bluuebunny/crossref_metadata_2025 huggingface dataset:
Hydrogen Exchange at the β‐Carbon of Amino Acids during Transamination
Authors: WALTER Ulrich , LUTHE Hilmar , SÖLING Hans‐Dieter , GERHART Fritz
Published: March 2005

The hydrogen exchange at the β‐carbon of l‐alanine, l‐glutamate and l‐aspartate with water has been examined during transamination catalyzed by glutamic‐oxaloacetic transaminase and by glutamic‐pyruvic transaminase. A significant hydrogen exchange at the β‐carbon has been demonstrated during incubation of l‐[3‐3H]alanine + glutamic‐pyruvic transaminase, l‐[3‐3H]alanine +α‐oxo‐glutarate + glutamic‐pyruvic transaminase, l‐[3‐3H]glutamate + glutamic‐oxaloacetic transaminase, l‐[3‐3H]glutamate + oxaloacetate + glutamic‐oxaloacetic transaminase, and l‐[3‐3H]glutamate + pyruvate + glutamic‐pyruvic transaminase as shown by the appearance of 3H2O.

No hydrogen exchange at the β‐carbon of l‐glutamate occurred during incubation of l‐[3‐3H]‐glutamate with glutamic‐pyruvic transaminase alone. The hydrogen exchange at the β‐carbon of l‐glutamate coincides with transamination as demonstrated by nuclear magnetic resonance studies of 2H2O‐l‐glutamate exchange during transamination by glutamic‐oxaloacetic transaminase and glutamic‐pyruvic transaminase.

No hydrogen exchange at the β‐carbon occurred during transamination of l‐aspartate by glutamic‐oxaloacetic transaminase as shown by nuclear magnetic resonance spectroscopy and confirmed by

The results are discussed with special reference to the different equilibria between the pyridoxal form and the pyridoxamine form of glutamic‐oxaloacetic transaminase and of glutamic‐pyruvic transaminase.
Pt(CN)2‐4 and Au(CN)−2 : Potential General Probes for Anion‐Binding Sites of Proteins
Authors: NORNE Jan‐Erik , LILJA Hans , LINDMAN Björn , EINARSSON Roland , ZEPPEZAUER Michael
Published: March 2005

Nuclear magnetic quadrupole relaxation appears to be a general method for studying the binding of anions to proteins. This is shown by the increase in transverse quadrupole relaxation rate of 35Cl− and 81Br− in the presence of horse liver alcohal dehydrogenase, lysozyme, trypsin, α‐chymotrypsin, human carbonic anhydrase, fructose‐1,6‐bisphosphate aldolase and human serum albumin. Of the many possible binding sites at the surface of a protein (e.g. positively charged amino acid side‐chains) only a few account for the main part of the relaxation enhancement. This is shown by the decrease in 35Cl− and 81Br− relaxation rate on addition of functional ligands.

Large, kinetically inert, complex anions like Pt(CN)2‐4 and Au(CN)−2 are found to act as strong competitors towards halogen ions for the high‐affinity anion binding sites of a number of proteins. Titrations with complex anions following the 35Cl− or 81Br− relaxation rates are found to be helpful in attempts to elucidate binding mechanisms. Especially, the complex anions may be useful probes for the discrimination between general and metallic anion binding sites in proteins and they also permit correlation of information from X‐ray investigations of crystals with that from physical measurements in solution.

From the change in halide ion quadrupole relaxation rate on addition of strongly binding ligands the quadrupole coupling constants of the high affinity Cl− and Br− binding sites are estimated using certain assumptions. It is found that for several proteins, comprising the metal‐free proteins but also alcohol dehydrogenase and Escherichia coli alkaline phosphatase, the 35Cl quadrupole coupling constants have approximately the same values. For some other metallo‐proteins like carbonic anhydrase and a zinc · serum‐albumin complex considerably greater quadrupole coupling constants were obtained. The estimated quadrupole coupling constants are used as a basis for a discussion of the interactions involved in anion‐protein interactions.
On the Mechanism of Ketogenesis and Its Control
Authors: HUTH Walter , JONAS Rainer , Ilona WUNDERLICH  , SEUBERT Werner
Published: March 2005

Two mitochondrial forms of acetoacetyl‐CoA thiolases designated as enzyme A and enzyme B were crystallized from ox liver. They could be shown to be homogenous by polyacrylamide gel electrophoresis.

In direction of acetoacetyl‐CoA cleavage enzyme A shows a double competitive substrate inhibition when acetoacetyl‐CoA is varied at different fixed CoA concentrations. With enzyme B a parallel kinetic pattern is obtained when acetoacetyl‐CoA is varied at different fixed CoA concentrations. In direction of acetoacetyl‐CoA synthesis both enzymes show linear reciprocal plots of initial velocities against acetyl‐CoA concentrations in absence of CoA.

These initial velocity kinetics in the forward and in the reverse direction are in accordance with a ping‐pong mechanism of reaction for both enzymes involving an acetyl‐S‐enzyme as intermediate.

Under saturating concentrations of substrate, the ratios of acetoacetyl‐CoA synthesis/acetoacetyl‐CoA cleavage is 0.31 for enzyme A and 0.08 for enzyme B. The maximum velocity in direction of acetoacetyl‐CoA synthesis of enzymes A and B are 0.43 μmol × min−1× unit thiolase−1and 0.10 μmol × min−1× unit thiolase−1, respectively.

Both enzymes show nearly the same affinity for acetyl‐CoA. TheKmvalues are 91 μM (enzyme A) and 80 μM (enzyme B).

Coenzyme A and acetoacetyl‐CoA both act as inhibitors in direction of acetoacetyl‐CoA synthesis: coenzyme A is a nonlinear competitive inhibitor of both enzymes. Acetoacetyl‐CoA exerts a negative cooperativity on enzyme A (nH= 0.63) and is a competitive inhibitor for enzyme B (Ki= 1.6μM).

