bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and Characterisation of a Membrane‐Bound Substance‐P‐Degrading Enzyme from Human Brain
Authors: LEE Chi‐Ming , SANDBERG Bengt E. B. , HANLEY Michael R. , IVERSEN Leslie L.
Published: March 2005

A membrane‐bound enzyme which degrades substance P (an undecapeptide) has been purified from human brain. The properties of this enzyme suggest that it may be involved in the physiological inactivation of the peptide by neural tissues. Enzyme activity was extracted from a membrane fraction of human diencephalon with a non‐ionic detergent, Brij 35, and activity was monitored by measuring the disappearance of added substance P using radioimmunoassay, bioassay or radiochemical assay. The enzyme was purified about 1000‐fold by chromatography on DEAE‐cellulose, hydroxyapatite and Sephadex gel filtration columns. To identify the cleavage sites in substance P, the peptide was incubated with the purified enzyme and the breakdown products were separated by reverse‐phase high‐performance liquid chromatography and identified by amino acid analysis. The results suggested that the enzyme preparation was functionally homogeneous and it cleaved substance P between Gln6–Phe7, Phe7–Phe8 and Phe8‐Gly9, with no exopeptidase action. The enzyme had a pH optimum in the range 7–9 and was strongly inhibited by metal‐chelating agents, but not affected by most other peptidase inhibitors; it can thus be classified as a neutral metallo‐endopeptidase. The enzyme was thermolabile and had a molecular weight of 40000–50000 as estimated by gel filtration, density‐gradient ultracentrifugation and sodium dodecylsulphate gel electrophoresis. The highly purified substance‐P‐degrading enzyme could be distinguished from previously described peptidases for which substance P is a substrate. An important feature was that substance P was the preferred substrate among various other neuropeptides tested.
Pyruvate Dehydrogenase Component of the Pyruvate Dehydrogenase Complex from Escherichia coli K12
Authors: SAUMWEBER Harald , BINDER Renate , BISSWANGER Hans
Published: March 2005

Free pyruvate dehydrogenase component of the Escherichia coli K12 pyruvate dehydrogenase complex was isolated from a mutant lacking the dihydrolipoamide transacetylase component. The procedure, employing three chromatographic steps, yields a product that is electrophoretically pure. The purified enzyme reassociates with the residual complex lacking this component to a fully active enzyme complex.

The kinetic characteristics of the free component were compared to that of the enzyme integrated into the native complex molecule. No essential differences could be detected regarding the behaviour of the catalytic reaction with variations in temperature, pH and substrate concentration. An inhibition, competitive to pyruvate, of the pyruvate dehydrogenase component (Ki= 18 μM) by fluoropyruvate was observed with both enzyme forms.

Pyruvate exerts a cooperative effect both upon the partial enzyme reaction of the pyruvate dehydrogenase component as well as upon the overall reaction of the native enzyme complex. However, there is a clear difference in the shape of the saturation curves of both types of reaction. Experiments with the free enzyme, with the component integrated into the native complex molecule and with enzyme complexes which are partially deficient in the pyruvate dehydrogenase component demonstrate that the type of saturation curves obtained were characteristic for the reaction observed rather than for the interaction of a high number of subunits of the pyruvate dehydrogenase complex.
The Major Components of the Mouse and Human Genomes
Authors: CUNY Gérard , SORIANO Philippe , MACAYA Gabriel , BERNARDI Giorgio
Published: March 2005

Main‐band DNA from mammals and birds can be resolved by density gradient centrifugation techniques into three or four families of fragments of different dG + dC contents. These major DNA components are similar in their buoyant densities and relative amounts in all species tested and are observed in DNA preparations ranging in Mr from 2 × 106 to over 200 × 106.

In the present work, the four major components of mouse and human DNAs were prepared and characterized in several basic properties: relative amounts, dG + dC contents, buoyant densities and compositional heterogeneity. The results obtained lead to the following conclusions: (a) the major DNA components of mouse and man form at least 85% and possibly the totality of the main bands of these DNAs; (b) they have very low compositional heterogeneities over a wide molecular weight range; (c) they derive from very large chromosomal DNA segments of fairly homogeneous base composition, for which the name ‘isochores’ is proposed.

A comparison of the compositional heterogeneity of main‐band DNAs from warm‐blooded and cold‐blooded vertebrates confirms our previous conclusion that these DNAs are characterized by a different sequence organization.
Sequence Diversity of Nuclear and Polysomal Polyadenylated and Non‐polyadenylated RNA in Normal and Regenerating Rat Liver
Authors: GRADY Leo J. , CAMPBELL Wayne P. , NORTH Arlene B.
Published: March 2005

A DNA probe purified from RNA · DNA hybrids of total sham‐operated liver RNA and non‐repetitive DNA was used to show that nuclear poly(A)‐rich RNA from sham‐operated liver and from 2.5‐h and 48‐h regenerating liver contains about 50% of the complexity of total liver RNA. It was further shown that the differences between normal and regenerating liver, reported earlier from this laboratory, occur in the poly(A)free fraction of nuclear RNA. At the polysome level it was found that polysomal RNA has one‐third of the sequence diversity of total RNA and of this, approximately 65 % can be accounted for by poly(A)‐rich and 55 by poly(A)‐free molecules. When DNA probes were prepared from hybrids formed using polysomal RNA from sham‐operated liver and regenerating liver at 2.5 h and 48 h post‐hepatectomy and then employed in reactions with homologous and heterologous RNAs, no differences were detectable between either normal and regenerating liver or regenerating liver at times during hypertrophy and hyperplasia.
Activation of Lymphocyte Populations with Concanavalin A or with Lipoprotein and Lipopeptide from the Outer Cell Wall of Escherichia coli Correlation of Early Membrane Changes with Induction of Macromolecular Synthesis
Authors: RESCH Klaus , BESSLER Wolfgang
Published: March 2005

