bluuebunny/crossref_metadata_2025 huggingface dataset:
Reinigung und Charakterisierung der durch Östrogene induzierbaren 17‐β‐Hydroxysteroid: NAD(P)‐Oxydoreduktase aus dem Myometrium von Kaninchen
Authors: Jütting G. , Thun K. J. , Kuss E.
Published: March 2005

The myometrium of the rabbit contains a 17‐β‐hydroxysteroid: NAD(P) oxidoreductase, whose specific activity depends on the functional state of the uterus. In pregnancy one observes a 25‐fold increase and after parenteral administration of oestradiol or stilboestrol an approximately 50‐fold increase compared to the normal adult rabbit.

The enzyme was purified from myometrium about 100‐fold by precipitation twice with ammonium sulfate and by gel filtration.

The enzyme was assayed by incubation with [16‐14C]oestradiol‐17β, determining oestron as the reaction product. Specific activity was expressed as n‐moles oestron/60 min/mg protein. The molecular weight of the 17‐β‐hydroxysteroid: NAD(P) oxidoreductase can be estimated from gel filtration studies in the range from 10,000 to 50,000.

The enzyme requires NAD+ or NADP+ as cofactor, NADP+ giving a 20% lower activity than an equimolar amount of NAD+. The optimal pH for the enzymatic oxidation of oestradiol to oestron is 9.4, and for the reduction of oestron to oestradiol 6.4. The Michaelis‐Menten constant for oestradiol is 7.6 × 10−6 M. The oxidation of testosterone is at least 100 times slower than that of oestradiol. In phosphate‐free buffer solutions, the addition of phosphate gives an increase of the enzyme activity.

p‐Chlormercuribenzoic acid (10−4M) and o‐iodosobenzoic acid (10−4M) inhibit the activity of 17‐β‐hydroxysteroid: NAD(P)oxidoreductase of the myometrium by 97% and 26% respectively.
Relation of Properties of Isolated Myosin to those of Intact Muscles of the Cat and Sloth
Authors: Bárány M. , Conover T. E. , Schliselfeld L. H. , Gaetjens E. , Goffart M.
Published: March 2005

Myosin was prepared from the extensor digitorum longus, gastrocnemius medialis and lateralis, tibialis anterior, flexor of the claws of the anterior limb, and diaphragm muscles of the cat and didactyl sloth (Choloepus hoffmanni Peters). Actin‐activated, Ca2+‐activated, and EDTA‐activated ATPase activities of the myosins from cat muscles were two to four times higher than those of the myosins from the same muscles of the sloth. The difference in the Ca2+‐activated ATPase activity was found in the pH range of 5.5 to 10.0, and in the KCl concentration range of 50 mM to 500 mM at pH 7.0.

The kinetic characteristics of the two myosins indicated that both the Km and the Vmax of the ATPase activities of the sloth myosin were lower than those of cat myosin by a factor of several‐fold.

Reconstituted cat actomyosin superprecipitated four to six times faster than sloth actomyosin. This difference in the superprecipitation did not vary appreciably when the concentration of ATP and the ratio of myosin to actin were varied. A correlation between the ATPase activity and the speed of superprecipitation of actomyosins of the cat and sloth was shown.

The contraction time of various muscles of the cat and sloth was found to be inversely proportional to the actin‐activated ATPase activity of their respective myosins. The speed constant of shortening of the muscles of the diaphragm appeared to be proportional to the actin‐activated ATPase activity of their myosin.

In contrast to the ATPase activity of myosin, which varied according to the speed of contraction, the actin‐binding ability of myosins of the cat and sloth was rather constant. The extent of superprecipitation of various reconstituted actomyosins of the cat and sloth was also the same. These properties of the isolated myosins were related to the similar tension output of the muscles of the cat and sloth.
Studies on Mutant Phenylalanyl RNA Synthetases of Escherichia coli
Authors: Böck A.
Published: March 2005

The temperature conditional mutant NP 37 of Escherichia coli differs from the wild type K 10 in that cell‐free extracts exhibit a 20‐fold reduced specific activity of phenylalanyl RNA synthetase. The mutant enzyme activity is characterized by an increased Km value for phenylalanine. Molecular weight determinations by sucrose density gradient centrifugation yield an approximate value of 180,000 for the wild type enzyme and of 100–110,000 for that of the mutant. Immunological analysis of the mutant extracts in these experiments, by means of antiserum prepared against purified phenylalanyl RNA synthetase, shows no cross‐reacting material at the position where the native enzyme sediments. However, an increased amount of cross‐reacting material is present in the fractions containing the mutant enzyme activity.

A phenotypic revertant of NP 37 was found to possess heat stability properties of the phenylalanyl RNA synthetase intermediate between the wild type and the NP 37 enzymes. Incubation of cell‐free extracts of such a results in preparations which show the catalytic properties of phenylalanyl RNA synthetase that are characteristic of mutant NP 37.

The results indicate that temperature inactivation of phenylalanyl RNA synthetase in NP 37 is a consequence of the dissociation of the enzyme into subunits with impaired catalytic properties.
Kupplungsreaktion an Lactatdehydrogenase nach reversibler Maskierung der SH‐Gruppen
Authors: Pfleiderer G. , Jeckel D.
Published: March 2005

For some time there have been indications of the importance of histidine and tyrosine for the catalytic activity of lactate dehydrogenase. The presence of reactive sulfhydryl groups in this enzyme has made it difficult to modify the histidine and tyrosine groups chemically without destroying the SH‐groups at the same time. We have thus attempted to mask all the sulfhydryl groups reversibly so that the reaction of diazotised [35S]sulfanilic acid with the protein could be examined.

