Lawrence Berliner - Academia.edu (original) (raw)
Papers by Lawrence Berliner
J Protein Chem, 1996
The active site of the prothrombin activation intermediate meizothrombin(desF1) was probed using ... more The active site of the prothrombin activation intermediate meizothrombin(desF1) was probed using several fluorosulfonylphenyl spin labels specific for the active serine hydroxyl of serine proteases. The mobilities of the thrombin species inhibited with the nitroxide spin labels m-IV [4-(2,2,6,6-tetramethyl-piperidine-1-oxyl) -m-(fluorosulfonyl)benzamide] and m-V [3-(2,2,5,5-tetramethyl-pyrrolidine-1-oxyl) -m-(fluorosulfonyl)benzamide], which are sensitive to differences between alpha- and gamma-thrombin, were quite similar to that of alpha-thrombin. That is, no major conformational differences between meizothrombin(desF1) and alpha-thrombin were observed in this region of the extended active site. On the other hand, p-IV [4-(2,2,6,6-tetramethyl piperidine-1-oxyl)-p-(fluorosulfonyl)benzamide], p-V [3-(2,2,5,5-tetramethylpyrrolidine-1-oxyl) -p-(fluorosulfonyl)benzamide], and m-VII [N-[m- (fluorosulfonyl)phenyl]-4-N-(2,2,6,6-tetramethyl- piperidine-1-oxyl)urea], which probe an apolar binding region of bovine thrombin, exhibited large differences in mobility between alpha-thrombin and meizothrombin(desF1). The conformational consequences of indole binding to spin-labeled thrombin species demonstrated that both species also possess an indole-binding site. However, the nitroxide mobility changes upon indole binding to the spin-labeled protein derivative were somewhat different between the two thrombin species under study. In addition, the effects of the benzamidine binding were quite similar for each labeled protein. Thus is appears that, while both species posses a fully functional active site, the region in meizothrombin(desF1) probed by spin labels p-IV, p-V, and m-VII, which corresponds to the apolar binding region, differs in conformation from alpha-thrombin.
Biochemistry Usa, 1987
The self-incorporation of apo-alpha-lactalbumin (alpha-LA) into single unilamellar vesicles (SUV)... more The self-incorporation of apo-alpha-lactalbumin (alpha-LA) into single unilamellar vesicles (SUV) of dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine was demonstrated by column chromatographic analyses on Sephadex G-200 (10 mM Tris-HCl, pH 7.4, 26-28 degrees C) and by intrinsic fluorescence emission of SUV-bound alpha-LA. It was shown that apo-alpha-LA slowly incorporated into the DMPC vesicle bilayer after equilibrating different mixtures of protein and SUV for several hours. The intrinsic fluorescence properties of the bound apo-alpha-LA were altered only slightly (lambda maxem = 333 nm vs. 337 nm in aqueous solution). The large blue shift in apo-alpha-LA fluorescence in solution induced by monovalent cations, such as Na(I), was almost completely prevented when apo-alpha-LA was membrane bound. Furthermore, the addition of calcium caused a slow conversion from apo-alpha-LA to Ca(II)-alpha-LA by a mechanism consistent with passive diffusion of Ca(II) into the bilayer interior to the (buried) calcium binding site. The release of Ca(II)-alpha-LA from the membrane is discussed with reference to alpha-LA release from the smooth endoplasmic reticulum in vivo.
Biochemistry, Feb 1, 1997
... Ming Chi Yang, Hong-Hsiang Guan, Jinn-Moon Yang, Cheng-Neng Ko, Ming-Yih Liu, Yih-Hung Lin, Y... more ... Ming Chi Yang, Hong-Hsiang Guan, Jinn-Moon Yang, Cheng-Neng Ko, Ming-Yih Liu, Yih-Hung Lin, Yen-Chieh Huang, Chun-Jung Chen and Simon JT Mao. ... Su-Ying Dong, Zhen-Wen Zhao, and Hui-Min Ma. Journal of Proteome Research 2006 5 (1), 26-31. ...
