THIOLS
ATTACHED
TO RAT
NUCLEAR
LIVER
NON-HISTONE
PROTEINS
M. GRONOW and F. A. LEWIS Department
ofExperimental
Pathology
and Cancer Research,
University
of Leeds, Leeds LS2 3AR, UK
SUMMARY Up to 88 % of the total thiol present in isolated rat liver nuclei can be extracted with 8 M urea 50 mM phosphate pH 7.6. There is approx. 5-10% disulphide material present in this extract. When the thiols were labelled with r4C-N-ethyl maleimide (l4C-NEM) the thiol material co-electrophoresed with the protein material. If a mixed disulphide was formed with 35S-labelled 5-thio-2-nitrobenzoic acid (Ellman’s reagent) the thiol compounds could be removed from the protein by isoelectric focusing in polyacrylamide gel. The mixed disulphides obtained could be resolved into at least 10 components on DEAE cellulose. One of the major components had an estimated molecular weight of 3 000 and did not contain peptide material.
Marsh, Ord & Stocken [l] reported that the disulphide content of isolated rat liver nuclei was small and that the amount of thiol present was increased about twofold in the presence of 4 M urea. Following up these observations it has been reported that approx. 88% of the total thiol can be extracted from isolated nuclei by 8 M urea 50 mM phosphate pH 7.6 [2]. Rat liver nuclear non-histone proteins (NHP) extracted by this procedure have a value, after removal of low molecular weight material (by dialysis), of 7Ok.5 nmoles of -SH/mg of protein. It is recognised that this value is higher than can be accounted for by the cysteine content of the NHP. As a result of our recent studies on nuclear thiol components we have found interesting tissue differences in the distribution of these thiol components of the nonhistone proteins [3]. In these studies we
used ‘4C-NEM as a labelling reagent, which forms a stable thioether link with thiol compounds. In an attempt to improve the separation of thiol components from other nuclear macromolecules (particularly by isoelectric focusing) we have made use of another reagent, namely 5’,5’-dithiohis-(Znitrobenzoic acid - DTNB), known as Ellman’s reagent. DTNB labelled with 35Swas successfully prepared according to Ellman’s original method [4]. On reaction of tissue thiols (X-SH) with an excess of this reagent the following reaction occurs:
Mixed disulphide Exptl Cell Res 9.3 (1975)
226
Gronow and Lewis
It is important that an excess of DTNB is present to prevent possible disulphide exchange [5], and that the reaction be carried out in high urea concentrations so that all ‘hidden’ -SH groups react. In this paper we wish to report our preliminary findings on the analysis of the X-S-35S-TNB found in rat liver nuclei. MATERIALS
AND METHODS
Preparation of 35S-labelled DTNB This was prepared as described by Ellman [4] from 2-nitro-S-chlorobenzoic acid (supplied by Koch-Light) and NA,3SS (Amersham SJ 2 1 original specific activity 2-10 mCi/mM, suitably diluted with cold sodium sulphide). The re-crystalhsed final product was checked for purity by thin layer chromatography in a variety of solvents and gave only one spot.
Labelling of nuclear thiols In order to check the validity of the result obtained using thiol reagents, the nuclear thiols were themselves labelled using YS-methionine (supplied by Amersham). Twenty-four rats were given 50 &i of the radiochemical in 1 ml of isotonic saline IP. Animals were killed after 1 h, the liver nuclei prepared and extracted as described below.
Preparation and extraction of nuclei Male Wistar SPF rats (250-350 g), obtained from A. Tuck & Son, Rayleigh, Essex, UK, were used for the preparation of liver nuclei [6]. Nuclei were then extracted with 8 M urea 50 mM phosphate pH 7.6 at 4-10°C containing a two-fold excess of labelled thiol reagent [3]. To ensure complete extraction a further two extractions were carried out, each consisting of half the initial volume of buffered urea. The combined extracts were then dialysed against 100vol of 8 M urea (deionized, AR quality) and this was repeated until very little further change OCcurred in the specific activity of the protein (NHP). Usually three changes of dialysing solution were sufficient.
