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Papers by Magdalena Frajnt

The effect of cAMP and cGMP on the activity and substrate specificity of protein kinase A from me... more The effect of cAMP and cGMP on the activity and substrate specificity of protein kinase A from methylotrophic yeast Pichia pastoris

The effect of vanadate on Pichia pastoris growth, protein

Protein kinases CKI and CKII are implicated in modification of ribosomal proteins of the yeast Tr... more Protein kinases CKI and CKII are implicated in modification of ribosomal proteins of the yeast Trichosporon cutaneum

Acta Biochimica Polonica, 2002

It was found that wild type yeast Pichia pastoris can tolerate vanadate concentration as high as ... more It was found that wild type yeast Pichia pastoris can tolerate vanadate concentration as high as 25 mM in the growth medium. Moreover, four vanadate-resistant P. pastoris strains designated JC100/1, JC100/3, JC100/9 and JC100/15 exhibiting tolerance up to 150 mM vanadate were selected. Growth of P. pastoris was correlated with vanadate to vanadyl reduction and its accumulation in the growth medium. In two selected strains, JC100/9 and JC100/15, protein kinase A activity was much higher in comparison to the wild type strain even without vanadate addition to the growth medium. Moreover, in the presence of vanadate, protein kinase A activity was significantly increased in the wild type and the vanadate-resistant JC100/1 and JC100/3 strains. It was also found that phosphorylation of a 40 kDa protein associated with ribosomes occured in all vanadate-resistant strains from the logarithmic, while in the wild type strain from the stationary growth phase. From the presented results it can be...

Acta Biochimica Polonica, 2002

Phosphorylation of acidic ribosomal proteins P1/P2-P0 is a common phenomenon in eukaryotic organi... more Phosphorylation of acidic ribosomal proteins P1/P2-P0 is a common phenomenon in eukaryotic organisms. It was found previously that in Trichosporon cutaneum, unlike in other yeast species, in addition to the two acidic ribosomal proteins, two other proteins of 15 kDa and 19 kDa of the small ribosomal subunit were phosphorylated. Here we describe two protein kinases: CKI and CKII, which are engaged in the modification of T. cutaneum ribosomal proteins. The acidic ribosomal proteins and the protein of 19 kDa were modified by CKII associated with ribosomes, while the protein of 15 kDa was modified by CKI. Protein kinase CKI was purified from cell-free extract (CKIC) and from ribosomal fraction (CKIR). The molecular mass of CKIC was established at 33 kDa while that of CKIR at 35-37 kDa. A protein of 40 kDa copurified with CKIR but not CKIC. Heparin significantly increased 40 kDa protein phosphorylation level by CKIR. Microsequencing analysis revealed the presence of CKI recognition motif...

Acta Biochimica Polonica, 2003

Cyclic AMP dependent protein kinase (PKA) from Pichia pastoris yeast cells was found to be activa... more Cyclic AMP dependent protein kinase (PKA) from Pichia pastoris yeast cells was found to be activated by either cAMP or cGMP. Analogs of cAMP such as 8-chloro-cAMP and 8-bromo-cAMP were as potent as cAMP in PKA activation while N6,2'-O-dibutyryl-cAMP did not stimulate the enzyme activity. It was shown that protamine sulfate was almost equally phosphorylated in the presence of 1-2 x 10(-6)M cAMP or cGMP while other substrates such as Kemptide, ribosomal protein S6, were phosphorylated to a lower extent in the presence of cGMP. It was demonstrated that pyruvate kinase is a substrate of PKA which co-purified with the P.pastoris enzyme. Moreover, pyruvate kinase was phosphorylated by PKA in the presence of cAMP and cGMP to comparable levels.

