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Utilisation of certain derivatives of alanine and arginine by yeasts
4I()
SHORT COMMUNICATIONS
BBA 93130
Utilisation of certain derivatives of alanlne and arginine by yeasts1 Peptidyl-nucleotidates have been isolated from yeasts 1-3 and other cells*,5 and are considered to be intermediates in protein synthesis s. One such compound, isolated from yeast 1, namely L-arginyl-L-alanyl-L-arginyl-L-alanyluridine-5'-phosphate has been synthesised, 7 and the opportunity has been taken to study the assimilation of this compound, of closely related compounds, and of intermediate peptide derivativesT,s by yeasts. The results now presented reveal that peptidylnucleotidates are not utilised under any of the conditions employed although the free peptides are able to support yeast growth. Two growth media were used: (a) a nitrogen-rich medium 9 containing amino acids but no purines to give information on the assimilation of the test compounds in the presence of amino acids, and the other (b) containing the trial substance as sole source of nitrogen 1°. Two yeast strains were employed, namely one of Saccharomyces cerevisiae (National Collection of Yeast Cultures No. 240), and the other of Pichia membranae/aciens (N.C.Y.C. No. 326). The latter was chosen since it possesses extracellular peptidase activity n (see later). The test compounds (Table I) were TABLE
I
GROWTH
ON, AND
UTILISATION
OF,
DRRIVATIVES
Derivative
OF ALANINE
AND
ARGININE
Growth (Spehker drum readings) in medium (b) (see text) with derivative as sole source o/
nitrog~ a/~r I8h L-Alanine (control) L-Arginine (control) L-Arginyl-L-alanine L-Alanyl-L-arginine L-Arginyl-L-alanyl-L-arginyl-L-alanine L-Alanyl-L-arginyl-L-alanyl-L-arginine D-Alanine L-Arginyl-D-alanine L-Arginyl-D-alanyl-L-arginyl-D-alanine L-Arginyl-L-alanyluridine-5'-phosphate L-Arginyl-L-alanyl-L-arginyl-L-alanyluridine-5'-phosphate
24h
54 h
6oh
o.oi o.II 0.49 o.oi o.13 0.59 O.Ol o . o 5 0 . 3 7 o.oi o.o6 o.33 o.oi o.o2 o.o8 o.ox o.oi 0.07 o.oi o.oi o.o2 o.oi o.oi o.oi o.oi o.oi o.o2 o.ox o.oi o.oi
0.98 1.17 o.78 o.69 o.37 0.35 0.02 o.o2 o.02 o.o2
1.24 toodense 0.9 ° o.84 0.45 o.41 0.02 o.oi 0.02 o.o2
o.oi
o.oi
O.Ol
o.oi
36h
o.oi
BY
S. cerevisiae
Utilisation (%) o/ derivatives 5 4 h alter inoculation in medium (a)
medium (b)
Not determined Not determined 87 88 4° 39 Not determined
88 95 73 7° 19 17 o
o
o
o
O
o
o
O
O
dissolved in water at a concentration of 1 % , the solutions Seitz-filtered and the resulting preparations added to sterilised growth media to give final concentrations of o.i %. The stock cultures were grown in medium (a) without test compound and a small inoculum (wire-loop) was transferred to the medium containing the test compound (IO ml), the culture being shaken at 25 ° for 6 days. Samples were withdrawn at daily intervals and growth of the yeast was followed by measurement of Biochim. Biophys. Acta, 119 ( I 9 6 6 ) 4 1 6 - 4 1 8
SHORT COMMUNICATIONS
417
turbidity, using a Spekker absorptiometer in conjunction with calibration curves prepared for each yeast strain. In addition, the cells were removed from each sample withdrawn b y centrifuging and the culture fluids examined b y paper chromatography and paper electrophoresis as described b y DAVIES AND HARRIS1. Of the compounds examined (Table I), only peptides composed of L-amino acid residues promoted growth of the yeasts when used as the sole source of nitrogen in medium (b) and were taken up from medium (a) during proliferation. The results for S. cerevisiae are given in Table I. Generally similar ones were obtained using P. membranae/aciens. Utilisation was considerably more rapid from medium (a) than from (b) (see Table I), the test compounds being determined using ninhydfin 1 after separating them from