Investigations on 8 M urea extract against SDS. The samples were dialysed against the starting buffer recommended for SDS electrophoresis by Weber & Osborn [7] without P-mercaptoethanol, i.e. 0.1% SDS 10 mM Tris-HCI buffer pH 7.0, initially with a view to performing SDS electrophoresis on these extracts. Isoelectric focusing in polyacrylamide gel. This was carried out basically as previously described [3] in 5 % polyacrvlamide gels, 2 % ampholine carrier amphobytes pH 3.5-10 (LKB Produkter AB, Stockholm, Sweden) in the presence of 8 M urea (deionized), the
Dialysis
Exptl Cetl Res 93 (1975)
sample being incorporated in the gel mtxture prior to polvmerisation. Instead of the original electrode solutions those employed by Righetti & Drysdale [8] were used, i.e. 10 mM H,PO, at the anode and 20 mM NaOH at the cathode. Focusing was continued longer than the usual 5 h Twenty-four hours (at 15°C) was required to complete the migration of X-S-35S-TNB from the gel to the anode solution.
Attempted measurement of-S-Scontent of 8 M urea extracts Reduction
using
Clelands
reagent
(dithiothreitol-DTT)
[9]. Nuclei were extracted with 8 M urea 50 mM phosphate pH 7.6 (as described above) and a IOO-fold excess of DTT added to the combined extracts. The mixture was then dialvsed aeainst two changes of 8 M urea 10 mM phosphate pHy.6 to remove rhe excess DTT and a larae excess of YS-labelled DTNB was added. After three dialysis changes against 100 vol of deionized 8 M urea the ?S and protein content of the mixture was measured. Reduction using sodium borohydride. This was carried out after reaction of available thiols with NEM. Duplicate samples (2 ml) of 8 M urea 0.05 M phosphate extracts of known thiol content were blocked with NEM and to these were added N sodium hydroxide (30 ~1) to bring the pH up to 8-9. Then, 200 ~1 of a solution of sodium borohydride in water (50 mg/ml) were added and the mixture incubated at 50°C for 45 min. The excess of borohydride was destroyed by the addition of N-hydrochloric acid (350 ~1) with cooling, and, after a brief centrifugation to reduce the froth,?he mixture was made up to4 ml with 1 M Tris buffer pH 7.5 containing excess DTNB (80 pg/ml). After 5-10 minAIIs was read against a suitable blank of DTNB.
Chromatography of X-S-35S-TNB released after isoelectric focusing The anode solution (10 mM phosphoric acid) after isoelectric focusine of the nuclear 8 M urea extracts described above was stirred for I h at room temperature with Amberlite XAD2 resin suoolied by BDH (three extractions each of 0.1 g XAD&l). The beads ” were filtered off, washed with water and the iabelled mixed disulphides removed with methanol. The latter solution was evaporated to dryness in a rotary evaporator and the solid obtained (partially crystalline) dissolved a 1 : 1 mixture of methanol, 10 mM phosphate. After adjusting the pH of the mixture to 7.6. the pale vellow solution was chromatographed on a 20x 1 cm column of DEAE cellulose (BioRad, Cellex D). usine a linear gradient of 0 to 0.2 M NaCl containing 10 mM phosphate pH 7.6. Further material was eluted using higher concentrations of NaCI. ,,
1
Specific activity measurements For the determination of dpm samples were dissolved in either Bray’s [IO] or Triton X-100 [I I] scintillator cocktails. Samples were counted in a Multimat (Multi
227
Rat liver nuclear thiols 8) scintillation counter (Intertechnique Ltd., Portslade, Sussex, UK), the dpm being computed from the data obtained from a set of quenched standards in these scintillator mixtures. As 9 has a half-life of only 86.7 days, the machine was programmed to compute all dpm calculations to the time when the initial specific activity had been calculated. Protein estimations were done by the Folin Lowry or Biuret methods using bovine serum albumin as a standard [ 121.