The occurrence of early membrane changes following the interaction of mouse lymphocytes with the B‐lymphocyte mitogens, lipoprotein or lipopeptide, and with the T‐lymphocyte mitogen concanavalin A was compared to the induction of RNA and DNA synthesis induced by the mitogens in several lymphocyte populations. Membrane alterations were determined by measuring the incorporation of phospholipid precursors into phosphatidylcholine after the addition of the mitogens. It was shown that both B‐cell and T‐cell mitogens caused an increase in the incorporation of [14C]oleate and [14C]acetate in T B‐cell mixtures after 4 h of stimulation. The B‐cell mitogens, lipoprotein and lipopeptide, induced a marked incorporation of phospholipid precursors in spleen and lymph node cells of athymic mice. Corresponding to their cell specificity for the induction of macromolecular synthesis, no membrane changes were induced by the bacterial mitogens in thymocytes. On the other hand, the T‐cell mitogen concanavalin A was active in causing marked membrane changes in thymocytes. Remarkably, however, concanavalin A also induced the incorporation of phospholipid precursors in spleen and lymph node cells of congenitally athymic mice. Since membrane events in response to concanavalin A were not found after treating normal spleen and lymph node cells with anti‐Thyl serum and complement, the positive response observed in athymic mice probably resulted from the presence of pre‐T‐lymphocytes.

Our results show that early alterations in membrane phospholipid metabolism in mature B or T lymphocytes are correlated with the subsequent induction of macromolecular synthesis, suggesting that these membrane changes are causally linked to cell activation. In addition the data suggest a similar molecular mechanism for the activation of lymphocytes by both T‐cell or B‐cell mitogens.
Multiple Forms of Rat‐Serum α1‐Protease Inhibitor
Authors: IKEHARA Yukio , MIYASATO Mihoko , OGATA Shigenori , ODA Kimimitsu
Published: March 2005

α1‐Protease inhibitor was purified from rat serum by two different methods, of which an immunoaffinity method should be preferentially used to obtain all of the multiple forms. The protein thus obtained showed a single protein band in dodecylsulphate/polyacrylamide gel electrophoresis corresponding to a molecular weight of 54000, and contained 13.2% carbohydrate by weight. By column isoelectric focusing in a pH 3.5–5.0 gradient the purified α1‐protease inhibitor was separated into five forms with pl values from 4.3–4.7. The amino acid composition of each form was identical, while sialic acid content was significantly different from each other. The most acidic form contained 6.7 residues/molecule, the most basic form, 5.1 residues/molecule, and three forms between them showed proportionally intermediate values between the two.

When α1‐protease inhibitor was treated with neuraminidase, the five forms were converted finally into three major forms with pl values of 5.3–5.7. In addition, the major form (band 3) of the inhibitor was also converted into three forms after complete removal of sialic acid. These results suggest that α1‐protease inhibitor originally exists as three forms with different charges, possibly due to modification of amino acids which might not be detectable by the amino acid composition analysis in the present study. A possible explanation was presented for involvement of sialic acid in appearance of multiple forms originating from three parental forms.
Photochemical Cross‐Linking of Elongation Factor G to 70‐S Ribosomes from Escherichia coli by 4‐(6‐Formyl‐3‐azidophenoxy)butyrimidate
Authors: MAASSEN J. Anthonie , MÖLLER Wim
Published: March 2005

Ribosomal proteins situated at or near the binding site of elongation factor G (EF‐G) on the Escherichia coli ribosome have been identified by use of the heterobifunctional cross‐linker 4‐(6‐formyl‐3‐azidophenoxy)butyrimidate. Four different preparations of EF‐G, in which the number of cross‐linker molecules coupled to EF‐G ranged from four to seven, all cross‐linked to 50‐S subunit proteins L1, L3 and L11 as well as to 30‐S subunit proteins S3 and S4.

Cross‐linking of EF‐G to ribosomal proteins was tested electrophoretically. In the case of L7/L12 and L11 immunological methods were also used.

Cross‐linking of EF‐G to L1, L3, L11, S3 and S4 is specific as judged from the fact that addition of unmodified EF‐G and of thiostrepton results in less cross‐linking. The cross‐linking data suggests that the binding site for EF‐G includes several proteins which are located at the interface between the 30‐S and 50‐S subunits.
A Bacterial Glucoamylase Degrading Cyclodextrins
Authors: BENDER Hans
Published: March 2005

A Flavobacterium species has been isolated which produces a cyclodextrin‐degrading glucoamylase. The inducible, cell‐bound enzyme was purified about 10‐fold to 75% purity in 57% yield. The action of the enzyme was studied with the main cyclodextrins (cyclohexaamylose, cycloheptaamylose and cyclooctaamylose) and with the typical glucoamylase substrates, respectively. The final degradation product with all the substrates was glucose. Small amounts of maltose, which could be detected in the course of cyclodextrin degradation, were hydrolyzed at a lower rate. V for cyclohexaamylase was found to be about 14–15 μmol glucose min−1 (mg pure protein)−1. the Km for cyclobexaamylose was 0.142 mM. Apparently the enzyme preferred shorter α‐d‐glucopyranosyl chains. Besides maltose, amylopectin and glycogen proved to be very poor substrates. Some properties of the enzyme have been described.
Circular Dichroism Study of Stacking Properties of Oligodeoxyadenylates and Polydeoxyadenylate
Authors: OLSTHOORN Cornelis S. M. , BOSTELAAR Lein J. , DE ROOIJ Jan F. M. , VAN BOOM Jacques H. , ALTONA Cornelis
Published: March 2005