The essential SH‐groups of the protein could be protected by adding a small excess of p‐chloromercuribenzoate or by forming a mixed disulphide with 5,5′‐dithiobis‐(2‐nitrobenzoic acid). The organo‐mercury derivative of lactate dehydrogenase, which had a residual activity of 13%, recovered 60% of the original enzymic activity on standing in pyrophosphate buffer, pH 8.5. The enzyme could not be fully reactivated on adding glutathione. Under the same conditions, there was a slow liberation of 2‐nitro‐5‐mercaptobenzoate from the enzyme which had reacted with 5,5′‐dithiobis‐(2‐nitrobenzoic acid). Coupling these modified lactate dehydrogenase preparations resulted in a fall in the SH‐titre of the enzyme. An attempt was thus made to mask all the sulfhydryl groups of the enzyme reversibly by means of the above mentioned reagents. When more than 1.5 g molecules of SH‐groups/36,000 g protein had reacted it was no longer possible to achieve 100% reactivation with cysteine.

In a final attempt, the complex of lactate dehydrogenase with mercuric chloride was prepared. This derivative was inactive and contained no free thiol groups. Both the original enzymic activity and SH‐titre were fully restored with cysteine. Like the native enzyme, Hg‐lactate dehydrogenase also binds 4 molecules of the NAD‐sulphite complex, albeit with an increased dissociation constant.

Hg‐lactate dehydrogenase was coupled with diazotised [35S]sulfanilic acid. The reaction was stopped with resorcinol and the reaction mixture was treated with cysteine. By this procedure the enzyme could be inhibited without loss of SH‐groups. The specific enzymic activity of the inhibited enzyme was proportional to the incorporation of 35S up to a value of 1.5 azoresidues/subunit. Azo‐lactate dehydrogenase migrated to the anode more rapidly than the native enzyme during electrophoresis at pH 8.5.
Effect of Oleic Acid on the Oxidation and Gluconeogenesis from [1‐14C]Pyruvate in the Perfused Rat Liver
Authors: Teufel H. , Menahan L. A. , Shipp J. C. , Böning S. , Wieland O.
Published: March 2005

Gluconeogenesis from [1‐14C]pyruvate in the perfused liver from fasted rats was stimulated by oleic acid as indicated by an increase in medium glucose, liver glycogen and specific activity of medium [14C]glucose. A balance of carbon from pyruvate revealed that in the presence of oleic acid 2 moles of pyruvate were utilized for each mole of glucose synthesized in contrast to a ratio 4:1 without fatty acid. From this and 14CO2 data it is concluded that oleic acid inhibited pyruvate oxidation markedly. Glucagon increased glucose formation from pyruvate but not from glycerol.
Preparation of Chemically Defined ɛN‐Acetylated Trypsin
Authors: Labouesse J. , Gervais M.
Published: March 2005

Acetylation of trypsin by acetic anhydride has been reinvestigated: it leads to a heterogeneous preparation, which can be fractionated by ammonium sulphate precipitation followed by chromatography on DEAE‐cellulose. The degree of substitution of ɛ and α‐amino groups, and that of phenolic hydroxyl groups was examined in fractions at different stages of purification, and it has been found that while all the ɛ‐aminogroups of lysine are acetylated the α‐aminogroup of the N‐terminal isoleucine remains free. The exposed tyrosyl residues of trypsin are also substituted, but the resulting ester bond is not very stable: these residues are slowly deacetylated on aging, even at slightly alkaline pH. This deacetylation brings about a decrease in the esterolytic activity of the enzyme. The final, homogeneous N‐ɛ‐acetyltrypsin obtained exhibits the same activity as native trypsin towards synthetic substrates.
On the Mechanisms of Photochemical Reductions of FAD and FAD‐Dependent Flavoproteins
Authors: McCormick D. B. , Koster J. F. , Veeger C.
Published: March 2005

The rate for photoreduction of riboflavin 5′‐phosphate is faster than flavin‐adenine dinucleotide which can be effectively photoreduced to the hydroquinone level by: ethylenediaminetetraacetate > ethylenediaminetetrapropionate > l‐methionine > nicotine > dimethylamino‐propanol > dimethylglycine ethyl ester > triethylamine. The same compounds can be used to photoreduce flavoproteins, the rates for which are: d‐amino acid oxidase > l‐amino acid oxidase > glucose oxidase > oxynitrilase > Shethna flavoprotein. The oxidation, reduction stage achieved is characteristics of the protein.

Addition of free flavin markedly enhances the photoreduction of those flavoproteins which have a readily dissociable flavin‐adenine dinucleotide. Urea enhances the photoreduction rate of flavoproteins in approximate corespondence to their ease of photoreduction in the absence of this reagent. Increase in temperature does not greatly enhance the rate of photoreduction of these flavoproteins. Benzoate decreases the rate of photoreduction of certain of the oxidases.
Formation and Metabolism of some Δ4‐Cholestenols in the Rat
Authors: Björkhem I. , Danielsson H.
Published: March 2005

The synthesis of unlabeled and tritium‐labeled cholest‐4‐ene‐3α, 7α‐diol and cholest‐4‐ene 3β, 7α‐diol is described. Both compounds were found to be efficiently converted into cheodeoxycholic acid and cholic acid in the bile fistula rat. The presence in liver of microsomal and soluble Δ4‐3‐hydroxysteroid dehydrogenases active on Δ4‐3‐hydroxysteroids of the C27 series was demonstrated. The soluble dehydrogenase(s) was more active on the Δ4‐3α‐hydroxysteroids than on the Δ4‐3β‐hydroxysteroids. The microsomal fraction contained dehydrogenase(s) capable of oxidizing both the Δ4‐3α‐hydroxysteroids and the Δ4‐3α‐hydroxysteroids in the presence of NAD or NADP, NAD being preferred. No significant conversion of cholest‐5‐ene‐3β, 7α‐diol to cholest‐4‐ene‐3β, 7α‐diol could be shown nor could any direct saturation of the double bond in cholest‐4‐ene‐3α, 7α‐diol or that in cholest‐4‐ene‐3α, 7α,12α‐triol be shown. Cholest‐4‐ene‐3α, 7α‐diol was hydroxylated in the 12α‐position more efficiently than cholest‐4‐ene‐3β, 7α‐diol. It is suggested that the Δ4‐cholestenols are converted to bile acids by first being oxidized to the corresponding Δ4‐3‐ketosteroids.
Studies on the Specificity of the Enzymic Synthesis of Polyribonucleotides
Authors: Farber F. E. , Chargaff E.
Published: March 2005

