Papers by Engelbert Buxbaum
International review of …, 1984
3. The precursors interact with a receptor in the outer mitochondrial membrane interaction being ... more 3. The precursors interact with a receptor in the outer mitochondrial membrane interaction being mediated by the presequences of the precursors. The presequences therefore act as addressing signals as well as possibly playing a role in one or all of (a) solubilization of precursors, ...
Fundamentals of Protein Structure and Function, 2015
All enzymes in a metabolic pathway operating in steady state have the same flux and maintain conc... more All enzymes in a metabolic pathway operating in steady state have the same flux and maintain concentrations of all intermediates. The system will respond to perturbation in such a way that the perturbation is counteracted. This results in homeostasis. Cells can change the flux through a pathway by changing enzyme concentration, switching enzymes on or off by posttranslational modification, allosteric control, and catalytic cycling.
Journal of Cell Science, 1996
Hsc70 was previously isolated by its ability to catalyse the uncoating of clathrin-coated vesicle... more Hsc70 was previously isolated by its ability to catalyse the uncoating of clathrin-coated vesicles from bovine brain. We have recently shown that Hsc70 is more active towards coated vesicles from brain than those from other tissues. In order to gain information on the mechanistic reason for this difference we have examined the ability of brain and placental coated vesicles to stimulate partial reactions during a single round of ATP turnover. The Hsc70-ATP complex is turned over to Hsc70-ADP center dot Pi, from which phosphate is slowly released. The resulting Hsc70-ADP complex exchanges ATP for ADP. Dissociation of ATP or ADP from Hsc70 does not seem to occur under physiological conditions. The hydrolysis of ATP is accelerated by the presence of clathrin-coated vesicles, with vesicles from brain being about twice as effective as vesicles from placenta. Additionally, it appears that brain, but not placental, coated vesicles can also stimulate the exchange of ADP for ATP.
Journal of Cell Science, 1995
Clathrin-coated vesicles from brain are primarily involved in synaptic vesicle recycling and are ... more Clathrin-coated vesicles from brain are primarily involved in synaptic vesicle recycling and are substrates for the constitutively expressed heat shock cognate hsc70 protein (uncoating ATPase). To investigate the regulation of clathrin coat turnover in other tissues the activity of hsc70 towards coated vesicles from other sources was examined. Concentrations of hsc70 which caused near-complete removal of clathrin from brain coated vesicles effected only partial uncoating of vesicles prepared from other tissues. The selective action of hsc70 could not be accounted for by tissue or species specificities of hsc70, but rather reflected differences in coat structure. Selective action was associated with two differences in the hsc70-dependent ATPase cycle. Firstly, uncoating of brain, but not placental vesicles, could occur under circumstances where ATP hydrolysis was prevented. Secondly, only brain coated vesicles could support multiple rounds of hsc70-dependent ATP hydrolysis. Implicati...
Fundamentals of Protein Structure and Function, 2015
Peptides and proteins are made by condensation of amino acids, forming peptide bonds. The sequenc... more Peptides and proteins are made by condensation of amino acids, forming peptide bonds. The sequence of amino acids in a protein is called its primary structure. Secondary structure is determined by the dihedral angles ; of the peptide bonds, the tertiary structure by the folding of protein chains in space. Association of folded polypeptide molecules to complex functional proteins results in quaternary structure. Proteins can be further modified by posttranslational addition of small molecules.
Transport from the ER through Golgi-apparatus to plasma membrane, secretory vesicles, or lysosome... more Transport from the ER through Golgi-apparatus to plasma membrane, secretory vesicles, or lysosomes occurs in specialised vesicles. Proteins in the plasma membrane, some with bound ligand, are recycled in clathrin-coated vesicles, which after uncoating fuse with the endosome. From there proteins can return to the plasma membrane or move on to the lysosome for degradation.
Biophysical Chemistry Of Proteins An Introduction To Laboratory Methods FREE DOWNLOAD BIOPHYSICAL... more Biophysical Chemistry Of Proteins An Introduction To Laboratory Methods FREE DOWNLOAD BIOPHYSICAL CHEMISTRY OF PROTEINS AN INTRODUCTION TO LABORATORY METHODS Interestingly, biophysical chemistry of proteins an introduction to laboratory methods that you really wait for now is coming. It's significant to wait for the representative and beneficial books to read. Every book that is provided in better way and utterance will be expected by many peoples. Even you are a good reader or not, feeling to read this book will always appear when you find it. But, when you feel hard to find it as yours, what to do? Borrow to your friends and don't know when to give back it to her or him. It's needed now to own this book by you. It is not as difficult as previously to find a book. The modern technology always is the best way to find something. As here, we are the website that always provides the book that you need. As biophysical chemistry of proteins an introduction to laboratory metho...
