bluuebunny/crossref_metadata_2025_split huggingface dataset:
Activation and Partial Proteolysis of Variant Glucocorticoid Receptors, Studied by Two‐Phase Partitioning
Authors: ANDREASEN Peter A. , GEHRING Ulrich 
Published: March 2005

In cultured lines of mouse lymphoma cells, resistance to glucocorticoids is frequently associated with the occurrence of glucocorticoid receptors with an abnormally low affinity (nt−) C an abnormally high affinity (nti) for nucleiand DNA. We have investigated whether the abnormal affinities for DNA are correlated with alterations in charge and surface properties of the receptors, that would be revealed through the partition coefficeint in aqueous dextran/poly(ethylene glycol) two‐phase systems.We have found that none of teh receptor variants is defective in the activation step per se, and that only the nti receptors are abnormal in partition properties. Partial proteolysis of wild‐type and nt− receptors with α‐chymotrypsin products forms which are indistinguishable from the nti receptors with respect to partition coefficeints, Upon α‐chymotrypsin treatment the wild‐type receptors attain DNA‐binding properties identical to those of the nti receptors, while the nt− receptors, in spite of some increase in DNA affinity, still bind less firmly to DNA than the α‐chymotrypsin‐treated wild‐type receptors, α‐Chymotrypsin treatment of the various receptor types also produces and increase in the inding to dextran sulphate, but the dextran sulphate affinity is higher and varies less between different receptor types than the DNA affinity. Trypsin‐treated receptors were found to be devoid of affinity for DNA and dextran sulphate.We conclude that the increased DNA affinity of the nti receptors is due to a gross change in teh structure of the receptors, while the decreased DNA affinity of the nt− receptors is caused by more subtle alterations in the polyanion binding domain of the receptors.
The Imidazole‐Promoted Inactivation of Horse‐Liver Alcohol Dehydrogenase
Authors: DAHL Knut H. , McKINLEY‐McKEE John S. 
Published: March 2005

The effect of imidazole on the inactivation of liver alohol dehydrogenase by alkylation of Cys‐46 with iodoacetate, bromoacetate, 2‐bromopropionate, 3‐bromopropionate, 2‐bromobutyrate and iodoacetamide has been studied at pH 7.0. Imidazole promoted inactivation with all the compounds but 2‐bromobutyrate.Enzyme inativation with haloacids was faster in a ternary enzyme‐imidazole‐haloacid complex compared to a binary enzyme‐haloacid complex. Inactivation with iodoacetamide, which is a direct bimolecular reaction, was faster with the binary enzyme‐imidazole complex as compared to the free enzyme. Results of haloacid and iodoaetamide inactivation in the presence of imidazole were fitted, by nonlinear regression analysis, to the rate expressions for the proposed mechanisms and the kinetic parameters resulted. Imidazole was also found to promote inactivation of the obalt‐substituted and cadmium‐substituted liver alcohol dehydrogenases.Cys‐46 is alkylated as a metal‐thiol complex. Imidazole, when binding to the active‐site metal, donates σ‐electrons to the metal atom, which distributes the inccreased electron density further to the other ligands. The increased nucleophilicity of the sulphur of Cys‐46 results in promoted alkylation. Proof that the imidazole promotion effect is caused by a displaced electron distribution in the active‐site coordination unit is provided by imidazole also promoting the alkylation of the model thiol, zinc‐meraptoethanol.
Lack of Evidence for a Tetrahedral Intermediate in the Hydrolysis of Nitroanilide Substrates by Serine Proteinases
Authors: MARKLEY John L. , TRAVERS Franck , BALNY Claude 
Published: March 2005