The catalytic and regulatory properties of the acetoacetyl‐CoA thiolases A and B are discussed in terms of their proposed role in regulating ketogenesis. Intracellular fluctuations of acetoacetyl‐CoA/3‐hydroxybutyryl‐CoA ratios, resulting in a suspension of inhibition of both enzymes at high NADH/NAD ratios, are postulated as a control mechanism of ketogenesis in addition to mechanisms already known.
Kinetic Studies Dealing with an Immobilized Bienzyme System
Authors: HERVAGAULT Jean‐François , JOLY Ghislaine , THOMAS Daniel
Published: March 2005

The binding of enzymes into artificial membranes makes possible a study of the interaction between membrane structure and enzyme kinetics within a simple context. Artificial protein membranes bearing a bienzyme system (xanthine oxidase, uricase) are produced by using a co‐crosslinking method. The inhibition of uricase was shown to be dependent not only on the concentration of inhibitor in the bulk solution, but also on the kinetic properties of the membrane‐bound enzymes. In the presence of xanthine oxidase inside the structure the uricase inhibition by xanthine is less important than in solution. Under defined conditions the activity was found to be higher in the presence of inhibitor than in its absence. Due to diffusion limitations this specific bienzyme system is more efficient when immobilized inside a membrane than when in solution.
Electron‐Spin Resonance of Nitrosyl Haemoglobins: Normal α and β Chains and Mutants Hb M Iwate and Hb Zürich
Authors: TRITTELVITZ Eberhard , GERSONDE Klaus , WINTERHALTER Kaspar H.
Published: March 2005

At 77 K the electron spin resonance (ESR) spectra of the NO derivatives of the mutant haemoglobins Hb M Iwate and Hb Zürich as well as of the isolated chains of normal haemoglobin were studied. Two types of ESR spectra differing in the g‐value and the hyperfine splitting at gzz were observed. The type II spectrum is characterized by a hyperfine structure at gzz= 2.005 with a splitting constant of ΔH= 23 G (14NO) or 32 G (15NO), respectively. In the type I spectrum the splitting constant of the hyperfine structure at gzz= 2.009 amounts to ΔH= 18 G (14NO) or 23 G (15NO), respectively. In some cases this hyperfine structure is coincident with another one at gxx= 2.064 with nearly identical splitting constant. In addition, the type I spectrum is characterized by an increased ESR absorption at gxx= 2.064.

At neutral pH the NO derivatives of the isolated chains as well as of the mutant haemoglobins give rise to a type II spectrum. In correspondence with previous results gained with normal NO haemoglobin, the ESR spectra of the NO‐α chains and NO‐Hb Zürich show a transition to type I in the acid region. This transition is favoured by binding of 2,3‐bisphosphoglycerate. On the other hand, the ESR spectra of the NO‐β chains and NO‐Hb M Iwate are of the type II also at acid pH. The NO‐β chains show a transition of the ESR spectrum from type II to type I only at alkaline pH. These results indicate that in the tetrameric NO haemoglobin only the a chains are responsible for the transition of the ESR spectrum from type II to type I in the acid region.

The two types of ESR spectra are interpreted in terms of two kinds of haem‐NO complexes differing in the iron‐NO and iron‐imidazole distances. The type II spectrum is attributed to a complex with a relatively short iron‐imidazole distance which is responsible for a weakened σ‐bond in trans position. The type I spectrum arises then from a complex with a larger iron‐imidazole bond leading to an approach of the NO molecule to the iron. The influence of the protein conformation upon the iron‐imidazole bond length is discussed with regard to the ESR spectra of the mutant NO haemoglobins and considering the influence of agents modifying the protein structure.
The Amino‐Acid Sequences of Three Cystine‐Free Cyanogen‐Bromide Fragments of Human Serum Transferrin
Authors: SUTTON Michael R. , MacGILLIVRAY Ross T. A. , BREW Keith
Published: March 2005

The amino acid sequences of three fragments obtained on cyanogen bromide cleavage of human transferrin have been determined. Two of the fragments are small (4 and 7 residues) and had not been isolated in previous studies of the CNBr fragments of transferrin. The sequence of the larger fragment (53 residues) was elucidated by examining peptides isolated from digests of the fragment with trypsin, chymotrypsin or thermolysin. This region of transferrin appears to contain the sites of three previously‐reported substitutions in the D1 and Dchi genetic variants.
Debranching Enzyme from Rabbit Skeletal Muscle
Authors: TAYLOR Carol , COX Anthony J. , KERNOHAN John C. , COHEN Philip
Published: March 2005

Debranching enzyme was purified 150‐fold from rabbit skeletal muscle by a three‐step procedure which utilised ammonium sulphate precipitation, ion‐exchange chromatography on DEAE‐cellulose and “hydrophobic” chromatography on Sepharose‐NH(CH2)4 NH2. The preparation was completed within three days, and 200 mg enzyme was isolated from 1000 g muscle, which represented an overall yield of 60 %. The preparation was homogeneous by the criteria of poly‐acrylamide gel electrophoresis and ultracentrifugal analysis. The sedimentation coefficient, s20,w, was 8.1 S. The amino acid composition was determined, and the absorption coefficient, A1%280, measured refractometrically was 17.5.

The subunit molecular weight determined by gel electrophoresis in the presence of sodium dodecyl sulphate was 166000 and this value was supported by sedimentation equilibrium in the presence of 6 M guanidinium chloride (155000). The molecular weight of the native enzyme measured by high‐speed sedimentation equilibrium was 164000, showing that the debranching enzyme is a monomeric protein at the concentrations which exist in muscle (0.7 mg/ml). The results indicate that the two different enzyme activities which are associated with debranching enzyme, 1,4‐glucan‐4‐glycosyltransferase and amylo‐1,6‐glucosidase, reside on the same polypeptide chain.

Protein‐glycogen particles isolated from skeletal muscle showed seven major protein‐staining components by polyacrylamide gel electrophoresis, one of which was identified as debranching enzyme. Four of the other components were the α and β subunits of phosphorylase kinase, glycogen phosphorylase and glycogen synthetase.