The temperature dependence of the circular dichroism (CD) spectra of a series of deoxyadenylates (dA)n, n= 2, 3, 6, 9, 12, ∞, in aqueous solution was studied. The data were interpreted on the basis of a new conformational model for the stacked state suggested by our previous proton NMR studies on (dA)2 and (dA)3 [C. S. M. Olsthoorn, L. J. Bostelaar, J. H. van Boom & C. Altona (1980) Eur. J. Biochem. 112, 95–110]. In this model the stacked regions of the single‐stranded oligomers consist of residues taking up a geometry resembling that of the B‐DNA genus of structures (all sugars S or C2′‐endo) except those residues at the 3′ end that do not ‘feel’ a following stacking interaction. The deoxyribose rings in the latter residues retain (or regain when melting out removes a stacking interaction somewhere along the chain) the conformational freedom (S ⇄ N, N = C3′‐endo) that these rings possess in the monomers 2′‐deoxvadenosine 5′‐methylphosphate or in 2′‐deoxyadenosine 3′,5′‐bis(methylphosphate). as the case may be. It is shown that this model allows (a) construction of the CD spectra of (dA)n, n= 3. 6. 9, 12. from those of the dimer and the polymer; (b) the separation of the weak CD displayed by the regular S‐S stacking mode and the far stronger CD exhibited by the 3′‐end S‐N stacking (the latter CD resembles that of the A‐DNA genus of structures); (c) delineation of the thermodynamics of stacking. The melting temperature remains constant and independent of chain length (about 50° C whereas ΔH0 and ΔS0 show a slight increase in absolute values on increasing n from 2 to ∞ owing to small cooperativity effects. Near 0° C the dimer occurs for about 90% in the stacked form, the oligomers attain even higher conformational purities. It is suggested that premelting phenomena observed in the CD spectra of double‐helical DNAs may also involve local transitions from the normal B‐like—‐S‐S‐S—‐stacking mode to an A‐like—‐S‐S‐N—‐stacking geometry.
The ATP Substrate Site of a Cyclic‐Nucleotide‐Independent Protein Kinase from Porcine Liver Nuclei
Authors: BAYDOUN Hassan , HOPPE Jürgen , FREIST Wolfgang , WAGNER Karl G.
Published: March 2005

The ATP substrate site of a second messenger‐independent protein kinase of the type NIT from porcine liver nuclei was mapped using a series of 30 ATP derivatives with modifications at the base, ribose or triphosphate moiety. Ki values for these derivatives were determined by competition with [γ‐32P]ATP; they range from 4 μM to 1.5 mM. For a comparison with data previously reported for the catalytic subunit of cAMP‐dependent protein kinase I from rabbit skeletal muscle, the Ki values were transformed into δΔG values. These values are related to the Ki value of unsubstituted ATP and indicate the decrease of affinity caused by the different substitutions.

With both enzymes the major binding affinity is derived from the interaction of the adenine base. The contributions of the two ribosyl OH groups are marginal and the triphosphate moiety interacts most strongly with its β‐phosphoryl group. Between the two enzymes the most striking differences, however, were observed for the specificity of the nucleobase interaction. While an unmodified N‐6 amino group is required in the case of the cAMP‐dependent protein kinase, the nuclear enzyme seems to tolerate extensive modification at this position, such as the introduction of a keto group or a bulky benzyl residue. Obviously, the ATP site of the nuclear kinase has an open cleft next to the N‐6 of the adenine and binding of the adenine occurs by hydrophobic interaction without the formation of hydrogen bonds to any of the adenine nitrogens.
Glycosaminoglycan Biosynthesis in Arterial Wall
Authors: LEVY Peggy , PICARD Jacques , BRUEL Arlette
Published: March 2005

Incubation of microsomal fractions with labelled 3′‐phosphoadenylyl sulfate results in incorporation of [35S]sulfate into endogenous glycosaminoglycans. Specific radioactivity observed incorporated into heparan sulfate chains is 10‐fold greater than that incorporated into chondroitin sulfate chains. This is in agreement with the results obtained for glycosylation of glycosaminoglycans in arterial wall membrane fractions. Sulfation of heparan sulfate was studied since it contains N‐ and O‐sulfate groups in contrast with the other sulfated glycosaminoglycans which contain only O‐sulfate groups.

Sulfation of heparan sulfate occurs rapidly, since sulfate incorporation is detected after exposure for only 0.5 min.

Heparan sulfate was identified on the basis of its resistance to hyaluronidase and chondroitin ABC lyase, its susceptibility to heparitinase, its sensitivity to nitrous acid and the presence of glucosamine as the only hexosamine.

The chemical composition of the purified heparan sulfate fractions provides evidence for the high degree of sulfation of its chains.

Studies into the distribution of sulfate residues on heparan sulfate at different times of sulfation indicate that N‐sulfate groups are not randomly introduced into the polymer.

The relationship between the processes of N‐ and O‐sulfation was studied. The present results demonstrate that preferential N‐sulfation is obtained for incorporation of labelled precursor over a short period, the O‐sulfation occurring on previously N‐sulfated heparan sulfate.
Changes in Amount of Hypo‐Modified tRNA Having Guanine in Place of Queuine during Erythroid Differentiation of Murine Erythroleukemia Cells
Authors: SHINDO‐OKADA Nobuko , TERADA Masaaki , NISHIMURA Susumu
Published: March 2005

The amounts of hypo‐modified tRNAs having guanine in place of queuine in murine erythroleukemic cells decreased markedly when the cells differentiated into mature erythroid cells. The amounts of these hypo‐modified tRNAs can be determined easily by measuring incorporation of labeled guanine into tRNA with Escherichia coli tRNA‐guanine transglycosylase. The decrease was detected at on early stage of erythroid differentiation: namely, before any detectable increase in the percentage of cells containing hemoglobin. The amount of guanine‐accepting tRNA species was nearly proportional to the percentage of undifferentiated cells in the population, regardless of the type of inducer used. Decrease in the amounts of hypo‐modified tRNAs in the cells was effectively blocked by 12‐O‐tetradecartoylphorbol 13‐acetate, which inhibits differentiation of these cells.