The purpose of this study was to ascertain whether enzymes having the same function (in this case, polynucleotide phosphorylase), but isolated from two very distant cell types, differed in their synthetic capacities. The enzymes were isolated from B. cereus, an organism possessing a DNA of a marked AT‐type (dissymmetry ratio of 1.70), and from M. lysodeikticus, whose DNA is of a pronounced GC‐type (dissymmetry ratio, 0.39). The approximate molecular weight of the B. cereus enzyme was found to be 210,000.

In both instances, and with several independent enzyme preparations from either source, the mixed polynucleotides synthesized from CDP and ADP as the precursors were investigated. Three conditions of the proportions of the precursor pool were imposed, with the input ratio of CDP/ADP as 2, 1 or 0.5.

Several criteria were employed for the study and the comparison of the synthetic polymers. The analysis of the base composition showed that, under equal conditions, the M. lysodeikticus enzyme incorporated a considerably higher proportion of CDP into the polynucleotides than did the B. cereus enzyme. The existence of concrete differences between polynucleotides synthesized by the two enzymes was emphasized by the study of the distribution of oligonucleotide fragments produced by the action of ribonuclease.

The statistical treatment of the frequency of adenylic acid runs of different length in the various polymers demonstrated that both enzymes produced heteropolymers composed of randomly arranged nucleotide sequences.

It is suggested that polynucleotide phosphorylase is species‐specific with respect to discrimination between precursors, but unspecific in its mode of action: the incorporated nucleotides are aligned in a random arrangement.
Regulation der Biosynthese der aromatischen Aminosäuren in Claviceps paspali
Authors: Lingens F. , Goebel W. , Uesseler H.
Published: March 2005

DAHP‐synthetase, chorismate mutase, prephenate dehydrogenase, prephenate dehydratase and anthranilate synthease were identified in extracts of Claviceps paspali mycelium. We were able to separate three DAHP‐synthetases, one sensitive to l‐phenylalanine, one sensitive to l‐tyrosine and one sensitive to l‐tryptophan. The l‐tryptophan sensitive isoenzyme provided the main part of the DAHP‐synthetase activity (about 60%). The inhibition due to l‐tryptophan was not removed by l‐tyrosine nor l‐phenylalanine. Chorismic acid did not inhibit these enzymes. Chorismate mutase was inhibited by l‐tyrosine and also by l‐phenylalanine, though these inhibitions were only detected after purification of the enzyme, and even then the highly purified enzyme (ca. 100‐fold) could not be separated into isoenzymes. The inhibition of the purified enzyme by l‐tyrosine and l‐phenylalanine could be removed entirely by l‐tryptophan.

Prephenate dehydratease was inhibited only by l‐phenylalanine and it could not be activated by l‐tyrosine nor l‐tryptophan.

Prephenate dehydrogenase showed an inhibiting effect by l‐tyrosin only after purification.

Anthranilate synthetase was not inhibited by l‐tryptophan even after purification.

Anthranilate synthetase was not inhibited by l‐tryptophan even after purification. With the simultaneous additions of l‐tyrosine, l‐phenylalanine and l‐tryptophan (in equal concentrations of 1 × 10−3 M) no repression effects could be produced.
The Solubilization of the Cytoplasmic Membrane of Bacillus subtilis by Sodium Dodecyl Sulphate
Authors: Bishop D. G. , Rutberg L. , Samuelsson B.
Published: March 2005

Cytoplasmie membranes of Bacillus subtilis have been solubilized by brief exposure to the anionic detergent, sodium dodecyl sulphate. Complete solubilization was obtained when the relative proportation of detergent to membrane protein was approximately 1:2.

The solubilized membrane preparation can be completely freed of detergent by gel filtration. The membrane components do not reaggregate, and membrane phospholipid remains associated with the protein during solubilization and subsequent removal of the detergent.

Model experiments showing that enzymes can be selectively solubilized by varying the concentration of sodium dodecyl sulphate, indicate that the solubilization of the membrane preparation by the detergent is not a random disaggregation.
Spectrométrie de masse de glycolipides
Authors: Adam A. , Senn M. , Vilkas E. , Lederer E.
Published: March 2005

This paper describes mass spectrometric studies of the peracetylated derivatives of synthetic and natural 6,6′‐diesters of trehalose containing acyl radicals varying from C22 to C90.

The peracetylated synthetic 6,6′‐dieicosnoyl trehalose (I) gave a molecular ion at m/e 1238. The synthetic 2,3,4,2′,3′,4′‐hexaacetyl‐6,6′‐di(α‐eicosanyl‐β‐acetoxy tetracosanoyl)‐trehalose (IIb) showed no molecular ion but a peak (M‐60) at m/e 1910 (Fig. 1). The cleavage of the glycosidic bond leads to typical oxonium ions which are at m/e 611 (A) for (I) and at m/e 977 (B2) for (IIb).

The three natural trehalose 6,6′‐diesters examined are the “cord factors” of a human virulent strain of Mycobacterium tuberculosis (strain Prévois), of a bovine attenuated strain (BCG) and of Mycobacterium butyricum.

The mycolic acids isolated after saponification of these compounds have also been studied by mass spectrometry. Formulae III, IV, V and VI are proposed for their methyl esters.

