bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural Elements of Methanopterin, a Novel Pterin
Authors: KELTJENS Jan T. , HUBERTS Marjan J. , LAARHOVEN Wim H. , VOGELS Godfried D. 
Published: March 2005

During short‐time labeling experiments, cells of Methanobacterium thermoautotrophicum incorporate a substantial part of 14CO2 in a yellow fluorescent compound (called YFC) [Daniels, L. & Zeikus, J. G. (1978) J. Bacteriol. 136, 75–84]. As the compound was present only in small amounts, its more abundant, metabolic precursor was identified, extracted and purified by column chromatography.The chromophore of this compound is 2‐amino‐4‐hydroxypteridine (pterin) as indicated by its ultraviolet‐visible‐light absorption and fluorescence properties, Decomposition studies revealed the presence of a number of structural elements, viz. glutamic acid, phosphate and a hexosamine. 1H‐NMR and 13C‐NMR spectra pointed to the presence of additional, as yet unidentified, elements. The compound is a complex, novel pterin derivative, which we have called methanopterin.
Immunochemical Demonstration of Zonal Growth of the Cell Envelope of Escherichia coli
Authors: DAVISON Michael T. , GARLAND Peter B. 
Published: March 2005

Many previous attempts to locate growth zones in the cell envelope of Escherichia coli or other gram‐negative enteric bacteria have been difficult to interpret due to lack of information on lateral diffusion in the outer membrane of markers used to distinguish between old and new surface. Using a fluorescence photobleaching recovery method we have shown in E. coli that the lateral diffusion coefficient (Dl) for surface‐bound antibodies directed to outer surface antigens (unspecified) is approx 10−11 cm2 s−1, with at least 50% recovery. Such diffusion would obliterate demarkation between old and new membrane. If the surface‐bound antibodies were cross‐linked with an anti‐IgG antibody, then Dl fell to < 10−13 cm2 s−1 in two of the three strains tested. Provided that Dl was lowered by this meanes, then growth following immunochemical labelling (either with fluorescent or ferritin label) led to the appearance of zones of old (labelled) and new (unlabelled) surface. In cephalexin‐induced filaments the new zones appeared as bands along the length of the cell at a repeat distance of 1—3 μm. In slowly growing non‐filamentous cells the new zone was unipolar, becoming bipolar around the time of septation. In fast growing non‐filamentous cells new zones appeared at several places.A number of features indicate that the growth‐dependent appearance of antibody‐labelled and unlabelled zones is distinct from the growth‐independent phenomenon known as antibody‐induced patching of surface antigens.We note that the antigens of the outer membrane of E. coli can be classified into those that are laterally mobile and those that are not, and that the latter class owes its immobility presumably to anchorage by peptidoglycan. We propose that immunochemical inhibition of lateral diffusion occurs by cross‐linking the mobile to immobile antigens and therefore indirectly to peptidoglycan. Accordingly the apparent growth zones that we observed must be assigned to growth zones of peptidoglycan. It does not follow that growth of the outer membrane is also zonal.
Alterations in Tubulin Immunoreactivity; Relation to Secondary Structure
Authors: ROUSSET Bernard , BERNIER‐VALENTIN Francoise , WOLF Jan , ROUX Bernard 
Published: March 2005