8‐Azaguanine, which is known to be substrate of tRNA—guanine transglycosylase. was incorporated almost exclusively into the first position of hypo‐modified tRNA in murine erythroleukemic cells when they were pulselabeled in culture with 8‐azaguanine, suggesting strongly that tRNA‐guanine transglycosylase in the cells is actually involved in incorporation of 8‐azaguanine into tRNA in vivo. The amount of 8‐azaguanine incorporated into tRNA in differentiated cells was one third of that in undifferentiated cells, the decrease being parallel with that in the amount of guanine‐accepting tRNA in these cells. The results suggest that the appearance of hypomodified tRNAs in the transformed cells was due to lack of substrate for queuosine biosynthesis in tRNA.
Different Transport Pathways of Individual Precursor Proteins in Mitochondria
Authors: ZIMMERMANN Richard , HENNIG Bernd , NEUPERT Walter
Published: March 2005

Transport of mitochondrial precursor proteins into mitochondria of Neurospora crassa was studied in a cellfree reconstituted system. Precursors were synthesized in a reticulocyte lysate programmed with Neurospora mRNA and transported into isolated mitochondria in the absence of protein synthesis. Uptake of the following precursors was investigated: apocytochrome c, ADP/ATP carrier and subunit 9 of the oligomycin‐sensitive ATPase.

Addition of high concentrations of unlabelled chemically prepared apocytochrome c (1–10 μM) inhibited the appearance in the mitochondrial of labelled cytochrome c synthesized in vitro because the unlabelled protein dilutes the labelled one and because the translocation system has a limited capacity [apparent V is 1–3 pmol × min−1× (mg mitochondrial protein)−1]. Concentrations of added apocytochrome c exceeding the concentrations of precursor proteins synthesized in vitro by a factor of about 104 did not inhibit the transfer of ADP/ATP carrier or ATPase subunit 9 into mitochondria. Carbonylcyanide m‐chlorophenylhydrazone, an uncoupler of oxidative phosphorylation, inhibited transfer in vitro of ADP/ATP carrier and of ATPase subunit 9, but not of cytochrome c.

These findings suggest that cytochrome c and the other two proteins have different import pathways into mitochondria. It can be inferred from the data presented that different ‘receptors’ on the mitochondrial surface mediate the specific recognition of precursor proteins by mitochondria as a first step in the transport process.
Large Globin RNA Molecules and Their Processing
Authors: SHAUL Yosef , KAMINCHIK Joseph , AVIV Haim
Published: March 2005

RNA containing β‐globin message sequences larger than 2000 nucleotides could be clearly detected in nuclei of murine erythroid cells using cloned β‐globin cDNA. Under steady‐state conditions, when nuclear RNA was separated on denaturing agarose gels and covalently bound to diazobenzyloxymethyl‐paper, a 4200‐nucleotide and a ∼ 3500‐nucleotide band could be seen. The presence of these large molecules could also be visualized under the electron microscope after hybridization to a β‐globin genomic DNA fragment. We suggest that these molecules are precursors to mature mRNAs. In addition to these large molecules, a series of molecules smaller than 2000 nucleotides were seen. These are postulated to be processing intermediates in the maturation of β‐globin mRNA.
Purifieation of a Soluble, Sodium‐Nitroprusside‐Stimulated Guanylate Cyclase from Bovine Lung
Authors: GERZER Rupert , HOFMANN Franz , SCHULTZ Günter
Published: March 2005

A soluble, sodium‐nitroprusside‐stimulated guanylate cyclase has been purified from bovine lung by DEAE cellulose chromatography, ammonium sulfate precipitation, chromatography on Blue Sepharose CL‐6B and preparative gel electrophoresis. Apparent homogeneity was obtained after at least 7000‐fold purification with a yield of 3%. A single stained band (Mr 72000) was observed after gel electrophoresis in the presence of sodium dodecyl sulfate. The purified enzyme migrated as one band also under non‐denaturing conditions in acrylamide gels (5–12%). The mobility of this band corresponded to an Mr of 145000. The enzyme sedimented on sucrose gradients with an S20.w of 7.0 S. Gel filtration yielded a Stokes' radius of 4.6 nm. These data suggest that the enzyme has an Mr of approximately 150000 and consists of two, presumably identical, subunits of Mr 72000. Sodium nitroprusside stimulated the purified enzyme 15‐fold and 140‐fold to specific activities of 8.5 and 15.7 pmol cGMP formed min−1 mg−1 in the presence of Mn2+ and Mg2+, respectively. Formation of cGMP was proportional to the incubation time and to the amount of enzyme added. The stimulatory effect of sodium nitroprusside was half‐maximal at about 2 μM. was observed immediately after addition and could be reversed either by dilution or by removal of sodium nitroprusside on a Sephadex G‐25 column. The purified enzyme in the absence of catalase was stimulated by sodium nitroprusside, N‐methyl‐N′‐nitro‐N‐nitrosoguanidine and 3‐morpholino‐sydnonimine and in the presence of catalase by sodium nitrite and sodium azide. In the presence of Mn2+ and sodium nitroprusside, the purified enzyme catalyzed the formation of cAMP from ATP at a rate of 0.6 μmol min−1 mg−1.
The Mechanism of Uncoupling by Picrate in Escherichia coli K‐12 Membrane Systems
Authors: MICHELS Monika , BAKKER Evert P.
Published: March 2005