Biochemistry Usa, 1981
ABSTRACT: Human a-and y-thrombins (clotting vs. nonclotting activities, respectively) were conjug... more ABSTRACT: Human a-and y-thrombins (clotting vs. nonclotting activities, respectively) were conjugated at their active-site serine with a series of 13 phenylsulfonyl fluoride spin-labeling reagents. One class (indole-site reagents), predominantly para-substituted phenylsulfonyl ...
J Am Chem Soc, 1980
ABSTRACT
Biochemistry Usa, 1985
Three new spin-labeled glycosides, spin-label I [ 1 -[4-(P-~-galactopyranosyloxy)phenyl] -3-(2,2,... more Three new spin-labeled glycosides, spin-label I [ 1 -[4-(P-~-galactopyranosyloxy)phenyl] -3-(2,2,6,6-tetramethyl-l-oxypiperidin-4-yl)-2-thiourea], spin-label I1 (2,2,6,64etramethyl-1-oxypiperidin-4-yl a-D-galactopyranoside), and spin-label I11 [1-(methyl 2-deoxy-a-~-galactopyranosid-2-yl)-3-(2,2,6,6tetramethyl-1 -oxypiperidin-4-yl)-2-thiourea], were investigated as structural probes of Griffoniu simplicifolia I isolectins (GS I) A4 and B4, respectively, by electron spin resonance (ESR) and inhibition of guaran isolectin precipitation. The p-aminophenyl P-galactoside spin-label I was strongly immobilized by the B4 isolectin (Kd = 0.42 mM; 2T,, = 54.0 f 0.3 G), while binding to the A4 isolectin was so weak (KI N 2 mM) that binding was undetectable by ESR. The preference for the B4 isolectin was indicative of a more extended hydrophobic binding locus adjacent to the carbohydrate-specific binding site. The a-galactosyl spin-label
Biochemistry Usa, 1985
The binding of the fluorescent probe 4,4'-bis[1-(phenylamino)-8-naphthalenesulfonate] (bi... more The binding of the fluorescent probe 4,4'-bis[1-(phenylamino)-8-naphthalenesulfonate] (bis-ANS) to bovine alpha-lactalbumin (alpha-LA) was investigated. A strong dependence of the Kd value with the bound calcium stoichiometry was found, with Kd values ranging from 6.2 +/- 0.4 to 64.6 +/- 5.9 microM for apo-alpha-LA and 1:1 Ca(II)-alpha-LA, respectively. A 350-fold enhancement of the bis-ANS emission was observed in the protein-bis-ANS complex, along with an approximately 33-nm blue shift. Both appeared to be related to the hydrophobicity of the binding site and were independent of the Ca(II) ion content. From the difference in bis-ANS affinity between apo-alpha-LA and Ca(II)-alpha-LA, we demonstrated that Zn(II) and Al(III) were able to "lock" the protein into a new "apo-like" conformation, which was similar to, but not identical with, the apo conformation. The protein could be interconverted between all three conformations in a Mn(II) titration. The first Mn(II) shifted the apoprotein to the Ca(II) conformation; at higher Mn(II) levels, binding to the second site shifted the protein toward the apo-like conformation. The same behavior was observed with calcium in large excess. The evidence supported a model for the mutually nonexclusive binding of metals both to site I ("calcium site") and to site II ("zinc site") simultaneously. The results suggest that alpha-lactalbumin possesses a hydrophobic surface that becomes somewhat less accessible upon 1:1 calcium binding in the absence of metals that also bind to the zinc site.
... CONTRIBUTORS Albert H. Beth Department of Molecular Physiology and Biophysics, Vanderbilt U... more ... CONTRIBUTORS Albert H. Beth Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232 Petr P. Borbat Baker Laboratory of Chemistry and Chemical Biology, Cornell University, 1thaca, New York 14853 Gary W. Brudvig ...