The immunoreactivity of tubulin varies with the conditions of preparation and storage of the protein. Four different antisera to tubulin detected less than or about 10% of the tubulin present in purified rat brain microtubule protein either immediately after preparation or after storage at ‐ 196°C. Addition of salt to the storage buffer led to a partial recovery of immunoreactivity. After storage at ‐ 20°C or after transfer from ‐ 196°C to ‐ 20°C, the recovery of tubulin immunoreactivity varied with the concentration of microtubule protein. At concentrations higher than 2.0–2.5 mg/ml, tubulin remained non‐immunoreactive. On decreasing the protein concentration, there was a progressive recovery of the immunoreactivity. Addition of salt to the storage buffer led to a full recovery whatever the protein concentration. Storage in 2 M glycerol prevented immunoreactivity in all the conditions. Phosphocellulose chromatography of microtubule protein lacking immunoreactivity yielded a pure tubulin that was fully immunoassayable.Transfer of microtubule protein from ‐ 20°C to ‐ 196°C led to a significant decrease of the amount of immunoassayable tubulin indicating that the alteration of tubulin immunoreactivity are reversible. Immunoreactivity of tubulin present in freshly prepared crude rat brain 100 000 × g supernatant was also imcompletely immunoreactive and increased after freezing at ‐ 20°C.Circular dichroic spectra showed that freezing at ‐ 20°C decreased the content of α helix from 41% (at ‐ 196°C) to 28% in purified microtubule protein. The percentage α helix was low in buffers of high ionic strength and high in the presence of glycerol irrespective of the storage temperature. Thus, the α helix content showed an inverse relationship to immunoreactivity and the availability of antigenic sites seems to be related to the conformation of tubulin. Although the ability of microtubule protein to polymerize was maintained after storage at ‐ 196°C and decreased after storage at ‐ 20°C, there was no direct relationship between immunoreactivity and the polymerization properties of tubulin.Our results show that care should be taken in the preparation of tubulin samples to be assayed by immunological techniques. Glycerol, which is often used to prepare and store tubulin, prevents the ability of tubulin to react with specific antibodies. Alterations of the availability of antigenic sites, observed in the presence of glycerol or salt or after freezing, seem to be related to changes of the conformation of tubulin.
Yeast Mutant with Thermolabile CDP‐choline Synthesis
Authors: NIKAWA Jun‐ichi , YONEMURA Kimie , YAMASHITA Satoshi 
Published: March 2005

A temperature‐sensitive mutant of Saccharomyces cerevisiae with a lesion in cholinephosphate cytidyltransferase was isolated from a choline auxotroph. This mutant grew in a medium supplemented with ethanolamine or N‐methylethanolamine, but at a much slower rate in a medium supplemented with choline or N,N‐dimethylethanolamine at 37 °C. At 23 °C, mutant cells grew at the same rate in any of these media.
Labelling experiments showed that the activity of phosphatidylcholine synthesis from choline in the mutant was thermolabile and rapidly lost on incubation of cells at 37 °C (half‐life, 4 min).
Determination of the intermediates and the enzyme activities of the CDP‐choline pathway indicated that mutant cells had a lesion in cholinephosphate cytidyltransferase.
Genetic analysis showed that the thermolabile phosphatidylcholine synthesis in the mutant arose from a single mutation in a gene coding for cholinephosphate cytidyltransferase.
The mutant strain was found to be defective in dCDP‐choline synthesis. Thus, a single enzyme is responsible for the synthesis of both CDP‐choline and dCDP‐choline.
Ethanolaminephosphate cytidyltransferase activity in the mutant was comparable to that in the parental strain. An alteration in cholinephosphate cytidyltransferase did not affect the incorporation of labelled ethanolamine into lipids. These results indicate that ethanolaminephosphate cytidyltranferase is coded for by a distinct gene from that for cholinephosphate cytidyltransferase.
The CDP‐choline pathway was not required by cells as long as phosphatidylcholine was supplied via the phosphatidylethanolamine methylation pathway. The phosphatidylethanolamine methylation pathway and the CDP‐choline pathway were found to be complementary to each other in phosphatidylcholine synthesis.
Fast Ionic Flux Activated by Cyclic GMP in the Membrane of Cattle Rod Outer Segments
Authors: CARETTA Antonio , CAVAGGIONI Andrea 
Published: March 2005

Guanosine 3/,5′‐monophosphate (3′,5'GMP) activates an influx of calcium ions into vesicles of rod outer segments washed free of water‐soluble proteins and filled with a hihg‐affinity ligand for calcium (EDTA, Arsenazo III, aequorin). Addition of 3′,5′GMP activates calcium inflix without any appreciable delay, studied with methods that resolve 40 ms.A direct interaction between the cyclic nucleotide and an intrinsic membrane component is considered.
Inactivation of the Calium‐Transport ATPase in the Sarcoplasmic Reticulum by the Combined Effect of Lasolicid and Triton X‐100
Authors: HASSELBACH Wihelm , LÜDI Hans , MIGALA Andrea 
Published: March 2005