The mechanism of action of the uncoupler picrate on intact cells and everted membrane vesicles of Escherichia coli K‐12 was investigated. Like in mitochondria [Hanstein, W. G. and Hatefi, Y. (1974) Proc. Natl Acad. Sci. USA, 71, 288–292], it was observed that picrate uncoupled energy‐linked functions only in everted, but not in intact membrane systems. In the vesicles picrate also decreased the magnitude of the transmembrane proton‐motive force at concentrations similar to those at which it caused uncoupling. Experiments with 14C‐labelled picrate showed that this compound bound both to deenergized intact cells and everted vesicles. However, upon energization of the membrane, picrate was extruded from the intact cell and taken up to a larger extent by the vesicles. These energy‐dependent changes in picrate uptake correlated with the magnitude of the transmembrane electrical potential, Jo. It is therefore proposed that picrate is a permeant uncoupler, that ΔΨ is the driving force for picrate movement across biological membranes, and that the uncoupling activity of picrate in everted membrane systems is due to its protonophoric action.
Biosynthesis and Export of Colicin A inCitrobacter freundiiCA31
Authors: VARENNE Stanislas , CAVARD Danielle , LAZDUNSKI Claude
Published: March 2005

Synthesis of colicin A after induction with mitomycin C was studied. Specific inhibition of chromosomal protein synthesis occurred very shortly after mitomycin addition. There was no coordinate synthesis of colicin A (61000Mr) and low‐molecular‐weight protein. Free and membrane‐bound polysome fractions were isolated from cells induced with mitomycin C. Colicin A is synthesizedin vitroin the free polysomes and not in the membranebound polysomes. Conditions are described which allow a practically specific labelling of colicin Ain vivo. By using this system it was possible to demonstrate that colicin A is not transferred cotranslationally across the cytoplasmic membrane. In contrast, this protein leaves the cell where it is made long after synthesis. Preliminary evidence. suggesting that pauses occur during synthesis of colicin A, is presented.
The Elongation Factor Tu · Guanosine Tetraphosphate Complex
Authors: PINGOUD Alfred , BLOCK Wolfgang
Published: March 2005

The elongation factor Tu (EF‐Tu) isolated from Escherichia coli cells that have undergone the stringent response is predominantly complexed with guanosine 5′‐diphosphate 3′‐diphosphate. This complex can be separated by anion‐exchange chromatography from EF‐Tu · GDP‐ Unlike EF‐Tu complexed with guanosine 5′‐triphosphate 3′‐diphosphate, the complex with guanosine 5′‐diphosphate 3′‐diphosphate cannot form a ternary complex with aminoacyl‐tRNAs as can be shown directly by hydrolysis protection experiments.
Concerning the Mode of Action of Micrococcin upon Bacterial Protein Synthesis
Authors: CUNDLIFFE Eric , THOMPSON Jill
Published: March 2005

The antibiotic, micrococcin, binds to complexes, formed between bacterial 23‐S ribosomal RNA and ribosomal protein L11 and, in doing so, inhibits the binding of thiostrepton. In assay systems simulating partial reactions of protein synthesis, micrococcin inhibits a number of processes believed to involve the ribosomal A site while stimulating GTP hydrolysis dependent upon ribosomes and elongation factor EF‐G. The latter effect is not observed upon ribosomes lacking a protein homologous with protein L11. Nor is it apparent upon those containing 23‐S RNA previously subjected to the action of a specific methylase known to render ribosomes resistant to thiostrepton. It is concluded that stimulation by micrococcin of factor‐dependent GTP hydrolysis results from the binding of the drug to its normal target site which involves 23‐S RNA and protein L11.
Biosynthesis of the Light‐Harvesting Chlorophyll a/b Protein
Authors: CUMING Andrew C. , BENNETT John
Published: March 2005

Antibodies raised against the 26000‐Mr polypeptides of the light‐harvesting chlorophyll a/b proteins of pea leaves specifically immunoprecipitated two 32000 ‐Mr polypeptides synthesized when pea leaf poly(A)‐containing RNA was translated in vitro. On the basis of immunochemical relatedness and by comparison of their partial tryptic digestion products, the 32000 ‐Mr products formed in vitro are identified as precursors to the authentic polypeptides of the light‐harvesting chlorophyll a/b complex.

The specificity of the immunoprecipitation permitted the development of an assay for the cellular levels of translationally active light‐harvesting protein mRNA in plants exposed to different light regimes. Low levels of the mRNAs were detectable in dark‐grown plants. Exposure to continuous illumination caused these levels to increase by at least ten‐fold and led to the appearance of large quantities of the light‐harvesting chlorophyll a/b complex. In plants exposed to intermittent illumination (2 min of white light every 2 h for 2 days), the light‐harvesting complex did not accumulate, although levels of mRNA specifying the polypeptides of the complex were high (50% of those in continuously illuminated plants).

Messenger RNAs encoding the light‐harvesting proteins were detected in polysomes of intermittently illuminated leaves. These polysomes were active in a wheat‐germ 100000 ×g supernatant ‘run‐off’ system, to form light‐harvesting protein precursors, under conditions when only nascent polypeptide chains initiated in vivo were elongated and terminated. These results demonstrate that the inability of intermittently illuminated leaves to accumulate the light‐harvesting proteins is not due to a selective inhibition of the translation of the corresponding mRNAs.