Proceedings of the National Academy of Sciences, 2004

Much research aimed at discovering the genetic bases of longevity focuses on the budding yeast Sa... more Much research aimed at discovering the genetic bases of longevity focuses on the budding yeast Saccharomyces cerevisiae. Unfortunately, yeast researchers use a definition of longevity not applied to other species. We propose here a method that makes it possible to estimate for yeast the same measures of longevity calculated for other species. We also show that the conventional method (equating longevity with the number of offspring) is only an approximate measure of true chronological lifespan. Our method will allow results for yeast to be compared more correctly with those for other species.

Proceedings of the National Academy of Sciences, 2004

Much research aimed at discovering the genetic bases of longevity focuses on the budding yeast Sa... more Much research aimed at discovering the genetic bases of longevity focuses on the budding yeast Saccharomyces cerevisiae. Unfortunately, yeast researchers use a definition of longevity not applied to other species. We propose here a method that makes it possible to estimate for yeast the same measures of longevity calculated for other species. We also show that the conventional method (equating longevity with the number of offspring) is only an approximate measure of true chronological lifespan. Our method will allow results for yeast to be compared more correctly with those for other species.

FEMS Microbiology Letters, 2001

Fractionation of Saccharomyces cerevisiae postribosomal extract on DEAE-cellulose revealed two fr... more Fractionation of Saccharomyces cerevisiae postribosomal extract on DEAE-cellulose revealed two fractions of cAMP-dependent protein kinase (PKA-1 and PKA-2). The presence of PKA in both fractions was confirmed by immunoblotting with anti-Bcy1 antibodies. Yeast pyruvate kinase Pyk1 identified by amino acid microsequencing analysis and immunoblotting with anti-Pyk1 antibodies copurified with the PKA-1 but not the-2 fraction. Pyk1 can be phosphorylated by yeast PKA in vitro in the presence of cAMP and cGMP. Two-dimensional gel electrophoretic analysis revealed four phosphorylated forms of Pyk1 modified by PKA. In phosphorylation of Pyk1 mainly the Tpk2 catalytic subunit of yeast PKA was involved.

Canadian Journal of Microbiology, 1999

Analysis of Saccharomyces cerevisiae genome revealed no sequence homologous to cyclic GMP (cGMP) ... more Analysis of Saccharomyces cerevisiae genome revealed no sequence homologous to cyclic GMP (cGMP) dependent protein kinase from other organisms. Here we demonstrate that cyclic AMP (cAMP) dependent protein kinase purified from S. cerevisiae was almost equally activated by cAMP and cGMP at 3 × 10-6 M concentrations of either nucleotide in the presence of Mg2+ ions. Interestingly, if Mn2+ ions were used instead of Mg2+, cGMP was only 30% as effective as cAMP in the activation of cAMP-dependent protein kinase. Analogs of cAMP such as 8-chloro-cAMP and 3':5'-cyclic monophosphate of ribofuranosylbenzimidazole were as potent as cAMP in the enzyme activation, while N6,2'-O-dibutyryl-cAMP activated the enzyme to a lower extent. It was also found that yeast cAMP-dependent protein kinase can be activated by limited proteolytic digestion. The results presented were obtained with protamine and ribosomal protein S10 used as phosphorylation substrates.Key words: cAMP-dependent protein ...

Biogerontology, 2006

Theories of the evolution of senescence state that symmetrically dividing organisms do not senesc... more Theories of the evolution of senescence state that symmetrically dividing organisms do not senesce. However, this view is challenged by experimental evidence. We measured by immunofluorescence the occurrence and intensity of protein carbonylation in single and symmetrically dividing cells of Schizosaccharomyces pombe. Cells of S. pombe show different levels of carbonylated proteins. Most cells have little damage, a few show a lot, an observation consistent with the gradual accumulation of carbonylation over time. At reproduction, oxidized proteins are shared between the two resulting cells. These results indicate that S. pombe does age, but does so in a different way from other studied species. Damaged cells give rise to damaged cells. The fact that cells with no or few carbonylated proteins constitute the main part of the population can explain why, although age is not reset to zero in one of the cells during division, the pool of young cells remains large enough to prevent the rapid extinction of the population.