A new titrimetric assay for debranching enzyme was developed; it was used to demonstrate that the maximum potential activity of debranching enzyme is only 5–10% that of phosphorylase at the concentrations of the two enzymes in skeletal muscle. Since the activity of debranching enzyme is unaffected by every mechanism which leads to the activation of glycogen phosphorylase and phosphorylase kinase, the evidence suggests that the hormonal control of muscle glycogenolysis by adrenalin might be confined to a stimulation of rate of degradation of the outermost branches of glycogen.
Microsomal 12α‐Hydroxylation of 7α‐[12α,12β‐2H2]Hydroxy‐4‐cholesten‐3‐one
Authors: BJÖRKHEM Ingemar
Published: March 2005

The synthesis of 7α‐[12α,12β‐2H2]hydroxy‐4‐cholesten‐3‐one is described. It was shown with different techniques that this compound was 12α‐hydroxylated by the microsomal fraction of a rat liver homogenate without marked isotope effect, indicating that cleavage of the C–H bond is not the rate‐limiting step in this hydroxylation. The rate of 12α‐hydroxylation was decreased by about 20% when performed in a medium containing deuterated water. The findings were discussed with reference to the specific properties of the 12α‐hydroxylating system and to the results of previous studies on rate‐limiting step in microsomal hydroxylation of steroids.
18‐Hydroxylation of Deoxycorticosterone by Reconstituted Systems from Rat and Bovine Adrenals
Authors: BJÖRKHEM Ingemar , KARLMAR Karl‐Eric
Published: March 2005

18‐Hydroxylation of deoxycorticosterone was studied with rat or bovine adrenal mitochondria or with reconstituted systems obtained from these fractions. The reconstituted systems consisted of a partially purified preparation of cytochrome P‐450 from rat adrenals and a partially purified NADPH‐cytochrome P‐450 reductase preparation from bovine adrenals. In some experiments, a soluble cytochrome P‐450 fraction from bovine adrenals was used. Adrenodoxine and adrenodoxine reductase were shown to be the active components of the NADPH‐cytochrome P‐450 reductase preparation.

Optimal assay conditions were determined for 18‐hydroxylation by the crude mitochondrial fraction as well as by the reconstituted systems. In the presence of excess NADPH‐cytochrome P‐450 reductase fraction, the rate of 18‐hydroxylation was linear with time and with the amount of cytochrome P‐450. In incubations with intact rat adrenal mitochondria to which Ca2+ and an excess NADPH had been added, NADPH‐cytochrome P‐450 reductase increased the rate of 18‐hydroxylation about 100%, indicating that NADPH‐cytochrome P‐450 reductase was to some extent rate‐limiting. The rate of 18–hydroxylation of deoxycorticosterone by the reconstituted system as well as by intact mitochondrial fraction was much higher than the rate of 18‐hydroxylation of corticosterone and progesterone. When the cytochrome P‐450 preparation from rat adrenals in the reconstituted system was substituted for cytochrome P‐450 from bovine adrenals, the rate of 18‐hydroxylation decreased considerably.

Under all experimental conditions, the 18‐hydroxylation of deoxycorticosterone occurred with a concomitant and efficient 11β‐hydroxylation. Provided the source of cytochrome P‐450 was the same, the ratio between 11β‐and 18‐hydroxylation was constant under all conditions and was not significantly different in the presence of metopirone, carbon monoxide, cytochrome c or different steroids. It is suggested that identical or at least very similar types of cytochrome P‐450 are involved in 11β‐and 18‐hydroxylation of deoxycorticosterone.
Comparative Studies on the Properties of Tryptophanase and Tyrosine Phenol‐lyase Immobilized Directly on Sepharose or by Use of Sepharose‐Bound Pyridoxal 5′‐Phosphate
Authors: FUKUI Saburo , IKEDA Sei‐ichiro , FUJIMURA Motoki , YAMADA Hideaki , KUMAGAI Hidehiko
Published: March 2005

Tryptophanase from Escherichia coli B/1t 7‐A and tyrosine phenol‐lyase (β‐tyrosinase) From Escherichia intermedia were immobilized on Sepharose 4B by several direct coupling reactions or through pyridoxal 5′‐phosphate previously bound to Sepharose. The most active preparation of immobilized tryptophanase was obtained by coupling tetrameric apoenzyme to pyridoxal‐P bound on Sepharose at the 6‐position through a diazo linkage. This immobilization procedure involves the formation of Schiff base linkage between 4‐formyl group of Sepharose‐bound pyridoxal‐P and the ɛ‐amino group of the lysine residue at the active center of one subunit of tetrameric apo‐tryptophanase, followed by the fixation of the Schiff base linkage by reduction with NaBH4. In the case of β‐tyrosinase having two catalytic centers, however, this method was not so suitable as the case of tryptophanase. Direct coupling of the apoenzyme to CNBr‐activated Sepharose or to a bromoacetyl derivative of Sepharose gave better results. In each case, the affinity for substrate or coenzyme was scarcely influenced by the immobilization. When used repeatedly in a batch system or continuously in a flow system in the absence of added pyridoxal‐P, immobilized holo‐tryptophanase or holo‐β‐tyrosinase gradually lost its original activity; however, supplement of pyridoxal‐P to the reaction system restored its initial activity. From the kinetic analyses of these phenomena, the rate constants of coenzyme dissociation from immobilized tryptophanase and β‐tyrosinase were calculated. Upon immobilization, the pH optima of both enzymes shifted 0.5 to 1.0 pH unit to the alkaline side. Both immobilized enzymes showed higher thermal stability and resistance to a denaturing agent such as guanidine‐HCl than their free counterpart. Furthermore, the reactivity of sulfhydryl group of β‐tyrosinase, in connection with its coenzynie‐binding property, was conveniently studied by use of the immobilized enzyme.
Extracellular Enzyme System Utilized by the Fungus Sporotrichum pulverulentum (Chrysosporium lignorum) for the Breakdown of Cellulose
Authors: ERIKSSON Karl‐Erik , PETTERSSON Bert
Published: March 2005

Five endo‐1,4‐β‐glucanases (EC 3.2.1.4) have been separated from culture solutions of the rot fungus Sporotrichum pulverulentum (formerly called Chrysosporium lignorum)grown on powder cellulose as the sole carbon source. They have been extensively purified and characterized with regard to some physicochemical properties. The purifications have been carried out on a quantitative basis, the purity of the enzymes being tested in several ways. After purification they all showed one single protein band in analytical polyacrylamide electrophoresis, on dodecylsulphate gels and in analytical isoelectric focusing on flat‐bed polyacrylamide gels. One exo‐1,4‐β‐glucanase has also been identified in the culture solution and separated from the endo‐1,4‐β‐glucanases.