Fundamentals of Protein Structure and Function
ABSTRACT
The long-term (34 weeks) effect of streptozotocin induced diabetes was assessed in Wistar rats.Na... more The long-term (34 weeks) effect of streptozotocin induced diabetes was assessed in Wistar rats.Na+/K+-ATPase activity was measured by ouabain inhibitable86Rb+-uptake into erythrocytes. No difference in the rate of Rb+-uptake, the Kmfor Rb+or the Kifor ouabain was detected between normal and diabetic rats. Thus, the change in Na+/K+-ATPase activity repeatedly described in short-term studies may not translate into a long term physiologically relevant change in ion flux through the sodium pump.Rats excrete ketone bodies mainly as β-hydroxybutyrate. This compound does not show up with nitroprusside sodium based test sticks, it can however be detected by coupled spectrophotometric assay with hydroxybutyrate dehydrogenase.Almost half of the diabetic animals reverted to a non-diabetic state during the experiment, followed by at least partial reversal of secondary diabetic damage.Abbreviations usedPKCProtein kinase CDAGDiacylglycerolEDTAEthylenediamine tetraacetic acidPIP3Phosphatidylinosit...
Biochemical Journal, 1996
Nucleotide binding to the 70 kDa heat-shock cognate protein (Hsc70) from mung bean seeds and pig ... more Nucleotide binding to the 70 kDa heat-shock cognate protein (Hsc70) from mung bean seeds and pig brain was investigated, as well as the clathrin uncoating activity of Hsc70 in the presence of these nucleotides. The two enzymes were found to behave identically. ATP bound to two different forms of Hsc70, with dissociation constants of 1.1±0.1 µM and 1.4±0.7 mM respectively at 25 °C. This corresponds to ΔG0´ = -34 and -16 kJ/mol respectively. From the temperature-dependence of the dissociation constant of the high-affinity site, ΔH0´ was calculated to -36±2 kJ/mol. This gives ΔS0´ = 6.7 J/mol per K. Adenosine 5´-[γ-thio]triphosphate, ADP, adenosine 5´-[β,γ-imino]triphosphate and adenosine 5´-[β,γ-methylene]triphosphate showed dissociation constants of 2.3, 11, 31 and 284 µM respectively. The order of affinities corresponded to the order of effectiveness in uncoating of pig brain coated vesicles. The implications of these findings for the mechanism of Hsc70 action are discussed.
Springer eBooks, 2011
If you want to study, say, the enzymes present in the liver, the first thing you have to do is ob... more If you want to study, say, the enzymes present in the liver, the first thing you have to do is obtain fresh liver tissue. This is done at the abattoir immediately after an animal has been slaughtered. The tissue is transported into the laboratory on ice to minimise proteolytic damage.
However, the precision for the parameters V max and K m determined this way is much lower than by... more However, the precision for the parameters V max and K m determined this way is much lower than by the conventional variation of [ S ]. Also, since all measurements are obtained from a single experiment, they are highly correlated and normal least-square fitting techniques give artificially low error estimates for the parameters. Therefore, this method is rarely used. However, [83, 304] give details on it, if the need arises.
European Journal of Biochemistry, 1991
Tetrammine cobalt(II1) phosphate [ C O (N H~)~P O~] inactivates Na'/K'-ATPase in the E2 conformat... more Tetrammine cobalt(II1) phosphate [ C O (N H~)~P O~] inactivates Na'/K'-ATPase in the E2 conformational state, dependent on time and concentration, according to Eqn (1): k 2 Co(NH3)4P04 + Ez Ez Co(NH3)4P04 + E' , ' Co(NH,),P04. Kd The inactivation rate constant k2 for the formation of a stable E;. C O (N H~)~P O~ at 37°C was 0.057 min-l; the dissociation constant, Kd = 300 pM. The activation energy for the inactivation process was 149 kJ/mol. ATP and the uncleavable adenosine 5'-[j,y-methylene]triphosphate competed with C O (N H~)~P O~ for its binding site with K, = 0.41 mM and 5 mM, respectively. MgP04 competed with C O (N H~)~P O~ linearly, with K, = 50 pM, as did phosphate (Ks = 16 mM) and Mg2+ (K, = 160 pM). It is concluded that the MgP04 analogue binds to the MgP0,binding subsite of the low-affinity ATP-binding site (of the E2 conformation). Also, Na' (Ks = 860 pM) protected the enzyme against inactivation in a competitive manner. From the intersecting (slope and intercept linear) noncompetitive effect of Na' against the inactivation by C O (N H~)~P O~, apparent affinities of K f for the free enzyme of 41 pM, and for the E. C O (N H ,)~P O~ complex of 720 pM, were calculated. Binding of C O (N H~)~P O~ to the enzyme inactivated N a f / K t-ATPase and K'-activated phosphatase, and, moreover, prevented the occlusion of 86Rb' ; however, the activity of the Na'-ATPase, the phosphorylation capacity of the high-affinity ATP-binding site and the ATP/ADP-exchange reaction remained unchanged. With C O (N H~)~~' P O~ a binding capacity of 135 pmol/unit enzyme was found. Phosphorylation and complete inactivation of the enzyme with C O (N