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An immunodiffusion activity stain for yeast histidinol dehydrogenase
4NAI
Sl
IC
AI
RIOCHt
LIISTRY
92,
189-
192 ( 1979)
mmunodiffusion Histidinol RAMLJNAS
A procedure enase-antibody system.
is described complexes
consisting
produces an the reaction intact HISN functional
Activity Stain for Yeast Dehydrogenase
BIGEI.IS
AND
for ‘detecting in immunodiffusion
of a tetrazolium
salt
G~KALD
enzymatic.Ay agar. and
insoluble formazan and stains is indicated by its dependence region. based on observations
The
a phenazine active precipitin on substrate with mutant
R.
FINK
active yeast histidinol dehydrogmethod employs a coupled dye methosulfate lines and forms
intermediate,
that
red. The specificity of by its dependence on an of the yeast HI.74 multi-
protein.
The HIS4ABC gene product in yeast is a 95,000 MW trifunctional protein that catalyzes steps 3, 2, and 10 of histidine biosynthesis (l-3). Antibody to the HIS4 protein and antibody to an ochre nonsense fragment with HIS4A and HtS4B activities cross-react strongly with crude extracts of wild-type yeast as judged by immunodiffusion in agar. Mutant proteins formed as a result of missense or certain nonsense or frameshift mutations can still retain partial function (4.5) and can still be recognized by both antibodies (6.7).. A sensitive technique based on staining for activity is described that detects the HIS4C activity, histidinol dehydrogenase. of wild-type and mutant proteins bound to antibody in immunodiffusion agar. The method employs a coupled dye system applicable to other dehydrogenases.
resuspended to a cell density of 100 Klett units, and then grown in minimal medium for 6 h to elevate the histidine biosynthetic enzymes. Harvested cells were resuspended in an equal volume of 0.05 M Tris-HCl. pH 7.5, 1.O M (NH&SO,, and 30% glycerol or 0.05 M Tris-HCl only and disrupted by vigorous vortexing in 30-m] Corex tubes (Kimax 8445) for 4 min using a threefold volume of 500-pm glass beads (Glasperlen 54170, Braun). The high ionic strength buffer was essential for the stability and detection of nonsense C and frameshift C HIS4 fragments (6) or missense C HIS4 proteins. Identical results were obtained with both buffers for all other strains tested. The high ionic strength conditions could have affected the conformation of the proteins with defects in the C region, reducing their susceptibility to proteolysis, or could have inhibited the proteases themselves. Antibody to the HIS4 protein and antibody to a 45,000 MW HIS4 ochre nonsense fragment (from a strain with nonsense mutation /zis4C864) was prepared by immunizing female New Zealand White rabbits and obtaining crude rabbit serum (R. Bigelis. J. Keesey. and G. R. Fink, unpublished
METHODS One-half-liter cultures of yeast cells were grown on yeast nitrogen base (Difco) (8), supplemented with 10 mg of histidine, at 30°C to a cell density of about 2150 Klett units (No. 52 filter), harvested, washed, 189
0003-269717910 Copynght All right\
IO I89-04$0?
.00/O
@ 1979 hy Aiademc Pres. Inc. of repmducmn ,n any form rcwrved
190
BIGFI.IS
i- histidinol FIG. I. lmmunodiffusion PMS dye solution with and extracts. to the reaction.
lower
wellh
- histidinol and
contained
Sidewells contained HIV protein. The
patterns without yeast
reacted histidinol. wild
in INTUpper type
crude
immune serum slide\ were dried
raised after
data). Both proteins had been purified to homogeneity by sodium dodecyl sulfate (SDS)-polyacrylamide’ gel electrophoresis prior to immunization. Ouchterlony (9) slides contained 1% agarose (Sigma), 0.05 M Tris-HCI. pH 7.5, and 0.15 M NaCl. After incubation at room temperature for 12 h, the slides were washed for about 12 h in the saline buffer before testing the activity of the precipitin lines. Preimmune serum did not produce an immunological reaction. The procedure of staining for activity was based on the coupled dye system phenazine methosulfate (PMS) (Sigma) and 2-p-iodo-3p - nitrophenyl - 5 - phenyl - 2H - tetrazolium chloride (INT) (Dajac Laboratories). The reduced NAD (Sigma) formed in the histidino1 dehydrogenase reaction was able to reduce INT nonenzymatically via the PMS intermediary to form an insoluble red INTformazan (IO). The dye solution was prepared fresh and was stored at 4°C in the dark. An l&ml preparation contained 10 ml of INT (3.2 mgiml). 2.0 ml of PMS (0.4 m&ml), 2.0 ml of a 0.2%’ gelatin solution. and 2.0 ml of NAD (20 mgiml) (I 1). The staining reaction was initiated by the addition of 20 ~1 of 0.1 M r-histidinol (Sigma) per milliliter of dye preparation. lmmunodiffusion slides were inverted in a dish containing the dye ’ Abbreviations used: SDS. PMS. phenazine mcthosulfatr: nitrophenyl-5-phenyl-:!H-tetrazolium
sodium dodecyl sulfate: INT. 2-/,+iodo-3-/>cho,ride.
