Biochbnica et Biophyxica Acta, 1120 (1992) 43-48 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00
43
BBAPRO 34139
Thymosin a I is a native peptide in several tissues Francisco J. Franco, Cristina Diaz, Miguel Barcia and Manuel Freire Departamento de Bioqubnica e Biolox[a Molecular, Facultade de Biolo.rkt, Unirersidade de Santiago de Compostela, Santiago tie Compostela (Spa#t) (Received 12 July 1991) (Revi.~d manuscript received 15 October 1991 )
Key words: Thymosin: Peptide: HPLC: IEF
Failure to detect thymosin a I (Ta,) in tissue extracts prepared by procedures that prevent proteolytic activity has hitherto supported the suggestion that Taj is not a natural peptide, but the product of uncontrolled proteolysis of prothymosin a (ProTa), a polypcptide that includes Ta~ at its NH, tcrmiau~. In t_hi~v,o~k, purification by isoelcctric focusing of a product with the same isoetcctric point as synthetic Ta~. and its (urther characterization, demonstrated that Ta~ is present as a native peptide in calf thymus and in several lymphoid and non-lymphoid rat tissues. Taj shows abnormal chromatographic behaviour which appears to be due to association with other components in tissue extracts. In all the tissues studied, Ta~ was present in higher concentration than ProTa (80-183 and 44-123 #g per gram of tissue, respectively). The ProTot/Ta t ratio did not change when no measures were taken to prevent protcolysis during tissue homogenization.
Introduction
Originally isolated from calf thymus, thymosin fraction 5 (TFS) [1] comprises a matrix of peptides considered to be involved in the maturation and differentiation of T lymphocytes by an extracellular mechanism [1,2]. Thymosin a~ (Ta I), an acidic peptide containing 28 amino acid residues, was the first component to be isolated from TF5 [3]. Later, two other TF5 peptides c l o s e l y related to T a ! were isolated, one (dcs[2428]Ta I) lacking four amino acid residues at the T a l C O O H terminus, and the other (thymosin a ~ ) containing seven additional residues at this terminus [4]. T a l has been reported to be the most active of the thymosin peptides in several systems for assaying enhancement of 'in vivo' or 'in vitro" immune function (for a review see Refs. 5 and 6). Structural analysis of the translation products of calf thymus m R N A has provided evidence that in this tissue Ta~ is initially synthesized as part of a larger peptide [7,8]. The 113-amino-acid polypeptide prothy-
Abbreviations: Ta t, tbymosin al: ProTa, prothymosin a; TFS, thymosin fraction 5: IF, isoelectric focusing: PIF, preparative isoelectric focusing. Correspondence: M. Freire, Departamento de Bioqulmica e Bioloxla Molecular. Facultade de Biolox[a, Universid~ide de Santiago de Compostela, Santiago de Cornpostela, Spain.
