bluuebunny/crossref_metadata_2025 huggingface dataset:
Affinity Purification and Properties of Cathepsin‐E‐Like Acid Proteinase from Rat Spleen
Authors: YAMAMOTO Kenji , KATSUDA Nobuo , KATO Keitaro
Published: March 2005

A unique acid proteinase different from cathepsin D was purified from rat spleen by a method involving precipitation at pH 3.5, affinity chromatography on pepstatin–Sepharose 4B and concanavalin A–Sepharose 4B, chromatography on Sephadex G‐100 and DEAE‐Sephacel, and isoelectric focusing. A purification of 4200‐fold over the homogenate was achieved and the yield was 11%. The purified enzyme appeared to be homogeneous on electrophoresis in polyacrylamide gels. The isoelectric point of the enzyme was determined to be 4.1–4.4. The enzyme hydrolyzed hemoglobin with a pH optimum of about 3.1. The molecular weight of the enzyme was estimated to be about 90000 by gel filtration on Sephadex G‐100. In sodium dodecylsulfate polyacrylamide gel electrophoresis, the purified enzyme showed a single protein band corresponding to a molecular weight of about 45000. The hydrolysis of bovine hemoglobin by the enzyme was much higher than that of serum albumin. Various synthetic and natural inhibitors of the enzyme were tested. The enzyme was inhibited by Zn2+, Fe3+, Pb2+, cyanide, p‐chloromercuribenzoate, iodoacetic acid and pepstatin, whereas 2‐mercaptoethanol, phenylmethyl‐sulfonyl fluoride and leupeptin showed no effect.
Studies on Polypeptide‐Chain‐Elongation Factors from an Extreme Thermophile, Thermus thermophilus HB8
Authors: NAKAMURA Shun , OHTA Shigeo , ARAI Ken‐ichi , ARAI Naoko , OSHIMA Tairo , KAZIRO Yoshito
Published: March 2005

Molecular properties of the polypeptide chain elongation factors from Thermus thermophilus HB8 have been investigated and compared with those from Escherichia coli.

As expected, the factors purified from T. thermophilus were exceedingly heat‐stable. Even free EF‐Tu not complexed with GDP was stable after heating for 5 min at 60°C.

GDP binding activity of T. thermophilus EF‐Tu was also stable in various protein denaturants, such as 5.5 M urea, 1.5 M guanidine‐HCl, and 4 M LiCl.

Amino acid compositions of EF‐Tu and EF‐G from T. thermophilus were similar to those from E. coli. On the other hand, amino acid composition of T. thermophilus EF‐Ts was considerably different from that of E. coli EF‐Ts.

In contrast to E. coli EF‐Tu, T. thermophilus EF‐Tu contained no free sulfhydryl group, but one disulfide bond. The disulfide bond was cleaved by sodium borohydride or sodium sulfite under native conditions. The heat stability of the reduced EF‐Tu · GDP, as measured by GDP binding activity, did not differ from that of the untreated EF‐Tu · GDP.

T. thermophilus EF‐Ts contained, in addition to one disulfide bond, a sulfhydryl group which could be titrated only after complete denaturation of the protein.

Under native conditions one sulfhydryl group of T. thermophilus EF‐G was titrated with p‐chloromercuribenzoate, while the rate of reaction was very sluggish. The sulfhydryl group appears to be essential for interaction with ribosomes, whereas the ability to form a binary GDP · EF‐G complex was not affected by its modification. The protein contained also one disulfide bond.

Circular dichroic spectra of EF‐Tu from T. thermophilus and E. coli were very similar. Binding of GDP or GTP caused a similar spectral change in both T. thermophilus and E. coli EF‐Tu. On the other hand, the spectra of T. thermophilus EF‐G and E. coli EF‐G were significantly different, the content of ordered structure being higher in the former as compared to the latter.
Physical Evidence for the Asembly of A and B Chains of Human Placental Collagen in a Single Triple Helix
Authors: BENTZ Hanne , BÄCHINGER Hans Peter , GLANVILLE Robert , KÜHN Klaus
Published: March 2005

Native collagen molecules containing A and B chains were isolated from pepsin‐solubilised human chorionic and amniotic membrane extracts by fractional salt precipitation and DEAE‐cellulose chromatography. They exhibited a circular dichroism spectrum, and a melting curve, characteristic for a triple‐helical structure. Electron microscopical investigations of their segment‐long‐spacing crystallites revealed a molecule similar to those of the interstitial types I, II and III collagens. After denaturation, the A and B chains were separated by DEAE‐cellulose chromatography and were consistently recovered in a ratio of 1:2. Renaturation experiments indicated that only the B chains are able to reform triple‐helical molecules which are stable under conditions in vivo. The data support a molecular formula A(B)2 for the native collagen molecule.
Structure and Synthesis of a Lipid‐Containing Bacteriophage
Authors: SCHÄFER Rolf , KÜNZLER Peter , LUSTIG Ariel , FRANKLIN Richard M.
Published: March 2005

The lipid‐containing bacteriophage PM2 was dissociated stepwise in 1 M NaCl (pH 7.2) with increasing urea concentrations. In 2 M urea the following substructures could be identified: (a) a nucleocapsid containing all of the viral lipid, the DNA, proteins III and IV, plus a fraction of protein II, and (b) a second substructure representing particles which contained all viral elements except protein I, the spike protein. In 4 M urea the viral nucleocapsid containing all of proteins III and IV, the DNA, plus a fraction of protein II, was isolated. Upon increasing the urea concentration further, this nucleocapsid is stable up to 8.5 M urea; in 9 M urea protein III was partly dissociated from the nucleocapsid. The nucleocapsid in 4–8.5 M urea is stabilized by the addition of 0.1–3 M NaCl but dissociates if the NaCl concentration is less than 0.1 M. The nucleocapsid was also dissociated in 4–8.5 M urea at pH 4.5.