The calcium‐transport ATPase of the sarcoplamic retulum membranes is ireversibly is irreversibly inactivated by the combined action of Lasolocid an Triton X‐100 at concentrations which separately do not interfere with the enzyme's activity. In the pesence of Lasolocid the enzyme is most suceptible to inactivatin when the Triton X‐100 concentration just exceeds its critical micellar concentratin, ∼ 0.2 mg · ml −1.Lasolocid becomes effective at a concentration of 10 μM and produces rapid intracivation at 100 μM. Phosphoprotein formation is less affected than phosphate liberation. The influence of the ATPase protein on the fluorescene intesity of Lasolocid passes a distinict maximim at the most effective Triton X‐100 concentration of 0.2 mg · ml−1.
Purification and Cahracyterization of te Brain Calmodulin‐Dependent Protein Kinase (Kinase II), Which Is involved in the Activtion of Tryptophan 5‐Monooxygnase
Authors: YAMAUCHI Takashi , FUJISAWA Hitoshi 
Published: March 2005

Calmodulin‐dependent protein kinase (kinase II) [Yamauchi, T. and Fujisawa, H. (1980) FEBS Lett. 116 141–144] which is involed in the activation of tryptophan 5‐monoosygenase was purified 720‐fold wiht a 36% yield from rat cerebvral cortex using ammonium sulfate and chromatography on Sepharose CL‐4B, Calmodulin‐Sepharose 4B and phosphocellulose. The purified enzyme showed one major protein band corresponding in to a molecular weight of 55000 and a faint band upo0n sodium dodecyl sufate/polyacrylamide disc gel electophoresis, wherase it gave a single protein band upon polyacrylamide gel electroporesis whithout soudium dodecyl sulfate. The molecualar weight ad th sedimentation coeffieient of the kinase were dtermined to be 540000 by sedimentation equilibrium and 16.5 S by sucrose density gradient cntrifugation.The Kinase required absolutely calmodulin and Ca2+ for its activity and the apparent Ka values for calmodulin and Ca+2 were 10 nM and 1.6 μM respectively. The Km values for ATP and Mg2+ wer calculated to be 0.06 mM and 1 mM, respectively. The concentration of tryptophan 5‐monooxygenase required to produece half‐maximal effects on ist activation by the kinase was estimated to be as low as 0.3 nM, on the basis of th finding that the molecular weight and the specific activity of trytophan 5‐monooxygenas were 245000 and 374 nmol min−1 mg oprotein−1 respectively [Nakata, H. and Fuisawa, H. (1982) Eur. J. Biochem. 122, 41–47]. The kinase phosphorylated casein, smooth muscle myosin light chain as well some endogenous protiens of brain cytosol. The enzyme did not phoisphorylte significantly histone, protamine, and phsphorlyase. b. Some other proterties of he Kinase were exiamined.
Solubiliztion and Partial Characterization of Alkylglycerol Monooxygenase from the Liver Mkcosomes
Authors: ISHIBASHI Teruo , IMAI Yoh 
Published: March 2005

The enzyme alkylgloycerol monooxygenase wa slubuilized ith 2% of Triton X‐100 and partially pufified approximatelkyt to 83–fold with a 36% yield after a procedure which included 6‐aminohexyl‐Sephaorse and DEAE cellulose column chromatography, followed by a surcros edensity gradient centrifugation. The moleucular weight of the native form was estimated to be appromiately 400000 as judged by Sepharose 6B column chromatography in the presence of Triton X‐100. The enzyme had a pH otimum nbear 8.5 and a Km of 0.66 for 1‐O‐hexadecylglycerol. intiaal velocity excepyt to maintiain it for a much longer longer period of the time.The purified enzyme required abslutely both molecular oxygen and reduced pteridine as an electron donor. Furthermore, reduced glutathoione glutathione an phospholipids wer necessary for expression of full enzyme activity. N‐Ethylmaleimide and p‐Chloromercuribenzoate singnificantly inhibited the enzyme activity, suggesting that alkylycerol monoxxygense is a ‐SH enzyme.
Analogues and Analogue Inhibitors of ppp(A2′p)nA
Authors: HAUGH Margaret C. , CAYLEY P. Jane , SERAFINOWSKA Halina T. , NORMAN David G. , REESE Colin B. , KERR Ian M. 
Published: March 2005