the bulk of the amino acids by paper electrophoresis1 in the case of medium (a) or directly when medium (b) was employed. Peptidyl-nucleotidates derived from both di- and tetrapeptides (Table I) and peptides containing D-alanine residues failed to promote yeast growth. Quantitative chromatographic analysis (see Table I) revealed that none of the compounds failing to promote growth on medium (b) were absorbed significantly by the yeast and all could be isolated from the medium in an unchanged condition. When larger inocula of yeast (IOO mg dry wt.) were added to the nitrogenfree medium (b) containing D-alanine or L-arginyl-n-alanine there were small uptakes (5-1o %) of these materials by the cells but no significant growth was observed. It appears possible that they were transported into the cell but are unable to initiate growth. When such yeast was resuspended in medium (a) it grew normally and no evidence of derangement was observed. In no case was hydrolysis of peptides or peptidyl-nucleotidates detected during culture of the yeasts in medium (b) and it appears, therefore, that the peptidases n excreted b y the Pichia strain used, are not able to attack the particular peptides employed. In this connection, aminopeptidase as prepared by DAVIES AND HARRIS1 readily attacked L-arginyl-L-alanyl-L-arginyl-L-alanine although it was without action on the corresponding isomer containing D-alanyl residues. The failure of peptidyl-nucleotidates to support growth or to be utilised under any conditions, confirmed recently b y other workers TM, suggests that no mechanism exists to introduce these compounds into the cell. Although natural purine and pyrimidine bases are readily taken up by yeasts TM, their ribosides are utilised only slowly and the corresponding ribotides almost not at all. Linking of the last-named to a peptide also fails to bring about entry into the cell even although mechanisms exist for the uptake of peptides themselves. No previous report has been published of the utilisation of tetrapeptides by yeast. DAMLI~ AND THORNE 14, predicted that these would not be readily assimilable in view of their findings that dipeptides are less easily taken up than the free amino acids comprising them. Although the present work (see Table I) confirms that the rate of utilisation varies in the order: amino acid > dipeptide > tetrapeptide, the last-named still supports adequate growth of yeast. The authors thank Dr. A. H. COOK, for his advice and encouragement given throughout the course of this work.
Brewing Industry Research Foundation, Nut/ield, Redhill, Surrey (England)
G. HARRIS I. C. MAcWILLIAM
Biochim. Biophys. Acta, 119 (1966) 416-418
418
SHORT COMMUNICATI< )NS
i J. "W. DAVIES AND G. HARRIS, Proc. Roy. Soe. London Set. B, 151 (196o), 5372 V. V. I~ONINGSBERGER, C. 0 . VAN DER GRINTEN AND J. TH. G. OVERBEEK, Biochim. Biophys. Acta, 26 (1957) 483 . 3 E. HASE, S. MIHARA, H. OTSUKA AND H. TAMIYA, Arch. Biochem. Biophys., 83 (1959) 17o. 4 ~k]-. A. MATHESON AND R. L. M. SYNGE, Biochem. J., 92 (1964) 48P. 5 M. F. I~HANINA, T. V. VENKSTERN AND A. A. BAEY, Biokhimiya, 29 (1964) 142. 6 A. ~VISEMAN, Organisation [or Protein Synthesis, B l a c k w e l l , Oxford, 1965, C h a p t e r i i. 7 O. HARRIS AND I. C. MACWILLIAM, J. Chem. Soc., (1961) 2053. 8 G. HARRIS AND I. C. MACWILLIAM, J. Chem. Soe., (1963) 5552. 9 N. S. CUTTS AND C. RAINBOW, J. Gen. Microbiol, 4 (195 °) 15°. IO G. HARRIS AND C. C. THOMPSON, J. Inst. Brewing, 66 (196o) 213. I1 G. HARRIS AND R. PARSONS, J. Inst. Brewing, 64 (1958) 3312 D. J. MILLIN AND M. J. SALTMARSH-ANDREW, Nature, 208 (1965) 468. 13 G. HARRIS in A. H. COOK, The Chemistry and Biology o[ Yeasts, A c a d e m i c Press, N e w Y ork, 1958 , c h a p t e r 9. 14 ~V. R. DAMLI~ AND R. S. W. THORNE, J. Inst. Brewing, 55 (1949) 13.