The molecular weights of the paracetylated “cord factors” are above 3000; no molecular ion could be detected. The oxonium ions, however, give significant peaks which are useful for structural determination. Oxonium ions for the glycolipid of the human virulent strain Prévois are found at m/e 1360–1528 (Fig.2). The peracetylated glycolipid of BCG contains two different series of homologous mycolic acids (acyl ions at m/e 1144–1284 and 1072–1184) and gives oxonium ions at m/e 1432–1572 for one series and at m/e 1360–1472 for the second one. The “cord factor” of M. butyricum gives homologous oxonium ions from m/e 1361 to 1445 corresponding to acyl ions from m/e 1073 to 1157.
Enzymatic Conversion of a Δ4‐3‐Ketosteroid into a 3α‐Hydroxy‐5β‐Steroid: Mechanism and Stereochemistry of Hydrogen Transfer from NADPH
Authors: Berséus O. , Björkhem I.
Published: March 2005

The conversion of [4‐14C]7α, 12α‐dihydroxycholest‐4‐en‐3‐one into 5β‐cholestane‐3α,7α,12α‐triol, catalyzed by Δ4‐3‐ketosteroid 5β‐reductase and 3α‐hydroxysteroid dehydrogenase from rat liver, has been studied in the presence of tritiated water and NADPH labeled with tritium in the A‐ and B‐positions. It was concluded that the reduction of the Δ4 double bond involves a direct hydride ion transfer from the A‐position of NADPH to the 5β‐position of the steroid and the addition of a proton to the 4‐position of the steroid. The reduction of the 3‐keto group was found to involve a direct transfer of hydrogen from the A‐position of NADPH to the 3β‐position of the steroid.
Metabolism of trans‐3‐Hexadecenoic Acid by Chlorella vulgaris and by Lettuce Leaf
Authors: Bartels C. T. , James A. T. , Nichols B. W.
Published: March 2005

The incubation of trans‐3‐hexadecenoic acid with cells of Chlorella vulgaris, and with lettuce leaf slices, resulted in reduction of the substrate to palmitic acid.

Before this reduction was complete, the trans‐acid entered all the acyl lipid classes present in the cell.

Consequently, the specific location of trans‐3‐hexadecenoic in the phosphatidyl glycerol fraction of photosynthetic tissues cannot be explained on the basis that lyso‐phosphatidyl glycerol is the only lipid capable of ‘accepting’ this acid. It is more likely that phosphatidyl glycerol or lyso‐phosphatidyl glycerol is a required substrate or cofactor for the synthesis of trans‐3‐hexadecenoic acid from palmitic acid.
A Study of DNA, Partially Denatured DNA and Protein‐DNA Complexes in the Polyethyleneglycol‐Dextran Phase System
Authors: Pettijohn D.
Published: March 2005

Partition properties of DNA and protein‐DNA complexes in the polyethyleneglycol‐dextran phase system are described which suggest several preparative and possibly analytical applications. The partition coefficients (K) of linear and circular double‐stranded DNAs are significantly reduced if relatively small region(s) of local denaturation are introduced into the DNA. The K value of polyoma DNA I, the twisted circular form, is approximately 10 fold less than the values of II and III, the open circular and linear DNA forms. DNA complexed with several different basic proteins or polyamines did not partition differently from free DNA; however, the complex with chymotrypsin or RNA polymerase had greatly reduced K values. The saturated RNA polymerase‐DNA complex was indistinguishable from heat denatured DNA in its partition properties.
A Method for Extracting Purified DNA or Protein‐DNA Complex from Escherichia coli
Authors: Favre J. , Pettijohn D. E.
Published: March 2005

A new technique for the extraction of DNA or protein‐DNA complex from bacteria has been developed, using multiple extractions in the polyethyleneglycol‐dextran two‐phase system. The procedure has several advantages over the usual methods for DNA extraction: purified DNA contains less than 0.05% RNA or 1 μmole arginine per mmole DNA phosphate; denatured and partially denatured DNA fragments are excluded; DNA precipitation and ribonuclease treatments are unnecessary; the method is fast, less than 8 hours being required for the complete procedure. The purified protein‐DNA complex contains about 0.5 mg protein/mg DNA and significant amounts of RNA polymerase activity.

A small amount of denatured DNA was separated from the double‐stranded DNA during the extraction. The size of this denaturated fraction depended upon the temperature and conditions of shear during lysis. Small amounts (0.2 to 3%) were found even at 30° under conditions where only inadvertant shears were introduced, while at higher temperatures with stirring the fraction was much larger. When purified T4 DNA was fragmented by shear to about the molecular weight of the final bacterial DNA fragments, denaturation of the T4 DNA was also detected. These results suggest that a shear‐induced denaturation of DNA can occur and that the fragments of denatured DNA found in bacterial lysates are produced at least in part by shear and may therefore have no direct biological significance.
Kinetics of Glycerol Kinases from Mammalian Liver and Candida mycoderma
Authors: Grunnet N. , Lundquist F.
Published: March 2005

Glycerol kinase (ATP: glycerol phosphotransferase, EC 2.7.1.30) has been purified from rat, beef and human liver. Glycerol kinase from Candida mycoderma was also investigated.

The kinetics with regard to glycerol are of the Michaelis‐type. The Lineweaver‐Burk plots with regard to ATP are non‐linear. The data obtained provide evidence for the ATP‐Mg2− complex as the true substrate and indicate that free ATP is a positive modifier for the enzymes.

Inhibition experiments showed no effect of ethanol, acetaldehyde, acetate, d,l‐glycerol‐3‐phosphate, fructose‐1,6‐diphosphate or NADH upon the reaction velocities. ADP and AMP are strong inhibitors of the enzyme.

The inhibition by AMP has been examined. It is concluded that AMP is a negative modifier for glycerol kinase. The inhibition by AMP is counteracted by free ATP. The interaction coefficient for AMP is about one.