Higher Oligomers of ppp(A2′p)n together with (A2′p)nA, (A2′p)2A3′OCH3, (A2′p)2A2′,3′CH2, (A2′p)2dA, (dA2′p)2dA, their 5′‐monophosphates and 5′‐S‐methylphorothioates have been investigated for relative stability and biological activity in mouse and human cells and mouse, human, and rabbit cell‐free systems. The oligomers from trimer to heptamer inhibited protein and DNA synthesis when introduced into intace mouse cells and activated the ppp(A2′p)n A‐dependent RNase at below nanomolar concentrations in mouse cell extracts.The 5′‐diphosphates pp(A2′p)2A and corresponding analogues were active both in cell‐free systems and on introduction into intact cells. The exception to this was the all 3′‐deoxyadenosine analogue pp(dA2′p)2 dA which failed to activate the ppp(A2′p)nA‐dependent nuclease in the mouse L and human (Daudi and HeLa) cell extracts tested. Of the active analogues the 3′‐OCH3 appeared to be the most stable in the cells and systems employed. On the other hand the non‐phosphorylated ‘core’ (A2′p)2A and its 3′‐sunstituted analogues were inactive in mouse L and Ehrlich acites tumours cell‐free systems and had to effect on intact (non‐permeabilised) 3T3 cells.The 5′‐monophoshate, p(A2′p)2A, can act as an inhibitor of ppp(A2′p)nA in mouse cell‐free systems [Torrence, Imai and Johnston (1981) Proc. Natl Acad. Sci. USA, 78, 5993‐59997]. Here in intact mouse L cells or extracts from interferon‐treated human (Daudi) cells, however, it mimicked the action of ppp(A2′p)2A, possibly through conversion to the 5′‐diphosphate or 5′‐S‐methylphosphorothioate derivatives of the 3′‐substituted analogues are both more stable to exonucleolytic cleavage and unlikely to be converted to the 5′‐diphosphated or 5′‐triphosphates. They are analogue inhibitors of ppp(A2′p)nA in mouse L cell extracts. How widely they will be effective in a variety of cell‐free systems and intact cells remains to be established.The 5′‐diphosphate pp(A2′p)2A and corresponding analogues were not equally active, nor was the CH3Sp(A2′p)2A2′,3′CH2 equally effective as an analogue inhibitor, in different cell‐free systems. This emphasises the apparent differences in the properdties of the ppp(A2′p)nA‐dependent RNases from different sources. Accordingly, in looking for a generally effective analogue inhibitor of ppp(A2′p)2A its activity in a variety of extracts should be tested and in any search for further analogues for potential clincial use human cells and extracts should be employed.
Chemical Modification of Lys‐6 in Phospolipase A2 from Naja melanoleuca Snake Venom
Authors: van EIJK Jan H. , VERHEIJ Hubertus M. , de HAAS Gerard H. 
Published: March 2005

Phospholopase A2 isolated form Naja melanoleuca snake venom was modified with 4‐chloro‐3,5‐dinitrobenzoate. The modification reaction was accelrated by the cofactor Ca2+. Micellar cncentrations of the substrate analog n‐tetradecylphosphocholine retarded th inactivation of the enzyme. Modification resulted in the incorporation of one moel dinitrobenzoate/mole of protein. The modified residue could be identified as Lys‐6 in the amino acid sequence of the enzyme. Although the modified enzyme still retiains high affinity for micellar substrate analogs an the cofactor Ca2+, it had lost nearly all its activity towars mice substrates.Upon reduction of the nitro groups in the modified protein the enzymatic activity could be restored by more than 50% Therfore Lys‐6 cannot be directly involved in enzymatic catalysis. It is conluded that introduction of the negatively chareged dinitrobenzoate group on Lys‐6 gieves rise to a distrotion of the active site of the anzyme.
FAD Analogues as Mechanistic and ‘Binding‐Domain’ Probes of Spinach Ferredoxin‐NADP+ Reductase
Authors: ZANETTI Giuliana , MASSEY Vincent , CURTI Bruno 
Published: March 2005