Intermittently illuminated leaves were labelled with [35S]methionine in darkness, and incorporation of radioisotope into the light‐harvesting proteins and their precursors was assayed immunologically. No pool of untransported or unprocessed 32000 ‐Mr precursor polypeptides could be detected in the soluble fraction (cytoplasm and stroma). However, low levels of the mature 26000 ‐Mr polypeptides were detected in the membrane fraction. It is concluded that the newly synthesized light‐harvesting chlorophyll a/b proteins fail to accumulate in intermittently illuminated leaves because they undergo rapid turnover. The site of light‐harvesting protein breakdown is probably the thylakoid membrane, and the cause of breakdown is probably the absence of chlorophyll a and chlorophyll b molecules that are required for eventual stabilization of the proteins within the photosynthetic membrane.
Mössbauer Spectroscopic Studies of the Nature of Centre X of Photosystem I Reaction Centres from the Cyanobacterium Chlorogloea fritschii.
Authors: EVANS E. Hilary , DICKSON Dominic P. E. , JOHNSON Charles E. , RUSH James D. , EVANS Michael C. W.
Published: March 2005

Reduced photosystem I samples, which give the electron paramagnetic resonance (EPR) signals associated with A, A and B, and A, B and X centres, have been studied using Mössbauer spectroscopy. The Mössbauer spectra obtained from each type of sample is different, which indicates that iron is associated with all three centres. The spectra are similar to those obtained from ferredoxins with 4Fe‐4S centres and were fitted with oxidized and reduced components, the relative proportions depending on the degree of reduction of the sample as monitored by EPR. The sample which gave only the A EPR signal showed about 26% of the reduced component, the sample which gave A and B EPR signals showed about 48% of the reduced component, whilethe sample which gave A, B and X EPR signals showed about 65% of the reduced component. The measurements are consistent with X being a 4Fe‐4S centre.
Energy Conservation in the Terminal Region of the Respiratory Chain of the Methylotrophic Bacterium Methylophilus methylotrophus
Authors: DAWSON Michael J. , JONES Colin W.
Published: March 2005

Reduced + CO minus reduced difference spectra of respiratory membranes prepared from methanol‐limited cultures of Methylophilus methylotrophus confirm the presence of three CO‐binding cytochromes i.e. cytochromes aa3, o and Cco. The kinetics of cyanide inhibition indicate that the respiratory chain of this organism is branched at the level of cytochrome c to two major terminal oxidases, viz. cytochromes aa3 and o; cytochrome Cco is probably not a physiologically significant oxidase. Determination of proton and charge translocation stoichiometries (→ H+/O and → K +/O quotients) during oxidation of ascorbate‐N,N,N′,N′ ‐tetramethyl‐p‐phenylenediamine shows that the terminal oxidase system of this organism exhibits a net inward translocation of 2e−, but no net proton translocation, when a pair of electrons are passed from cytochrome c to oxygen. The use of appropriate concentrations of cyanide to selectively inhibit cytochrorne o indicates that the overall translocation stoichiometries are achieved by the two oxidases, aa3 and o, functioning similarly. These and other results suggest that methanol oxidase is organised as a simple redox arm with the metnanol oxidation site and the oxygen consumption site(s) on the periplasmic and cytoplasmic faces of the inner membrane respectively.
Synergism Between Coenzyme and Carboxylate Binding to Liver Alcohol Dehydrogenase
Authors: ANDERSSON Pia , KVASSMAN Jan , OLDÉN Bertil , PEJTERSSON Göata
Published: March 2005

The interaction of decanoate and benzoate with the substrate‐binding site in liver alcohol dehydrogenase has been characterized by fluonmetric equilibrium binding studies, using auraimne O as a reporter ligand.

The affinity of the enzyme for the carboxylates examined decreases about 50‐fold on complex formation with NADH. The coenzyme‐competitive inhibitors ADP‐ribose and Pt(CN)2−4 have a similar destabilizing effect on decanoate binding, indicating that the effect of NADH derives from its negatively charged pyrophosphate group.

Carboxylate binding to free enzyme requires the protonated form of an ionizing enzymic group with pKa 9.2, while there is no corresponding effect of pH on binary complex formation with auramine O. Carboxylate binding to the enzyme · NADH+ complex exhibits no significant dependence on pH over the pH range 7–10.

These results, combined with previously reported data for decanoate binding to the enzyme · NAD+ complex, provide clear evidence that carboxylates (in contrast to auramine O) are bound at the active‐site zinc ion of the enzyme in a process regulated by the ionization state of zinc‐bound water. The synergistic effect of NADH and NAD+ on carboxylate binding can be qualitatively and quantitatively explained in terms of electrostatic interactions analogous to those proposed to account for the effect of coenzymes on the pKa of zinc‐bound water.
Specifically Primed Synthesis in vitro of Full‐Length DNA Complementary to Potato‐Spindle‐Tuber Viroid
Authors: ROHDE Wolfgang , SCHNÖLZER Martina , RACKWITZ Hans‐Richard , HAAS Bernd , SELIGER Hartmut , SÄNGER Heinz L.
Published: March 2005

Potato spindle tuber viroid (PSTV) RNA is transcribed in vitro by reverse transcriptase into complementary DNA in the presence of synthetic oligodeoxyribonucleotides as primers. In the case of priming with the pentadecadeoxyribonucleotide d(T‐T‐C‐T‐T‐T‐T‐T‐T‐C‐T‐T‐T‐T‐C) complementary to PSTV RNA from nucleotides 49 to 63, specificity of transcription initiation allows rapid sequencing of part of the viroid genome using chain‐terminating dideoxyribonucleoside triphosphates. The DNA transcripts obtained represent distinct molecular species with the largest product being a full‐length copy of the viroid RNA template. Molecular hybridization with 32P–labeled complementary DNA detects sequence homologies among different viroid species.
Localization of the Phosphoester Bond Hydrolyzed by the Major Apurinic/Apyrimidinic Endodeoxyribonuclease from Rat‐Liver Chromatin
Authors: VERLY Walter G. , COLSON Pierre , ZOCCHI Germaine , GOFFIN Colette , LIUZZI Michel , BUCHSENSCHMIDT Gisèle , MULLER Marc
Published: March 2005