We have used a stopped‐flow apparatus to reinvestigate reports, based on the observation of ‘burst’ kinetics, of an intermediateprior to the acyl‐enzyme complex in hydrolyis reactions of anilides catalyzed by trypsin and elastase [M.W. Hunkapiller, M.D. Forgac and J.H. Richards (1976) Biochemistry 15, 5581–5588; D.D. Petkov (1978) Biochim. Biophys. Acta, 523, 538–541; A.L. Fink and P. Meehan (1979) Proc. Natl Acad. Sci. USA, 76, 1566–1569; P. Compton and A.L. Fink (1980) Biochem. Biophys. Res. Commun. 93, 427–431]. We studied the hydrolysis of seferal anilide substrates by bovien and porcine elastase between ‐30°C and 20°C. In no case did we record true ‘burst’ kinetics. We show that confusing spectral changes can arise from incomplete mixing, thermal gradients, or heterogeneity of the substrate. We conclude that there is no solid spectroscopic evidence at present for the existence of a tetrahedral intermediate in the hydrolysis of amides by serine proteinases.The substrate N‐acetyl‐l‐alanyl‐l‐prolyl‐l‐alanine 4‐nitroanilide is a mixture of two isomers trans and cis about the l‐alanyl‐l‐propyl peptide bond. It appears that elastase hydrolyzes the cis isomer more rapidly than the trans isomer and this could lead to false ‘burst’ kinetics.We describe the construction of the stopped‐flow apparatus designed for cryoenzymology used for this work that has novel features and is adaptable to a variety of spetrophotometers. Solutions can be handled under anaerobic conditions. A window allows teh drive syringes to be observed or exposed to light for photochemical experiments. The apparatus operates over the temperature range −35°C to +25°C. The dead time is under 5 ms. A recording system is described that permits one to follow reations over a wide time scale covering half‐times of the order of several milliseconds to hours.
Regulation of Protein Synthesis in the Plasmodial Phase of Physarum polycephalum
Authors: TURNOCK Geoffrey , CHAMBERS Jane , BIRCH Barbara 
Published: March 2005

The rate of protein synthesis changes very little during the first 2–3 h (S phase) of the nuclear division cycle in plasmodia of Physarum polycephalum and then increases continuously during G2 phase, so that by the end of the cycle the rate has doubled relative to that in S phase. Protein synthesis appears to continue during mitosis.
Fractionation of extracts of plasmodia, labeled with [3H]lysine for 1h, by two‐dimensional electrophoresis indicated that most if not all proteins are synthesized throughout the nuclear division cycle. However, two metabolically stable polypeptides, the synthesis of which occurs predominantly in G2 phase, were detected.
Using a double‐labelling procedure, the differential rates of synthesis of 30 relatively abundant polypeptides were measured in relation to the nuclear division cycle. As a group, their differential rates of synthesis increase during the cycle so that their actual rates of synthesis increase 4–6‐fold. This implies that their synthesis is regulated over and above any simple change due to a doubling in the number of genes during S phase.
An Investigation of the Active Site of Lactate Dehydrogenase with NAD+ Analogues
Authors: SAMAMA Jean‐Pierre , MARCHAL‐ROSENHEIMER Nicole , BEIELLMANN Jean‐Francois , ROSSMANN Michael G. 
Published: March 2005

The kinetic properties of 18 NAD+ analogues, with alterations to the nicotinamide moiety, have been studied with respect to dogfish M4, rabbit M4 and beef H4 lactate dehydrogenases.The size of the groups present at C‐3 of the pyridinium can be increased quite extensively without loss of coenzyme activity. Groups tested were thioamide, methyl, ethyl, diazoketone and chloroacetyl.Substitutions at positions C‐4 and C‐5 prevent proper positioning for hydride transfer and can hinder binding to the enzyme. The kinetic properties of pyridine‐adenine dinucleotide and its 3‐iodo derivative reveal the bidning role of the amide at C‐3 whereas 3‐cyanopyridine‐adenine dinculeotide is a strong inhibitor.
The Function fo the Subunits of the Fumarate Reductase Complex of Vibrio succinogenes
Authors: UNDEN Gottfried , KRÖGER Achim 
Published: March 2005

The membrance‐bound fumarate reductase complex of Vibrio succinogenes catalyzes the reduction fo fumarate by 2,3‐dimethyl‐1,4‐naphthohydrquinone (dimethylnaphthohydroquinone) and consists of three different keptides (Mr 79000, Mr 31000 and Mr 25000), the smallest of which is cytochrome b [Unden, G., Hackenberg, H. and Kröger, A.(1980) Biochem. Biophys.Acta 591, 275–288]. The complex was cleaved with guanidinium chloride, the resulting subunits characterized and their functions within the complex investigated by reconstitutional experiments.