The kinetic properties of the glycerol kinases examined are to some extent similar to those of phosphoglycerate kinase. The possible physiological significance of the inhibition by AMP is discussed.
Reaktionsschritte der Methionin‐Synthese bei Escherichia coli
Authors: Stavrianopoulos J. , Jaenicke L.
Published: March 2005

Methionine synthetase has been purified 860 fold from Escherichia coli. The homogeneous red protein has a particle weight of 135,000 and is a vitamin B12 protein, containing one mole of dimethylbenzimidazole‐cobalamin per mole of protein. Several of its properties: Michaelis constants, spectrum, pH‐optimum, and stability have been determined, and studies on its mechanism of action performed by exchange and isolation experiments. In the presence of S‐adenosylmethionine and a reducing agent, the enzyme‐bound B12 is methylated by methyltetrahydrofolate. Its methyl group then is transferred to homocysteine to yield methionine. The one reaction product, tetrahydrofolate, is only loosely bound to the enzyme, whereas S‐adenosylmethionine is tightly attached to the protein, though not to its B12‐site. The other product, enzyme‐bound methyl‐cobalamin, was isolated after enzymic hydrolysis, and identified. Its labelling corresponded to the labelling of the methionine in the overall reaction. Relative to the methionine formed, S‐adenosylmethionine and the reducing system—in this instance a NADH/FAD‐reductase—are required in catalytic amounts. The former can act as methylating agent of the enzyme‐bound B12 if used in stoichiometric concentration. However, under physiological conditions, it is not used as methyl donor for methionine. On the other hand, the enzyme also catalyzes the transfer of a methyl group from S‐adenosyl methionine to tetrahydrofolate, yielding 5‐methyl tetrahydrofolic acid, thus accounting for the observed dilution of label. The ratio of synthesis to transfer is about 1200. By following the stoichiometry of reduction and methionine synthesis together with the lack of exchange of methyl groups from methyl‐B12 to tetrahydrofolate on irradiation, it is concluded that the enzyme reaction proceeds by ionic steps. A plausible reaction scheme is given in which, however, the rôle of S‐adenosylmethionine is still a matter of conjecture. It seems possible that the enzyme participates in the quaternization of the methyl donor, 5‐methyltetrahydrofolate, to facilitate the dissociation of its methyl group.
Specificity of the Constitutive Hexose Transport in Yeast
Authors: Heredia C. F. , Sols A. , Fuente G. Dela
Published: March 2005

A method has been developed based on the osmotic sensitivity of protoplasts, that permits turbidimetrical estimation of the penetration of non‐metabolizable compounds into yeast protoplasts. Using this method a wide variety of sugars and related compounds have been tested as presumptive substrates for the constitutive hexose transport system in baker's yeast. An integration of the results obtained by this method with those obtained by other approaches has led to the establishment of the structural requirements that a compound has to meet in order to be transported. The basic structural requirement seems to be met by a pyranose ring for glucose and a furanose ring for fructose. With compounds that can be regarded as structurally related to d‐glucopyranose, there is a broad tolerance for modifications at carbons 1 and 2, and somewhat less so for modifications at carbon 3. Similar requirements, except for carbon 2, apply to compounds that can be regarded as structurally related to D‐fructofuranose.

Physical diffusion of sugars, and related compounds, through the cell membrane of baker's yeast is very low, so that they only can enter into the cell at measurable rates by means of specific transport devices. The factor by which the entrance of glucose is increased over its physical diffusion, at concentrations in the millimolar range, is of the order of 106.
A Comprehensive Fractionation Procedure for Avian Erythrocyte Histones
Authors: Vidali G. , Neelin J. M.
Published: March 2005

A fractionation procedure for avian erythrocyte histones was devised to give the six known, reproducible histone fractions from a single nuclear preparation, with limited manipulation and little wastage. The proposed method includes selective, two‐step acid extraction, followed by cation‐exchange chromatography and exclusion chromatography. The fractions were identified by amino acid analysis and by starch gel electrophoresis. The method is flexible in scale and permits a continuous balance of compositions and activities in metabolic experiments.

Factors contributing to the relative extractability of histones from chromatin in acid were considered. The essential role of this step was the separation of erythrocyte‐specific, serine‐rich histone V, and arginine‐rich histones III, IV, which otherwise would be eluted together from Amberlite CG‐50 by guanidinium chloride. Two other arginine‐rich components, more easily eluted in chromatograms of other tissues, were absent from the usual extracts of mature erythrocytes. However, similar components were produced from histones III, IV by treatment with β‐mercaptoethanol.

The conditions for good resolution of sub‐fractions of moderately lysine‐rich histones and of arginine‐rich histones by exclusion chromatography on Bio‐Gel P‐60 and P‐30 were examined. Reversible adsorption appeáred to be more significant than pore size of the gel; the sulphates of arginine‐rich histones were resolved better than the chlorides.
Thioredoxin: 3. Amino Acid Sequences of the Peptic Peptides from S‐Aminoethylated Peptide B
Authors: Holmgren A. , Perham R. N. , Baldesten A.
Published: March 2005

Peptide B, the N‐terminal fragment of thioredoxin obtained by cyanogen bromide treatment was aminoethylated and digested with pepsin. Eight peptides were separated by high voltage paper electrophoresis and chromatography and characterized with respect to amino acid sequence. The results account for all 37 residues of peptide B. The N‐terminal sequence of peptide B was determined as: Ser‐Asp‐Lys‐Ile‐Ile‐His‐Leu‐Thr‐Asp‐Asp‐Ser‐Phe.
Thioredoxin: 4. Amino Acid Sequence of Peptide B
Authors: Holmgren A.
Published: March 2005

Tryptic and chymotryptic digestion of peptide B, the N‐terminal fragment obtained by cyanogen bromide cleavage of thioredoxin, provided the information required to determine the contiguity of the peptic fragments studied previously. The complete sequence of the 37 residues of peptide B could thus be established. Peptide B contains the active center of thioredoxin. Its amino acid sequence was found to be: . This sequence supports earlier conclusions drawn from fluorescence studies of thioredoxin, which indicated a close relationship between one or both tryptophan residues and the disulfide bridge of the active site.