The nucleocapsid structures and some intermediate morphological subunits have been analysed by physical methods, enabling us to draw some conclusion about the structure and hydration of the virus.
RNA Hybridization to DNA Coupled with Cyanogen‐Bromide‐Activated Sephadex
Authors: SIDDELL Stuart G.
Published: March 2005

A method for purifying RNA by hybridization to complementary DNA immobilized on Sephadex is described and is illustrated by the purification of polyoma mRNA. Denatured DNA can be immobilized on cyanogen‐bromide‐activated Sephadex whilst remaining freely available for hybridization. However, after activation with cyanogen bromide, followed by the coupling of DNA and deactivation with ethanolamine, Sephadex acts as an ion‐exchange resin. It is therefore necessary to elute RNA from DNA‐Sephadex by a combination of formamide buffers containing NaCl and elevated temperatures, conditions which dissociate DNA · RNA hybrids and also prevent the binding of RNA to the resin by ion exchange.

Polyoma DNA, or restriction fragments thereof, were bound to Sephadex and used to purify viral mRNA. RNA purified from polyadenylated RNA isolated from the cytoplasm of mouse cells 28–30 h after infection with polyoma virus sedimented at values between 19 and 16 S in sucrose/formamide gradients and, in a wheat germ cell‐free translation system, directed the synthesis of three polypeptides; these were identified by gel mobility, immunoprecipitation and fingerprinting as the three structural proteins of polyoma virus. Viral mRNA could also be purified by hybridization directly from total cytoplasmic infected‐cell RNA without prior selection of polyadenylate‐containing RNA.

The advantages and limitations of DNA attached to cyanogen‐bromide‐activated Sephadex as an affinity matrix for RNA purification are discussed.
Purification and Properties of Brain Cathepsin B
Authors: SUHAR Alojz , MARKS Neville
Published: March 2005

CM‐cellulose chromatography of calf brain extracts revealed the presence of several enzymes hydrolysing benzoyiarginyl‐β‐naphthylamide (Bz‐Arg‐Nap) one of which resembled cathepsin B (EC 3.4.22.1). The latter was purified to homogeneity and showed an absolute dependence on thiol compounds and was optimally active at pH 6.5. The molecular weight of the purified enzyme was 27000 ± 1500.

Purified enzyme was inhibited by leupeptin, antipain, iodoacetate, Hg2+, zn2+, and partially by chymostatin and phenylmethylsulfonyl fluoride but was unaffected by pepstatin, puromycin, bestatin and bacitracin. The Km with Bz‐Arg‐Nap was 0.53 mM, Kcat 4.5 min−1. Dipeptidyl and tripeptidyl‐acrylamide substrates were hydrolysed also by the brain enzyme but with lower Kcat/Km ratios.

Comparison of the kinetics of inhibition using leupeptin (Ac‐l‐Leu‐Leu‐arginal) with Boc‐d‐Phe‐Pro‐arginal showed that the latter was non‐competitive and the former competitive with Ki of 1.5*10−8 M and 8*10−7 M respectively.

Purified enzyme hydrolysed histones (lysine‐rich and type‐1), myelin basic protein, porcine β‐lipotropin, human β‐endorphin and protamine. Hydrolysis of β‐lipotropin was accompanied by the generation of a 8000‐Mr fragment.

Cathepsin‐B‐like enzymes were present in highest concentration in rat pituitary, and lower in cerebellum, hypothalamus, medulla oblongata, striatum and spinal cord.
Routes of Thrombin Action in the Production of Proteolytically Modified, Secondary Forms of Antithrombin‐Thrombin Complex
Authors: FISH Wayne W. , ORRE Kerstin , BJÖRK Ingemar
Published: March 2005

The reaction between thrombin and antithrombin results in the formation of an inactive, stable, equimolar complex between the two proteins. However, under most reaction conditions several secondary complex forms, which have lower apparent molecular weights in dodecyl sulfate/polyacrylamide gel electrophoresis, appear concomitantly with or immediately following the production of the primary form of the complex. Purification of nascent, intact complex and treatment of this complex form with thrombin demonstrated that these subsidiary forms of antithrombin‐thrombin complex may arise by proteolysis of the nascent complex by excess thrombin. Dissociation of such proteolytically modified complex preparations by hydroxylamine, and examination of the dissociation products by dodecyl sulfate/polyacrylamide gel electrophoresis suggested that degradation occurs primarily in the thrombin part of the complex, and only after prolonged proteolysis in its antithrombin moiety also. Incubation of antithrombin with several autolytically modified thrombin preparations showed that formation of subsidiary complex forms can also occur by an alternative route, i.e. between premodified thrombin forms and the inhibitor. In contrast, complex formation between thrombin and active forms of antithrombin, which have been modified by thrombin before complex formation, is unlikely, since no such active forms of antithrombin could be demonstrated.
Reaction of β‐d‐Glucosidase A3 from Aspergillus wentii with d‐Glucal
Authors: LEGLER Günter , ROESER Kurt‐Rainer , ILLIG Heinrich‐Konrad
Published: March 2005

d‐Glucal acts as mixed competitive/non‐competitive inhibitor against β‐d‐glucosidase A3 from Aspergillus wentii with slow approach to the steady‐state rate of substrate hydrolysis. The same rate is reached whether d‐glucal competes directly with the substrate or the substrate displaces d‐glucal that has been bound by preincubation in the absence of substrate. The Ki for competitive inhibition and the rate constants for the approach to the steady state are in agreement with the kinetic constants for the hydration of d‐glucal to 2‐deoxy‐d‐glucose.