Neuroscience, Feb 1, 2004
The major aim of this study was to elucidate the role of nitric oxide (NO) in the development of ... more The major aim of this study was to elucidate the role of nitric oxide (NO) in the development of pentylenetetrazole (PTZ)-kindling as an animal model of primary generalized epilepsy. The daily administration of PTZ is associated with an increase in the amount of neuronal nitric oxide synthase (nNOS). NO generation was measured directly by in vivo and ex vivo electron paramagnetic resonance on rodents undergoing progressive convulsions. We found that primary generalized epilepsy is caused by NO induction during the persistent up-regulation of nNOS expression, but that NO induction is not associated with severe generalized seizures following long-term kindling phenomena after PTZ withdrawal. Morphological changes in the brain structure of rats were measured by magnetic resonance imaging during epileptic convulsions induced by repetitive administration of PTZ. Cerebellum volume for kindled rats decreased 20% but not in rats treated with the nNOS inhibitor, 3Br-7NI, suggesting that generation of NO in the cerebellum is related to decrease in cerebellum volume following PTZ-kindling.
Biochemistry Usa, 1997
The functional role of previously identified calcium binding residues in alpha-lactalbumin (alpha... more The functional role of previously identified calcium binding residues in alpha-lactalbumin (alpha-LA) was investigated by site-directed mutagenesis. Mutation of D82 to alanine did not effect the binding affinity for calcium, the protein structure, or its function in the lactose synthase assay, suggesting that this aspartate side chain is not essential for calcium binding or structural stabilization. In contrast, mutation of either D87 or D88 to alanine completely eliminated the strong calcium binding and altered alpha-LA as shown by several spectroscopically derived properties such as near- and far-UV CD and intrinsic fluorescence studies. These latter two mutants displayed significantly reduced abilities to stimulate lactose synthase activity (<3.5% of the maximal rate). Additionally, residues K79 and D84, which chelate calcium by backbone carbonyls, were mutated to alanine. K79A lost approximately 50% of its tertiary structure and stability (as determined by CD) but retained full calcium binding activity, indicating that at least the lysine side chain does not influence the carbonyl-mediated calcium coordination. In contrast, D84A lost approximately 25% of its tertiary structure and stability which was accompanied by a modest reduction in calcium affinity. Both mutants were able to stimulate normal lactose synthase activity. The triple mutant, D82A/D87A/D88A alpha-LA, lost its ability to bind calcium, similar to D87A and D88A. These studies clearly demonstrate the importance and variation of side chain interactions, which might be the seminal event in the establishment of the correct calcium binding loop conformation, possibly to stabilization and final folding of the overall protein structure.
Biochemistry Usa, Feb 1, 1988
... Giovanni Musci,*,f Lawrence J. Berliner,$ and Charles T. Esmon*,Il Department of Chemistry, T... more ... Giovanni Musci,*,f Lawrence J. Berliner,$ and Charles T. Esmon*,Il Department of Chemistry, The Ohio State University, Columbus, Ohio 4321 0, and ... 2T,, = 48-70 G), which are related to the tumbling motion of the nitroxide moiety (Shimshick & McConnell, 1974). ...
Magnetic Resonance in Medicine, Mar 1, 1989
Low-field in vivo electron spin resonance (ESR) has been used to follow the course of metabolism ... more Low-field in vivo electron spin resonance (ESR) has been used to follow the course of metabolism and distribution of a paramagnetic spin probe, 3-carboxamido-2,2,5,5tetramethylpyrrolidine-I-oxyl. Sprague-Dawley rats (250-300 g) were catheterized in the jugular vein and given serial doses ofthe nitroxide spin probe above. The decrease in the tail blood nitroxide ESR spectrum with the time was followed. This reflects both normal distribution/excretion and a significant metabolic step-reversible reduction of the nitroxide group to its hydroxylamine. The studies serve as important and useful comparisons to the nitroxide reduction kinetics in vitro while offering the important advantage of avoiding those non-steady-state artifacts frequently expected in ex vivo procedures. This study represents the first report of systematic in vivo phannacokinetics of a paramagnetic probe.