The native flavin, FAD, of spinach ferredoxin—NADP+ reductase, has been replaced by a number of FAD analogues with modifications of the isoalloxazine ring system. The apoenzyme binds 8‐mercapto‐FAD in its thiolate anion form and 6‐hydroxy‐FAD in its neutral form. These results are consistent with classification of this enzyme as a dehydrogenase/electron transferase, an ascription originally made on the basis of its physiological function and in common with other properties of this class, e. g. stabilization of the neutral flavin semiquinone.The chemical reactivity toward methylmethanethiolsulfonate of the 8‐mercapto‐FAD enzyme clearly shows that the flavin 8‐position is exposed to solvent. On the other hand, the lack of reactivity with the 2‐thio‐FAD enzyme indicates that the pyrimidine subnucleus of the flavin is buried within the protein molecule. The seven modified flavins examined all support NADPH‐ferricyanide reductase activity, the catalytic velocity being directly proportional to the redox potential of the flavin. No such linear free energy relationship was found between redox potential and activity with ferredoxin or iodonitrotetrazolium as acceptor.
The Regulation of the Na+ ‐Ca2+ Exchanger of Heart Sarcolemma
Authors: CARONI Pico , CARAFOLI Ernesto 
Published: March 2005

The Na+/Ca2+ ‐exchange of calf‐heart sarcolemma is activated by a treatment with ATP, Mg2+, and Ca2+, and deactivated by a treatment with phosphorylase phosphatase. The effect of th e latter can be substituted by a treatment with Mg2+, Ca2+, and calmodulin. the activating treatment does not require added calmodulin, but is inhibited by calmodulin antagonists. Evidently, engdogenous calmodulin is required and sufficient.Activation is half‐maximal at about 2μM Ca2+. Added clamodulin, however, decreases the Km (Ca2+) of the activating process to about 0.8 μM. Deactivation is half‐maximal, at optimal calmodulin concentrations, at about 1.5 μM Ca2+.Experiments with adenosine 5′‐[γ‐thio]triphosphate have shown that the activating treatment is mediated by a kinase and the deactivating treatment by a phosphatase. The concerted operation of the two enzymes is made possible by their different Ca2+ affinity. At saturating Ca2+ concentrations, the level of ATP may also influence the balance of the two enzymes.
Destruction of Phosphilipids and Respiratory‐Chain Activity in Pig=Heart Submitochondrial Particles Induced by an Adriamycin‐Iron Complex
Authors: Erland J. F. DEMANT  , JENSEN Poul. K. 
Published: March 2005

Ferric ion complexes of the antibiotic and antitumor agent adriamycin support peroxidation of phospholipids in pig heart submitochondrial particles as judged by disappearance of polyunsaturated fatty acids, loss in extractable phospholipid, adramycin‐Fe3+‐deoebdent oxygen consumption and formation of material with ultraviolet absorption.Peroxidation was stimulated to an extent of about 100% at low rates of NADH oxidation in the submitochondrial particles, whereas oxidation rateds higher than 50 nmol NADH oxidized X min−1× mg protein−1 appeared to protect the membrane lipids.NADH oxidase, NADH‐cytocyrme c reductase as well as cytochrome c oxidase activities in the submitochondrial particles were progeressively inactivated during incubation with abriamycin in the presence of Fe3+ ions, whereas NADH‐ferricyanide reductase activity remained fully stable.
Purification and Characterization of a New Sodium‐Transport Decarboxylase
Authors: HILPERT Wilhelm , DIMROTH Peter 
Published: March 2005