Peptide maps of the tryptic digests of carboxymethylated thioredoxin and of the two cyanogen bromide fragments are described. Amide nitrogen analyses of thioredoxin and the two cyanogen bromide fragments demonstrate that all seven amide groups of thioredoxin are localized in the C‐terminal fragment (peptide A).
The Proflavin‐Induced Increase in the Catalytic Activity of Ficin
Authors: Hollaway M. R.
Published: March 2005

The dye proflavin (3,6 diaminocaridine) binds to a single site on the ficin molecule (EC 3.4.4.12).

The value of the dissociation constant (Kp) for the enzyme‐dye complex was 3.5 ± 1.8 × 10−4 M measured spectrophotometrically and 1.55 ± 0.61 × 10−4 by equilibrium dialysis.

Interaction with proflavin increased the catalytic constant (kcat) for the ficin‐catalysed hydrolysis of benzoyl‐L‐arginine ethyl ester although the value of KM app was unaltered.

Dye‐binding did not significantly affect the value of kcat for the ficin‐catalysed hydrolysis of p‐nitrophenyl hippurate, a substrate for which acylation is not rate‐limiting in catalysis.

The dependence of initial velocity of benzoyl‐l‐arginine ethyl ester hydrolysis on dye concentration enabled the computation of a value of Kp= 1.61 × 10−4 M assuming a kinetic model involving formation of a ternary dye‐enzyme‐substrate complex in which substrate is more rapidly hydrolysed than in the binary enzyme‐substrate complex.

Dye‐binding leads to an enhanced nucleophilicity of the essential thiol group of ficin.

It is concluded that deacylation is not the rate‐limiting step in the ficin‐catalysed hydrolysis of benzoyl‐L‐arginine ethyl ester.

Possible explanations of the proflavin‐induced acceleration of benzoyl L‐arginie ethyl ester hydrolysis by ficin are discussed, including consideration of an allosteric modification mechanism.
Studies on the Interaction of F‐Actin with Tropomyosin
Authors: Drabikowski W. , Nowak E.
Published: March 2005

The interaction of tropomyosin with F‐actin was studied under various conditions. The maximum ratio of tropomyosin bound to actin was found by ultracentrifugation to be 1:2, by weight. Viscometric study showed, however, no further increase of viscosity due to formation of the actin‐tropomyosin complex when the above ratio exceeded 1:5. Higher ionic strength caused dissociation of the complex.

The comparison of the rate of polymerization of tropomyosin free actin under various salt conditions with that in the presence of tropomyosin showed that the latter partially modified some properties of actin.

The pattern of polymerization of actin extracted at room temperature (RT‐actin) resembled that of the mixtures of actin with tropomyosin, although some differences were observed indicating tighter binding in the case of RT‐actin, possibly due to the presence of troponin.

The results obtained are discussed from the point of view of the possible localization of tropomyosin in thin flaments.
Initiation de chaînes de RNA par la RNA polymérase in vitro
Authors: Sentenac A. , Ruet A. , Fromageot P.
Published: March 2005

In the presence of cordycepin triphosphate (3′‐deoxyadenosine triphosphate), an analogue of ATP, RNA polymerase synthesizes very short RNA chains in vitro. These chains, labelled on the first nucleotide with [γ32P]GTP, are acid soluble but are retained on nitrocellulose filters. After alkaline hydrolysis, chromatography on Dowex 1 (CI−)shows that the 32P‐labelled molecules behave like guanosine tetraphosphate.

When filtered on Sephadex G‐75 the oligonucleotides synthesized are associated with a structure of high molecular weight, which is probably the complex DNA‐enzyme, and remain so after ribonucleases or deoxyribonuclease treatment.

The number of nascent oligonucleotides measured by incorporation of [γ32P]GTP, represents only 50% of the chains initiated by guanosine in the control. This observation, together with the fact that nascent oligonucleotides are not sensitive to pancreatic or T1 ribonuclease, shows that they are involved in a structure which determines their stability. This structure could be an Enzyme‐(DNA‐RNA hybrid) complex at the growing point of the RNA chains.

The kinetics of initiation of RNA are the same whether chain propagation is prevented or not.

The number of RNA chains initiated per phage T7 DNA molecule has been measured with [γ32P]ATP and [γ32P]GTP. It increase linearly with the amount of RNA polymerase added although the amount of RNA made tends towards a maximum. On the other hand, the number of nascent oligonucleotides made does not increase with an excess of enzyme, which shows the existence of a finite number of initiation sites. These results are thought to indicate that initiation sites are reutilized during normal RNA synthesis.

The number of chains initiated by adenine and guanine per phage T7 DNA at the point where RNA synthesis, as a function of RNA polymerase concentration, departs from proportionality, is around 50.
Excess of α‐Globin Synthesis in Homozygous β‐Thalassemia Its Cytoplasmic Molecular Forms
Authors: Conconi F. , Bargellesi A. , Pontremoli S.
Published: March 2005

The cytoplasmic distribution of the excess newly synthesized α‐globin, present in the red cell lysate of β‐thalassemic subjects has been investigated.

The labelled globins present in the cytoplasm, as well as in purified Hb A and Hb F, were separated by CM‐cellulose chromatography, and the ratios 3Hα/3Hβ and 3Hα/3Hγ were determined.