RESULTS AND DISCUSSION Similar specific activities were obtained for rat liver nuclear NHP thiol content using the two methods described. Using 35S-TNB of specific activity 17.5 mCi/mM, the 8 M urea soluble proteins contained 2.65 x IO6 dpm/mg protein corresponding to a value of 68.5 nmoles of -SH/mg: if 14C-NEM of specific activity 2.4 mCi/mM was used, a value of 3 .%x lo5 dpm/mg equivalent to 75.0 nmoles -SH/mg was obtained. Of the thiol material solubilized in the buffered 8 M urea solutions approx. 2030% can be dialysed off immediately or separated on Sephadex columns [2]. If the higher molecular weight protein bound thiol is reacted with NEM the thiol remains attached to the protein complex and separates into components when analysed by the techniques of IEF and SDS electrophoresis [3]. These patterns have been consistently obtained. However, when the thiol is labelled with TNB to form a mixed disulphide it can be separated from the nonhistone protein by isoelectric focusing and in the presence of SDS; the latter finding was observed on trying to analyse the mixture by SDS electrophoresis. That these lower molecular weight components are real and not an artefact produced by the reagent is indicated by the data shown in tables 1 and 2. If the NHP was labelled with 3H-leucine, negligible loss of radioactivity was found after dialysis against SDS.
Table 1. Loss of nuclear thiol material
by SDS 10 mM Tris-
dialysis against 0.1% HCl buffer pH 7.0
Form of radiolabel 1) X-W5S-TNB Expt 1 Expt 2 2)” X-Y&S-STNB Expt 1 Expt 2 3) X-S-W-NEM (2 expts)
Radioactivity released (%I
TNB lost” (So)
65.0 67.5
66.0 70.0
47 54
70.0 75.2
0
a Animals given 50 &i of Y&methionine (Amersham SJ123) in 1 ml of isotonic saline. Killed after 1 h. b Measured by change in A412 after the addition of DTT to release coloured anion.
Attempts to measure and label the disulphide components using 35SDTNB gave the unexpected result shown in table 2. It is evident that DTT is also capable of removing some of the protein bound thiol. A different result was obtained with regenerating liver (shown in brackets) in which the nuclear thiol content doubles at 18-24 h, and this phenomenon is being investigated further. However, it is evident that more disulphide is present than in normal rat liver and that DTT probably does not strip the sulphur compounds from the nuclear protein complex of regenerating liver. Table 2. Sulphydryl-disulphide 8 M urea 50 mM phosphate nuclei Method of reduction
1) By DTT (Cleland’s Reagent)
content of
of rat liver
SH of reduced extract Xl00 Original -SH Expt 1 Expt 2 55.5 P531 110
63.5
11611 2) By borohydtide
106
Figures in brackets are those obtained from samples of regenerating liver (24 h). ExprlCellRes93
(1975)
228
Gronow und Letcis
Table 3. Polyacrylamide isoelectric focusing of8 M ureu extructs (from rut lit,er) thiols labelled with 35S-TNB. Approximate percentage recovery of ?S applied to gel Gel slices extracted Bottom electrode solution, 10 mM H,PO,
(1) with 8 M urea 50 mM phosphate pH 7.6
(2) N sodium hydroxide
Top electrode solution, 20 mM NaOH
Percentage recovery
56-60
IO-1 I
24
0.3-0.5
72-7x
In addition,
-3 % was present in the initial gel overlay
The data shown in table 3 show that the even after hydrolysis in 6 N HCl at 100°C bulk of the labelled mixed disulphide com- indicating the absence of amino acids. pounds are released into the anode solution From the specific activity of the solid ob(10 mM H,PO,) on prolonged electrofocus- tained, assuming one -SH per mole, the ing of the 8 M urea extracts. The ionization molecular weight was estimated as approx. of the TNB carboxyl group is suppressed in 3.000. A similar DEAE pattern has been obthe acid solution and up to 75% of the disulphides can be adsorbed onto the XAD2 tained from the labelled disulphides obtained from chromatin preparations and furresin. Recovery of this material in methanol ther investigations are under way to see if was high (
ATTACHED
TO RAT
NUCLEAR
LIVER
NON-HISTONE
PROTEINS
M. GRONOW and F. A. LEWIS Department
ofExperimental
Pathology
and Cancer Research,
University
of Leeds, Leeds LS2 3AR, UK
SUMMARY Up to 88 % of the total thiol present in isolated rat liver nuclei can be extracted with 8 M urea 50 mM phosphate pH 7.6. There is approx. 5-10% disulphide material present in this extract. When the thiols were labelled with r4C-N-ethyl maleimide (l4C-NEM) the thiol material co-electrophoresed with the protein material. If a mixed disulphide was formed with 35S-labelled 5-thio-2-nitrobenzoic acid (Ellman’s reagent) the thiol compounds could be removed from the protein by isoelectric focusing in polyacrylamide gel. The mixed disulphides obtained could be resolved into at least 10 components on DEAE cellulose. One of the major components had an estimated molecular weight of 3 000 and did not contain peptide material.