Febs Lett, 2000
Small milk protein α-lactalbumin (α-LA), a component of lactose synthase, is a simple model Ca2+ ... more Small milk protein α-lactalbumin (α-LA), a component of lactose synthase, is a simple model Ca2+ binding protein, which does not belong to the EF-hand proteins, and a classical example of molten globule state. It has a strong Ca2+ binding site, which binds Mg2+, Mn2+, Na+, and K+, and several distinct Zn2+ binding sites. The binding of cations to the Ca2+ site increases protein stability against action of heat and various denaturing agents, while the binding of Zn2+ to the Ca2+-loaded protein decreases its stability. Functioning of α-LA requires its interactions with membranes, proteins, peptides and low molecular weight substrates and products. It was shown that these interactions are modulated by the binding of metal cations. Recently it was found that some folding variants of α-LA demonstrate bactericidal activity and some of them cause apoptosis of tumor cells.
The functional role of previously identified calcium binding residues in alpha-lactalbumin (alpha... more The functional role of previously identified calcium binding residues in alpha-lactalbumin (alpha-LA) was investigated by site-directed mutagenesis. Mutation of D82 to alanine did not effect the binding affinity for calcium, the protein structure, or its function in the lactose synthase assay, suggesting that this aspartate side chain is not essential for calcium binding or structural stabilization. In contrast, mutation of either D87 or D88 to alanine completely eliminated the strong calcium binding and altered alpha-LA as shown by several spectroscopically derived properties such as near- and far-UV CD and intrinsic fluorescence studies. These latter two mutants displayed significantly reduced abilities to stimulate lactose synthase activity (<3.5% of the maximal rate). Additionally, residues K79 and D84, which chelate calcium by backbone carbonyls, were mutated to alanine. K79A lost approximately 50% of its tertiary structure and stability (as determined by CD) but retained full calcium binding activity, indicating that at least the lysine side chain does not influence the carbonyl-mediated calcium coordination. In contrast, D84A lost approximately 25% of its tertiary structure and stability which was accompanied by a modest reduction in calcium affinity. Both mutants were able to stimulate normal lactose synthase activity. The triple mutant, D82A/D87A/D88A alpha-LA, lost its ability to bind calcium, similar to D87A and D88A. These studies clearly demonstrate the importance and variation of side chain interactions, which might be the seminal event in the establishment of the correct calcium binding loop conformation, possibly to stabilization and final folding of the overall protein structure.
J Protein Chem, 1996
The active site of the prothrombin activation intermediate meizothrombin(desF1) was probed using ... more The active site of the prothrombin activation intermediate meizothrombin(desF1) was probed using several fluorosulfonylphenyl spin labels specific for the active serine hydroxyl of serine proteases. The mobilities of the thrombin species inhibited with the nitroxide spin labels m-IV [4-(2,2,6,6-tetramethyl-piperidine-1-oxyl) -m-(fluorosulfonyl)benzamide] and m-V [3-(2,2,5,5-tetramethyl-pyrrolidine-1-oxyl) -m-(fluorosulfonyl)benzamide], which are sensitive to differences between alpha- and gamma-thrombin, were quite similar to that of alpha-thrombin. That is, no major conformational differences between meizothrombin(desF1) and alpha-thrombin were observed in this region of the extended active site. On the other hand, p-IV [4-(2,2,6,6-tetramethyl piperidine-1-oxyl)-p-(fluorosulfonyl)benzamide], p-V [3-(2,2,5,5-tetramethylpyrrolidine-1-oxyl) -p-(fluorosulfonyl)benzamide], and m-VII [N-[m- (fluorosulfonyl)phenyl]-4-N-(2,2,6,6-tetramethyl- piperidine-1-oxyl)urea], which probe an apolar binding region of bovine thrombin, exhibited large differences in mobility between alpha-thrombin and meizothrombin(desF1). The conformational consequences of indole binding to spin-labeled thrombin species demonstrated that both species also possess an indole-binding site. However, the nitroxide mobility changes upon indole binding to the spin-labeled protein derivative were somewhat different between the two thrombin species under study. In addition, the effects of the benzamidine binding were quite similar for each labeled protein. Thus is appears that, while both species posses a fully functional active site, the region in meizothrombin(desF1) probed by spin labels p-IV, p-V, and m-VII, which corresponds to the apolar binding region, differs in conformation from alpha-thrombin.