Received J a n u a r y 7th, 1966 Biochim. Biophys. Acta, 119 (1966) 4 1 6 - 4 1 8
SC 93125
A gentle method for isolation of D N A and histones from the same small tissue sample Although various methods for the isolation of small quantities of DNA from mammalian tissues are already available in the literature z, none of these techniques describes a method which allows for the purification of the nucleic acid without employing chemical reagents such as detergents or phenol. Due to the m a n y physical and biological studies that are being conducted on both the histones and the nucleoprotein complex 2, it would be advantageous to combine the DNA isolation with the preparation of the conjugate protein (histone) fraction. In the method presented here, the DNA is separated from the protein b y ultracentrifugation in the presence of high salt concentrations and finally purified b y the use of Sephadex. The histone portion, free of nucleic acid impurities, remains suspended during the course of the centrifugation. Thus uncontaminated DNA a n d h i s t o n e are made available from the same small tissue sample. The thymus glands of young adult albino rats were obtained and homogenized with a Waring blender at slow speed for 3 min using 15 ml of o.15 M NaCl-o.oI M sodium citrate (pH 7.1) per gram of wet weight of tissue. All operations were carried out at 0-4 °. The homogenate was centrifuged at 2000 x g for 20 rain. The sediment was washed in the same solvent five times b y repeated homogenization with a Potter homogenizer and centrifugation at the same speed. The washed sediment was finally taken up in 20 ml of 2.0 M NaCl-o.oI M sodium citrate per gram of tissue and stirred for 9 ° min to dissociate the deoxyribonucleoprotein complex. This was centrifuged at 27 o o o x g for 35 min to remove any undissolved materials. The soluble material was then subjected to ultracentrifugation at 198 ooo x g for 8 h to sediment the DNA and a small amount of contaminating protein. The histones, which remained suspended during this ultracentrifugation, were found to be uncontaminated by DNA as determined from ultraviolet spectrum as shown in Fig. I. Biochim. Biophys. Acta, 119 (1966) 4 1 8 - 4 2 o
SHORT COMMUNICATIONS
BBA 93130
Utilisation of certain derivatives of alanlne and arginine by yeasts1 Peptidyl-nucleotidates have been isolated from yeasts 1-3 and other cells*,5 and are considered to be intermediates in protein synthesis s. One such compound, isolated from yeast 1, namely L-arginyl-L-alanyl-L-arginyl-L-alanyluridine-5'-phosphate has been synthesised, 7 and the opportunity has been taken to study the assimilation of this compound, of closely related compounds, and of intermediate peptide derivativesT,s by yeasts. The results now presented reveal that peptidylnucleotidates are not utilised under any of the conditions employed although the free peptides are able to support yeast growth. Two growth media were used: (a) a nitrogen-rich medium 9 containing amino acids but no purines to give information on the assimilation of the test compounds in the presence of amino acids, and the other (b) containing the trial substance as sole source of nitrogen 1°. Two yeast strains were employed, namely one of Saccharomyces cerevisiae (National Collection of Yeast Cultures No. 240), and the other of Pichia membranae/aciens (N.C.Y.C. No. 326). The latter was chosen since it possesses extracellular peptidase activity n (see later). The test compounds (Table I) were TABLE
I
GROWTH
ON, AND
UTILISATION
OF,
DRRIVATIVES
Derivative
OF ALANINE
AND
ARGININE
Growth (Spehker drum readings) in medium (b) (see text) with derivative as sole source o/
nitrog~ a/~r I8h L-Alanine (control) L-Arginine (control) L-Arginyl-L-alanine L-Alanyl-L-arginine L-Arginyl-L-alanyl-L-arginyl-L-alanine L-Alanyl-L-arginyl-L-alanyl-L-arginine D-Alanine L-Arginyl-D-alanine L-Arginyl-D-alanyl-L-arginyl-D-alanine L-Arginyl-L-alanyluridine-5'-phosphate L-Arginyl-L-alanyl-L-arginyl-L-alanyluridine-5'-phosphate
24h
54 h
6oh
o.oi o.II 0.49 o.oi o.13 0.59 O.Ol o . o 5 0 . 3 7 o.oi o.o6 o.33 o.oi o.o2 o.o8 o.ox o.oi 0.07 o.oi o.oi o.o2 o.oi o.oi o.oi o.oi o.oi o.o2 o.ox o.oi o.oi
0.98 1.17 o.78 o.69 o.37 0.35 0.02 o.o2 o.02 o.o2