The Mr‐79000 subunits catalyzed the reduction fo fumarate by benzylviologen radicals as well as the oxidation of succinate by methylene blue, but not fumarate reduction by dimethylnaphthohydroquinone.
The spectral and the redox properties of the isolated cytochrome b(Mr 25000) were equivalent to those of the high‐protential cytochrome b of the bacteria. The isolated cytochrome b had a midpoint potential of ‐15 mV and was reducible by dimethylnaphthohydroquinone in the absence of the other subunits.
The Mr‐31000 subunit did not catalyze any of the ractions mentioned above. For the reduction of cytochrome b by succinate in the presence of the Mr‐79000 subunits, an amount of the Mr‐31000 subunits was required which was equimolar to cytochrome b.
The activity of fumarate reduction by dimethylnaphthohydroquinone could be restord by coprecipitation of the three subunits.It is concluded that the fumarate reductase complex has two different reactive sites, which are essential for its function in the phosphorylative electron transport of the bacterium. The site reacting with the substrates fumarate and succinate is situated on the Mr‐79000 subunit, and that reacting with dimethylnaphthohydroquinone is cytochrome b. The Mr‐31000 subunit mediates the electron transport between cytochrome b and the Mr‐31000 subunits mediates the electron transport between cytochrome b and the Mr‐79000 subunit.
cAMP Receptor Proteins and protein Kinases in Human Lymphocytes: Fundamental Alterations in Chronic Lymphocytic Leukemia Cells
Authors: WEBER Wolfgang , SCHWOCH Grehild , WIELCKENS Klaus , GARTEMANN Axel , HILZ Helmuth 
Published: March 2005

To study the significance of cAMP receptor proteins and protein kinase activities in tumor dys‐differentiation peripheral blood lymphocytes from normal donors and from patients with chronic lymphocytic leukemia were isolated by Percoll gradient centrifugation, and analyzed, Both normal and tumor lymphocyte populations were devoid of cell proliferation.Normal human lymphocytes contained about equal activities of histone kinase and ‘casein kinase’, Of the total histone kinase, cAMP‐dependent activity in normal and tumor lymphocytes comprised about 50% as shown by the use of the heat‐stable inhibitor. The tumor lymphocytes, however, exhibited drastically reduced values of all three protein kinase activities, the most pronounced decrease (to 7% of the normal control cells) being observed with the ‘casein kinase’.Total high‐affinity camp‐binding sites (= regulatory subunits R of camp‐dependent protein kinases type I and II) in leukemic lymphocytes were also strongly reduced (to <20%) Immunotitration with specific antibodies raised against rabbit muscle RI regulatory subunits in both normal and leukemic lymphocytes. Immuno‐reactive RII components comprised about 40% of total binding sites in lymphocytes o chronic lymphocytic leukemia, but only 20% in normal cells, the residual 20% being represented by RI protein of low immunoreactivity. Combination of gel elctrophoretic analysis of the camp‐binding proteins labeled with [32P]n3camp and binding to the R‐specific antibodies allowed the identification fo individual type I and type II R proteins: Besides the regulatory subunit RI of 49 kDa present in both cell types, additional isoproteins wwere found, the predominat RII form being a 52kDa subunit in normal lymphocytes and a 50‐kDa subunit in leukemic lymphocytes.The findings in lymphocytes of chronic lymphocytic leukemia cannot be explained by the fact that they are B‐type lymphocytes.Control experiments reveled that normal B cell do not differ significantly from total blood lymphocytes with respect to the parameters mentioned above.Biochemical correlates of the dys‐differentiated status of these non‐proliferating (G0) cell from patients with chronic lymphocytic leukemia, then, are represented by reduced levels of protein kinases, of basal cAMP as well as by changes of the R‐protein pattern. The data indicate that both protein kinases type I and type II may relate to the state of differentiation, independently from cell proliferation.
Stereochemical Investigation of the Phosphoketolase Reaction
Authors: MERKLER Ingeborg , RÉTEY János 
Published: March 2005

d‐(1R)‐(1‐2H1)[1‐3H] Fructose 6‐phosphate was prepared by the action of glucosephosphate isomerase on d‐(1‐2H)glucose 6‐phosphate in tritiated water. Using the same enzyme but interchanging the labeling pattern of the substrate d‐(1S)‐(1‐2H)[1‐3H] fructose 6‐phosphate was also obtained.
The doubly labeled samples of d‐fructose 6‐phophate were converted into chiral acetylphosphate on phosphoketolase from Bifidium globosum (ATCC 25864), and these were reacted in situ with phosphotransacetylase and malate synthase in the presence of coenzyme A and glyoxylate.
The steric distributin of the tritium in the samples of malate, as shown by the action of fumarase, revealed that d‐(1R)‐and d‐(1S)‐(1‐2H1)[1‐3H] fructose 6‐phosphate was converted into (r) and (S)‐(2H1)[3H] acetylphosphate, repectively.
The results are discussed in terms of the mechanism of action of phosphoketolase.
The Fatty Acid Composition of Subcellular Membranes of Rat Liver, Heart, and Brain : Diet‐Induced Modifications
Authors: TAHIN Quivo S. , BLUM Max , CARAFOLI Ernesto 
Published: March 2005