The concentration dependence of the inhibition shows that one molecule of d‐glucal (I) binds with complete inhibition but the biphasic dissociation kinetics of the enzyme‐glucal complex and labeling studies point to an additional binding site. An EI2 complex can be isolated at pH 6 and 0°C by rapid ion‐exchange chromatography. This complex can be reactivated at pH 4 and room temperature; all the d‐glucal is released as 2‐deoxy‐d‐glucose.

After denaturation of the EI2 complex one molecule of d‐glucal remains bound to the enzyme. The binding site was identified (by isolation and structure determination of a radioactive peptide) as the same aspartate residue that had been labeled with the active‐site‐directed inhibitor, con‐duritol B epoxide, in a previous study.
Structure and Properties of the Putrescine Carbamoyltransferase of Streptococcus faecalis
Authors: WARGNIES Brigitte , LAUWERS Nadine , STALON Victor
Published: March 2005

Ornithine and putrescine carbamoyltransferases from Streptococcus faecalis ATCC11700 have been purified and their structural properties compared. The molecular weight of native ornithine Carbamoyltransferase, measured by moleclar sieving, is 250000. It is composed of six apparently identical subunits with a molecular weight of 39000, as determined by cross‐linking with the bifunctional reagent glutaraldehyde followed by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Using the same method, putrescine Carbamoyltransferase is a trimer of 140000 consisting of three identical subunits with a molecular weight of 40000.

Ornithine Carbamoyltransferase displays a narrow specificity towards its substrate, ornithine. In contrast, putrescine Carbamoyltransferase carbamoylates ornithine and several diamines (diaminopropane, diaminohexane, spermine, spermidine, cadaverine) in addition to its preferred substrate, putrescine, but with a considerably lower efficiency than for putrescine.

The kinetic mechanism of putrescine Carbamoyltransferase has been investigated. Initial velocity studies yield intersecting plots using either putrescine or ornithine as substrate, indicating a sequential mechanism. The patterns of protection of the enzyme by the reactants during heat inactivation as well as the results of product and dead‐end inhibition studies provide evidence for a random addition of the substrates. The putrescine inhibition that is induced by phosphate does, however, suggest that a preferred pathway exists in which carbamoylphosphate is the leading substrate. The different kinetic constants have been established.

The properties of putrescine Carbamoyltransferase are compared to the known properties of other carbarnoyltransferases. The evolutionary implications of this comparison are discussed.
Guinea‐Pig α1 Microglobulin
Authors: ÅKERSTRÖM Bo , BERGGÅRD Ingemar
Published: March 2005

α1 microglobulin has been isolated from urine of guinea‐pigs with renal damage, induced by sodium chromate. Ultrafiltration, gel chromatography, zone electrophoresis and ion‐exchange chromatography were used for the purification. The protein was shown to be homologous to human α1 microglobulin, as the antiserum against this protein binds guinea‐pig α1 microglobulin. The molecular weight of the protein was 26000 when determined on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, 23000 as determined by gel chromatography in 6 M guanidine hydrochloride, and 24000 estimated from sedimentation and diffusion data. The amino acid content of guinea‐pig α1 microglobulin was similar to that of human α1 microglobulin. The carbohydrate content was 16% and composed of sialic acid, glucosamine and neutral hexoses. The protein was markedly heterogeneous in charge. It was shown to consist of a number of subgroups with different isoelectric points, which showed extreme conservation between the guinea‐pig and human α1 microglobulin. The heterogeneity apparently does not reside in the carbohydrate moiety, since digestion with neuraminidase did not reduce the heterogeneity when tested on agarose gel electrophoresis. Only one band was observed when guinea‐pig α1 microglobulin was subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing conditions. The native protein has a tendency to form dimers. A brown substance is strongly bound to the protein and caused absorbance of light in the ultraviolet and visible regions. Fluorescence was observed in the high‐frequency visible region. α1 microglobulin was present in the serum of guinca‐pigs at a mean concentration of 26.4 mg/ml as determined with radioimmunoassay. The antigenically active α1 microglobulin was eluted as four components of different size when serum of guinea‐pigs was chromatographed on a Sephadex G‐200 column.
A Study of p‐Hydroxybenzoate Hydroxylase from Pseudomonas fluorescens
Authors: MÜLLER Franz , VOORDOUW Gerrit , VAN BERKEL Willem J. H. , STEENNIS Pieter J. , VISSER Servaas , VAN ROOIJEN Peter J.
Published: March 2005

The purification procedure for p‐hydroxybenzoate hydroxylase has been modified by replacement of the DEAE‐cellulose (DE‐32) column in the original procedure by a Sephadex‐Cibacron‐blue affinity column. In this way the yield of enzyme could be improved from 16% to about 40–50%. Preparative gel chromatography indicated that the enzyme does not exist as a monomeric species as earlier believed but mainly as a dimer. Sodium dodecyl sulfate gel electrophoresis of purified enzyme revealed a minimum relative molecular mass (Mr) of 43000–45000. Analytical gel chromatography, sedimentation equilibrium and sedimentation velocity experiments showed that the enzyme exists in solution mainly as a dimer but also in higher‐order quaternary structures (presumably tetramer and hexamer). Temperature dependence of the distribution of the oligomers suggests that the association is of hydrophobic nature. The amino acid composition of the enzyme is also presented. The enzyme contains no disulfide but five sulfhydryl groups. In the native state of the enzyme only one sulfhydryl group is accessible to N‐ethylmaleimide or 5, 5′‐dithiobis(2‐nitrobenzoic acid). The iso‐electric point of the enzyme was found to be 5.8.
Immobilized Soybean Trypsin Inhibitor in the Stabilization, Resolution and Purification of Adenosine‐3′: 5′‐monophosphate‐Dependent Protein Kinases
Authors: ALHANATY Eytan , SHALTIEL Shmuel , BASHAN Nava , MOSES Shimon
Published: March 2005