1.24 toodense 0.9 ° o.84 0.45 o.41 0.02 o.oi 0.02 o.o2
o.oi
o.oi
O.Ol
o.oi
36h
o.oi
BY
S. cerevisiae
Utilisation (%) o/ derivatives 5 4 h alter inoculation in medium (a)
medium (b)
Not determined Not determined 87 88 4° 39 Not determined
88 95 73 7° 19 17 o
o
o
o
O
o
o
O
O
dissolved in water at a concentration of 1 % , the solutions Seitz-filtered and the resulting preparations added to sterilised growth media to give final concentrations of o.i %. The stock cultures were grown in medium (a) without test compound and a small inoculum (wire-loop) was transferred to the medium containing the test compound (IO ml), the culture being shaken at 25 ° for 6 days. Samples were withdrawn at daily intervals and growth of the yeast was followed by measurement of Biochim. Biophys. Acta, 119 ( I 9 6 6 ) 4 1 6 - 4 1 8
SHORT COMMUNICATIONS
417
turbidity, using a Spekker absorptiometer in conjunction with calibration curves prepared for each yeast strain. In addition, the cells were removed from each sample withdrawn b y centrifuging and the culture fluids examined b y paper chromatography and paper electrophoresis as described b y DAVIES AND HARRIS1. Of the compounds examined (Table I), only peptides composed of L-amino acid residues promoted growth of the yeasts when used as the sole source of nitrogen in medium (b) and were taken up from medium (a) during proliferation. The results for S. cerevisiae are given in Table I. Generally similar ones were obtained using P. membranae/aciens. Utilisation was considerably more rapid from medium (a) than from (b) (see Table I), the test compounds being determined using ninhydfin 1 after separating them from the bulk of the amino acids by paper electrophoresis1 in the case of medium (a) or directly when medium (b) was employed. Peptidyl-nucleotidates derived from both di- and tetrapeptides (Table I) and peptides containing D-alanine residues failed to promote yeast growth. Quantitative chromatographic analysis (see Table I) revealed that none of the compounds failing to promote growth on medium (b) were absorbed significantly by the yeast and all could be isolated from the medium in an unchanged condition. When larger inocula of yeast (IOO mg dry wt.) were added to the nitrogenfree medium (b) containing D-alanine or L-arginyl-n-alanine there were small uptakes (5-1o %) of these materials by the cells but no significant growth was observed. It appears possible that they were transported into the cell but are unable to initiate growth. When such yeast was resuspended in medium (a) it grew normally and no evidence of derangement was observed. In no case was hydrolysis of peptides or peptidyl-nucleotidates detected during culture of the yeasts in medium (b) and it appears, therefore, that the peptidases n excreted b y the Pichia strain used, are not able to attack the particular peptides employed. In this connection, aminopeptidase as prepared by DAVIES AND HARRIS1 readily attacked L-arginyl-L-alanyl-L-arginyl-L-alanine although it was without action on the corresponding isomer containing D-alanyl residues. The failure of peptidyl-nucleotidates to support growth or to be utilised under any conditions, confirmed recently b y other workers TM, suggests that no mechanism exists to introduce these compounds into the cell. Although natural purine and pyrimidine bases are readily taken up by yeasts TM, their ribosides are utilised only slowly and the corresponding ribotides almost not at all. Linking of the last-named to a peptide also fails to bring about entry into the cell even although mechanisms exist for the uptake of peptides themselves. No previous report has been published of the utilisation of tetrapeptides by yeast. DAMLI~ AND THORNE 14, predicted that these would not be readily assimilable in view of their findings that dipeptides are less easily taken up than the free amino acids comprising them. Although the present work (see Table I) confirms that the rate of utilisation varies in the order: amino acid > dipeptide > tetrapeptide, the last-named still supports adequate growth of yeast. The authors thank Dr. A. H. COOK, for his advice and encouragement given throughout the course of this work.