mosin a (ProTa), which fits the characteristics of the proposed precursor and includes T a c r e l a t e d peptides at its NH 2 terminus, has been isolated from rat thymus extracts prepared by a procedure which avoids proteolytic modification [9,10]; and studies of ProTa cDNA [11,12] and the ProTot gene family [13] confirm that ProTa is the precursor from which Tcr, and Ta~-related peptides are derived. However, the proteinase activity responsible for the generation of both Ta~ and Tat l by hydrolysis of Asn-Gly bonds at positions in the ProTa sequence showing heterogeneity [10], has not yet been detected in animal tissues. The ubiquitous distribution of ProTa [14,15], and the absence of a signal peptide in the transcript of the ProTa gene [11,12] are aspects of a-thymosin biology which are incompatible with the traditional attribution to these peptides of hormonal function executed specifically in the immune system. In keeping with this discrepancy, there are data suggesting that ProTa has an intracellular function [16,17] linked to cell proliferation [11,15]. Another controversial aspects of a-thymosins has consisted in the failure to detect Ta~ in several tissues, including thymus, when proteolysis has been prevented during isolation of ProTa [9]. This has led to the suggestion that Tat is not a natural peptide, but an artefact produced by proteolysis during extraction of the endogenous peptide ProTa. In contradiction with this suggestion, Tat has been located in mouse thymus cells using a Ta~-specific monoclonal antibody [18]; a
peptide with thymosin immunoreactivity and the chromatographic properties of Ta t has been found in denaturing extracts of IEC-6 small intestinal rat cells l lq!; and a component with the same i,~oelectric point as Ta t is present in caif thymocyte extracts prepared in conditions that prevent proteolysis [20,21]. In this article we provide further evidence that T a t is a native peptide in calf thymocytes and in several rat tissues. Materials and Methods
Obtention of tissues and cells. Thymus from calf and rat and liver, lung, kidney, heart and spleen from rat were removed and frozen in liquid nitrogen immediately after the animals were killed. Thymocytes from freshly slaughtered calves and rats were obtained by free diffusion from thymus fragments: briefly, the thymus was cut into small pieces and stirred in RPMI-1640, thymic fragments were removed by passage through a nylon mesh, and the thymocytes were pelleted by centrifugation. Rat spleen iymphocytes were obtained by diffusion from spleen fragments and centrifugation on Ficoll density gradients; T lymphocytes were separed from lg + cells on antibody-coated plates as described by Mage [22]. Thymocytes and spleen T lymphocytes were frozen in liquid nitrogen just after obtention. Extraction and fractionation of peptides. Extracts under denaturing conditions were prepared by the procedure previously described [21] with slight modifications. Briefly, frozen tissues and cells were pulverized under liquid nitrogen with a chilled pestle and mortar, dispersed in 10 vol. of boiling 0.15 M NaCI, and homogenized in a Waring blender. The homogenates were ultracentrifuged, the supernatants brought to pH 2.5, and insoluble material removed by centrifugation. The resulting supernatant was diluted up to 70 mM NaC1 and made 50 mM with respect to Tris, adjusted to pH 8 and loaded in a DEAE-cellulose chromatography column, being the acidic peptides eluted with 0.4 M NaCI. Alternatively, .calf thymocytes were homogenized isotonically in 0.15 M NaCI (5 mi per g of wet cells) in a glass-teflon homogenizer and the supernatant resulting from centrifugation at 100000xg for 1 h was heated for 10 min in a water bath at 80°C, with stirring. Insoluble material was removed by centrifugation and the supernatant was fractionated by DEAE-cellulose chromatography as above. lsoelectric focusing, lsoelectric focusing was carried out as previously described [9]. Briefly, samples were applied to PAG plates with a pH range of 4.0-6.5 (LKB-Bromma) and electrofocused for 2.5 h (2000 V, 25 mA, 25 W) using a LKB Multiphor isoelectric focusing cell termostatted at 10 o C. The electrofocused gels were fixed in trichloroacetic/sulphosalicilic acid and stained sequentially in Coomassie/ ethanol/ acetic acid and Coomassie/propanol/acetic acid solutions.