Biochemistry Usa, 1987
The self-incorporation of apo-alpha-lactalbumin (alpha-LA) into single unilamellar vesicles (SUV)... more The self-incorporation of apo-alpha-lactalbumin (alpha-LA) into single unilamellar vesicles (SUV) of dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine was demonstrated by column chromatographic analyses on Sephadex G-200 (10 mM Tris-HCl, pH 7.4, 26-28 degrees C) and by intrinsic fluorescence emission of SUV-bound alpha-LA. It was shown that apo-alpha-LA slowly incorporated into the DMPC vesicle bilayer after equilibrating different mixtures of protein and SUV for several hours. The intrinsic fluorescence properties of the bound apo-alpha-LA were altered only slightly (lambda maxem = 333 nm vs. 337 nm in aqueous solution). The large blue shift in apo-alpha-LA fluorescence in solution induced by monovalent cations, such as Na(I), was almost completely prevented when apo-alpha-LA was membrane bound. Furthermore, the addition of calcium caused a slow conversion from apo-alpha-LA to Ca(II)-alpha-LA by a mechanism consistent with passive diffusion of Ca(II) into the bilayer interior to the (buried) calcium binding site. The release of Ca(II)-alpha-LA from the membrane is discussed with reference to alpha-LA release from the smooth endoplasmic reticulum in vivo.
Biochemistry, Feb 1, 1997
... Ming Chi Yang, Hong-Hsiang Guan, Jinn-Moon Yang, Cheng-Neng Ko, Ming-Yih Liu, Yih-Hung Lin, Y... more ... Ming Chi Yang, Hong-Hsiang Guan, Jinn-Moon Yang, Cheng-Neng Ko, Ming-Yih Liu, Yih-Hung Lin, Yen-Chieh Huang, Chun-Jung Chen and Simon JT Mao. ... Su-Ying Dong, Zhen-Wen Zhao, and Hui-Min Ma. Journal of Proteome Research 2006 5 (1), 26-31. ...
Biochemistry Usa, 1981
ABSTRACT: Human a-and y-thrombins (clotting vs. nonclotting activities, respectively) were conjug... more ABSTRACT: Human a-and y-thrombins (clotting vs. nonclotting activities, respectively) were conjugated at their active-site serine with a series of 13 phenylsulfonyl fluoride spin-labeling reagents. One class (indole-site reagents), predominantly para-substituted phenylsulfonyl ...
J Am Chem Soc, 1980
ABSTRACT
Biochemistry Usa, 1985
Three new spin-labeled glycosides, spin-label I [ 1 -[4-(P-~-galactopyranosyloxy)phenyl] -3-(2,2,... more Three new spin-labeled glycosides, spin-label I [ 1 -[4-(P-~-galactopyranosyloxy)phenyl] -3-(2,2,6,6-tetramethyl-l-oxypiperidin-4-yl)-2-thiourea], spin-label I1 (2,2,6,64etramethyl-1-oxypiperidin-4-yl a-D-galactopyranoside), and spin-label I11 [1-(methyl 2-deoxy-a-~-galactopyranosid-2-yl)-3-(2,2,6,6tetramethyl-1 -oxypiperidin-4-yl)-2-thiourea], were investigated as structural probes of Griffoniu simplicifolia I isolectins (GS I) A4 and B4, respectively, by electron spin resonance (ESR) and inhibition of guaran isolectin precipitation. The p-aminophenyl P-galactoside spin-label I was strongly immobilized by the B4 isolectin (Kd = 0.42 mM; 2T,, = 54.0 f 0.3 G), while binding to the A4 isolectin was so weak (KI N 2 mM) that binding was undetectable by ESR. The preference for the B4 isolectin was indicative of a more extended hydrophobic binding locus adjacent to the carbohydrate-specific binding site. The a-galactosyl spin-label
Biochemistry Usa, 1985
The binding of the fluorescent probe 4,4'-bis[1-(phenylamino)-8-naphthalenesulfonate] (bi... more The binding of the fluorescent probe 4,4'-bis[1-(phenylamino)-8-naphthalenesulfonate] (bis-ANS) to bovine alpha-lactalbumin (alpha-LA) was investigated. A strong dependence of the Kd value with the bound calcium stoichiometry was found, with Kd values ranging from 6.2 +/- 0.4 to 64.6 +/- 5.9 microM for apo-alpha-LA and 1:1 Ca(II)-alpha-LA, respectively. A 350-fold enhancement of the bis-ANS emission was observed in the protein-bis-ANS complex, along with an approximately 33-nm blue shift. Both appeared to be related to the hydrophobicity of the binding site and were independent of the Ca(II) ion content. From the difference in bis-ANS affinity between apo-alpha-LA and Ca(II)-alpha-LA, we demonstrated that Zn(II) and Al(III) were able to "lock" the protein into a new "apo-like" conformation, which was similar