end . physiological implications 
nitrate tolerance is a complex 
  and almost certainly multifactorial phenomenon , with potential contributions 
  from impaired bioactivation , oxidative stress , sgc desensitization , and 
  physiological counter - regulation 
  ( 3 , 
  6 , 
  14 , 
  15 ) .
the role of impaired gtn 
  bioactivation depends on the physiologically relevant mechanism of gtn 
  biotransformation . because decomposition of gtn by aldh2 results in oxidation 
  of the active site thiol that must subsequently be reduced to sustain 
  catalysis , the proposed involvement of aldh2 in the bioactivation of gtn 
  revived the early proposal that ascribed nitrate tolerance to thiol depletion 
  ( 34 ) .
however , whereas the 
  mechanism of bioactivation involves reversible thiol oxidation , an 
  irreversible or slowly reversible process must underlie nitrate tolerance . the 
  irreversible inhibition reported in the present study
may , therefore , 
  contribute to nitrate tolerance in vivo . in the absence of dtt
the rate of irreversible inactivation can be 
  estimated from the observed inactivation rates ( reversible + irreversible ) and 
  the relative activity after reactivation .
s1 
  yields an apparent inhibition rate constant of 3  10 
  m s with a limit of 0.040.06 
  s. according to figs . 
  1 and 
  2 a large fraction of the 
  inactivation ( 70% ) will be irreversible , suggesting that the rate constant 
  for irreversible inactivation is of similar magnitude . for gtn concentrations 
  in the physiological range ( 0.010.1 m )
this would result 
  in lifetimes for active aldh2 between 6 and 60 h. for the inactivation in the 
  presence of dtt a rate constant of 4  10 
  s can be derived ( fig . 
 
2 ) , but this rate corresponds to saturating concentrations of gtn 
  and dtt and can not be easily extrapolated to physiologically relevant levels . 
  moreover ,
the present model does not account for the small but significant 
  fraction of irreversible inhibition ( 7% ) that seems to be refractory to 
  dtt ( fig .
s2 ) . a crucial question regarding the physiological significance of irreversible 
  aldh2 inactivation concerns the applicability of the observations to gtn 
  reduction .
the results presented in fig . 
  7 suggest that gtn reduction behaves like aldehyde dehydrogenation 
  in this respect . in the absence of dtt
the enzyme turned over only once and 
  after recovery no more gtn was reduced , in line with the hypothesis that the 
  reaction with gtn results in oxidation of the active site thiol . in the same 
  time frame the enzyme underwent multiple turnovers ( 5.35 times ) in the 
  presence of dtt .
however , activity diminished by 33 and 51% after 
  preincubation with 2 m gtn in the absence and presence of dtt , 
  respectively .
considering that , during preincubation in the presence of dtt , 
  aldh2 underwent 5.35 turnovers on average , the 51% inhibition after recovery 
  corresponds to a loss of active enzyme of 12.5% per turnover .
comparison with 
  the 33% inhibition during one turnover in the absence of dtt indicates that 
  dtt provides partial protection against irreversible inactivation by gtn . 
 
the central observation of the present paper is that in addition to the 
  previously noticed thiol - reversible inhibition , there is an irreversible 
  aspect to the inactivation of aldh2 by gtn .
this irreversible component , which 
  affects dehydrogenase activity as well as gtn reductase activity , may 
  contribute to the phenomenon of nitrate tolerance .
in line with this 
  hypothesis , thiols are often unable to reverse nitrate tolerance completely 
  ( 14 , 
  30 ) . in summary
, the present 
  study allows the following conclusions : ( i ) in the absence of dtt , gtn 
  oxidizes the active site thiol of mitochondrial aldh resulting in complete 
  inactivation of dehydrogenation , ester hydrolysis , and gtn reduction ; ( ii ) the 
  rate of inactivation is affected by the concentrations of substrate and 
  inhibitor , indicative of competitive binding , but the apparent dissociation 
  constants ( in the order of 10 m ) are much higher than 
  reported km values for the respective reactions , which 
  suggests that competition takes place outside of the high - affinity binding 
  site ; ( iii ) dehydrogenase activity is only restored to a modest extent by dtt , 
  indicating that a large fraction of inactivation is irreversible ; ( iv ) gtn 
  reduction is irreversibly inactivated in a similar fashion as dehydrogenase 
  activity , suggesting a role for irreversible aldh2 inactivation in the 
  development of nitrate tolerance .
 mitochondrial aldehyde dehydrogenase ( aldh2 ) may be involved in the 
  biotransformation of glyceryl trinitrate ( gtn ) , and the inactivation of aldh2 
  by gtn may contribute to the phenomenon of nitrate tolerance . 
 we studied the 
  gtn - induced inactivation of aldh2 by uv / visible absorption spectroscopy . 
  
 dehydrogenation of acetaldehyde and hydrolysis of 
  p - nitrophenylacetate ( p - npa ) were both inhibited by gtn . 
 the 
  rate of inhibition increased with the gtn concentration and decreased with the 
  substrate concentration , indicative of competition between gtn and the 
  substrates . 
 inactivation of p - npa hydrolysis was greatly enhanced in 
  the presence of nad+ , and , to a lesser extent , in the presence of 
  nadh . in the presence of dithiothreitol ( dtt ) inactivation of aldh2 was much 
  slower . 
 dihydrolipoic acid ( lpa - h2 ) was less effective than dtt , 
  whereas glutathione , cysteine , and ascorbate did not protect against 
  inactivation . 
 when dtt was added after complete inactivation , dehydrogenase 
  reactivation was quite modest ( 16% ) . 
 the restored dehydrogenase activity 
  correlated inversely with the gtn concentration but was hardly affected by the 
  concentrations of acetaldehyde or dtt . 
 partial reactivation of dehydrogenation 
  was also accomplished by lpa - h2 but not by gsh . 
 we conclude that , 
  in addition to the previously documented reversible inhibition by gtn that can 
  be ascribed to the oxidation of the active site thiol , there is an 
  irreversible component to aldh inactivation . 
 importantly , aldh2-catalyzed gtn 
  reduction was partly inactivated by preincubation with gtn , suggesting that 
  the inactivation of gtn reduction is also partly irreversible . 
 these 
  observations are consistent with a significant role for irreversible 
  inactivation of aldh2 in the development of nitrate tolerance . 
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
Supplementary Material