AND
FINK
solution and substrate and incubated for10 min at 37’C in the dark. Color appearance was rapid with crude extracts and immediate with partially purified preparations. The reaction was terminated by immersion of the slide in 5? methanol-7.!ic; acetic acid. The distinct crimson precipitin lines were stable but changed to reddishpurple only after prolonged immersion. Washing overnight in methanol-acetic acid removed unreacted dye and allowed the slides to be dried. Such stained, pressed. and subsequently dried slides were stable for at least a year when stored in the dark. RESULTS AND DISCUSSION The INT-PMS staining procedure described allowed the detection of histidinol dehydrogenase activity after its interaction with antibody raised to the Hf.9 multifunctional protein. The specificity of the staining reaction in immunodiffusion agar was indicated by its dependence on r.-histidino1 (Fig. I). Antigen-antibody precipitation lines accumulated red dye and formed dark patterns only when substrate was added to the dye solution. Since the slides in Fig. I were not washed prior to incubation in the dye system. reaction occurred in the upper and lower crude extract wells, the intensity being above the background without histidinol. Crude serum alone, present in the sidewell. did not promote reaction of the dye system. The specificity of the reaction was also demonstrated by its requirement for an intact, undamaged C region of the HIS4 trifunctional protein. Figure 2 reveals the types of mutations employed and their relative locations within the HIS4ABC region. The derivation of these mutations and strains bearing them has been described elsewhere (1.2,4,5,12). Figure 3 shows a double-diffusion pattern employing crude serum and extracts of various yeast strains. Polar mutations nonsense A385 or frameshift A38 did not allow the production of antigenic material. Precipitin lines were
IMMUNODIFFUSION
FIG. locations functional
2. Genetic maps of the of frameshift mutations
HIS3
region. the
(above
ACTIVITY
ta) The line1 and
relative nonsense
191
STAIN
locations mutati~ms
of missense mutations. (b) The relative (below the line). A. B. and C‘ represent
segments.
formed by extracts of wild-type yeast and extracts of strains with a missense mutation in the A. B, or C region. However, only those precipitin lines formed by antigen with HZSIC function produced the red INTformazan and resulted in a dense line. Missense A and missense B mutant extracts produced both a precipitin line and a dense formazan line after activity staining. Missense C extracts. however, cross-reacted but did not produce a precipitin line that possessedenzymatic activity (Fig. 3). Similarly. as tabulated in Table 1. HIS4 fragments generated by nonsense mutations C.52 and C864 and frameshift mutation C712 cross-reacted with antibody but did not allow red dye accumulation. A wild-type
wild
yeast crude extract where the HfS4C histidinol dehydrogenase activity had been destroyed by freezing and thawing also retained the ability to complex with antibody but was unable to reduce INT. In contrast to missense. nonsense, or frameshift mutations in the C region, the extremely polar mutations in A listed in Table 1. nonsense A385 and A86 and frameshift A38 and A5lY. did not allow the production of CRM (crossreacting material) and consequently the expression of HISlC function based on the staining reaction. The results in Table 1 were the same regardless of whether serum from rabbits immunized with the 95.000 MW HIS4 multifunctional protein was used or whether
type
38 280
a FIG.