The protein kinase dependent on adenosine 3′: 5′‐monophosphate (cAMP) in homogenates of rat intestinal mucosa was found to be remarkably unstable during its purification due (at least in part) to proteolysis. Since soybean trypsin inhibitor was found to protect the kinase from inactivation, an attempt was made to use this inhibitor, coupled to Sepharose, in order to extract and remove the proteolytic enzyme(s) responsible for the instability at an early stage of the purification. Indeed, this column excluded the undissociated form of cAMP‐dependent protein kinase (R2C2) in a considerably more stable form, illustrating the use of immobilized proteolytic inhibitors for stabilizing proteins in crude extracts containing potent proteases. But unexpectedly the same column retarded a protein kinase which was not activated by cAMP. The latter enzyme was identified as the free catalytic subunit (C) of cAMP‐dependent protein kinase since it was found to be inhibited by the Walsh‐Krebs specific protein inhibitor. On the basis of these observations, a method was developed for the purification of C from rabbit muscle which is based on (a) passage of the crude extract on the column and (b) biospecific dissociation of R2C2 by cAMP and rechromatography of the extract on the same column. This procedure yields > 300‐fold purification of C in the last step (with a recovery of 34%) and a rapid resolution between R2C2 and C. The same column can also be used for the resolution of type I and type II cAMP‐dependent protein kinases, and for > 40 fold purification of the type II form of the enzyme, in one step. The theoretical and practical implications of these results with regard to the use and abuse of biorecognition in chromatography are discussed.
Chloroplast Elongation Factors are Synthesized in the Chloroplast
Authors: CIFERRI Orio , DI PASQUALE Giuseppe , TIBONI Orsola
Published: March 2005

The elongation factor G (EF‐Gchl) and elongation factor Tu (EF‐Tuchl) present in spinach chloroplasts become labelled when isolated chloroplasts are incubated in the light with radioactive methionine. EF‐Gchl and EF‐Tuchl account for approximately 0.04% and 0.2% respectively of the total radioactivity incorporated by isolated organelles.
Uncoupling Action of Amytal in Membrane Vesicles from Escherichia coli
Authors: BOONSTRA Johannes , OTTEMA Sander , SIPS Herman J. , KONINGS Wil N.
Published: March 2005

The barbiturate amytal (5‐ethyl‐5‐isopentylbarbituric acid) has been shown to inhibit amino acid transport in membrane vesicles from anaerobically grown Escherichia coli. Amytal has no effect on the activity of the enzymes of the nitrate respiration system, nor on electron transfer in this system. However, addition of amytal to the membrane vesicles results in a decrease of the membrane potential from −90 mV to −72 mV, and to a decrease of the pH‐gradient of ‐61 mV to undetectable values. Furthermore, amytal causes an increase in the rate of ferricyanide reduction in liposomes, indicating that amytal increases the proton permeability of phospholipid membranes. These results demonstrate that amytal acts as an uncoupler in membrane vesicles from anaerobically grown E. coli.
Mechanism of Carbamoyl‐Phosphate Synthetase
Authors: BRITTON Hubert Greenslade , RUBIO Vicente , GRISOLIA Santiago
Published: March 2005

Carbamoyl phosphate synthethase I synthesizes carbamoyl phosphate from ammonia, HCO−3 and two molecules of ATP, one of which, ATPA, yields Pi while the other, ATPB, yields the phosphoryl group of carbamoyl phosphate. Pulse‐chase experiments with [γ‐32P]ATP without added HCO−3 demonstrate separate binding sites for ATPA and ATPB. Bound ATPA dissociates readily from its site (t1/2∼ 1–2 s) and the Kd is 0.2–0.7 mM. For the ATPB binding site the t1/2 for dissociation is 5–12 s and the Kd∼ 10 μM. Kd for ATPA seems to increase with enzyme concentration whereas Kd for ATPB does not change. HClO4 releases the ATP unchanged from the enzyme · ATPB and enzyme · ATPB· ATPA complexes.

In the presence of HCO−3, ATP and N‐acetylglutamate, an enzyme · ATPB· HCO−3· ATPA complex is formed. Its formation by the addition of HCO−3 to the enzyme · ATPB· ATPA complex appears to involve an initial bimolecular addition reaction followed by an isomerization. Treatment with HClO4 releases Pi from ATPA but ATPH is released unchanged. Spontaneous hydrolysis of ATPA is responsible for the ATPase activity of the enzyme. Thus, a covalent bond may form between HCO−3 and ATPA. However, ATPA can dissociate rapidly (t1/2≪ 10 s). The Kd for ATPA is approximately 0.2 mM.

ATPB appears unable to dissociate from the enzyme. ATPB· HCO−3· ATPA complex since the t1/2 for dissociation of ATPB from the enzyme is lengthened about five times in the presence of 19 mM HCO−3 and at 1 mM ATP. ATPA may also hydrolyse in this complex and be replaced by another molecule of ATP in the absence of exchange of ATPB. However, the ATPA binding site must be occupied to prevent ATPB release. ATPB may be bound in a pocket which becomes inaccessible to the solution when HCO−3 and ATPA also bind. In contrast, HCO−3 does not inhibit the binding of ATPB to the enzyme.