After destaining, the gels were dried at room temperature and ~canned in a He wlett-Pa~kard dcr~itamctcr. Frcparative isoelectric focusing of acidic peptides obtained by DEAE-ceilulose chromatography of denaturing extracts was carried out in the same conditions as above, loading 220/.tg per channel (6 mg per plate). Pure calf ProTa (60 /xg) and synthetic T a r (50 ttg) were run in parallel. After isoelectric focusing, the visible opalescent bands at the p l of synthetic T a t and calf ProTa were sliced, ground and extracted with 5 ml of 1% ammonium bicarbonate containiLg 0.075% sodium dodecyl sulfate (SDS). Insoluble material was removed by centrifugation and the supernatant was brought to a K2HPO 4 concentration of 20 mM to remove SDS [23] and kept on ice for 30 min. The solution was centrifuged again and the resulting supernatant dialysed and concentrated by lyophilization. Protein determination. Protein was determined by the methods of Lowry [24], Bradford [25] and Dzugaj [26] using bovine serum albumin as standard. Results
Ta I in calf thymocytes lsoelectric focusing (IF) of acidic products fractionated from calf thymocyte extracts prepared in conditions preventing proteolysis detected a component with the same p l as synthetic T a t (Fig. 1, inset, lane 9). However, this component did not separate under reverse-phase HPLC (Fig. 1). IF analysis of the HPLC peak fractions (Fig. 1, inset), showed that while ProTa was recovered pure, the Tat-like product was spread among several HPLC peaks together with other longretention components. Similar behaviour of the Tallike product was observed in the 1F analysis of the acidic fractions of thymocyte denaturing extracts separated by reverse-phase HPLC using diverse C r 8 columns, ion-exchange HPLC, gel filtration on Sephacryl S-300 and SDS-urea PAG electrophoresis (data not shown). In view of these results, we used preparative isoelectric focusing (PIF) in PAG plates to separate acidic peptides in the denaturing extracts of calf thymocytes. Extraction of the gel zone corresponding to the Tat-like product yielded a component with the same HPLC retention time (Fig. 2A) and p l (inset in Fig. 2A) as synthetic Tar2. The tryptic peptides derived from this product had identical HPLC retention times as those of synthetic T a r (Fig. 2B). Analysis of the amino acid composition of the tryptic peptides (not shown in the paper) confirmed that the acidic peptide obtained by PIF was T a t. The concentration of T a t (estimated by densitometric scanning of the stained gel using synthetic T a t as reference) was 184.5/~g per g of wet cells, while the amount recovered from the gels was 6 5 / z g / g , a yield of 35%. The data for ProTa showed a similar yield of
45
-
-
ProTa/Ta~ ratio did not vary when crude isotonic thymocyte homogenates were incubated for 4 or 8 h at room temperature.
-Ti
"tttiP! E m
1 2 3
0,1
4
5 6
1
7 8
T a t in rat tissues A c i d i c p r o d u c t s s e p a r a t e d by i o n - e x c h a n g e c h r o m a t o g r a p h y from extracts of frozen rat tissues in which
9
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2
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0 7
the presence of ProTa [14,27] and ProTot mRNA [15] have been reported were analysed by IF. The compositions of the various fractions were very similar, especially as regards to a-thymosins (Fig. 4). Separation of the acidic fractions by HPLC resulted in patterns similar to those obtained for thymocyte products. In particular, ProTa was efficiently separated by reverse-phase HPLC and recovered in a concentration which, as
TIME, minutes
Fig. l. Separation by reverse-phase HPLC of ~lcitlic products obtained from denaturing thymocyle extracts. Aliquots of acidic products (l mg) eluted with 0.4 M NaCI from DEAE-ceilulose columns were di.~solved in 100 p.I of ().lC~ Irifluoroacetic acid (TFA), applied to a UItrapore-C3 RPSC column (10xZS0 ram) and eluted with 0.1t~ TFA in water with a programmed gradient (pecked line) of n-propanol in 0.1~ TFA, Fractions were collected every minute and those corresponding to peaks I to 8 were concentrated by lyophilization. The arrow indicates the elution time of synthetic Ta~ and peak l corresponds to the elution position of calf thymus ProTa. inset: IF analysis of about 70/.tg of each HPLC peak fraction as indicated in Materials and Methods. Lane 9 shows the acidic products applied to the HPLC column, and arrows indicate the positions where synthetic T a t (pl 3.85) (T) and calf thymocyte ProTa (p/3.55) (P) obtained as described [21], electrofocuse, p l values were determined by measuring the pH in the extracts of the I mm slices in which the IF gel was
0.3,
0,2
i a
b
c
0,1
d
l=
ZO.~
....'