Aging Cell, 2009

For the species that have been most carefully studied, mortality rises with age and then plateaus... more For the species that have been most carefully studied, mortality rises with age and then plateaus or declines at advanced ages, except for yeast. Remarkably, mortality for yeast can rise, fall and rise again. In the present study we investigated (i) if this complicated shape could be modulated by environmental conditions by measuring mortality with different food media and temperature; (ii) if it is triggered by biological heterogeneity by measuring mortality in stationary phase in populations fractionated into subpopulations of young, virgin cells, and replicatively older, non-virgin cells. We also discussed the results of a staining method to measure viability instead of measuring the number of cells able to exit stationary phase and form a colony. We showed that different shapes of age-specific death rates were observed and that their appearance depended on the environmental conditions. Furthermore, biological heterogeneity explained the shapes of mortality with homogeneous populations of young, virgin cells exhibiting a simple shape of mortality in conditions under which more heterogeneous populations of older cells or unfractionated populations displayed complicated death rates. Finally, the staining method suggested that cells lost the capacity to exit stationary phase and to divide long before they died in stationary phase. These results explain a phenomenon that was puzzling because it appeared to reflect a radical departure from mortality patterns observed for other species.

Acta Biochimica Polonica, 1999

We have found that heparin has a different effect on Trichosporon cutaneum ribosomal protein phos... more We have found that heparin has a different effect on Trichosporon cutaneum ribosomal protein phosphorylation by CKI and by CKII. In the presence of heparin, modification of 13 kDa, 19 kDa and 38 kDa proteins catalyzed by CKII was inhibited, while in the case of CKI, in addition to protein of 15 kDa, phosphorylation of 20 kDa and 35 kDa proteins was detected. It was also found that, in the presence of heparin, phosphorylation of P proteins (13 kDa and 38 kDa) by ribosome-bound protein kinases was inhibited. Moreover at the same conditions modification of 40 kDa protein was observed in all four yeast species tested.

Journal of Basic Microbiology, 1997

The effect of cAMP and cGMP on the activity and substrate specificity of protein kinase A from me... more The effect of cAMP and cGMP on the activity and substrate specificity of protein kinase A from methylotrophic yeast Pichia pastoris

The effect of vanadate on Pichia pastoris growth, protein

Protein kinases CKI and CKII are implicated in modification of ribosomal proteins of the yeast Tr... more Protein kinases CKI and CKII are implicated in modification of ribosomal proteins of the yeast Trichosporon cutaneum

Acta Biochimica Polonica, 2002

It was found that wild type yeast Pichia pastoris can tolerate vanadate concentration as high as ... more It was found that wild type yeast Pichia pastoris can tolerate vanadate concentration as high as 25 mM in the growth medium. Moreover, four vanadate-resistant P. pastoris strains designated JC100/1, JC100/3, JC100/9 and JC100/15 exhibiting tolerance up to 150 mM vanadate were selected. Growth of P. pastoris was correlated with vanadate to vanadyl reduction and its accumulation in the growth medium. In two selected strains, JC100/9 and JC100/15, protein kinase A activity was much higher in comparison to the wild type strain even without vanadate addition to the growth medium. Moreover, in the presence of vanadate, protein kinase A activity was significantly increased in the wild type and the vanadate-resistant JC100/1 and JC100/3 strains. It was also found that phosphorylation of a 40 kDa protein associated with ribosomes occured in all vanadate-resistant strains from the logarithmic, while in the wild type strain from the stationary growth phase. From the presented results it can be...