Marsh, Ord & Stocken [l] reported that the disulphide content of isolated rat liver nuclei was small and that the amount of thiol present was increased about twofold in the presence of 4 M urea. Following up these observations it has been reported that approx. 88% of the total thiol can be extracted from isolated nuclei by 8 M urea 50 mM phosphate pH 7.6 [2]. Rat liver nuclear non-histone proteins (NHP) extracted by this procedure have a value, after removal of low molecular weight material (by dialysis), of 7Ok.5 nmoles of -SH/mg of protein. It is recognised that this value is higher than can be accounted for by the cysteine content of the NHP. As a result of our recent studies on nuclear thiol components we have found interesting tissue differences in the distribution of these thiol components of the nonhistone proteins [3]. In these studies we
used ‘4C-NEM as a labelling reagent, which forms a stable thioether link with thiol compounds. In an attempt to improve the separation of thiol components from other nuclear macromolecules (particularly by isoelectric focusing) we have made use of another reagent, namely 5’,5’-dithiohis-(Znitrobenzoic acid - DTNB), known as Ellman’s reagent. DTNB labelled with 35Swas successfully prepared according to Ellman’s original method [4]. On reaction of tissue thiols (X-SH) with an excess of this reagent the following reaction occurs:
Mixed disulphide Exptl Cell Res 9.3 (1975)
226
Gronow and Lewis
It is important that an excess of DTNB is present to prevent possible disulphide exchange [5], and that the reaction be carried out in high urea concentrations so that all ‘hidden’ -SH groups react. In this paper we wish to report our preliminary findings on the analysis of the X-S-35S-TNB found in rat liver nuclei. MATERIALS
AND METHODS
Preparation of 35S-labelled DTNB This was prepared as described by Ellman [4] from 2-nitro-S-chlorobenzoic acid (supplied by Koch-Light) and NA,3SS (Amersham SJ 2 1 original specific activity 2-10 mCi/mM, suitably diluted with cold sodium sulphide). The re-crystalhsed final product was checked for purity by thin layer chromatography in a variety of solvents and gave only one spot.
Labelling of nuclear thiols In order to check the validity of the result obtained using thiol reagents, the nuclear thiols were themselves labelled using YS-methionine (supplied by Amersham). Twenty-four rats were given 50 &i of the radiochemical in 1 ml of isotonic saline IP. Animals were killed after 1 h, the liver nuclei prepared and extracted as described below.
Preparation and extraction of nuclei Male Wistar SPF rats (250-350 g), obtained from A. Tuck & Son, Rayleigh, Essex, UK, were used for the preparation of liver nuclei [6]. Nuclei were then extracted with 8 M urea 50 mM phosphate pH 7.6 at 4-10°C containing a two-fold excess of labelled thiol reagent [3]. To ensure complete extraction a further two extractions were carried out, each consisting of half the initial volume of buffered urea. The combined extracts were then dialysed against 100vol of 8 M urea (deionized, AR quality) and this was repeated until very little further change OCcurred in the specific activity of the protein (NHP). Usually three changes of dialysing solution were sufficient.