From the data obtained during the quantitative purification it has been possible to calculate that the ratio of activity between the five endoglucanases T1, T2a, T2b, T3a and T3b in the culture solutions is 4:1:1:1:1. It has also been calculated that the weight ratio endoglucanase protein to exoglucanase protein is approximately 1:1.

Flat‐bed isoelectric focusing has been used for the identification of the individual endoglucanases and a new zymogram technique, useful for studies of carbohydrases in general, has been developed.

The molecular weights, determined by ultracentrifugation, and calculated on the basis of a knowledge of the amino acid composition and carbohydrate content vary between 28300 and 37 500. Small but significant differences in the amino acid compositions of the different endoglucanases have been found.

The carbohydrate content varies between 0 and 10.5%, all but one of the enzymes being glycoproteins. For two of these the exact carbohydrate composition has been determined. Enzyme T1contains 2 glucose and 19 mannose units per enzyme molecule while enzyme T2b contains 5 mannose, 7 galactose, 1 glucose and 1 arabinose unit per molecule.
Extracellular Enzyme System Utilized by the FungusSporotrichum pulverulentum (Chrysosporium lignorum)for the Breakdown of Cellulose
Authors: ALMIN Karl Erik , ERIKSSON Karl‐Erik , PETTERSSON Bert
Published: March 2005

A complementary discussion is given of an earlier presented theory for viscometric determination of enzymic activity towards polymer substrates.

Five different, purified endo‐1,4‐β‐glucanases from the fungusSporotrichum pulverulentumhave been characterised with respect to their molecular activities (Am) and Michaelis‐Menten constants (Km).

The errors in theAmandKmvalues are discussed. TheAmvalue seems to be more valuable as a distinguishing parameter than theKmvalue.
Purine Nucleoside Phosphorylase from Escherichia coli and Salmonella typhimurium
Authors: JENSEN Kaj Frank , NYGAARD Per
Published: March 2005

The purine nucleoside phosphorylases from Escherichia coli and from Salmonella typhimurium have been purified to electrophoretic homogeneity and crystallized.

Comparative studies revealed that the two enzymes are very much alike. They obey simple Michaelis‐Menten kinetics for their substrates with the exception of phosphate for which they show negative cooperativity. Gel filtration on Sephadex G‐200 of the native enzymes revealed a molecular weight for both enzymes of 138000 ± 10%. By use of dodecylsulphate gel electrophoresis a subunit molecular weight of 23700 ± 5% was determined, suggesting that both enzymes consist of six subunits of equal molecular weight.

When the subunits were partially crosslinked with dimethyl suberimidate before dodecylsulphate electrophoresis six protein bands were observed in agreement with the proposed oligomeric state of the enzyme, consisting of six subunits of equal molecular weight. Analysis of the amino acid composition also indicates that the subunits are identical.

6 M guanidinium chloride dissociates the enzymes; association experiments with native and succinylated enzymes suggested that only the hexameric form is active.

Both enzymes could be dissociated into subunits by p‐chloromercuribenzoate; this dissociation is prevented by the substrates: the nucleosides, the pentose 1‐phosphates, and mixtures of phosphate and purine bases.
Étude de l'activité de transfert de glucose, à partir d'UDP‐glucose, dans les membranes microsomiques des hépatocytes de rat
Authors: BERTHILLIER Gisèle , AZZAR Gérard J.‐C. , GOT René
Published: March 2005

Transfer of Glucose from UDP‐Glucose in Microsomal Membranes of Rat Hepatocytes

Microsomal preparations from rat liver mediate transfer of glucosyl units from UDP‐glucose to three different kinds of acceptors: an endogenous glycoprotein, exogenous glycogen and collagen. Both glucosyl transferases work at acidic pH, 6.5 for transfer on endogenous acceptor and glycogen and at pH 5.5 for transfer on collagen. None of these enzymes require divalent cations for activity. While transfers on endogenous acceptor and glycogen are inhibited by the presence of a non‐ionic detergent, Triton X‐100, the transfer on collagen is activated by the same detergent. Glycogen‐synthase activity requires glucose 6‐phosphate at an optimal concentration of 1 mM. The Km values for UDP‐glucose are respectively: 0.5 mM, 0.33 mM, and 1 mM for transfer on endogenous acceptor, glycogen and collagen.

Characterisation of the product indicates that a protein‐bound α1–4 glucan is formed when no primer is added. Enzymatic and acidic hydrolyses of radioactive glycogen and collagen show only glucose as a radioactive sugar identified by thin layer chromatography on cellulose.