The major apurinic/apyrimidinic (AP) endodeoxyribonuclease from rat liver chromatin, an enzyme specific for AP sites in DNA, cleaves the phosphodiester bridge which is the immediate neighbour of the AP site on its 5′ side leaving 3′‐hydroxyl and 5′‐phosphate ends. In contrast with Escherichia coli endonuclease VI, this chromatin enzyme is inactive on reduced AP sites.
The Specific Velocity Plot
Authors: BAICI Antonio
Published: March 2005

A new method is described for plotting kinetic results for inhibited enzymatic reactions. The plot is particularly designed for the analysis of hyperbolic, mixed‐type inhibitors, but can be applied to any other linear system. The method consists of plotting experimental data in a normalized way, as v0/vi versus the specific velocity σ/(1 +σ), where v0/vi represents the ratio of the initial velocities for the non‐inhibited and inhibited reactions at a given substrate concentration [S], and σ is the [S]/Km ratio. The procedure provides a simple way of determining the inhibition constatnts Ki and K′i, i.e. the dissociation constants of the EI and ESI complexes, respectively, and the velocity saturation values.
A Comparison of Purified Poly (ADP‐ribose) Polymerases from Ehrlich Ascites Tumor Cells, Pig Thymus, and HeLa S3 Cells
Authors: HOLTLUND Jostein , KRISTENSEN Tom , ØSTVOLD Anne‐Carine , LALAND SØren G.
Published: March 2005

Poly(ADP‐ribose) polymerases, from Ehrlich ascites tumor cells, pig thymus, and HeLa S3 cells were purified by chromatography on DNA‐agarose and Blue Sepharose. A molecular mass of 112000 Da was found for all three polymerases. Fragmentation of polyacrylamide‐gel‐embedded polymerases with cyanogens bromide, and with regard to fragment sizes. The amino acid composition of the pig thymus enzyme was very similar to that of the polymerase from Ehrlich ascites tumor cells, and the terminal amino group appeared to be blocked. The HeLa polymerase electrofocused in two peaks at pH 8.8 and 5.5, while the Ehrlich ascites tumor cell and the pig thymus enzyme focused in single peaks at pH 9.4 and 9.6, respectively. Removal of residual DNA by treatment with hydroxyapatite abolished these differences in apparent isoelectric points: all three plymerases focused at pH 9.8. No important differences were found with regard to the effect of a number of substances on the synthesis of poly(ADP‐ribose). Apparent Michaelis constants for NAD of 41μM, 48μM, and 34μM were found for polymerase from Ehrlich ascites tumor cells, pig thymus, and HeLa S3 cells, respectively. All these results indicate that the three polymerases, which represented the major poly(ADP‐ribose) polymerase activity in the organism investigated, are closely related proteins.
The Synthesis of Ricinus communis Agglutin
Authors: ROBERS Lynne M. , LORD J. Michael
Published: March 2005

Polyadenylated RNA isolated from the endosperm tissue of maturing castor been seeds was translated in a call‐free rabbit reticulocyte lysate syste. Rabbit antibodies raised against Ricinus communis agglutinin were used to identify nascent agglutinin chains. In contrast to the authentic agglutinin polypeptides with molecular weights of 31000 (A chains) and 37000 (glycosylated B chains), immunoreactive translational products of Mr 33500 and 59000 were observed. The inclusion of canine pancreatic microsomes in the translational system resulted in the contranslational segregation of these immunoreactive products into the lumen of the vesicles and their modification, to molecular weights of 32000 and 66000–69000 respectively. These contranslational size modifications resulted from the cleavage of leader sequences and, in the case of the larger product, concomittant core glycosylation. 32000‐Mr and 66000–69000‐Mr proteins were also observed amonst the immunoreactive products initially formed during the labeling of intact endosperm tissue in vivo, together with 37000‐Mr and 39000‐Mr proteins. Pulse‐chase experiments showed that 66000–69000‐Mr proteins slowly disappeared while the smaller proteins were further cleaved to chains of Mr 31000 (authentic A chain), 34000 and 37000 (authentic glycosylated B chains).

It was concluded that R. communis agglutinin polypeptides were synthesized in precursor form, possibly as a ‘giant’ precursor in the case of the B chain, on membrane‐bound polysomes. Contranslational translocation across the endoplasmic reticulum membrane was accompanied by proteolysis to remove leader sequences and, where appropriate, core glycosylation. Even after contranslational processing agglutinin polypeptides were still in precursor form. Processing to authentic size appeared to occur posttranslationally.
Characterisation of a Membrane‐Bound Serine‐Specific Casein Kinase Isolated from Lactating Guinea‐Pig Mammary Gland
Authors: PASCALL John C. , BOULTON Anthony P. , CRAIG Roger K.
Published: March 2005

Serine‐specific and threonine‐specific casein kinase activities have been identified in a Golgi‐enriched membrane fraction isolated from the lactating guinea‐pig mammary gland. The serine‐specific casein kinase has been purified 2000‐fold by affinity chromatography on ATP‐agarose. The enzyme has an estimated Mr of 100000 as determined by sucrose gradient centrifugation and phosphorylates the serine residues of dephosphorylated guinea‐pig caseins A and B in a qualitatively and quantitatively identical manner to caseins A and B secreted by lactating mammary gland explants in organ culture. The enzyme also phosphorylates casein C at serine, but not threonine residues. Studies on the relative location of the enzyme within a Golgi‐enriched membrane fraction show that it is an integral component of the membrane, either in the form of a transmembrane protein or exposed on the luminal side of the membrane. Although casein kinase activity is not associated with the endoplasmic reticulum, it remains to be proven whether it is truly a Golgi enzyme, since analysis of subcellular membrane components fractioned by sucrose gradient centrifugation shows that the particulate protein kinase activity of the lactating mammary gland does not cosediment with galactosyl transferase, possibly a reflection of the heterogeneous nature of mammary gland golgi apparatus. It seems likely that the serine‐specific casein kinase activity described is responsible for the phosphorylation of caseins in the lactating guinea‐pig mammary gland, and that this occurs after the sequestration of processed but unphosphorylated caseins within the lumen of the endoplasmic reticulum.
The Regulation of Potassium Fluxes for the Adjustment and Maintenance of Potassium Levels in Escherichia coli
Authors: MEURY Jean , KEPES Adam
Published: March 2005

