bluuebunny/crossref_metadata_2025_split huggingface dataset:
Immunochemical Studies on Tyrosinase Induction in Neurospora
Authors: KATAN Talma , GALUN Esra , ARNON Ruth 
Published: March 2005

An immunoassay for tyrosinase, using the modified bacteriophage technique, was developed: Tyrosinase of Neurospora was conjugated to bacteriophage T4 using glutaraldehyde as a cross‐linking agent. The conjugated phage that survived the coupling process could be inactivated by antiserum raised in rabbits against pure tyrosinase, but not by normal serum. This inactivation was specifically inhibited by pure Neurospora tyrosinase, and the degree of inhibition was proportional to the concentration of tyrosinase within the range of 30 – 150 ng/ml. Crude mycelial extracts possessing tyrosinase activity could similarly inhibit the inactivation of the conjugated phage by the antiserum. To evaluate the tyrosinase content of crude extracts, their inhibitory capacity was compared to that of known amounts of pure tyrosinase, and the amounts thus calculated agreed with those predicted from an enzymatic assay.The tyrosinase‐bacteriophage immunoassay was used for the quantitation of tyrosinase‐antigen in crude extracts of Neurospora cultures that had been induced to form tyrosinase by the addition of ethionine. Enzymatic activity appeared after a lag of several hours, increased for 2 – 3 days and then declined. Immunological assays of these cultures showed: (a) serologically reactive protein started to accumulate upon culture starvation and was evident during the lag period; (b) specific activity (units per mg antigen) was constant throughout induction; (c) at the phase of decrease in mycelial enzyme content, increasing amounts of serologically reactive protein were detected in the medium, indicating that some enzyme was eventually excreted. These results show that the lag is not a qualitatively distinct period, and support the previously forwarded notion that tyrosinase is synthesized de novo upon induction.
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 byThe 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.
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.
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.
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.
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.
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.