to, but not identical with, the apo conformation. The protein could be interconverted between all three conformations in a Mn(II) titration. The first Mn(II) shifted the apoprotein to the Ca(II) conformation; at higher Mn(II) levels, binding to the second site shifted the protein toward the apo-like conformation. The same behavior was observed with calcium in large excess. The evidence supported a model for the mutually nonexclusive binding of metals both to site I ("calcium site") and to site II ("zinc site") simultaneously. The results suggest that alpha-lactalbumin possesses a hydrophobic surface that becomes somewhat less accessible upon 1:1 calcium binding in the absence of metals that also bind to the zinc site.
... CONTRIBUTORS Albert H. Beth Department of Molecular Physiology and Biophysics, Vanderbilt U... more ... CONTRIBUTORS Albert H. Beth Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232 Petr P. Borbat Baker Laboratory of Chemistry and Chemical Biology, Cornell University, 1thaca, New York 14853 Gary W. Brudvig ...
Neuroscience, Feb 1, 2004
The major aim of this study was to elucidate the role of nitric oxide (NO) in the development of ... more The major aim of this study was to elucidate the role of nitric oxide (NO) in the development of pentylenetetrazole (PTZ)-kindling as an animal model of primary generalized epilepsy. The daily administration of PTZ is associated with an increase in the amount of neuronal nitric oxide synthase (nNOS). NO generation was measured directly by in vivo and ex vivo electron paramagnetic resonance on rodents undergoing progressive convulsions. We found that primary generalized epilepsy is caused by NO induction during the persistent up-regulation of nNOS expression, but that NO induction is not associated with severe generalized seizures following long-term kindling phenomena after PTZ withdrawal. Morphological changes in the brain structure of rats were measured by magnetic resonance imaging during epileptic convulsions induced by repetitive administration of PTZ. Cerebellum volume for kindled rats decreased 20% but not in rats treated with the nNOS inhibitor, 3Br-7NI, suggesting that generation of NO in the cerebellum is related to decrease in cerebellum volume following PTZ-kindling.
Biochemistry Usa, 1997
The functional role of previously identified calcium binding residues in alpha-lactalbumin (alpha... more The functional role of previously identified calcium binding residues in alpha-lactalbumin (alpha-LA) was investigated by site-directed mutagenesis. Mutation of D82 to alanine did not effect the binding affinity for calcium, the protein structure, or its function in the lactose synthase assay, suggesting that this aspartate side chain is not essential for calcium binding or structural stabilization. In contrast, mutation of either D87 or D88 to alanine completely eliminated the strong calcium binding and altered alpha-LA as shown by several spectroscopically derived properties such as near- and far-UV CD and intrinsic fluorescence studies. These latter two mutants displayed significantly reduced abilities to stimulate lactose synthase activity (<3.5% of the maximal rate). Additionally, residues K79 and D84, which chelate calcium by backbone carbonyls, were mutated to alanine. K79A lost approximately 50% of its tertiary structure and stability (as determined by CD) but retained full calcium binding activity, indicating that at least the lysine side chain does not influence the carbonyl-mediated calcium coordination. In contrast, D84A lost approximately 25% of its tertiary structure and stability which was accompanied by a modest reduction in calcium affinity. Both mutants were able to stimulate normal lactose synthase activity. The triple mutant, D82A/D87A/D88A alpha-LA, lost its ability to bind calcium, similar to D87A and D88A. These studies clearly demonstrate the importance and variation of side chain interactions, which might be the seminal event in the establishment of the correct calcium binding loop conformation, possibly to stabilization and final folding of the overall protein structure.