Investigations on 8 M urea extract against SDS. The samples were dialysed against the starting buffer recommended for SDS electrophoresis by Weber & Osborn [7] without P-mercaptoethanol, i.e. 0.1% SDS 10 mM Tris-HCI buffer pH 7.0, initially with a view to performing SDS electrophoresis on these extracts. Isoelectric focusing in polyacrylamide gel. This was carried out basically as previously described [3] in 5 % polyacrvlamide gels, 2 % ampholine carrier amphobytes pH 3.5-10 (LKB Produkter AB, Stockholm, Sweden) in the presence of 8 M urea (deionized), the
Dialysis
Exptl Cetl Res 93 (1975)
sample being incorporated in the gel mtxture prior to polvmerisation. Instead of the original electrode solutions those employed by Righetti & Drysdale [8] were used, i.e. 10 mM H,PO, at the anode and 20 mM NaOH at the cathode. Focusing was continued longer than the usual 5 h Twenty-four hours (at 15°C) was required to complete the migration of X-S-35S-TNB from the gel to the anode solution.
Attempted measurement of-S-Scontent of 8 M urea extracts Reduction
using
Clelands
reagent
(dithiothreitol-DTT)
[9]. Nuclei were extracted with 8 M urea 50 mM phosphate pH 7.6 (as described above) and a IOO-fold excess of DTT added to the combined extracts. The mixture was then dialvsed aeainst two changes of 8 M urea 10 mM phosphate pHy.6 to remove rhe excess DTT and a larae excess of YS-labelled DTNB was added. After three dialysis changes against 100 vol of deionized 8 M urea the ?S and protein content of the mixture was measured. Reduction using sodium borohydride. This was carried out after reaction of available thiols with NEM. Duplicate samples (2 ml) of 8 M urea 0.05 M phosphate extracts of known thiol content were blocked with NEM and to these were added N sodium hydroxide (30 ~1) to bring the pH up to 8-9. Then, 200 ~1 of a solution of sodium borohydride in water (50 mg/ml) were added and the mixture incubated at 50°C for 45 min. The excess of borohydride was destroyed by the addition of N-hydrochloric acid (350 ~1) with cooling, and, after a brief centrifugation to reduce the froth,?he mixture was made up to4 ml with 1 M Tris buffer pH 7.5 containing excess DTNB (80 pg/ml). After 5-10 minAIIs was read against a suitable blank of DTNB.
Chromatography of X-S-35S-TNB released after isoelectric focusing The anode solution (10 mM phosphoric acid) after isoelectric focusine of the nuclear 8 M urea extracts described above was stirred for I h at room temperature with Amberlite XAD2 resin suoolied by BDH (three extractions each of 0.1 g XAD&l). The beads ” were filtered off, washed with water and the iabelled mixed disulphides removed with methanol. The latter solution was evaporated to dryness in a rotary evaporator and the solid obtained (partially crystalline) dissolved a 1 : 1 mixture of methanol, 10 mM phosphate. After adjusting the pH of the mixture to 7.6. the pale vellow solution was chromatographed on a 20x 1 cm column of DEAE cellulose (BioRad, Cellex D). usine a linear gradient of 0 to 0.2 M NaCl containing 10 mM phosphate pH 7.6. Further material was eluted using higher concentrations of NaCI. ,,
1
Specific activity measurements For the determination of dpm samples were dissolved in either Bray’s [IO] or Triton X-100 [I I] scintillator cocktails. Samples were counted in a Multimat (Multi
227
Rat liver nuclear thiols 8) scintillation counter (Intertechnique Ltd., Portslade, Sussex, UK), the dpm being computed from the data obtained from a set of quenched standards in these scintillator mixtures. As 9 has a half-life of only 86.7 days, the machine was programmed to compute all dpm calculations to the time when the initial specific activity had been calculated. Protein estimations were done by the Folin Lowry or Biuret methods using bovine serum albumin as a standard [ 121.