H~)~~' P O~ or the 32P-labelled tetramminecobalt ATP ([y-32P]Co(NH3)4ATP} at the low-affinity ATP-binding site, allowed (independent of the purity of the Na' /K'-ATPase preparation) a further incorporation of radioactivity from 32P-labelled tetraaquachromium(II1) ATP ([y-32P]CrATP) to the highaffinity ATP-binding site with unchanged phosphorylation capacity. However, inactivation and phosphorylation of Na+/K'-ATPase by [y-32P]CrATP prevented the binding of C O (N H~)~~' P O , or [y-32P]Co(NH3)4ATP to the enzyme. [Y-~'P]CO(NH~)~ATP and C O (N H~)~~' P O~ are mutually exclusive. The data are consistent with the assumption of a cooperation of catalytic subunits within an (a,&diprotomer, which change their interactions during the Na'/K+-pumpimg process. Our findings seem not to support a symmetrical Repke and Stein model of enzyme action. Na'/K+-ATPase, which transports Na' and K f against an electrochemical gradient through cell membranes, is believed to work according to the Albers-Post model [l-31. It assumes that the overall Na'/K'-ATPase reaction is the sum of partial reactions caused by this enzyme. However, this widely accepted model has some unsolved problems. The intrinsic rate constant of Na+/K+-ATPase is about 30 times higher than that of Na'-ATPase [4]; the Na +-ATPase reaction Correspondence to E. Buxbaum, Institut fur Biochemie und Endokrinologie, Fachbereich Veteriniirmedizin, Justus-Liebig-Universitat Giessen, Frankfurter Strasse 100, W-6300 Ciessen, Federal Republic of Germany Abbreviations. CrATP, 1,y-bidentate chromium(II1)-ATP; CO(NH~)~ATP, tetramminecobalt-ATP; C O (N H~)~P O~, tetramminecobalt phosphate; AdoPP[CH,]P, adenosine 5-[P,y-methylene]triphosphate; TNP-ATP, 2',3'-0-(2,4,6-trinitrocyclohexadienyIidene)adenosine 5'-triphosphate; Ks, half-maximal concentration of a ligand of Na+/K+-ATPase in kinetic studies. Enzyme. Na+/K+-ATPase, Na+/K+-transporting ATPase (EC 3.6.1.3 7). is believed to represent a partial reaction of the overall Na'] K f activity [I-31. The activation energy changes during the
Biophysical Chemistry of Proteins, 2010
Aliphatic amines ( e -amino group of Lys) are moderately nucleophilic when deprotonated (beyond p... more Aliphatic amines ( e -amino group of Lys) are moderately nucleophilic when deprotonated (beyond pH 9 for the free amino acid, but often much lower in proteins); on the other hand, the inactivation of the labelling reagent by water increases with pH. The α -amino group has a pKa of 7. 6–8. 5, thus the N-terminus of a protein can be labelled specifically around pH 7. Aromatic amines require very reactive probes (thiocyanate, sulphonyl chloride), but can be labelled at any pH > 4. The nitrogen of peptide bonds and Gln, Asn, Arg and His side-chains, and of guanosine and adenosine are almost unreactive. Amino-sugars in glycoproteins may also react.
Biophysical Chemistry of Proteins, 2010
The reactivity of alcohols in aqueous solutions is low, most reagents prefer to react with more n... more The reactivity of alcohols in aqueous solutions is low, most reagents prefer to react with more nucleophilic groups like amine or thiol.
Springer eBooks, 2011
This type of investigation is quite important for the characterisation of an enzyme: $$\Delta {G&... more This type of investigation is quite important for the characterisation of an enzyme: $$\Delta {G'}^{0} = -R {_\ast} T {_\ast}\ln (1/{K}_{\mathrm{ d}})\qquad \mathrm{at}\,\,25\mathrm{\!\circ \mathrm{C}}$$ (44.1) $$\ln ({K}_{\mathrm{d}}) = -\dfrac{\Delta {H'}^{0}} {R} {_\ast} \dfrac{1} {T} + A$$ (44.2) $$\Delta {S'}^{0} = \dfrac{\Delta {H'}^{0} - \Delta {G'}^{0}} {T}$$ (44.3) Such experiments can be performed on a Forbes-bar (see Fig. 44.1), that allows measurements at different temperatures in parallel. The standard Gibbs free energy of ligand binding1 Δ G ′ 0 can be determined from the dissociation constant at standard temperature (25∘C)
Biophysical Chemistry of Proteins, 2010
In the last chapter we have seen how to determine the sequence of a protein. For many purposes it... more In the last chapter we have seen how to determine the sequence of a protein. For many purposes it would be useful to synthesise proteins of a given sequence. The solid-phase approach of Merrifield (Fig. 31.1) serves this purpose.
Methods in molecular biology (Clifton, N.J.), 2012
Certain transition metal complexes show intensive fluorescence when bound to proteins. They can b... more Certain transition metal complexes show intensive fluorescence when bound to proteins. They can be used to stain gels after electrophoresis with a sensitivity approaching that of silver staining, but in a much simpler and more reproducible procedure. Stains can be prepared easily and at a fraction of the cost of commercially available reagents.Hydrophobic dyes can be used to stain gels without fixing; they do not interfere with later blotting or electro-elution.
Progress in clinical and biological research, 1988
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Papers by Engelbert Buxbaum