The results obtained show that an intracellular exchange occurs between non radioactive α‐chains of existing hemoglobins and the newly synthesized ones. Since the amount of labelled α‐globin chains which is found in combination with Hb A or Hb F does not account for all the newly synthesized α‐globin, it is concluded that part of this α‐globin must exist in a free form. A series of experimental data support the conclusion that this “free fraction” is probably composed of non heminated α‐globin molecules.
Studies on the Metabolism of C19 Steroids in Rat Liver
Authors: Gustafsson J.‐Å. , Lisboa B. P. , Sjövall J.
Published: March 2005

The formation of polar, saturated C19steroids with a 3‐keto‐5α‐configuration after incubation of 17β‐hydroxyandrost‐4‐en‐3‐one (testosterone) with 105,000 ×g microsomes of adult male rat liver was investigated. The metabolites were isolated by thin‐layer chromatography and were identified as 2β‐,6β‐(tentatively), 7α‐, and 16α‐hydroxy‐5α‐dihydrotestosterone by means of gas chromatography‐mass spectrometry. The same compounds were also isolated after incubation with 2β‐,6β‐,7α‐, and 16α‐hydroxytestosterone, respectively. Incubation with 5α‐dihydrotestosterone, however, resulted in the formation of 2β‐,7α‐, and 16α‐hydroxy‐5α‐dihydrotestosterone whereas no 6β‐hydroxy‐5α‐dihydrotestosterone could be found.
La Tyrosyl‐tRNA synthétase de Escherichia coli
Authors: Chousterman S. , Sonino F. , Stone N. , Chapeville F.
Published: March 2005

Partially purified tyrosyl‐tRNA synthetase from Escherichia coli B was incubated either in the presence of [14C]tyrosine and ATP or [14C]tyrosyl‐tRNA and AMP, or [3H]tyrosinyl‐AMP, a structural analogue of tyrosyl‐AMP. After filtration on a Sephadex G‐75 column, [14C]tyrosyl‐AMP: Enzyme and [3H]tyrosinyl‐AMP: Enzyme complexes were isolated. The [14C]tyrosine is transferred directly from [14C]tyrosyl‐AMP: Enzyme to the tRNA; no such transfer was observed with the [3H]tyrosinyl‐AMP:Enzyme. The stability of the tyrosyl‐AMP:Enzyme complex was examined under different conditions: (a) its half‐life at pH 7.0 is 150 minutes at 0°, 35 minutes at 20°, and 5 minutes at 37°; this instability is due to the non‐enzymatic hydrolysis of the anhydride bond; (b) its stability is affected neither by the presence of homologous or heterologous tRNAs which do not accept tyrosine, nor by the presence of ATP and/or tyrosine; (c) when the preformed complex is incubated with tyrosinyl‐AMP in conditions where this analogue inhibits completely tyrosyl‐AMP: Enzyme formation from ATP and tyrosine, tyrosyl‐AMP is not exchanged with tyrosinyl‐AMP. The stability of tyrosinyl‐AMP: Enzyme was also studied: after incubation for 30 minutes at 37°, or for several days at 4°, 90% of the [3H]tyrosinyl‐AMP was found associated with the enzyme after gel filtration. The complex remains unaffected when incubated in the presence of inorganic pyrophosphate, ATP or tRNA, but in the presence of tyrosinyl‐AMP, tyrosinol and ATP or tyrosine and ATP, only 20% of [3H]tyrosinyl‐AMP remained bound to the enzyme.

These results show that tyrosyl‐AMP does not dissociate from the enzyme while tyrosinyl‐AMP does. It follows that, despite the great similarity of tyrosyl‐AMP and tyrosinyl‐AMP, the differences between the anhydride and the ester bond seem to influence decisively the stability of the complexes formed with the enzyme.
Oxidative Metabolism in Cells Isolated from Brown Adipose Tissue
Authors: Prusiner S. B. , Cannon B. , Lindberg O.
Published: March 2005

Brown fat cells exhibit a classical mitochondrial electron transport system.

The respiration of brown fat cells can be stimulated by nor‐epinephrine and this hormonally mediated respiration can be mimicked by oleate.

Both nor‐epinephrine and oleate‐stimulated respiration can be blocked by rotenone, antimycin, azide and malonate, and the antimycin block can be by‐passed by tetramethyl‐p‐phenylenediamine.

α‐Glycerol phosphate and succinate were found to stimulate respiration.

The potent α‐glycerol phosphate oxidase cannot quantitatively contribute to the respiration evoked during catecholamine induced thermogenesis.
Stereoselective Alkylation with Asymmetric Reagents
Authors: Eisele B. , Wallenfels K.
Published: March 2005

Yeast alcohol dehydrogenase is inactivated at different rates by the antipodes of α‐iodopro‐pionic acid and its amide; the d(+) antipodes react faster. The influence of coenzyme, substrate and pH on the reaction velocity were investigated. Under all conditions second order kinetics have been observed. Activation parameters for the alkylation of the SH groups with the different antipodes were calculated from the temperature dependence of the reaction rates. Large differences in the activation enthalpies (ΔΔH= 6.8 kcal/mole and 2.3 kcal/mole, respectively) were found for the reaction of yeast alcohol dehydrogenase with the different antipodes of α‐iodopropionic acid and α‐iodopropionamide. These were compensated for by differences in activation entropy of —18.4 and —5.6 entropy units, respectively.

These results are interpreted by assuming highly dissymmetric surroundings of the reacting SH groups favouring the d(+) antipodes in the transition state of the alkylation reaction. The negative activation entropy indicates that this favourable transition state may be attained only by restriction of the degrees of freedom.