Pre‐treatment of microsomal membranes by α‐amylase demonstrates that glucosyltransferases are not adsorbed on endogenous glycogen and seem to be really membranous enzymes.
The Nucleotide Sequence of a Methionine tRNA which Functions in Protein Elongation in Mouse Myeloma Cells
Authors: PIPER Peter W.
Published: March 2005

The major form of methionine tRNA operational in the elongation of protein synthesis in mouse myeloma cells was purified from these cells after they had been cultured in the presence of [32P]‐phosphate. This [32P]tRNA4Met species was then digested with T1 RNase or pancreatic RNase so as to obtain both complete and partial RNase digestion products. The nucleotide sequences of these fragments were analysed to enable the derivation of the complete primary structure of this tRNA. tRNA4Met of mouse myeloma cells is 76 nucleotides in length and contains 15 modified nucleotides. It is the only tRNA yet sequenced which has been found to possess the minor nucleoside 2‐methylguanosine (m2G) within the amino acid (a) stem, and also to have an anticodon (c) stem of only 4 and not 5 base‐pairs. The loop IV sequence of eukaryotic initiator methionine tRNA (tRNAfMet) species, ‐A‐U‐C‐G‐m1 A‐A‐A‐, is not found in tRNA4Met and is therefore absent from at least one of the methionine tRNAs functioning in polypeptide elongation in mammalian cells. This is consistent with the suggested importance of this loop structure in the initiator function of tRNAfMet in eukaryotic organisms. Three distinct regions of the tRNA cloverleaf, the (b) stem, the anticodon loop (loop II), and loop III, are substantially conserved in structure between tRNAfMet and tRNA4Met of mouse myeloma cells. These regions of the structures of mammalian methionine tRNAs probably do not determine whether a certain tRNA4Met will function in the initiation or elongation of protein synthesis, although they might be important in tRNAMet recognition if the different cytoplasmic tRNAMet species of mammalian cells are aminoacylated by a single activating enzyme.
Paramagnetic and Fluorescent Probes Attached to “Buried” Sulfhydryl Groups in Human Carbonic Anhydrases. Application to Inhibitor Binding, Denaturation and Refolding
Authors: CARLSSON Uno , AASA Roland , JONSSON Bengt‐Harald , LINDSKOG Sven , HENDERSON Louis E.
Published: March 2005

The single–SH groups in the human carbonic anhydrases B and C have been modified under denaturing conditions. The modified enzymes recover catalytic activity after dilution of the denaturing medium with buffer. By this method a spin label and a fluorescent probe were specifically introduced into the molecules.

The modified and reactivated enzymes have similar kinetic properties, inhibitor‐binding constants, circular dichroism spectra, and stabilities towards guanidine hydrochloride as the native enzymes. However, the esterase activity of the modified C enzyme is reduced to about 50%.

The spectra associated with the probes are insensitive to inhibitor binding in case of the B enzyme, whereas changes of electron paramagnetic resonance spectrum and fluorescence intensity respectively, were observed for the probe‐containing C enzymes. The cysteines are located in different parts of the tertiary structures of the homologous B and C enzymes, and these observations suggest that small conformational changes accompanying inhibitor binding are localized to regions of the molecules near the active‐site cavity.

During denaturation of the spin‐labeled B enzyme in 1.7 M guanidine hydrochloride a transient mobilization of the probe occurs, but the mobility is ultimately reduced to a low level. This observation supports previous evidence that denaturation under these conditions, in or near the transition region, mainly yields incorrectly folded molecules rather than stable intermediates between native and fully denatured molecules.

During refolding of fully denatured, spin‐labeled B and C enzymes the mobility of the probe is drastically reduced within less than 0.1 s after dilution. This would reflect a very short lifetime of the randomly coiled state under these conditions.
Preparation and Some Properties of Strongly Acidic Proteins from Calf‐Thymus Nucleohistone
Authors: HACHA Renee , FREDERICQ Eugène
Published: March 2005

The isolation of acidic proteins from calf‐thymus nucleohistone (starting from purified nuclei) is reported. The method involves dissociation in 1 M KCl solution. Denaturating agents were not used at all. After electrophoresis on polyacrylamide gel, fractions containing a small number of components were obtained.

The fractions display high ratios of acidic to basic amino acids, the ratios ranging from 4.0 to 1.5. In all fractions, the major components were of molecular weights in the ranges 12000–15000 and 24000–28000 as determined by gel‐disc electrophoresis in dodecylsulphate and by equilibrium ultracentrifugation. Minor components of high molecular weights were also present.

Amino‐acid analyses are also reported. The tryptophan content was determined by a fluorometric method. Circular dichroism spectra depict a very low content of α‐helicity that did not increase at higher ionic strength. A marked RNA‐polymerase activity was found in one fraction.
Biospecific Affinity Chromatography in Aqueous‐Organic Cosolvent Mixtures
Authors: LOWE Christopher R. , MOSBACH Klaus
Published: March 2005

The effect of the weak polarity‐reducing agent, ethylene glycol, on the binding of pig heart lactate dehydrogenase to N6‐(6‐aminohexyl)‐AMP‐Sepharose has been investigated. In the absence of the reagent and under the conditions used, a non‐specific interaction of the enzyme with the adsorbent led to recoveries of enzyme activity as low as 60% when the enzyme was eluted from the columns with a linear NADH gradient. The inclusion of low concentrations of ethylene glycol in column irrigants considerably improved the recovery of enzyme activity with quantitative recoveries being obtained in the presence of 20–30%.

Concentrations of ethylene glycol above 35% altered the native conformation of lactate dehydrogenase and led to a decreased affinity for the immobilised ligand. Under these conditions, the altered protein fluorescence and decreased ability to bind NADH could be correlated with the chromatographic behaviour of the enzyme on columns of N6‐(6‐aminohexyl)‐AMP‐Sepharose. These effects were exploited to elute the enzyme from a column of immobilised‐AMP with a linear gradient of ethylene glycol.
A Precursor of Glycogen Biosynthesis: α‐1,4‐Glucan‐Protein
Authors: KRISMAN Clara R. , BARENGO Renée
Published: March 2005

The mechanism of glycogen biosynthesis in the absence of added primers takes place at least in two different steps. The first step could be initiated by a new enzyme named glycogen intiator synthase that catalyzes the transfer of glucose from UDP‐glucose to an acceptor protein. This step takes place in vitro only in the presence of some salts at high concentration. The complex product from this reaction has been isolated and demonstrated to act as a precursor for the synthesis of glycogen which takes place in the second step and is catalyzed by the already known glycogen synthase.
Diaminobenzidine an Electron Donor to Photosystem 1 and to Photosystem 2 in Chloroplasts
Authors: BEN‐HAYYIM Gozal , DRECHSLER Ziwia , GOFFER Judith , NEUMANN Joseph
Published: March 2005

3,3′‐Diaminobenzidine was shown to serve as an electron donor to photosystem 1 [in the presence of 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea. In Tris‐treated chloroplasts diaminobenzidine serves as an electron donor to photosystem 1 and to photosystem 2; the latter is sensitive to 3‐(3,4‐dichloro‐pheny 1)‐1,1‐dimethylurea.