Various intermediate steps in the formation of the enzyme · ATPB· HCO−3· ATPA complex are discussed. Additional evidence is presented that the ATPB binding site is only periodically accessible to ATP in solution and that ATPB in the steady‐state reaction binds when the products leave. Since ⋝ 1.3 mol ATPB and ⋝ 1.8 mol ATPA bind/mol enzyme dimer, the enzyme monomer may be an active species.
The Polysomal Proteins of L Cells
Authors: CAZILLIS Michèle , HOUSSAIS Jean‐Fançois
Published: March 2005

Three groups of proteins can be clearly discriminated in the total protein of L cell polysomes by selective labelling in the presence of low doses of actinomycin D and two‐dimensional polyacrylamide/dodecylsulfate gel electrophoresis followed by autoradiography: (a) structural ribosomal proteins which are not labelled in the presence of actinomycin D and form stained non‐radioactive spots in gels; (b) exchangeable ribosomal proteins which are labelled in the presence of actinomycin D and form stained radioactive spots; (c) non‐ribosomal proteins which are detectable only by autoradiography of gels.

The large and small subunits of L cell ribosomes contain respectively 45 and 34 ribosomal proteins with molecular weights ≤ 50000; seven of the large subunit proteins and nine of the small subunit proteins are exchangeable. Most of the non‐ribosomal proteins migrate in the region of the second‐dimension gel slab corresponding to the molecular weight range 50000 –120000. Problems related to the separation of the ribosomal proteins of mammalian cells and the possible significance of the presence of non‐ribosomal proteins in polysomes are discussed.
Localization of the High‐Affinity ATP Site in Adenosine‐3′:5′‐monophosphate‐Dependent Protein Kinase Type I
Authors: HOPPE Jürgen , FREIST Wolfgang
Published: March 2005

8‐Azido‐adenosine 5′‐triphosphate (n38ATP) appeared to be a suitable photoaffinity label for the protein kinase dependent on adenosine 3′:5′‐monophosphate (cAMP). It competes with ATP for the high‐affinity ATP site in the undissociated form of the kinase and in the phosphotransferase reaction catalyzed by the catalytic subunit. Furthermore, it is accepted as a substrate in the phosphotransfer reaction.

n38ATP incorporated into the holoenzyme is covalently bound upon irradiation. Protection experiments with ATP indicated that this covalent attachment occurs in the high‐affinity ATP site of the enzyme. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate shows that n38ATP is bound to the catalytic subunit.

After irradiation the enzyme was dissociated by cAMP. Proportional to the incorporated [γ‐32P]n38ATP, a loss in phosphotransferase activity was found. These results support our model that both ATP sites coincide with respect to their adenine binding part. Thus binding of the regulatory subunit to the catalytic subunit would then transform the low‐affinity catalytically active ATP site into a high‐affinity inactive site.
An Investigation of the Conformational Properties of Ribosomes Using N‐Ethylmalemide as a Probe
Authors: GHOSH Nandini , MOORE Peter B.
Published: March 2005

The reactivity of ribosomal proteins towards N‐ethylamaleimide has been examined in a variety of ribosome and ribosomal subunit preparations from Escherichia coli. The data show that samples which would be regarded as equivalent operationally can differ significantly in conformation, as judged by reactivity, depending on the method of preparation. The washing of ribosomes with high concentrations of salt has a particularly dramatic effect on protein reactivity. The implications of these results for our understanding of ribosome conformation and for the further study of conformation by chemical reactivity are discussed.
Purification and Characterization of 3,4‐Dihydroxyphenylalanine Decarboxylase from Pig Kidney
Authors: BORRI VOLTATTORNI Carla , MINELLI Alba , VECCHINI Paola , FIORI Anna , TURANO Carlo
Published: March 2005

A procedure for 3,4‐dihydroxyphenylalanine decarboxylase from pig kidney purification is described in detail.

The preparation has no detectable impurity on elecrophoresis and on ultracentrifugation and has a coenzyme content and a specific activity comparable with the same enzyme purified by other authors.

However two significant differences are observed: a different stimulation of activity by added pyridoxal 5′‐phosphate and a nearly complete decarboxylation of L‐3,4‐dihydroxyphenylalanine in absence of added coenzyme.

Absorption, fluorescence and circular dichroism properties of the coenzyme‐apoenzyme interaction are also described.

The results are consistent with the existence of at least four coenzyme‐apoenzyme complexes, three of them active.
Purification and Characterization of the Messenger RNA Coding for Bovine Corticotropin/β‐Lipotropin Precursor
Authors: KITA Toru , INOUE Akira , NAKANISHI Shigetada , NUMA Shosaku
Published: March 2005

The mRNA coding for the common precursor of corticotropin and β‐lipotropin has been purified to homogeneity from neurointermediate lobes of bovine pituitaries. The homogeneity of the mRNA preparation is evidenced by analysis of its translation product, electrophoresis on polyacrylamide gel in the presence of formamide and analysis of the kinetics of hybridization with its cDNA. The purification procedure involves the isolation of RNA from membrane‐bound polysomes, chromatography on oligo(dT)‐cellulose and on poly(U)‐Sepharose and sucrose density gradient centrifugation. The mRNA has a molecular weight of approximately 450000, equivalent to approximately 1360 nucleotides in length, and contains a polyadenylate sequence with an average length of 68 nucleotides. The size of the mRNA is sufficiently large to encode the corticotropin/β‐lipotropin precursor.
Mitochondrial GTP‐AMP Phosphotransferase
Authors: TOMASSELLI Alfredo G. , SCHIRMER R. Heiner , NODA Lafayette H.
Published: March 2005