Upon resolution of the particulate cell fraction of Veilionella aclcalescens by chromatography, membranes and ribosomes were celarly resolved. Methylmalony‐CoA decarboxylase was bound to the membranes and not to ribosomes as reported earler.Membrane vesicels containing mentylmalonyl‐CoA decarboxylase were prepared by disrupting V. alcalescens cells with French pressure chdmaber. About 64% of the decaroxylase was oriented in these vesicles with the sbstrated binding site facing to the outside. The vesicels perforemd a rapid acdumulation of Na+ ions in respon se to the decarboylatino of methylmalonyl‐CoA.Decarbosylation and transport wer highly uncouped. The effciency of the transport was considerably increased if methylmalonyl‐CoA cecarbosylation was retared by using a low temperature of by slowaly genrating the substrate enzymically form priopionyl‐CoA. Under optimized coditions Na+ was concentrated inside the inverted vesicles eight‐times than in the incuabaion medium.Mehtylmalonyl‐CoA decarboxylase was solubilized formt eh membranes with Tritom X‐100 and purified about 20‐fp;d nu affinity chromatography on monomeric avidin‐Sepharose coulmns. The decarboxylase was specifically activated by Na+ ions (apparend Km∼ 0.6 mM). Wherase (S)‐methylmalonyl‐CoA was the superior substrte. (apparent km∼ 7 μM). The decarboxylation of methylmalonyl‐CoA yielded CO2 and not HCO3− as th primary reaction product.Analysis of the prurified enzyme by dodecylsulfate gel electrophoresis inducated the presence of four differnet polypetides α, β, γ, δ with Mr 60 0000, 33 0000, 18 500 and 14000. The letter of these polypetides was elearly visible only after stiaing but not after staining with Coomassie brilliant blue. A low moleualr weight polypetide wit similar staaining properties also found in oxaloacetate decarbvoxylase Methylmalonyl‐CoA decarboxlase contained about 1 mol convalently bound biotin per 125 500 g protien which was localized exculsively in the γ‐subunit. This subunit therfore represents th biotin carboysl carier protein of methylmalonyl‐CoA decarboxylase. A new very senstivitymethod for the dtection of biotin containg proteins is described.
Study of the Interaction of Yeast Arginyl‐tRNA Synthetase with Yeast tRNA2Arg and tRNA3Arg by Partial Digestions with Cobra Venom Ribonuclease
Authors: GANGLOFF Jean , JAOZARA Raymond , DIRHEIMER Guy 
Published: March 2005

Yeast tRNA2Arg and tRNA3Arg are two isoacceptors which show similar V and Km for yeast arginyl‐tRNA synthetase despite important differences in their primary structures. Fragments resulting from the partial digestion of 3′ or 5′ end‐labelled tRNA2Arg and tRNA3Arg in the presence or absence of arginyl‐tRNA synthetase by cobra venom ribonuclease, an enzyme whiich cuts preferentially in double‐stranded regions, were analysed by electrophoresis on polyacrylamide gels.In the absence of arginyl‐tRNA synthetase, major cuts were observed in tRNA2Arg and tRNA3Arg at the end of the 3′ part of the acceptor stem and in the 5′ part of the anticodon stem, whereas the 5′ part of the acceptor stem and the 3′ part of the anticodon stem are only slightly cleaved. The D and the T stems are almost fully resistant to cobra venom ribonuclease attack confirming the strong tertiary structural organization of this region.In the presence of arginyl‐tRNA synthetase the two or three last sites of the 3′ halves of the acceptor stems and the sites in the 3′ halves of the anticodon stems are almost completely protected against ribonuclease hydrolysis in both tRNAs; 31–69% protection of the sites located in the 5′ halves of the anticodon stem is also observed. However, the cleavage levels are enhanced for the three head positions in the 3′ halves of the acceptor stems and a new cut appears at the first position of this region in the case of tRNA3Arg.The similarity of the protection patterns of tRNA2Arg and tRNA3Arg suggests that both molecules interact in nearly the same manner with arginyl‐tRNA synthetase, which in turn implies great similarities in their tertiary structure when involved in the complex. If this tertiary organization is like that described for tRNAPhe, all protected sites are located in the inside of its l‐shaped model.