_E
I
Ol
B 0,2
cut.
recovery from PIF (33%), but a somewhat lower concentration, 120 p.g/g. The amount of ProTa recovered by HPLC purification (peak 1 in Fig. 1, channel 1 in t h e inset) was 128 F g / g which is in good agreement with the densitometrieally estimated concentration. All these data are means of two experimental values differing by less than 12%. Since the presence of Ta t in thymus extracts has been considered to be the result of proteolytic degradation of ProTa during the preparation of isotonic homogenates for isolation of thymosin F5 [9], we investigated the concentrations of both Ta t and ProTa in both isotonic and denaturing calf thymocyte homogenates. As before, reverse-phase HPLC was unable to separate Ta t from the acidic components of the various thymocyte extracts that, as shown in Fig. 3, presented a very similar IF pattern. Densitometric scanning of the IF gels (Table I) showed a quite similar content of Tat and ProTa in the acidic components of isotonic and denaturing thymocyte extracts. The agreement between the ProTot concentration estimated either by densitometry or by HPLC, as indicated above, makes trustworthy the densitometric data. The
0,1
2O
4O
6O
Time, minutes
Fig. 2. (A) Reverse-phase HPLC analysis of the Toq-like product obtained by preparative isoelectric focusing of acidic peptides in denaturing extracts of calf thymocytes. 201) #g of the product obtained by PIF were applied to a Vydac Ct~ column (5 #m, 5x250 mm) and eluted with 0.1t2~ TFA in water with a programmed gradient (pecked line) of n-propanol in (Lit?; TFA. Fractions were ct~lleeted every minute; those corresponding to the main peak were concentrated by lyophilization. Inset show:: the result of IF analysis of (a) the acidic products (220 p,g) subjected to PIF; (b) the product (40 pg) from the HPLC peak; (c) calf thymocyte ProTa (40 p.g); and (d) synthetic Ta t (80 #g). Arrow indicates the retention time of synthetic T a t. (B) Separation by reverse-phase HPLC of the tryptic peptides of the PIF product with the retention time of synthetic Ta~. An aliquot of 100 p,g of the product recovered from the marked HPLC peak (panel A) was digested with trypsin and the tryptic peptides applied to an ODS-C ts column (5/tin: 4.6 x 250 ram) eluled with 0.19~, TFA with a programmed gradient (dashed line) of acelonitrile in 0.1~ TFA. Arrows indicate the retention times of Ihc tryptic peptides derived from synthetic Ta I.
46 LIVER ....... ~
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43
BBAPRO 34139
Thymosin a I is a native peptide in several tissues Francisco J. Franco, Cristina Diaz, Miguel Barcia and Manuel Freire Departamento de Bioqubnica e Biolox[a Molecular, Facultade de Biolo.rkt, Unirersidade de Santiago de Compostela, Santiago tie Compostela (Spa#t) (Received 12 July 1991) (Revi.~d manuscript received 15 October 1991 )
Key words: Thymosin: Peptide: HPLC: IEF
Failure to detect thymosin a I (Ta,) in tissue extracts prepared by procedures that prevent proteolytic activity has hitherto supported the suggestion that Taj is not a natural peptide, but the product of uncontrolled proteolysis of prothymosin a (ProTa), a polypcptide that includes Ta~ at its NH, tcrmiau~. In t_hi~v,o~k, purification by isoelcctric focusing of a product with the same isoetcctric point as synthetic Ta~. and its (urther characterization, demonstrated that Ta~ is present as a native peptide in calf thymus and in several lymphoid and non-lymphoid rat tissues. Taj shows abnormal chromatographic behaviour which appears to be due to association with other components in tissue extracts. In all the tissues studied, Ta~ was present in higher concentration than ProTa (80-183 and 44-123 #g per gram of tissue, respectively). The ProTot/Ta t ratio did not change when no measures were taken to prevent protcolysis during tissue homogenization.