GTP‐AMP phosphotransferase has been purified 116‐fold with a yield of 24% from beef heart mitochondria using freeze‐thawing, alkali and acid treatment and successive column chromatography on phosphocellulose, Sephadex G‐100 and blue‐dextran–Sepharose. It has crystallized from poly(ethylene glycol) and is essentially homogeneous by sodium dodecylsulfate electrophoresis and isoelectrofocusing. The specific activity of the crystalline preparation was 290 U/mg. The molecular weight was found to be 26000 and the isoelectric point to be 9.8. Amino acid analysis showed 21 aspartic acid or asparagine, 19 threonine, 12 serine, 26 glutamic acid or glutamine, 15 proline, 16 glycine, 14 alanine, 15 valine, 4 methionine, 12 isoleucine, 28 leucine, 7 tyrosine, 7 phenylalanine, 5 histidine, 14 lysine, 16 arginine, 2 tryptophan, no –SS– bonds or free –SH. Guanosine(5′)pentaphospho(5′)adenosine is a very strong inhibitor similar to adenosine(5′)pentaphospho(5′)adenosine as an inhibitor of cytosolic adenylate kinase.
Singlet Oxygen Formation by a Peroxidase, H2O2 and Halide System
Authors: PIATT John , O'BRIEN Peter J.
Published: March 2005

Evidence for singlet oxygen formation has been obtained for the lactoperoxidase, H2O2 and bromide system by monitoring 2,3‐diphenylfuran and diphenylisobenzofuran oxidation, O2 evolution, and chemiluminescence. This could provide an explanation for the cytotoxic and microbicidal activity of peroxidases and polymorphonuclear leukocytes. Evidence for singlet oxygen formation included the following. (a) Chemiluminescence accompanying the enzymic reaction was doubled in a deuterated buffer and inhibited by singlet oxygen traps. (b) The singlet oxygen traps, diphenyl furan and diphenylisobenzofuran, were oxidized to their known singlet oxygen oxidation products in the presence of lactoperoxidase, hydrogen peroxide and bromide. (c) The rate of oxidation of diphenylfuran and diphenylisobenzofuran was inhibited when monitored in the presence of known singlet oxygen traps or quenchers. (d) Oxygen evolution from the enzymic reaction was inhibited by singlet oxygen traps but not by singlet oxygen quenchers. (e) The traps or quenchers which were effective inhibitors in the experiments above did not inhibit peroxidase activity, were not competitive peroxidase substrates and did not react with the hypobromite intermediate since they did not inhibit hydrogen peroxide consumption by the enzyme.

Using these criteria, various biological molecules were tested for their reactivity with singlet oxygen. Furthermore, by studying their effect on oxygen release by the enzymic reaction, it could be ascertained whether they were acting as singlet oxygen traps or quenchers.
Elongation‐Factor‐2‐Induced Sensitization of Ribosomes to Modeccin
Authors: OLSNES Sjur , ABRAHAM Abraham K.
Published: March 2005

Isolated rabbit reticulocyte ribosomes were treated with the toxic lectin modeccin for increasing periods of time. The toxin treatment was stopped by adding specific antitoxin and the residual activity of the ribosomes was tested in a polyphenylalanine‐synthesizing system.

With increasing time of toxin treatment the ability of the ribosomes to support polymerization of phenylalanine decreased. Addition of anti‐toxin immediately stopped further inactivation of the ribosomes. The presence of elongation factor 2 (EF‐2) strongly increased the rate of ribosome inactivation by modeccin. There was no similar effect with EF‐1. Maximal rate of inactivation was observed when the molar ratio of EF‐2 to ribosomes was about 1. The rate of ribosome inactivation was about 4 ribosomes min−1 (molecule of modeccin)−1 under optimal conditions.

The ability of EF‐2 to sensitize ribosomes to modeccin was strongly descreased in the presence of GTP, GDP and guanosine 5′‐(β,γ‐methylene]triphosphate. EF‐2, which had been ADP‐ribosylated in the presence of diphtheria toxin, was fully active in sensitizing the ribosomes whereas N‐ethylmaleimide‐treated EF‐2 had lost this ability.

Isolated 60‐S subunits were sensitized to modeccin by EF‐2 in a similar way as whole ribosomes.

The data indicate that modeccin inactivates ribosomes catalytically and that in the absence of nucleotides it binds to the 60‐S subunit in a specific way. The possibility of two binding sites for EF‐2 on the ribosomes is discussed.
Translation of Vitellogenin mRNA in the Presence of 7‐Methylguanosine 5′‐Triphosphate
Authors: WILLEMS Margherita , WIERINGA Bé , MULDER Janny , AB Geert , GRUBER Max
Published: March 2005

Messenger RNAs for total estrogenized chicken liver proteins and for two of their major constituents (vitellogenin and serum albumin), globin mRNA and Mengo virion RNA were translated in the wheat germ system and in the mRNA‐dependent reticulocyte lysate in the presence of 7‐methylguanosine 5′‐triphosphate. Protein synthesis inhibition was studied as a function of the concentration of cap analog, mRNA and translational factors. The following observations were made. (a) Synthesis directed by total poly(A)‐containing RNAs, purified vitellogenin and serum albumin mRNAs could be completely inhibited by cap analogs; messenger RNA in poysomal RNA preparations was less efficiently inhibited than purified mRNAs. (b) Monitoring immunologically translation of the population of liver messengers, vitellogenin mRNA was found to be more inhibited than serum albumin mRNA. (c) Cap analogs inhibited translation of the capped globin mRNA competitively in the wheat germ system. (d) By increasing the concentration of translational factors inhibition could be reversed. (e) Cap analogs did not inhibit translation of the uncapped Mengo virion RNA in the reticulocyte lysate but did do so, partially and not competitively, in the wheat germ system. (f) By increasing the concentration of wheat germ extract the inhibitable portion of mengo‐directed synthesis increased.