Addition of diaminobenzidine to Tris‐treated chloroplasts causes an increase in fluorescence yield.

Diaminobenzidine‐dependent electron transport mediated by photosystem 2 is coupled to synthesis of ATP even in the absence of an electron acceptor. This phosphorylation which is presumably supported by cyclic electron flow, is sensitive to 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea.

Diaminobenzidine‐dependent ATP formation, in Tris‐treated chloroplasts exhibits the red‐drop phenomenon.

The diaminobenzidine‐induced cyclic photophosphorylation (mediated by photosystem 2) is resistant to a large extent to KCN‐treatment which is known to inhibit reactions catalyzed by photosystem 1.On the other hand ATP formation supported by electron transport from diaminobenzidine to methyl viologen in the presence of 3‐(3,4‐dichlorophenyl)‐1,1‐dirnethylurea] is largely inhibited by KCN‐treatment. This observation suggests that there are two coupling sites of ATP formation, one catalyzed by diaminobenzidine as a donor to photosystem 1 (in the presence of 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea), and the other supported by diaminobenzidine which acts both as a donor to photosystem 2 (in Tris‐treated chloroplasts) and as an acceptor (in its oxidized form) from a carrier located between the two photosystems.
Synthesis and Hydrolysis by Pepsin and Trypsin of a Cyclic Hexapeptide Containing Lysine and Phenylalanine
Authors: TERADA Shigeyuki , KATO Tetsuo , IZUMIYA Nobuo
Published: March 2005

A cyclic hexapeptide, cyclo(‐Gly2‐Phe2‐Gly‐Lys‐), and the corresponding open‐chain hexa‐peptides, Gly2‐Phe2‐Gly‐Lys and Phe‐Gly‐Lys‐Gly2‐Phe2 have been synthesized and their susceptibilities to the hydrolytic action of pepsin and trypsin were determined.

The cyclic peptide was hydrolyzed slowly by trypsin to a hexapeptide Gly2‐Phe2‐Gly‐Lys2 the value of the Michaelis constant for this reaction being Km= 0.010 M, whereas Phe‐Gly‐Lys‐Gly2‐Phe was hydrolyzed rapidly to yield Phe‐Gly‐Lys and Gly2‐Phe; Km= 0.00022 M.

The cyclic peptide was not cleaved by pepsin at all, but Gly2‐Phe2‐Gly‐Lys was hydrolyzed rapidly at a Phe‐Phe bond; Km= 0.0091 M.

The cyclic peptide inhibits the hydrolysis of Gly2‐Phe2‐Gly‐Lys by pepsin in a linear non‐competitive manner, the value of the inhibition constant being Ki= 0.004 M.
The Binding of Atractylate and Carboxy‐atractylate to Mitochondria
Authors: KLINGENBERG Martin , GREBE Karin , SCHERER Burkhard
Published: March 2005

35S‐labelled atractylate and carboxy‐atractylate are produced biosynthetically and used for studying the binding of these specific ligands to the ADP, ATP carrier in beef heart mitochondria. The following results are obtained.

Inhibition of translocation activity goes parallel to the increase of binding by [35S]atractylate. No additional binding is observed after full inhibition of translocation is reached giving evidence that atractylate binds exclusively to the carrier.

The maximum number of binding sites of both atractylates is about 1.6 μmol/g protein in beef heart mitochondria and decreases on treatment of the membrane by Pi, freezing, ageing, etc. The dissociation constants of the binding are approximately for atractylate Kd= 5.10−8M and for carboxy‐atractylate Kd= 10−8M. The mass action plots of the concentration dependence for the binding are nonlinear‐convex in particular with carboxy‐atractylate and more linear with atractylate. Nonlinearity appears to be caused by some retardation of equilibration in the case of very high affinity binding.

The binding of atractylate and carboxy‐atractylate is relatively fast in intact mitochondria and slower in aged membranes. There is a slower and a faster binding portion.

The atractylates remove ADP in a nearly 1:1 stoichiometry from untreated mitochondria. In aged and Pi‐treated membranes the ratio Δ; ADP/Δatractylate approaches 0. Obviously binding of carrier sites to ADP is more sensitive to alterations then that of the atractylates. The assumption is maintained that the binding site for atractylate is identical with that for ADP and ATP.

Bongkrekate prevents binding of both atractylates. However, when added after, it only removes atractylate but not the carboxy compound because of its different tight binding. The removal of atractylate depends on the synergistic effect of bongkrekate with ADP.

The binding studies with [35S]atractylate and in particular the interaction with bongkrekate support the reorienting carrier model in which atractylate as an impermeable ligand fixes the binding site of the carrier outside while with bongkrekate the carrier site is turned to the inside.
Hybridization in vitro of Subunits of the DNA‐Dependent RNA Polymerase from Escherichia coli and Micrococcus luteus
Authors: LILL Ute I. , BEHRENDT Eva M. , HARTMANN Guido R.
Published: March 2005