The regulation of K+ fluxes has been studied in Escherichia coli after depletion of K+ by an osmotic shock or at steady state of potassium accumulation.

In the absence of a carbon source, bacteria accumulate K+ to an intracellular level near 0.1 M about half of actively metabolizing cells. During uptake the rate of net unidirectional influx decreased with time down to zero as a plateau was reached and no efflux was observed. Accumulated K+ was not exchanged with external K+ under these conditions. In the presence of a carbon source bacteria took up potassium to an intracellular concentration near 0.2 M. Efflux was delayed: it started only when 50% of final cellular K+ has been taken up. At steady state cellular K+ was exchangeable with external K+. The removal of the carbon source or the addition of respiratory inhibitors immediately stopped the K+ influx but did not affect efflux until the cellular K+ concentration has dropped to a level near 0.1 M; residual potassium was no longer exchangeable with external potassium. Maintaining such an impermeability to K+ ion does not require energy (ΔΨ or ATP), even though residual K+ is concentrated 100‐fold compared to K+ in the medium. These results suggest that K+ efflux is dependent on active metabolism and on the concentration of intracellular K+ above a threshold. Unidirectional influx is also regulated by intracellular K+ according to a different concentration dependence. It appears that the trkA system, which is the only functional system under these experimental conditions is not a ‘pump and leak’ type of transport system.
Superoxide Release by Zymosan‐Stimulated Rat Kupffer Cells in vitro
Authors: BHATNAGAR Rakesh , SCHIRMER Rüdiger , ERNST Martin , DECKER Karl
Published: March 2005

Kupffer cells were isolated from pronase‐perfused rat livers and were maintained as a monolayer culture in a state of high purity and viability. Immediately after contact with zymosan particles, O2 uptake of the Kupffer cells increased fivefold; about 50% of the net oxygen consumed was accounted for as superoxide released into the medium. Concomitantly, a transient burst of luminol‐dependent chemiluminescence, an increased activity of NAD(P)H oxidase and a stimulation of the flow of glucose through the hexose monophophate shunt were observed. Chemiluminescence and O2 production were almost completely inhibited by superoxide dismutase and iodoacetate. Zymosan‐induced chmiluminesceance was not inhibited in the presence of the non‐penetrating thiol reagents, 5,5′‐dithio‐bis‐2‐nitrobenzoate and iodoacetyl‐sepharose. Iodoacetate acted on the cytosolic glucose‐6‐phosphate dehydrogenase rather than on NAD(P)H oxidase of the cell membrane.
Molecular Forms and Hydrodynamic Properties of Acetylcholine Receptor from Electric Tissue
Authors: RÜCHEL Reinhard , WATTERS Diane , MAELICKE Alfred
Published: March 2005

We have studied purified acetylcholine receptor proteins from Electrophorus electricus and Torpedo marmorata which function in both binding and reconstitution experiments. The molecular properties of these receptor‐channel complexes were analyzed under non‐denaturing conditions by polyacrylamide gradient gel electrophoresis, ultracentrifugation sedimentation studies and laser light scattering.

The purified receptor proteins exist in different interconvertible forms depending on both the type and concentration of detergent present, and the presence or absence of an intersubunit disulfide bridge. Receptor purified in the absence of sulfhydryl‐blocking agents exists in two monomeric and two dimeric forms at very low detergent concentrations (0.01‐0.05% Tween 80). At intermediate detergent concentrations (0.4% Triton X‐100) one monomeric and one dimeric form are present. Only the monomeric form remains at high levels of detergent (2% Triton X‐100). This form has a sedimentation coefficient of 9.29 S, as measured by ultra‐centrifugation using Schlieren optics. If receptor is purified in the presence of sulfhydryl‐blocked agents, conververt dimmers into monomers by high concentrations of detergent does not occur. Disulfide‐reducing agents convert dimmers into monomers independent of whether the receptor's free sulfhydryl groups are blocked or not. These findings imply that dimmer formation is primarily due to hydrophobic interaction between monomers. When these interactions are reduced by high levels of detergent the sulfhydryl group(s) have remained unblocked.

Monomers and dimmers bind α‐cobratoxin with the same affinity and kinetics. Dimers of disulfide‐linked monomers are not required for the reconstitution of a functional ion‐translocation system. The presence of EDTA during the purification of receptor proteins (as recommended by many groups to inhibit proteclysis) adversely influences the activity of the receptor in channel gating. These observations are discussed in terms of the requirements for a purification procedure yielding receptor preparations unaffected by proteolysis and functioning in both binding and ion translocation.

The molecular weight of the receptor monomer was determined by several independent techniques and yielded values in the range of 250‐300×103. With a translational diffusion constant D20, w=2.95%10−7 cm2s−1 and a sedimentation coefficient s20, w=9.29 S, the frictional coefficient ratio f/fo=1.15 was calculated for the receptor monomer from Torpedo marmorata. This indicates a considerable asymmetry in the shape of the detergent‐solubilized receptor.