The relationship between the stereoselective chemical reactivity and the stereospecificity of the enzyme is discussed.
Simulierung einer mikrobiologischen Hydroxylierung mit dem Analogcomputer
Authors: Riemann J. , Röpke H. , Kieslich K. , Koch H.‐J. , Gibian H.
Published: March 2005

A simple kinetic model of the 11β‐hydroxylation of the 6α‐fluor‐21‐acetoxy‐16α‐methyl‐4‐pregnen‐3,20‐dione was conceived to comprehend the complex processes of an enzymatic reaction. Although eleven parameters were varied in these experiments, it was possible to simulate the reaction‐curves in all cases with the analog computer and to characterise the microbiological processes by only two kinetic parameters.
Adenine Nucleotide Translocation of Mitochondria
Authors: Pfaff E. , Klingenberg M.
Published: March 2005

The adenine nucleotide translocation in mitochondria has previously been established as an exchange between exogenous and endogenous adenine nucleotides across the inner membrane. The specificity and the control of the exchange are examined with the following major results:

The adenine nucleotide translocation is relatively specific for exogenous ADP and ATP, AMP being nearly inactive. Among other nucleotides tested, only dADP and dATP exchange with a noticeable activity.

In the controlled state ADP exchanges 2–4 times faster than ATP. If simultaneously added, ADP and ATP compete for the exchange, with ADP being about tenfold more active than ATP.

The specificity of the exit of adenine nucleotides in the exchange is similar to the specificity of the entrance with the difference that ADP and ATP are released with equal activity in proportion to their intramitochondrial content. AMP is released only after a slow conversion to ADP. Therefore the short time exchange is limited by the endogenous content of ADP plus ATP.

The exchange is influenced by the metabolic state of the mitochondria. The ATP exchange is more variable than the ADP exchange. Two effects are elucidated: (a) the influence of the metabolic state on the relative content of AMP which inhibits both the ADP and ATP exchange (b) the coupling of the energy transfer system which inhibits only the ATP exchange. An example for case (a) is the inhibition of the ADP and ATP exchange by arsenate and an example for case (b) is the strong increase of the ATP exchange on uncoupling.

The following effects are relevant to the mechanism of the control of the exchange by ATP. The stimulation of the ATP exchange by uncoupler has the same concentration dependence as the uncoupling of oxidative phosphorylation (Km [CCP] = 0.08 μM, where CCP = carbonyl‐cyanide‐phenylhydrazone). Oligomycin does not abolish the uncoupler effect on the ATP exchange. “Endogenous uncoupling” on aging of mitochondria also stimulates the ATP exchange. Valinomycin plus K+ only slightly stimulate the ATP exchange. Anaerobiosis stimulates the ATP exchange to a smaller extent than uncoupling.

In competition with ADP the effects of energy transfer on ATP exchange are more strongly revealed. On uncoupling the more than tenfold preference for ADP is fully abolished.

It is concluded that basically the exchange for ADP and ATP has equal specificity in forward and reverse reaction. In the controlled state a superimposed force makes the specificity asymmetric and inhibits the entrance of ATP. This control of the ATP exchange is concluded to be based on the anionic character of the adenine nucleotides. Thus the ATP4‐ex–ADP3‐in exchange is inhibited unless the charge difference is compensated for by an uncoupler stimulated H+ movement across the membrane. Furthermore an electric potential gradient appears to be effective in the controlled state which is abolished on uncoupling.
Porphyrin Biosynthesis
Authors: Tomio J. M. , Grinstein M.
Published: March 2005

The enzymic system that catalyzes the total synthesis of protoporphyrin IX has been found in the Harderian gland of the rat. The presence of the enzyme δ‐aminolevulinic acid synthetase was demonstrated in mitochondria by the formation of [14C]δ‐aminolevulinic acid. On incubation of homogenates of the Harderian glands with δ‐aminolevulinic acid and porphobilinogen, varying conditions such as temperature, time, aero‐or anaerobiosis and cofactor and substrate concentration, porphyrins with a carboxyl content ranging from 8–2 were formed. Thus, the presence of δ‐aminolevulinate dehydratase, porphobilinogenase, decarboxylase and coproporphyrinogenase systems were demonstrated.
δ‐Aminolevulinate Dehydratase from Rat Harderian Gland Purification and Properties
Authors: Tomio J. M. , Tuzman V. , Grinstein M.
Published: March 2005

δ‐Aminolevulinate dehydratase which catalyzes the second step in the biosynthesis of protoporphyrin has been purified from Harderian gland homogenates of normal rats. The enzyme preparation has a specific activity of 860 units per mg of protein at 37°, and about a 150‐fold purification from the crude extract has been achieved. The purified material appears to be homogeneous in starch gel and polyacrylamide gel disc electrophoretic studies. Partially purified enzyme preparations have an absolute requirement for cysteine or analogous thiol reagents. Effect of anions and cations is discussed.
On the Analysis of Melting Curves of Stacked Polynucleotides
Authors: Richards E. G.
Published: March 2005

A method of analysing melting profiles of stacked polynucleotides is described. This method assumes that the melting process is non‐cooperative and that the optical parameter is proportional to the degree of stacking, but requires no assumption concerning the properties of the polymer in the fully stacked or fully unstacked conformation. It is equally applicable to absorption, optical rotation or circular dichroism data. The method has been applied to a high precision absorbance‐temperature profile of alkaline polyadenylic acid and has enabled the calculation, subject to the assumptions noted above, of the absorption spectra of this material in both the fully stacked conformation and in the random coil conformation. It is suggested that not all the hypochromism of the polymer with respect to the monomer can be accounted for in terms of stacking.
A Comparison of some Properties of Alternating Poly(A‐U) and the two Stranded Complex of Poly A and Poly U
Authors: Richards E. G. , Simpkins H.
Published: March 2005

Some optical and thermodynamic properties of the double helical form of the alternating copolymer poly(A‐U) and the two stranded complex of poly A and poly U are compared. It is shown that their thermal difference spectra are closely similar when that of the latter complex is corrected for the hypochromicity of the poly A formed on denaturation. Similarly differences in their Tm and pHm values can be accounted for in terms of the free energy of stacking of the poly A formed on denaturation. It is suggested that sequence effects are of only marginal consequence in determining the optical and thermodynamic properties of two stranded helical polynucleotides.