RNA polymerase core enzyme from the gram‐positive bacterium Micrococcus luteus is composed of three different types of subunits, very similar to the equivalent enzyme from the gram‐negative Escherichia coli although the overall net charge and the molecular size of the subunits is different. As in the E. coli enzyme the property of rifampicin resistance resides completely in the second largest subunit p. To investigate the functional correspondence of the subunits, intergeneric polymerase hybrids have been prepared in vitro from the isolated subunits. Active hybrids were obtained from mixtures of subunit α (from M. luteus) with β and β′ (from E. coli), of subunits α and β (from E. coli) with β′ (from M. luteus), of subunit α (from E. coli) with β and β' (from M. luteus), of subunit α and β′ (from M. luteus) with β (from E. coli). Rifampicin resistance and sensitivity of the hybrids containing subunit β from E. coli was solely determined by the resistance and sensitivity respectively of the E. coli RNA polymerase from which subunit β was isolated. The rate of formation of these hybrids is distinctly slower than the reconstitution of the parent E. coli RNA polymerase from subunits. The final activity reached during incubation is different for the various hybrids. All hybrids were able to transcribe natural and synthetic DNA and to catalyse the DNA‐directed synthesis of poly(A), whereas they were inactive in the de novo synthesis of poly(A) · poly(U) and poly(I‐C) · poly(I‐C) in contrast to the reconstituted E. coli enzyme. These results indicate a common structural and functional organization of bacterial RNA polymerases.
The Specificity of Five DNAases as Studied by the Analysis of 5′‐Terminal Doublets
Authors: BERNARDI Alberto , GAILLARD Claire , BERNARDI Giorgio
Published: March 2005

The 5′‐terminal dinucleotides released by five deoxyribonucleases (spleen acid DNase, snail acid DNase, pancreatic DNase, Escherichia coli endonuclease I and crab DNase) have been determined on E. coli DNA (51% dG + dC) digests having different average sizes (P̄n) in the range 50 to 10. It has been shown that the composition of the 5′‐terminal dinucleotide (a) is independent upon the degradation level, at least in the range explored; (b) is strongly different from the composition of E. coli DNA doublets, these differences being characteristic for each enzyme; (c) is very significantly different from the statistical composition of 5′‐terminal dinucleotides, as calculated from the composition of 5′‐terminal and penultimate nucleotides. A calculation of the statistical composition of the trinucleotides split by each enzyme, using the 3′‐terminal nucleotide data in conjunction with the 5′‐terminal dinucleotide results provided a qualitative “specificity spectrum” for each enzyme.
Kinetics of Binding of Bovine Trypsin‐Kallikrein Inhibitor (Kunitz) in which the Reactive‐Site Peptide Bond Lys‐15‐Ala‐16 is Cleaved, to α‐Chymotrypsin and β‐Trypsin
Authors: QUAST Ulrich , ENGEL Jürgen , STEFFEN Erna , MAIR Gerhard , TSCHESCHE Harald , JERING Helmut
Published: March 2005

Equilibrium measurements of the binding of reactive‐site‐cleaved (modified) bovine trypsin‐kallikrein inhibitor (Kunitz) to α‐chymotrypsin and β‐trypsin show a stoichiometric 1:1 association with high binding constants. At least in the case of chymotrypsin much evidence is presented that the reaction with modified inhibitor leads to the same complex as the reaction with virgin inhibitor does. The association rate constant of modified inhibitor with chymotrypsin at pH 7, 22.5° C is 15.8 M−1 s−1. This is about 2×104 times slower than the binding of virgin inhibitor to that enzyme. In the analogous reaction of modified inhibitor with β‐trypsin, however, the association rate constant (1.2 × 104 M−1 s−1 at pH 6.9, 22.5° C) is of about the same order of magnitude as it is in the reaction of virgin inhibitor and trypsin. These and analogous phenomena observed in the reactions of virgin and modified soybean trypsin inhibitor (Kunitz) with α‐chymotrypsin and β‐trypsin suggest that the specificity of both inhibitors to trypsin is strongly reflected in the association rate constants of the modified forms.

The dissociation rate constants of the complexes of trypsin‐kallikrein inhibitor with chymotrypsin or with trypsin towards the modified inhibitor are estimated to be unmeasurably slow (half‐life times of 45 or 1.5 × 104 years, respectively).
The Effect of Cleaving the Reactive‐Site Peptide Bond Lys‐15‐Ala‐16 on the Conformation of Bovine Trypsin‐Kallikrein Inhibitor (Kunitz) as Revealed by Solvent‐Perturbation Spectra, Circular Dichroism and Fluorescence
Authors: QUAST Ulrich , ENGEL Jürgen , STEFFEN Erna , TSCHESCHE Harald , JERING Helmut , KUPFER Sigrid
Published: March 2005

Spectroscopic measurements of virgin bovine trypsin‐kallikrein inhibitor and its modified species (in which the reactive‐site peptide bond Lys‐15–Ala‐16 is split) indicate a conformational difference between both proteins. The inhibitor contains four tyrosines but no tryptophan. In the modified inhibitor a tyrosyl blue shift is seen in the difference absorption spectrum of modified against virgin inhibitor. The solvent perturbation spectra show an increase of the fraction of exposed tyrosyls from 0.45 in the virgin inhibitor to 0.59 in the modified form. Comparison of the circular dichroism spectra of the modified and virgin inhibitors reveals a decrease of the mean residue ellipticity in the tyrosine and peptide bond region of the modified inhibitor. In the fluorescence spectra a 50% increase in the quantum yield of the tyrosine fluorescence is observed in the modified inhibitor. All these spectroscopic data support the idea, which is also evidenced by the X‐ray crystallographic model, that in the modified inhibitor up to five residues from Ala‐16 to Arg‐20 gain rotational freedom.
The Amino‐Acid Sequence of a Peptide (PS‐1) from Drosophila funebris: A Paragonial Peptide from Males which Reduces the Receptivity of the Female
Authors: BAUMANN Heinz , WILSON Kenneth J. , CHEN Pei Shen , HUMBEL René E.
Published: March 2005