The influence of diets having different fatty acids composition on the fatty acid content of (the phospholipids) of rat liver mitochondria and microsomes, heart mitochondria, brain mitochondria and microsonies has been analyzed. It has been found that each organelle has its own peculiar composition in fatty acids. This composition may be profoundly influenced by the diet, but to different degrees in different organelles. Those of brain are most resistant.The changes observed are rather rapid, being generally already maximal after three weeks of treatment. The parallel between fatty acid composition of diets, and the changes observed in the organelles, is not strict, and is probably influenced by the metabolic competition among oleic acid, linoleic acid, linolenic acid. Unusual fatty acids like crucic acid. trans‐oleic acid. and trans‐linoleic acid can also become incorporated into the membranes of cell organelles.
Larger Precursors of Mitochondria1 Translation Products in Neurospora crassa
Authors: Van't SANT Peter , MAK J. Frdncisca C , KROON Albert M. 
Published: March 2005

Specific labeling in vivo of the formylated N termini of mitochondrial translation products revealed that some mitochondrially synthesized proteins were not labeled this way. As a consequence, it was worthwhile considering that larger precursor proteins of mitochondrial translation products exist. Although we used a rapid isolation procedure, only after 2‐h of labeling in the presence of cycloheximide, could three additional mitochondrial translation products (molecular mass 45, 36 and 25 kilodaltons) be detected. Preincubation with cycloheximide indicated that these proteins might be larger precursors which were no longer processed due to the prolonged presence of cycloheximide.To prevent processing of the precursors during isolation, cells of the slime mutant were directly lysed in boiling sodium dodecyl sulphate solution. In this way, the same three additional mitochondrial translation products were detected after a pulse‐labeling of 1 min. These proteins behave in a precursor‐like fashion. Labeling at 9 °C resulted in a partial accumulation of the three additional proteins.Finally protein blots treated with antibodies and 251‐labeled protein A, support the idea that the 45‐kDa protein is a precursor of subunit 1 of cytochrome c oxidase; 50–80% of this precursor could be detected in the post‐mitochondria1 supernatant, indicating that this polypeptide is not tightly bound to the membrane.
Mössbauer Studies on the Active Fe …[2Fe‐2S] Site of Putidamonooxin, Its Electron Transport and Dioxygen Activation Mechanism
Authors: BILL Eckhard , BERNHARDT Frithjof‐Hans , TRAUTWEIN Alfred Xaver 
Published: March 2005

Putidamonooxin, the oxygenase of a 4‐methoxybenzoate monooxygenase enzyme system, catalyzes the oxidative O‐demethylation of the substrate 4‐methoxybenzoate in conjunction with the NADH: putidamonooxin oxidoreductase. Putidamonooxin is a conjugated iron‐sulfur protein which needs iron ions as cofactors for its enzymatic activity.Putiamonooxin was isolated from Pseudornonus putida, which was grown on a 57Fe‐enriched culture medium. Thus putidamonooxin was enriched in vivo with 57Fe up to about 80%.During our Mössbauer study of putidamonooxin a number of parameters have been varied: (a) the oxidation state of putidamonooxin (oxidized, reduced and aerobically reoxidized); (b) the substrate bound to putidamonooxin (Cmethoxybenzoate, benzoate, 4‐tert‐butylbenzoate); (c) the temperature between 2.7 K and 245 K; (d) the applied magnetic field between 0 and 0.1 T and (e) the amount of iron cofactor.From our M&ouml;ssbauer results it is obvious that the iron‐sulfur centers of putidamonooxin are [2 Fe‐2 S] clusters similar to those of the plant‐type ferredoxins. Further, we have evidence for the existence of iron ions (one per [2 Fe‐2S] cluster), which serve as cofactors for the dioxygen activation, functioning as the dioxygen binding site and mediating the electron flow from the [2 Fe‐2 S] cluster to dioxygen.
Analysis of Proteinase A Function in Yeast
Authors: MECHLER Bernd , WOLF Dieter H. 
Published: March 2005