3. A double-diffusion
slide
reacted
b with
revealed by darh-field photography. tb) Precipitin center well contained immune rabbit serum raised of various yeast strains. In addition to a wild-type missense hi.c4ASM, missense hi.s4E33/. missense
INT-PMS
after
immunological
reaction.
lines activity-stained with the INT-PMS to the HIS4 protein. The outer v.ells contained strain. mutant strains bearing these lesions his4C280. frameshift his4A.18. and nonsense
(a)
Precipitin dye
lines
system. The crude extracts were employed:
his4A3Ri.
192
BIGELIS
AND
TABLE
FINK 1
Activity Strain Wild type Wild type Missense Missense Missense Nonsense Nonsense Frameshift Frameshift
or genetic
defect
a Presence or absence of a precipitin line based antibody to a HIS4 ochre nonsense fragment. b Inactivation by freezing and thawing.
with
+ Histidinol
CRM”
(inactivated”) A his4A588. hi.\JAl I I B his4B33/. his4ESY4 C his4C280. his4C,‘YO A his4A38.5. hi.t4A86 C his4C864, his4CSZ A his4A38. his4A5/Y c‘ hi.s4C7/-7
stain
+ + + + +
+
+ -
-
INT-PMS ~ Histidinol -
+ +
-
+ on interaction
serum from rabbits immunized with a 45,000 MW ochre nonsense fragment with only HIS4A or HIS4B activities was used. However, INT-PMS staining reactions were stronger when the latter serum was tested suggesting that antibody to the 95,000 MW HIS4 protein may have. to some degree. inhibited or occluded the histidinol dehydrogenase active site. Even though both antibodies were to SDS-denatured HIS4 proteins, both antibodies recognized native wild-type and missense HIS4 proteins and allowed enzymatic function of all of those with an undamaged HIS4C portion. The data obtained by this procedure is consistent with the recent determination of the multifunctional nature of the HIS4 protein (3) and also with the recent determination of the colinearity of the peptide map with the genetic map (based on the size of nonsense termination fragments) (6). The INT-PMS staining procedure is sensitive and significantly increases the intensity of precipitin lines (Fig. 3). especially if the incubation procedure is prolonged. Such a procedure allows the simultaneous determination of immunological activity and enzymatic activity and should be a convenient test for the activity of many dehydrogenase-antibody complexes. Similar procedures employing coupled dye systems
with
antibody
to the HIS-1
protein
or with
could be used to detect individual activities of enzyme aggregates complexed to antibody raised to only one component of the aggregate. REFERENCES I. Fink. G. R. (1964) .Sc~ic~,lc~c,146, 525-597. 2. Finh. G. R. t 1966) Gcrrctic.v 53, 445-459. 3. Bigelis. R.. Keesey. J.. and Fink. G. R. t 1977) iti Molecular Approaches to Eucaryotic Genetic Systems (Wilcox, G.. and Abelson, J.. eds.). ICN-UCLA Symposia. Academic Press. New York, Vol. VIII. l79- 187. 4. Shaffer. B.. Rytka. J.. and Fink. G. R. (1969) f’roc~. sYtrr. Ac,d. SI i. U..\.A. 63, 119% 1105, Fink. G. R.. and Styles. C. A. (1974) (;r,r/c,ric \ 77. 23 I-244. 6. Bigelis. R., and Burridge. K. (1978) Bicjchc,m. Bic~phys. Rr.s. Comw~ur~. 82. 312-327. 7. Bigelis. R.. and Fink, G. R. (1978) ./. f~~/~~~rr~~r~/. Mrrho1l.s. 22, 393-395. 8. Wickerham, L. J. (1946l.1. &r(~r~,ri<~/. 52, 193-301. 9. Ouchterlony. 0. ( 1949) Ac.ru Ptrfhcd. blic,rc&cl/. S<~trrrtf. 26, 507-5 15. 10. Nachlas, M. M.. Margulies. S. 1.. Goldberg. J. D.. and Seligman. A. M. (1960) Aocll. Bi~~c~lr~~m. 1. 317-326. I I. Martin. R. G.. Berberich. M. A., Ames. B. N.. Davis. W. W.. Goldberger. R. F., and Yourno. J. D. t 1971 )BI Methods in Enzymology (Tahor, H.. and Tabor. C. W.. eds.). Vol. 178. pp. 3-46. Academic Press. New York. 12. Culbertson. M. R., Charnas. L., Johnson. M. T.. and Fink. G. R. (1977) G;cucfic..s 86, 745-764.