The substance, PS‐1, produced in the paragonial gland of adult male Drosophila funebris influences the mating behavior of virgin female flies after injection. The substance was isolated and characterized as a 27‐residue peptide. The complete amino acid sequence was determined by manual sequence analysis of tryptic peptides, automated Edman degradation, and carboxypeptidase A digestion. The sequence is Asp‐ValLeu‐Pro‐Ser‐Ala‐Asn‐Ala‐Asn‐Ala‐Asn‐Asn‐Gln‐Arg‐Thr‐Ala‐Ala‐Ala‐Lys‐Pro‐Gln‐Ala‐Asn‐Ala‐Glu‐Ala‐Ser‐Ser. The ratio of Val: Leu in the second position of the sequence is 7:3. This is the first detailed report on an insect peptide which causes a biological response in the opposite sex following mating.
Complexes of Metal Phthalocyanines with Globin as the Models of Heme Proteins
Authors: PRZYWARSKA‐BONIECKA Helena , TRYNDA Liliana , ANTONINI Eraldo
Published: March 2005

The reaction between iron and cobalt tetrasulfonated phthalocyanines and globin results in the formation of the green complexes, as has been proved by difference spectroscopy. Spectrophotometric titration data indicate the formation of those complexes at the molar ratio 1:1. The complexes of ferrous, ferric and cobaltous tetrasulfonated phthalocyanines with globin have been isolated from the reaction mixtures by separation on Sephadex G‐50 and precipitation of the protein fractions with ammonium sulfate. The visible spectra of these complexes are characterised by the main intensive peak at 641 nm, 678 nm, and 675 nm for ferric, ferrous and cobaltous derivatives, respectively. The new globin complexes have the property of reversible combination with oxygen and coordination with cyanide ions.

It is evident from the results of the spectrophotometric titrations of hemoglobin and methemoglobin with cobaltous tetrasulfonated phthalocyanine that iron protoporphyrins are displaced by this cobalt derivative; this suggests that phthalocyanine and porphyrin are bonded in a similar manner.
31P Nuclear‐Magnetic‐Resonance Studies of Pyridoxal and Pyridoxamine Phosphates
Authors: MARTINEZ‐CARRION Marino
Published: March 2005

The 31P nuclear magnetic resonance (NMR) spectrum of the phosphate in free pyridoxal or pyridoxamine phosphate reveals a resonance signal that is coupled to the methylene protons of the 5‐CH2 with JHP of 6.0 ± 0.3 Hz. Proton noise decoupling results in a single with a pH‐dependent chemical shift with deprotonation of the phosphate resulting in a shift of the 31P resonance to lower fields. A single 31P NMR signal at a frequency corresponding to fully ionized phosphate monoesters is observed in aspartate‐transaminase‐bound pyridoxal or pyridoxamine phosphate. The 31P resonance in the holotransaminase is pH‐independent and is unaffected by saturating concentrations of substrates or inhibitors. Only denaturation with 6 M guanidine · HCl results in changes in the 31P of the holoenzyme. It appears that the phosphate group of pyridoxal phosphate is bound to a positive pocket in the holoenzyme and remains fully ionized in the pH range of 5.6 to 9.2. The phosphate‐binding properties are present even in the apoenzyme which is able to bind inorganic phosphate which then can be displaced by pyridoxal or pyridoxamine phosphate in the process of holoenzyme formation.
Metabolism of the Phosphatidylglycerol Molecular Species in Escherichia coli
Authors: KITO Makoto , ISHINAGA Masataka , NISHIHARA Masateru , KATO Michie , HATA Tadao , SAWADA Seiji
Published: March 2005

The fractionation, turnover and biosynthesis of the phosphatidylglycerol molecular species of Escherichia coli were studied. Monoacetyldiglycerides derived from phosphatidylglycerol were separated into five subfractions; cis‐vaccenyl‐palmitoleyl, cis‐vaccenyl‐cis‐vaccenyl, palmityl‐palmitoleyl, palmityl‐cis‐vaccenyl and the disaturated molecular species on a silica gel plate impregnated with silver nitrate. Individual molecular species had different turnover rates. The palmityl‐cis‐vaccenyl species was metabolized faster than the others. Disaturated species were rather stable. Various phosphatidylglycerol molecular species were synthesized in the presence of sn‐glycerol 3‐phosphate, palmityl‐CoA, palmitoleyl‐CoA, cis‐vaccenyl‐CoA and CTP by the E. coli membrane particulate fraction. When only the proportion of palmityl‐CoA among the three acyl‐CoAs was increased, the molecular species containing the palmityl residue were increased. Similar results were obtained with the other acyl‐CoAs. However, a temperature‐sensitive incorporation of unsaturated and saturated fatty acids into phosphatidylglycerol molecular species was observed with no change in the proportions of the three acyl‐CoAs, completely reflecting the in vivo unsaturated/saturated ratio.
The Mitochondrial ATPase
Authors: FERGUSON Stuart J. , LLOYD William J. , LYONS Michael H. , RADDA George K.
Published: March 2005

Evidence is presented which indicates that inactivation of the mitochondrial ATPase from bovine heart by the reagent 4‐chloro‐7‐nitrobenzofurazan results from modification of one tyrosine residue per enzyme molecule. Activity can be restored by a variety of sulphydryl reagents.

In sodium dodecyl sulphate, the nitrobenzofurazan group on tyrosine is transferred to newly exposed sulphydryl groups on the enzyme.

The rate of transfer of the nitrobenzofurazan moiety from the enzyme to sulphydryl compounds is compared with that for transfer from the model compound N‐acetyl‐tyrosine‐O(7‐nitrobenzofurazan) ethyl ester, the synthesis and properties of which are also described.

The ligands ATP and ADP exert a protective effect on the rate of reaction between the mitochondrial ATPase and 4‐chloro‐7‐nitrobenzofurazan. The variation in rate of this reaction with change in pH has also been examined and a pKa of 9.5 estimated for the tyrosine residue.

The modification does not prevent substrate binding as judged by changes in the fluorescence of aurovertin, an antibiotic with specific affinity for mitochondrial ATPases.

When the ATPase activity of submitochondrial particles is inhibited by 4‐chloro‐7‐nitrobenzofurazan, there is a parallel decrease in the extent of the energy‐linked fluorescence enhancement of 1‐anilino‐naphthalene‐8‐sulphonate induced by ATP hydrolysis. Both ATPase activity and the fluorescence enhancement are restored by sulphydryl reagents.