Introduction
Originally isolated from calf thymus, thymosin fraction 5 (TFS) [1] comprises a matrix of peptides considered to be involved in the maturation and differentiation of T lymphocytes by an extracellular mechanism [1,2]. Thymosin a~ (Ta I), an acidic peptide containing 28 amino acid residues, was the first component to be isolated from TF5 [3]. Later, two other TF5 peptides c l o s e l y related to T a ! were isolated, one (dcs[2428]Ta I) lacking four amino acid residues at the T a l C O O H terminus, and the other (thymosin a ~ ) containing seven additional residues at this terminus [4]. T a l has been reported to be the most active of the thymosin peptides in several systems for assaying enhancement of 'in vivo' or 'in vitro" immune function (for a review see Refs. 5 and 6). Structural analysis of the translation products of calf thymus m R N A has provided evidence that in this tissue Ta~ is initially synthesized as part of a larger peptide [7,8]. The 113-amino-acid polypeptide prothy-
Abbreviations: Ta t, tbymosin al: ProTa, prothymosin a; TFS, thymosin fraction 5: IF, isoelectric focusing: PIF, preparative isoelectric focusing. Correspondence: M. Freire, Departamento de Bioqulmica e Bioloxla Molecular. Facultade de Biolox[a, Universid~ide de Santiago de Compostela, Santiago de Cornpostela, Spain.
mosin a (ProTa), which fits the characteristics of the proposed precursor and includes T a c r e l a t e d peptides at its NH 2 terminus, has been isolated from rat thymus extracts prepared by a procedure which avoids proteolytic modification [9,10]; and studies of ProTa cDNA [11,12] and the ProTot gene family [13] confirm that ProTa is the precursor from which Tcr, and Ta~-related peptides are derived. However, the proteinase activity responsible for the generation of both Ta~ and Tat l by hydrolysis of Asn-Gly bonds at positions in the ProTa sequence showing heterogeneity [10], has not yet been detected in animal tissues. The ubiquitous distribution of ProTa [14,15], and the absence of a signal peptide in the transcript of the ProTa gene [11,12] are aspects of a-thymosin biology which are incompatible with the traditional attribution to these peptides of hormonal function executed specifically in the immune system. In keeping with this discrepancy, there are data suggesting that ProTa has an intracellular function [16,17] linked to cell proliferation [11,15]. Another controversial aspects of a-thymosins has consisted in the failure to detect Ta~ in several tissues, including thymus, when proteolysis has been prevented during isolation of ProTa [9]. This has led to the suggestion that Tat is not a natural peptide, but an artefact produced by proteolysis during extraction of the endogenous peptide ProTa. In contradiction with this suggestion, Tat has been located in mouse thymus cells using a Ta~-specific monoclonal antibody [18]; a
peptide with thymosin immunoreactivity and the chromatographic properties of Ta t has been found in denaturing extracts of IEC-6 small intestinal rat cells l lq!; and a component with the same i,~oelectric point as Ta t is present in caif thymocyte extracts prepared in conditions that prevent proteolysis [20,21]. In this article we provide further evidence that T a t is a native peptide in calf thymocytes and in several rat tissues. Materials and Methods
Obtention of tissues and cells. Thymus from calf and rat and liver, lung, kidney, heart and spleen from rat were removed and frozen in liquid nitrogen immediately after the animals were killed. Thymocytes from freshly slaughtered calves and rats were obtained by free diffusion from thymus fragments: briefly, the thymus was cut into small pieces and stirred in RPMI-1640, thymic fragments were removed by passage through a nylon mesh, and the thymocytes were pelleted by centrifugation. Rat spleen iymphocytes were obtained by diffusion from spleen fragments and centrifugation on Ficoll density gradients; T lymphocytes were separed from lg + cells on antibody-coated plates as described by Mage [22]. Thymocytes and spleen T lymphocytes were frozen in liquid nitrogen just after obtention. Extraction and fractionation of peptides. Extracts under denaturing conditions were prepared by the procedure previously described [21] with slight modifications. Briefly, frozen tissues and cells were pulverized under liquid nitrogen with a chilled pestle and mortar, dispersed in 10 vol. of boiling 0.15 M NaCI, and homogenized in a Waring blender. The homogenates were ultracentrifuged, the supernatants brought to pH 2.5, and insoluble material removed by centrifugation. The resulting supernatant was diluted up to 70 mM NaC1 and made 50 mM with respect to Tris, adjusted to pH 8 and loaded in a DEAE-cellulose chromatography column, being the acidic peptides eluted with 0.4 M NaCI. Alternatively, .calf thymocytes were homogenized isotonically in 0.15 M NaCI (5 mi per g of wet cells) in a glass-teflon homogenizer and the supernatant resulting from centrifugation at 100000xg for 1 h was heated for 10 min in a water bath at 80°C, with stirring. Insoluble material was removed by centrifugation and the supernatant was fractionated by DEAE-cellulose chromatography as above. lsoelectric focusing, lsoelectric focusing was carried out as previously described [9]. Briefly, samples were applied to PAG plates with a pH range of 4.0-6.5 (LKB-Bromma) and electrofocused for 2.5 h (2000 V, 25 mA, 25 W) using a LKB Multiphor isoelectric focusing cell termostatted at 10 o C. The electrofocused gels were fixed in trichloroacetic/sulphosalicilic acid and stained sequentially in Coomassie/ ethanol/ acetic acid and Coomassie/propanol/acetic acid solutions.