RESULTS AND DISCUSSION Similar specific activities were obtained for rat liver nuclear NHP thiol content using the two methods described. Using 35S-TNB of specific activity 17.5 mCi/mM, the 8 M urea soluble proteins contained 2.65 x IO6 dpm/mg protein corresponding to a value of 68.5 nmoles of -SH/mg: if 14C-NEM of specific activity 2.4 mCi/mM was used, a value of 3 .%x lo5 dpm/mg equivalent to 75.0 nmoles -SH/mg was obtained. Of the thiol material solubilized in the buffered 8 M urea solutions approx. 2030% can be dialysed off immediately or separated on Sephadex columns [2]. If the higher molecular weight protein bound thiol is reacted with NEM the thiol remains attached to the protein complex and separates into components when analysed by the techniques of IEF and SDS electrophoresis [3]. These patterns have been consistently obtained. However, when the thiol is labelled with TNB to form a mixed disulphide it can be separated from the nonhistone protein by isoelectric focusing and in the presence of SDS; the latter finding was observed on trying to analyse the mixture by SDS electrophoresis. That these lower molecular weight components are real and not an artefact produced by the reagent is indicated by the data shown in tables 1 and 2. If the NHP was labelled with 3H-leucine, negligible loss of radioactivity was found after dialysis against SDS.
Table 1. Loss of nuclear thiol material
by SDS 10 mM Tris-
dialysis against 0.1% HCl buffer pH 7.0
Form of radiolabel 1) X-W5S-TNB Expt 1 Expt 2 2)” X-Y&S-STNB Expt 1 Expt 2 3) X-S-W-NEM (2 expts)
Radioactivity released (%I
TNB lost” (So)
65.0 67.5
66.0 70.0
47 54
70.0 75.2
0
a Animals given 50 &i of Y&methionine (Amersham SJ123) in 1 ml of isotonic saline. Killed after 1 h. b Measured by change in A412 after the addition of DTT to release coloured anion.
Attempts to measure and label the disulphide components using 35SDTNB gave the unexpected result shown in table 2. It is evident that DTT is also capable of removing some of the protein bound thiol. A different result was obtained with regenerating liver (shown in brackets) in which the nuclear thiol content doubles at 18-24 h, and this phenomenon is being investigated further. However, it is evident that more disulphide is present than in normal rat liver and that DTT probably does not strip the sulphur compounds from the nuclear protein complex of regenerating liver. Table 2. Sulphydryl-disulphide 8 M urea 50 mM phosphate nuclei Method of reduction
1) By DTT (Cleland’s Reagent)
content of
of rat liver
SH of reduced extract Xl00 Original -SH Expt 1 Expt 2 55.5 P531 110
63.5
11611 2) By borohydtide
106
Figures in brackets are those obtained from samples of regenerating liver (24 h). ExprlCellRes93
(1975)
228
Gronow und Letcis
Table 3. Polyacrylamide isoelectric focusing of8 M ureu extructs (from rut lit,er) thiols labelled with 35S-TNB. Approximate percentage recovery of ?S applied to gel Gel slices extracted Bottom electrode solution, 10 mM H,PO,
(1) with 8 M urea 50 mM phosphate pH 7.6
(2) N sodium hydroxide
Top electrode solution, 20 mM NaOH
Percentage recovery
56-60
IO-1 I
24
0.3-0.5
72-7x
In addition,
-3 % was present in the initial gel overlay
The data shown in table 3 show that the even after hydrolysis in 6 N HCl at 100°C bulk of the labelled mixed disulphide com- indicating the absence of amino acids. pounds are released into the anode solution From the specific activity of the solid ob(10 mM H,PO,) on prolonged electrofocus- tained, assuming one -SH per mole, the ing of the 8 M urea extracts. The ionization molecular weight was estimated as approx. of the TNB carboxyl group is suppressed in 3.000. A similar DEAE pattern has been obthe acid solution and up to 75% of the disulphides can be adsorbed onto the XAD2 tained from the labelled disulphides obtained from chromatin preparations and furresin. Recovery of this material in methanol ther investigations are under way to see if was high (