These results support speculation that cap analogs compete with mRNAs on the basis of affinity for initiation complex formation. Implications regarding the behaviour of the intact reticulocyte lysate and of uncapped viral RNAs in presence of cap analog are discussed.
Determination of the Interaction of ADP and dADP with Copper(II), Manganese(II) and Lanthanide(III) Ions by Nuclear‐Magnetic‐Resonance Spectroscopy
Authors: FAZAKERLEY G. Victor , REID David G.
Published: March 2005

The aqueous solution conformation of the 1:1 complexes of ADP and dADP bound to a lanthanide ion have been determined by examination of the dipolar shifts and induced relaxation at pH 6.4. Apparent inconsistencies in the observed data are interpreted in terms of a gradually changing lanthanide‐oxygen bond length from Pr3+ to Yb3+. The conformations of ADP and dADP are very similar showing an extended diphosphate, a 2E ribose conformation and with the adenine base displaying a small syn contribution. Relaxation data obtained from Mn(II) titrations are readily interpreted in terms of bidentate coordin ation to α and β phosphates with the nucleotides retaining the same overall conformation found from the lanthanide study. No evidence to support an intramolecular water‐bridged background structure is observed. The interaction with Cu(II) is more complex, coordination is observed not only at the diphosphate but also at two sites on the base, N‐1. and the chelate formed between N‐7 and the amino group. The relative importance of these sites is different for ADP and dADP and is also pH‐dependent for ADP.
Poly(adenylic acid)‐Containing RNA of Euglena gracilis during Chloroplast Development
Authors: VERDIER Gérard
Published: March 2005

During light‐induced chloroplast development of dark‐grown Euglena gracilis, changes in the polyadenylated RNA distribution have been studied by hybridization of complementary DNA (cDNA) to poly(A)‐containing RNA from different preparations: total cell polyribosomes of dark‐grown cultures illuminated for 0, 1, 6, 24 h, and isolated chloroplasts extracted after 24 h of illumination.

The hybridization kinetics revealed three or four frequency classes in the different poly(A)‐containing RNA populations. The high complexity of the poly(A)‐containing RNA sequences synthesized after only 1 h of illumination suggested a transcription mainly from nuclear genome origin. The disribution of poly(A)‐containing RNA was strongly modified after 24 h of illumination and a new class appeared corresponding to abundant sequences. At this stage of the greening, 73% of the poly(A)‐containing RNA sequences were common to the poly(A)‐containing RNA from‐grown cultures; so the sequences synthesized after illumination represented qualitatively a large diversity, but quantitatively, a low proportion of the poly(A)‐containing RNA population.

The majority of poly(A)‐containing RNA sequences obtained from isolated chloroplasts was also present in total cell polyadenylated RNA population after 24 h of greening. However. a class corresponding to the most abundant RNA sequences represented 50% of poly(A)‐containing RNA from isolated chloroplasts and only 11% of total cell poly(A)‐containing RNA preparation. The estimated complexities, either for this specific class or for the total polyadenylated RNA population from isolated chloroplasts, suggested the presence of RNA sequences from nuclear origin at the level of chloroplast preparations.
The Dicyclohexylcarbodiimide‐Binding Protein of the Mitochondrial ATPase Complex from Neurospora crassa and Saccharomyces cerevisiae
Authors: SEBALD Walter , GRAF Thomas , LUKINS Harold Bruce
Published: March 2005

Incubation of mitochondria from Neurospora crassa and Saccharomyces cerevisiae with the radioactive ATPase inhibitor [14C]dicyclohexylcarbodiimide results in the irreversible and rather specific labelling of a low‐molecular‐weight polypeptide. This dicyclohexylcarbodiimide‐binding protein is identical with the smallest subunit (Mr8000) of the mitochondrial ATPase complex, and it occurs as oligomer, probably as hexamer, in the enzyme protein.

The dicyclohexylcarbodiimide‐binding protein is extracted from whole mitochondria with neutral chloroform/methanol both in the free and in the inhibitor‐modified form. In Neurospora and yeast, this extraction is highly selective and the protein is obtained in homogeneous form when the mitochondria have been prewashed with certain organic solvents. The bound dicyclohexylcarbodiimide label is enriched in the purified protein up to 50‐fold compared to whole mitochondria.

Based on the amino acid analysis, the dicyclohexylcarbodiimide‐binding protein from Neurospora and yeast consists of at least 81 and 76 residues, respectively. The content of hydrophobic residues is extremely high. Histidine and tryptophan are absent. The N‐terminal amino acid is tyrosine in Neurospora and formylmethionine in yeast.
A Histone Programme during the Life Cycle of the Sea Urchin
Authors: BRANDT Wolf F. , STRICKLAND Walter N. , STRICKLAND Marie , CARLISLE Lesley , WOODS Derek , VON HOLT Claus
Published: March 2005

The histone complement of chromatin from early gastrula, late gastrula and from fully differentiated gut cells of the sea urchin Parechinus angulosus has been fractionated by molecular sieve and ion‐exchange chromatography. Several of the subfractions thus isolated have been characterized by amino acid composition and partial amino acid sequences as a series of variants of the histones H1, H2A and H2B. Specific histone variants are present in chromatin at specific stages of differentiation.
Lipid Composition of Human Malignant Melanoma Tumors at Various Levels of Malignant Growth
Authors: PORTOUKALIAN Jacques , ZWINGELSTEIN Georges , DORÉ Jean‐François
Published: March 2005