After destaining, the gels were dried at room temperature and ~canned in a He wlett-Pa~kard dcr~itamctcr. Frcparative isoelectric focusing of acidic peptides obtained by DEAE-ceilulose chromatography of denaturing extracts was carried out in the same conditions as above, loading 220/.tg per channel (6 mg per plate). Pure calf ProTa (60 /xg) and synthetic T a r (50 ttg) were run in parallel. After isoelectric focusing, the visible opalescent bands at the p l of synthetic T a t and calf ProTa were sliced, ground and extracted with 5 ml of 1% ammonium bicarbonate containiLg 0.075% sodium dodecyl sulfate (SDS). Insoluble material was removed by centrifugation and the supernatant was brought to a K2HPO 4 concentration of 20 mM to remove SDS [23] and kept on ice for 30 min. The solution was centrifuged again and the resulting supernatant dialysed and concentrated by lyophilization. Protein determination. Protein was determined by the methods of Lowry [24], Bradford [25] and Dzugaj [26] using bovine serum albumin as standard. Results
Ta I in calf thymocytes lsoelectric focusing (IF) of acidic products fractionated from calf thymocyte extracts prepared in conditions preventing proteolysis detected a component with the same p l as synthetic T a t (Fig. 1, inset, lane 9). However, this component did not separate under reverse-phase HPLC (Fig. 1). IF analysis of the HPLC peak fractions (Fig. 1, inset), showed that while ProTa was recovered pure, the Tat-like product was spread among several HPLC peaks together with other longretention components. Similar behaviour of the Tallike product was observed in the 1F analysis of the acidic fractions of thymocyte denaturing extracts separated by reverse-phase HPLC using diverse C r 8 columns, ion-exchange HPLC, gel filtration on Sephacryl S-300 and SDS-urea PAG electrophoresis (data not shown). In view of these results, we used preparative isoelectric focusing (PIF) in PAG plates to separate acidic peptides in the denaturing extracts of calf thymocytes. Extraction of the gel zone corresponding to the Tat-like product yielded a component with the same HPLC retention time (Fig. 2A) and p l (inset in Fig. 2A) as synthetic Tar2. The tryptic peptides derived from this product had identical HPLC retention times as those of synthetic T a r (Fig. 2B). Analysis of the amino acid composition of the tryptic peptides (not shown in the paper) confirmed that the acidic peptide obtained by PIF was T a t. The concentration of T a t (estimated by densitometric scanning of the stained gel using synthetic T a t as reference) was 184.5/~g per g of wet cells, while the amount recovered from the gels was 6 5 / z g / g , a yield of 35%. The data for ProTa showed a similar yield of
45
-
-
ProTa/Ta~ ratio did not vary when crude isotonic thymocyte homogenates were incubated for 4 or 8 h at room temperature.