Sl
IC
AI
RIOCHt
LIISTRY
92,
189-
192 ( 1979)
mmunodiffusion Histidinol RAMLJNAS
A procedure enase-antibody system.
is described complexes
consisting
produces an the reaction intact HISN functional
Activity Stain for Yeast Dehydrogenase
BIGEI.IS
AND
for ‘detecting in immunodiffusion
of a tetrazolium
salt
G~KALD
enzymatic.Ay agar. and
insoluble formazan and stains is indicated by its dependence region. based on observations
The
a phenazine active precipitin on substrate with mutant
R.
FINK
active yeast histidinol dehydrogmethod employs a coupled dye methosulfate lines and forms
intermediate,
that
red. The specificity of by its dependence on an of the yeast HI.74 multi-
protein.
The HIS4ABC gene product in yeast is a 95,000 MW trifunctional protein that catalyzes steps 3, 2, and 10 of histidine biosynthesis (l-3). Antibody to the HIS4 protein and antibody to an ochre nonsense fragment with HIS4A and HtS4B activities cross-react strongly with crude extracts of wild-type yeast as judged by immunodiffusion in agar. Mutant proteins formed as a result of missense or certain nonsense or frameshift mutations can still retain partial function (4.5) and can still be recognized by both antibodies (6.7).. A sensitive technique based on staining for activity is described that detects the HIS4C activity, histidinol dehydrogenase. of wild-type and mutant proteins bound to antibody in immunodiffusion agar. The method employs a coupled dye system applicable to other dehydrogenases.
resuspended to a cell density of 100 Klett units, and then grown in minimal medium for 6 h to elevate the histidine biosynthetic enzymes. Harvested cells were resuspended in an equal volume of 0.05 M Tris-HCl. pH 7.5, 1.O M (NH&SO,, and 30% glycerol or 0.05 M Tris-HCl only and disrupted by vigorous vortexing in 30-m] Corex tubes (Kimax 8445) for 4 min using a threefold volume of 500-pm glass beads (Glasperlen 54170, Braun). The high ionic strength buffer was essential for the stability and detection of nonsense C and frameshift C HIS4 fragments (6) or missense C HIS4 proteins. Identical results were obtained with both buffers for all other strains tested. The high ionic strength conditions could have affected the conformation of the proteins with defects in the C region, reducing their susceptibility to proteolysis, or could have inhibited the proteases themselves. Antibody to the HIS4 protein and antibody to a 45,000 MW HIS4 ochre nonsense fragment (from a strain with nonsense mutation /zis4C864) was prepared by immunizing female New Zealand White rabbits and obtaining crude rabbit serum (R. Bigelis. J. Keesey. and G. R. Fink, unpublished
METHODS One-half-liter cultures of yeast cells were grown on yeast nitrogen base (Difco) (8), supplemented with 10 mg of histidine, at 30°C to a cell density of about 2150 Klett units (No. 52 filter), harvested, washed, 189
0003-269717910 Copynght All right\
IO I89-04$0?
.00/O
@ 1979 hy Aiademc Pres. Inc. of repmducmn ,n any form rcwrved
190
BIGFI.IS
i- histidinol FIG. I. lmmunodiffusion PMS dye solution with and extracts. to the reaction.
lower
wellh
- histidinol and
contained
Sidewells contained HIV protein. The
patterns without yeast
reacted histidinol. wild
in INTUpper type
crude
immune serum slide\ were dried
raised after
data). Both proteins had been purified to homogeneity by sodium dodecyl sulfate (SDS)-polyacrylamide’ gel electrophoresis prior to immunization. Ouchterlony (9) slides contained 1% agarose (Sigma), 0.05 M Tris-HCI. pH 7.5, and 0.15 M NaCl. After incubation at room temperature for 12 h, the slides were washed for about 12 h in the saline buffer before testing the activity of the precipitin lines. Preimmune serum did not produce an immunological reaction. The procedure of staining for activity was based on the coupled dye system phenazine methosulfate (PMS) (Sigma) and 2-p-iodo-3p - nitrophenyl - 5 - phenyl - 2H - tetrazolium chloride (INT) (Dajac Laboratories). The reduced NAD (Sigma) formed in the histidino1 dehydrogenase reaction was able to reduce INT nonenzymatically via the PMS intermediary to form an insoluble red INTformazan (IO). The dye solution was prepared fresh and was stored at 4°C in the dark. An l&ml preparation contained 10 ml of INT (3.2 mgiml). 2.0 ml of PMS (0.4 m&ml), 2.0 ml of a 0.2%’ gelatin solution. and 2.0 ml of NAD (20 mgiml) (I 1). The staining reaction was initiated by the addition of 20 ~1 of 0.1 M r-histidinol (Sigma) per milliliter of dye preparation. lmmunodiffusion slides were inverted in a dish containing the dye ’ Abbreviations used: SDS. PMS. phenazine mcthosulfatr: nitrophenyl-5-phenyl-:!H-tetrazolium
sodium dodecyl sulfate: INT. 2-/,+iodo-3-/>cho,ride.