Acta Biochimica Polonica, 2002

Phosphorylation of acidic ribosomal proteins P1/P2-P0 is a common phenomenon in eukaryotic organi... more Phosphorylation of acidic ribosomal proteins P1/P2-P0 is a common phenomenon in eukaryotic organisms. It was found previously that in Trichosporon cutaneum, unlike in other yeast species, in addition to the two acidic ribosomal proteins, two other proteins of 15 kDa and 19 kDa of the small ribosomal subunit were phosphorylated. Here we describe two protein kinases: CKI and CKII, which are engaged in the modification of T. cutaneum ribosomal proteins. The acidic ribosomal proteins and the protein of 19 kDa were modified by CKII associated with ribosomes, while the protein of 15 kDa was modified by CKI. Protein kinase CKI was purified from cell-free extract (CKIC) and from ribosomal fraction (CKIR). The molecular mass of CKIC was established at 33 kDa while that of CKIR at 35-37 kDa. A protein of 40 kDa copurified with CKIR but not CKIC. Heparin significantly increased 40 kDa protein phosphorylation level by CKIR. Microsequencing analysis revealed the presence of CKI recognition motif...

Acta Biochimica Polonica, 2003

Cyclic AMP dependent protein kinase (PKA) from Pichia pastoris yeast cells was found to be activa... more Cyclic AMP dependent protein kinase (PKA) from Pichia pastoris yeast cells was found to be activated by either cAMP or cGMP. Analogs of cAMP such as 8-chloro-cAMP and 8-bromo-cAMP were as potent as cAMP in PKA activation while N6,2'-O-dibutyryl-cAMP did not stimulate the enzyme activity. It was shown that protamine sulfate was almost equally phosphorylated in the presence of 1-2 x 10(-6)M cAMP or cGMP while other substrates such as Kemptide, ribosomal protein S6, were phosphorylated to a lower extent in the presence of cGMP. It was demonstrated that pyruvate kinase is a substrate of PKA which co-purified with the P.pastoris enzyme. Moreover, pyruvate kinase was phosphorylated by PKA in the presence of cAMP and cGMP to comparable levels.

Proceedings of the National Academy of Sciences, 2004

Much research aimed at discovering the genetic bases of longevity focuses on the budding yeast Sa... more Much research aimed at discovering the genetic bases of longevity focuses on the budding yeast Saccharomyces cerevisiae. Unfortunately, yeast researchers use a definition of longevity not applied to other species. We propose here a method that makes it possible to estimate for yeast the same measures of longevity calculated for other species. We also show that the conventional method (equating longevity with the number of offspring) is only an approximate measure of true chronological lifespan. Our method will allow results for yeast to be compared more correctly with those for other species.

Proceedings of the National Academy of Sciences, 2004

Much research aimed at discovering the genetic bases of longevity focuses on the budding yeast Sa... more Much research aimed at discovering the genetic bases of longevity focuses on the budding yeast Saccharomyces cerevisiae. Unfortunately, yeast researchers use a definition of longevity not applied to other species. We propose here a method that makes it possible to estimate for yeast the same measures of longevity calculated for other species. We also show that the conventional method (equating longevity with the number of offspring) is only an approximate measure of true chronological lifespan. Our method will allow results for yeast to be compared more correctly with those for other species.

FEMS Microbiology Letters, 2001

Fractionation of Saccharomyces cerevisiae postribosomal extract on DEAE-cellulose revealed two fr... more Fractionation of Saccharomyces cerevisiae postribosomal extract on DEAE-cellulose revealed two fractions of cAMP-dependent protein kinase (PKA-1 and PKA-2). The presence of PKA in both fractions was confirmed by immunoblotting with anti-Bcy1 antibodies. Yeast pyruvate kinase Pyk1 identified by amino acid microsequencing analysis and immunoblotting with anti-Pyk1 antibodies copurified with the PKA-1 but not the-2 fraction. Pyk1 can be phosphorylated by yeast PKA in vitro in the presence of cAMP and cGMP. Two-dimensional gel electrophoretic analysis revealed four phosphorylated forms of Pyk1 modified by PKA. In phosphorylation of Pyk1 mainly the Tpk2 catalytic subunit of yeast PKA was involved.