-Ti
"tttiP! E m
1 2 3
0,1
4
5 6
1
7 8
T a t in rat tissues A c i d i c p r o d u c t s s e p a r a t e d by i o n - e x c h a n g e c h r o m a t o g r a p h y from extracts of frozen rat tissues in which
9
II|
..~"
2
__ ""
0 7
the presence of ProTa [14,27] and ProTot mRNA [15] have been reported were analysed by IF. The compositions of the various fractions were very similar, especially as regards to a-thymosins (Fig. 4). Separation of the acidic fractions by HPLC resulted in patterns similar to those obtained for thymocyte products. In particular, ProTa was efficiently separated by reverse-phase HPLC and recovered in a concentration which, as
TIME, minutes
Fig. l. Separation by reverse-phase HPLC of ~lcitlic products obtained from denaturing thymocyle extracts. Aliquots of acidic products (l mg) eluted with 0.4 M NaCI from DEAE-ceilulose columns were di.~solved in 100 p.I of ().lC~ Irifluoroacetic acid (TFA), applied to a UItrapore-C3 RPSC column (10xZS0 ram) and eluted with 0.1t~ TFA in water with a programmed gradient (pecked line) of n-propanol in 0.1~ TFA, Fractions were collected every minute and those corresponding to peaks I to 8 were concentrated by lyophilization. The arrow indicates the elution time of synthetic Ta~ and peak l corresponds to the elution position of calf thymus ProTa. inset: IF analysis of about 70/.tg of each HPLC peak fraction as indicated in Materials and Methods. Lane 9 shows the acidic products applied to the HPLC column, and arrows indicate the positions where synthetic T a t (pl 3.85) (T) and calf thymocyte ProTa (p/3.55) (P) obtained as described [21], electrofocuse, p l values were determined by measuring the pH in the extracts of the I mm slices in which the IF gel was
0.3,
0,2
i a
b
c
0,1
d
l=
ZO.~
....'
_E
I
Ol
B 0,2
cut.
recovery from PIF (33%), but a somewhat lower concentration, 120 p.g/g. The amount of ProTa recovered by HPLC purification (peak 1 in Fig. 1, channel 1 in t h e inset) was 128 F g / g which is in good agreement with the densitometrieally estimated concentration. All these data are means of two experimental values differing by less than 12%. Since the presence of Ta t in thymus extracts has been considered to be the result of proteolytic degradation of ProTa during the preparation of isotonic homogenates for isolation of thymosin F5 [9], we investigated the concentrations of both Ta t and ProTa in both isotonic and denaturing calf thymocyte homogenates. As before, reverse-phase HPLC was unable to separate Ta t from the acidic components of the various thymocyte extracts that, as shown in Fig. 3, presented a very similar IF pattern. Densitometric scanning of the IF gels (Table I) showed a quite similar content of Tat and ProTa in the acidic components of isotonic and denaturing thymocyte extracts. The agreement between the ProTot concentration estimated either by densitometry or by HPLC, as indicated above, makes trustworthy the densitometric data. The
0,1
2O
4O
6O
Time, minutes
Fig. 2. (A) Reverse-phase HPLC analysis of the Toq-like product obtained by preparative isoelectric focusing of acidic peptides in denaturing extracts of calf thymocytes. 201) #g of the product obtained by PIF were applied to a Vydac Ct~ column (5 #m, 5x250 mm) and eluted with 0.1t2~ TFA in water with a programmed gradient (pecked line) of n-propanol in (Lit?; TFA. Fractions were ct~lleeted every minute; those corresponding to the main peak were concentrated by lyophilization. Inset show:: the result of IF analysis of (a) the acidic products (220 p,g) subjected to PIF; (b) the product (40 pg) from the HPLC peak; (c) calf thymocyte ProTa (40 p.g); and (d) synthetic Ta t (80 #g). Arrow indicates the retention time of synthetic T a t. (B) Separation by reverse-phase HPLC of the tryptic peptides of the PIF product with the retention time of synthetic Ta~. An aliquot of 100 p,g of the product recovered from the marked HPLC peak (panel A) was digested with trypsin and the tryptic peptides applied to an ODS-C ts column (5/tin: 4.6 x 250 ram) eluled with 0.19~, TFA with a programmed gradient (dashed line) of acelonitrile in 0.1~ TFA. Arrows indicate the retention times of Ihc tryptic peptides derived from synthetic Ta I.
46 LIVER ....... ~
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< P SPLEEN