AND
FINK
solution and substrate and incubated for10 min at 37’C in the dark. Color appearance was rapid with crude extracts and immediate with partially purified preparations. The reaction was terminated by immersion of the slide in 5? methanol-7.!ic; acetic acid. The distinct crimson precipitin lines were stable but changed to reddishpurple only after prolonged immersion. Washing overnight in methanol-acetic acid removed unreacted dye and allowed the slides to be dried. Such stained, pressed. and subsequently dried slides were stable for at least a year when stored in the dark. RESULTS AND DISCUSSION The INT-PMS staining procedure described allowed the detection of histidinol dehydrogenase activity after its interaction with antibody raised to the Hf.9 multifunctional protein. The specificity of the staining reaction in immunodiffusion agar was indicated by its dependence on r.-histidino1 (Fig. I). Antigen-antibody precipitation lines accumulated red dye and formed dark patterns only when substrate was added to the dye solution. Since the slides in Fig. I were not washed prior to incubation in the dye system. reaction occurred in the upper and lower crude extract wells, the intensity being above the background without histidinol. Crude serum alone, present in the sidewell. did not promote reaction of the dye system. The specificity of the reaction was also demonstrated by its requirement for an intact, undamaged C region of the HIS4 trifunctional protein. Figure 2 reveals the types of mutations employed and their relative locations within the HIS4ABC region. The derivation of these mutations and strains bearing them has been described elsewhere (1.2,4,5,12). Figure 3 shows a double-diffusion pattern employing crude serum and extracts of various yeast strains. Polar mutations nonsense A385 or frameshift A38 did not allow the production of antigenic material. Precipitin lines were
IMMUNODIFFUSION
FIG. locations functional
2. Genetic maps of the of frameshift mutations
HIS3
region. the
(above
ACTIVITY
ta) The line1 and
relative nonsense
191
STAIN
locations mutati~ms
of missense mutations. (b) The relative (below the line). A. B. and C‘ represent
segments.
formed by extracts of wild-type yeast and extracts of strains with a missense mutation in the A. B, or C region. However, only those precipitin lines formed by antigen with HZSIC function produced the red INTformazan and resulted in a dense line. Missense A and missense B mutant extracts produced both a precipitin line and a dense formazan line after activity staining. Missense C extracts. however, cross-reacted but did not produce a precipitin line that possessedenzymatic activity (Fig. 3). Similarly. as tabulated in Table 1. HIS4 fragments generated by nonsense mutations C.52 and C864 and frameshift mutation C712 cross-reacted with antibody but did not allow red dye accumulation. A wild-type
wild
yeast crude extract where the HfS4C histidinol dehydrogenase activity had been destroyed by freezing and thawing also retained the ability to complex with antibody but was unable to reduce INT. In contrast to missense. nonsense, or frameshift mutations in the C region, the extremely polar mutations in A listed in Table 1. nonsense A385 and A86 and frameshift A38 and A5lY. did not allow the production of CRM (crossreacting material) and consequently the expression of HISlC function based on the staining reaction. The results in Table 1 were the same regardless of whether serum from rabbits immunized with the 95.000 MW HIS4 multifunctional protein was used or whether
type
38 280
a FIG.