Brewing Industry Research Foundation, Nut/ield, Redhill, Surrey (England)
G. HARRIS I. C. MAcWILLIAM
Biochim. Biophys. Acta, 119 (1966) 416-418
418
SHORT COMMUNICATI< )NS
i J. "W. DAVIES AND G. HARRIS, Proc. Roy. Soe. London Set. B, 151 (196o), 5372 V. V. I~ONINGSBERGER, C. 0 . VAN DER GRINTEN AND J. TH. G. OVERBEEK, Biochim. Biophys. Acta, 26 (1957) 483 . 3 E. HASE, S. MIHARA, H. OTSUKA AND H. TAMIYA, Arch. Biochem. Biophys., 83 (1959) 17o. 4 ~k]-. A. MATHESON AND R. L. M. SYNGE, Biochem. J., 92 (1964) 48P. 5 M. F. I~HANINA, T. V. VENKSTERN AND A. A. BAEY, Biokhimiya, 29 (1964) 142. 6 A. ~VISEMAN, Organisation [or Protein Synthesis, B l a c k w e l l , Oxford, 1965, C h a p t e r i i. 7 O. HARRIS AND I. C. MACWILLIAM, J. Chem. Soc., (1961) 2053. 8 G. HARRIS AND I. C. MACWILLIAM, J. Chem. Soe., (1963) 5552. 9 N. S. CUTTS AND C. RAINBOW, J. Gen. Microbiol, 4 (195 °) 15°. IO G. HARRIS AND C. C. THOMPSON, J. Inst. Brewing, 66 (196o) 213. I1 G. HARRIS AND R. PARSONS, J. Inst. Brewing, 64 (1958) 3312 D. J. MILLIN AND M. J. SALTMARSH-ANDREW, Nature, 208 (1965) 468. 13 G. HARRIS in A. H. COOK, The Chemistry and Biology o[ Yeasts, A c a d e m i c Press, N e w Y ork, 1958 , c h a p t e r 9. 14 ~V. R. DAMLI~ AND R. S. W. THORNE, J. Inst. Brewing, 55 (1949) 13.
Received J a n u a r y 7th, 1966 Biochim. Biophys. Acta, 119 (1966) 4 1 6 - 4 1 8
SC 93125
A gentle method for isolation of D N A and histones from the same small tissue sample Although various methods for the isolation of small quantities of DNA from mammalian tissues are already available in the literature z, none of these techniques describes a method which allows for the purification of the nucleic acid without employing chemical reagents such as detergents or phenol. Due to the m a n y physical and biological studies that are being conducted on both the histones and the nucleoprotein complex 2, it would be advantageous to combine the DNA isolation with the preparation of the conjugate protein (histone) fraction. In the method presented here, the DNA is separated from the protein b y ultracentrifugation in the presence of high salt concentrations and finally purified b y the use of Sephadex. The histone portion, free of nucleic acid impurities, remains suspended during the course of the centrifugation. Thus uncontaminated DNA a n d h i s t o n e are made available from the same small tissue sample. The thymus glands of young adult albino rats were obtained and homogenized with a Waring blender at slow speed for 3 min using 15 ml of o.15 M NaCl-o.oI M sodium citrate (pH 7.1) per gram of wet weight of tissue. All operations were carried out at 0-4 °. The homogenate was centrifuged at 2000 x g for 20 rain. The sediment was washed in the same solvent five times b y repeated homogenization with a Potter homogenizer and centrifugation at the same speed. The washed sediment was finally taken up in 20 ml of 2.0 M NaCl-o.oI M sodium citrate per gram of tissue and stirred for 9 ° min to dissociate the deoxyribonucleoprotein complex. This was centrifuged at 27 o o o x g for 35 min to remove any undissolved materials. The soluble material was then subjected to ultracentrifugation at 198 ooo x g for 8 h to sediment the DNA and a small amount of contaminating protein. The histones, which remained suspended during this ultracentrifugation, were found to be uncontaminated by DNA as determined from ultraviolet spectrum as shown in Fig. I. Biochim. Biophys. Acta, 119 (1966) 4 1 8 - 4 2 o