Canadian Journal of Microbiology, 1999

Analysis of Saccharomyces cerevisiae genome revealed no sequence homologous to cyclic GMP (cGMP) ... more Analysis of Saccharomyces cerevisiae genome revealed no sequence homologous to cyclic GMP (cGMP) dependent protein kinase from other organisms. Here we demonstrate that cyclic AMP (cAMP) dependent protein kinase purified from S. cerevisiae was almost equally activated by cAMP and cGMP at 3 × 10-6 M concentrations of either nucleotide in the presence of Mg2+ ions. Interestingly, if Mn2+ ions were used instead of Mg2+, cGMP was only 30% as effective as cAMP in the activation of cAMP-dependent protein kinase. Analogs of cAMP such as 8-chloro-cAMP and 3':5'-cyclic monophosphate of ribofuranosylbenzimidazole were as potent as cAMP in the enzyme activation, while N6,2'-O-dibutyryl-cAMP activated the enzyme to a lower extent. It was also found that yeast cAMP-dependent protein kinase can be activated by limited proteolytic digestion. The results presented were obtained with protamine and ribosomal protein S10 used as phosphorylation substrates.Key words: cAMP-dependent protein ...

Biogerontology, 2006

Theories of the evolution of senescence state that symmetrically dividing organisms do not senesc... more Theories of the evolution of senescence state that symmetrically dividing organisms do not senesce. However, this view is challenged by experimental evidence. We measured by immunofluorescence the occurrence and intensity of protein carbonylation in single and symmetrically dividing cells of Schizosaccharomyces pombe. Cells of S. pombe show different levels of carbonylated proteins. Most cells have little damage, a few show a lot, an observation consistent with the gradual accumulation of carbonylation over time. At reproduction, oxidized proteins are shared between the two resulting cells. These results indicate that S. pombe does age, but does so in a different way from other studied species. Damaged cells give rise to damaged cells. The fact that cells with no or few carbonylated proteins constitute the main part of the population can explain why, although age is not reset to zero in one of the cells during division, the pool of young cells remains large enough to prevent the rapid extinction of the population.

Aging Cell, 2009

For the species that have been most carefully studied, mortality rises with age and then plateaus... more For the species that have been most carefully studied, mortality rises with age and then plateaus or declines at advanced ages, except for yeast. Remarkably, mortality for yeast can rise, fall and rise again. In the present study we investigated (i) if this complicated shape could be modulated by environmental conditions by measuring mortality with different food media and temperature; (ii) if it is triggered by biological heterogeneity by measuring mortality in stationary phase in populations fractionated into subpopulations of young, virgin cells, and replicatively older, non-virgin cells. We also discussed the results of a staining method to measure viability instead of measuring the number of cells able to exit stationary phase and form a colony. We showed that different shapes of age-specific death rates were observed and that their appearance depended on the environmental conditions. Furthermore, biological heterogeneity explained the shapes of mortality with homogeneous populations of young, virgin cells exhibiting a simple shape of mortality in conditions under which more heterogeneous populations of older cells or unfractionated populations displayed complicated death rates. Finally, the staining method suggested that cells lost the capacity to exit stationary phase and to divide long before they died in stationary phase. These results explain a phenomenon that was puzzling because it appeared to reflect a radical departure from mortality patterns observed for other species.

Acta Biochimica Polonica, 1999

We have found that heparin has a different effect on Trichosporon cutaneum ribosomal protein phos... more We have found that heparin has a different effect on Trichosporon cutaneum ribosomal protein phosphorylation by CKI and by CKII. In the presence of heparin, modification of 13 kDa, 19 kDa and 38 kDa proteins catalyzed by CKII was inhibited, while in the case of CKI, in addition to protein of 15 kDa, phosphorylation of 20 kDa and 35 kDa proteins was detected. It was also found that, in the presence of heparin, phosphorylation of P proteins (13 kDa and 38 kDa) by ribosome-bound protein kinases was inhibited. Moreover at the same conditions modification of 40 kDa protein was observed in all four yeast species tested.

Journal of Basic Microbiology, 1997