3. A double-diffusion
slide
reacted
b with
revealed by darh-field photography. tb) Precipitin center well contained immune rabbit serum raised of various yeast strains. In addition to a wild-type missense hi.c4ASM, missense hi.s4E33/. missense
INT-PMS
after
immunological
reaction.
lines activity-stained with the INT-PMS to the HIS4 protein. The outer v.ells contained strain. mutant strains bearing these lesions his4C280. frameshift his4A.18. and nonsense
(a)
Precipitin dye
lines
system. The crude extracts were employed:
his4A3Ri.
192
BIGELIS
AND
TABLE
FINK 1
Activity Strain Wild type Wild type Missense Missense Missense Nonsense Nonsense Frameshift Frameshift
or genetic
defect
a Presence or absence of a precipitin line based antibody to a HIS4 ochre nonsense fragment. b Inactivation by freezing and thawing.
with
+ Histidinol
CRM”
(inactivated”) A his4A588. hi.\JAl I I B his4B33/. his4ESY4 C his4C280. his4C,‘YO A his4A38.5. hi.t4A86 C his4C864, his4CSZ A his4A38. his4A5/Y c‘ hi.s4C7/-7
stain
+ + + + +
+
+ -
-
INT-PMS ~ Histidinol -
+ +
-
+ on interaction
serum from rabbits immunized with a 45,000 MW ochre nonsense fragment with only HIS4A or HIS4B activities was used. However, INT-PMS staining reactions were stronger when the latter serum was tested suggesting that antibody to the 95,000 MW HIS4 protein may have. to some degree. inhibited or occluded the histidinol dehydrogenase active site. Even though both antibodies were to SDS-denatured HIS4 proteins, both antibodies recognized native wild-type and missense HIS4 proteins and allowed enzymatic function of all of those with an undamaged HIS4C portion. The data obtained by this procedure is consistent with the recent determination of the multifunctional nature of the HIS4 protein (3) and also with the recent determination of the colinearity of the peptide map with the genetic map (based on the size of nonsense termination fragments) (6). The INT-PMS staining procedure is sensitive and significantly increases the intensity of precipitin lines (Fig. 3). especially if the incubation procedure is prolonged. Such a procedure allows the simultaneous determination of immunological activity and enzymatic activity and should be a convenient test for the activity of many dehydrogenase-antibody complexes. Similar procedures employing coupled dye systems
with
antibody
to the HIS-1
protein
or with
could be used to detect individual activities of enzyme aggregates complexed to antibody raised to only one component of the aggregate. REFERENCES I. Fink. G. R. (1964) .Sc~ic~,lc~c,146, 525-597. 2. Finh. G. R. t 1966) Gcrrctic.v 53, 445-459. 3. Bigelis. R.. Keesey. J.. and Fink. G. R. t 1977) iti Molecular Approaches to Eucaryotic Genetic Systems (Wilcox, G.. and Abelson, J.. eds.). ICN-UCLA Symposia. Academic Press. New York, Vol. VIII. l79- 187. 4. Shaffer. B.. Rytka. J.. and Fink. G. R. (1969) f’roc~. sYtrr. Ac,d. SI i. U..\.A. 63, 119% 1105, Fink. G. R.. and Styles. C. A. (1974) (;r,r/c,ric \ 77. 23 I-244. 6. Bigelis. R., and Burridge. K. (1978) Bicjchc,m. Bic~phys. Rr.s. Comw~ur~. 82. 312-327. 7. Bigelis. R.. and Fink, G. R. (1978) ./. f~~/~~~rr~~r~/. Mrrho1l.s. 22, 393-395. 8. Wickerham, L. J. (1946l.1. &r(~r~,ri<~/. 52, 193-301. 9. Ouchterlony. 0. ( 1949) Ac.ru Ptrfhcd. blic,rc&cl/. S<~trrrtf. 26, 507-5 15. 10. Nachlas, M. M.. Margulies. S. 1.. Goldberg. J. D.. and Seligman. A. M. (1960) Aocll. Bi~~c~lr~~m. 1. 317-326. I I. Martin. R. G.. Berberich. M. A., Ames. B. N.. Davis. W. W.. Goldberger. R. F., and Yourno. J. D. t 1971 )BI Methods in Enzymology (Tahor, H.. and Tabor. C. W.. eds.). Vol. 178. pp. 3-46. Academic Press. New York. 12. Culbertson. M. R., Charnas. L., Johnson. M. T.. and Fink. G. R. (1977) G;cucfic..s 86, 745-764.