The lipid pattern of thirteen human melanoma tumors from various tissues were investigated. In seven of the tumors, an estimate was given about the proportion of malignant melanocytes to the total cell population, and a reverse correlation was determined between the proportion of malignant cells in these tumors and their neutral lipid content. The phospholipids did not show any modification, nor did the cholesterol in the cancerous tissues. The ganglioside pattern was found to be similar in all analyzed samples, with GM3, GM2 and GD3 as major components, although no correlation was found between the malignant level and the ganglioside content of the tumors.
Immunochemical Studies of Partially Hydrolyzed Lipopolysaccharide from Fusobacterium nucleatum Fev1
Authors: HOFSTAD Tor , FREDRIKSEN Gjert
Published: March 2005

Fusobacterium nucleatum Fev1 lipopolysaccharide was split by hydrolysis with 1% acetic acid into acid‐soluble polysaccharide and lipid A. Gel filtration of the polysaccharide on Bio‐Gel P‐60 gave a high‐molecular‐weight fraction eluted with the void volume, and a fraction eluted at 2.4 ×V0. The high‐molecular‐weight fraction contained L‐glycero‐D‐manno‐heptose in relatively large amounts, glucose, glucosamine, an unknown amino compound and small amounts of (or no) D‐glycero‐D‐manno‐heptose. Phosphorus and 3‐deoxy‐D‐manno‐octulosonic acid were not detected. The other fraction contained L‐ and D‐glycero‐D‐manno‐heptose, glucose, glucosamine, 3‐deoxy‐D‐manno‐octulosonic acid and phosphorus. Further fractionation experiments and serological investigations indicated that the high‐molecular‐weight fraction carried the O‐antigenic side chains, whereas the material eluted from Bio‐Gel P‐60 at 2.4 ×V0 represented the core oligosaccharide.
Limited Proteolysis of the Pyruvate Dehydrogenase Multienzyme Complex of Escherichia coli
Authors: HALE Geoffrey , PERHAM Richard N.
Published: March 2005

Incubation of the pyruvate dehydrogenase multienzyme complex of Escherichia coli with trypsin at pH 7.0 leads to limited proteolysis of the constituent polypeptide chains and, more slowly, to loss of enzymic activity. The core component, lipoate acetyltransferase, is the most susceptible to tryptic cleavage, whereas the lipoamide dehydrogenase component is apparently unaffected. The susceptiable peptide bonds in lipoate acetyltransferase involve lysine residues, probably on exposed loops of polypeptide chain.

No protection against the action of trypsin is afforded to the individual enzymes by their self‐assembly into the complex, suggesting that no gross conformational changes in the subunits take place on assembly.

The principal large fragments of lipoate acetyltransferase chains produced by limited tryptic digestion of native complex have molecular weights (estimated by sodium dodecylsulphate/polyacrylamide gel electrophoresis) of about 39000 and 36000. The fragment of molecular weight 36000 appears to be the ultimate main product. This fragment, presumably a folding domain of some kind, may be represented twice in the lipoate acetyltransferase chain (assumed molecular weight 83000).

The major product of tryptic digestion of the native complex remains of high molecular weight. Similarly, the proteolysed lipoate acetyltransferase/lipoamide dehydrogenase subcomplex retains its ability to form a complex of high molecular weight with pyruvate decarboxylase, the third component enzyme. The large folding domains of the lipoate acetyltransferase chains appear to be of major importance in the maintenance of subunit interactions in the enzyme complex.

These results with trypsin provide a satisfying rationale for the degradation of the native complex by endogenous proteinases encountered in earlier work.
Proteolysis of Human Ceruloplasmin
Authors: MOSHKOV Kirill A. , LAKATOS Susan , HAJDU János , ZÁVODSZKY Péter , NEIFAKH Solomon A.
Published: March 2005

Highly purified human ceruloplasmin was isolated from fresh donor blood in the presence of inhibitors of proteolysis and from stored retroplacental blood serum both with and without inhibitors of proteolysis. According to the data of electrophoresis. ultracentrifuge sedimentation velocity and sedimentation equilibrium, all the ceruloplasmin samples were homogeneous, their molecular weight being 130 000.

The dissociation of the samples treated by dodecylsulphate, guanidine · HCl, and urea was studied by means of quantitative analytica and preparative electrophoresis, and sedimentation equilibrium. The dissociation patterns depended on whether inhibitors were used in the isolation procedure. Polypeptides with molecular weights of 130 000, 112 000 and 16 000 (minor component) were obtained, if phenylmethylsulfonyl fluoride and/or 6‐aminohexanoic acid were used; if these compounds were not used, the dissociation yielded additional polypeptides of molecular weights of 100 000 (minor component), 64 000 and 48 000. Under proteolysis‐favouring conditions the relative amount of these polypeptides increased. Prolonged storage of samples under sterile conditions without inhibitors of proteolysis resulted in a decrease of the relative amount of polypeptides with molecular weights of 130 000, 112 000, 100 000, 64 000 and 48 000, contrary to that of the 16 000‐Mr polypeptide, which increased. At the same time new polypeptides appeared with molecular weights of 42 000 and 21 500–23 000.

Spontaneous specific fragmentation of the ceruloplasmin molecule is due to trace amounts of proteases, which seem to originate from blood plasma.

Limited tryptic hydrolysis of the ceruloplasmin globule resulted in the appearance of polypeptides with the same molecular weights which were observed in spontaneous fragmentation.

A conclusion is drawn that the ceruloplasmin molecule in vivo is a single polypeptide chain with at least five bonds which in vitro are the points of specific proteolytic fragmentation, yielding six principal fragments.