Yeast mutants lacking proteinase A were isolated. One of these mutants (Hb I) is characterized in detail. The mutation called pral segregates 2:2 in meiotic tetrads indicating a single gene mutation. No anti‐(proteinase A) cross‐reacting material can be detected. Diploids heterozygous for pral show gene dosage. Thus, it appears that PRAI might be the structural gene for proteinase A.Results obtained with this mutant show that proteinase A is not a vital component of the vegetative cell cycle. The mutant exhibits normal mitotic growth under rich and poor growth conditions and shows normal mating. Enzymes subject to carbon catabolite inactivation and inactivation of NADP‐dependent glutamate dehydrogenase, processes which were proposed to be of proteolytic nature, are not affected by the absence of proteinase A. However, protein degradation under sporulation conditions is about 30;(reduced in proteinasc A mutant cells. The differentiation process of spordation is also disturbed leading to a 40 reduced sporulation frequency in mutant cells.
Modulation of Phosphofructokinase Behavior by Chemical Modifications during the Immobilization Process
Authors: CAMBOU Bernard , LAURENT Michel , HERVAGAULT Jean‐François , THOMAS Daniel 
Published: March 2005

Phosphofructokinase was immobilized within a protein membrane or on soluble protein polymers using glutaraldehyde as cross‐linking reagent. The native enzyme was also modified chemically, using the cross‐linking reagent alone. A comparative kinetic investigation of these preparations was carried out.The catalytic activity of the chemically modified enzyme and its affinity towards fructose 6‐phosphate decreased significantly; the modified enzyme lost its cooperative properties and the allosteric regulation by AMP was affected. When the chemical treatment was performed in the presence of effectors (AMP or ATP) the allo‐ steric transition induced by AMP was restored, suggesting that the cross‐linking reagent modified the AMP regulatory sites, albeit no higher‐substrate‐affinity enzyme conformation was frozen. Molecular data showed that glutaraldehyde produced intramolecular then intermolecular bonds as its concentration increased.When the enzyme was immobilized into protein membranes or on soluble polymers, the enzyme behavior was quite similar: decrease of affinity towards fructose 6‐phosphate but no changes in cooperative properties and modifications of allosteric transition induced by AMP. When AMP was present during the immobilisation process, the enzyme immobilized in this way was no longer sensitive to effectors, either AMP or ATP. It showed Michaelian behavior and higher substrate affinity quite similar to that of the native enzyme. The data suggested that a higher‐ substrate‐affinity enzymatic form was most probably stabilized by immobilization.
Equilibrium and Kinetic Measurements of the Binding of Pyridoxal 5′‐Phosphate to Hybrid Tryptophan Synthase from Escherichia coli
Authors: BALK Hubert , FRANK Albert , BARTHOLMES Peter , JAENICKE Rainer 
Published: March 2005

Hybrid tryptophan synthase was prepared that contains a β, subunit with one functional active site while the internal aldimine between cofactor and enzyme of the other protomer was reduced with sodium borohydride (α,ββ*). The modified enzyme shows a specific activity for the l‐serine+indole to l‐tryptophan reaction of about 50% compared to the native enzyme [Hathaway, G. M., Kida, S. and Crawford, I. P., Biochemistry 8, 989—997 (1969)]. The binding of pyridoxal 5′‐phosphate (pyridoxal‐P) to the α2apo ββ* complex and to the apo ββ* subunit was studied by equilibrium methods and rapid mixing experiments.The equilibrium binding curves for the hybrid species are hyperbolic (i. e. non‐cooperative), yielding apparent microscopic dissociation constants of 1.6±O.2 × 10−6 M for the α2 holoββ* subunit.The rate of formation of the internal aldimine in the unmodified active site was followed by fast reduction with sodium borohydride, L‐tryptophan synthesis was used to monitor the rate of formation of active enzyme.Cofactor binding to the α2apo ββ* complex is characterized by three sequential steps of decreasing rate: formation of a non‐covalent initial complex, which reacts to an enzymatically inactive internal aldimine, followed by a conformational change leading to the active α2holo ββ* bienzyme complex. A similar series of consecutive steps with decreasing reaction rates is observed for the binding of pyridoxal‐P to the apoββ* subunit. The formation of active enzyme, however, is biphasic. The faster process, which parallels the formation of the internal aldimine and which accounts for 85% of the total amplitude, obviously involves an already active high‐affinity state of the hybrid molecule whereas the second phase parallels the activation process as observed for cofactor binding to the native dimer.These findings are discussed with respect to the recently proposed mechanism for pyridoxal‐P binding to the unmodified apoβ2 subunit [Bartholmes, P. Balk, H. and Kirschner, K, Biochemistry 19, 4527–4533 (1980]