Biochemistry Usa, Feb 1, 1988
... Giovanni Musci,*,f Lawrence J. Berliner,$ and Charles T. Esmon*,Il Department of Chemistry, T... more ... Giovanni Musci,*,f Lawrence J. Berliner,$ and Charles T. Esmon*,Il Department of Chemistry, The Ohio State University, Columbus, Ohio 4321 0, and ... 2T,, = 48-70 G), which are related to the tumbling motion of the nitroxide moiety (Shimshick & McConnell, 1974). ...
Magnetic Resonance in Medicine, Mar 1, 1989
Low-field in vivo electron spin resonance (ESR) has been used to follow the course of metabolism ... more Low-field in vivo electron spin resonance (ESR) has been used to follow the course of metabolism and distribution of a paramagnetic spin probe, 3-carboxamido-2,2,5,5tetramethylpyrrolidine-I-oxyl. Sprague-Dawley rats (250-300 g) were catheterized in the jugular vein and given serial doses ofthe nitroxide spin probe above. The decrease in the tail blood nitroxide ESR spectrum with the time was followed. This reflects both normal distribution/excretion and a significant metabolic step-reversible reduction of the nitroxide group to its hydroxylamine. The studies serve as important and useful comparisons to the nitroxide reduction kinetics in vitro while offering the important advantage of avoiding those non-steady-state artifacts frequently expected in ex vivo procedures. This study represents the first report of systematic in vivo phannacokinetics of a paramagnetic probe.
Febs Lett, 2000
Small milk protein α-lactalbumin (α-LA), a component of lactose synthase, is a simple model Ca2+ ... more Small milk protein α-lactalbumin (α-LA), a component of lactose synthase, is a simple model Ca2+ binding protein, which does not belong to the EF-hand proteins, and a classical example of molten globule state. It has a strong Ca2+ binding site, which binds Mg2+, Mn2+, Na+, and K+, and several distinct Zn2+ binding sites. The binding of cations to the Ca2+ site increases protein stability against action of heat and various denaturing agents, while the binding of Zn2+ to the Ca2+-loaded protein decreases its stability. Functioning of α-LA requires its interactions with membranes, proteins, peptides and low molecular weight substrates and products. It was shown that these interactions are modulated by the binding of metal cations. Recently it was found that some folding variants of α-LA demonstrate bactericidal activity and some of them cause apoptosis of tumor cells.
The functional role of previously identified calcium binding residues in alpha-lactalbumin (alpha... more The functional role of previously identified calcium binding residues in alpha-lactalbumin (alpha-LA) was investigated by site-directed mutagenesis. Mutation of D82 to alanine did not effect the binding affinity for calcium, the protein structure, or its function in the lactose synthase assay, suggesting that this aspartate side chain is not essential for calcium binding or structural stabilization. In contrast, mutation of either D87 or D88 to alanine completely eliminated the strong calcium binding and altered alpha-LA as shown by several spectroscopically derived properties such as near- and far-UV CD and intrinsic fluorescence studies. These latter two mutants displayed significantly reduced abilities to stimulate lactose synthase activity (<3.5% of the maximal rate). Additionally, residues K79 and D84, which chelate calcium by backbone carbonyls, were mutated to alanine. K79A lost approximately 50% of its tertiary structure and stability (as determined by CD) but retained full calcium binding activity, indicating that at least the lysine side chain does not influence the carbonyl-mediated calcium coordination. In contrast, D84A lost approximately 25% of its tertiary structure and stability which was accompanied by a modest reduction in calcium affinity. Both mutants were able to stimulate normal lactose synthase activity. The triple mutant, D82A/D87A/D88A alpha-LA, lost its ability to bind calcium, similar to D87A and D88A. These studies clearly demonstrate the importance and variation of side chain interactions, which might be the seminal event in the establishment of the correct calcium binding loop conformation, possibly to stabilization and final folding of the overall protein structure.