bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein Kinases of Rat Liver during Hepatocarcinogenesis Induced by an Azo Dye
Authors: WEBER Anne , COTTREAU Dominique , HENRY Joelle , PHAN DINH TUY Francoise , SKALA Henriette , KAHN Axel
Published: March 2005

We studied phosphoproteins and protein kinases in livers from normal rats and rats fed a diet containing 3′‐methyl‐p‐dimethylaminoazobenzene, an azo‐dye carcinogen.

Endogenous phosphophortein patterns obtained by incubation of soluble and insoluble cell fractions with [γ‐32P]ATP were different for livers from rats submitted to carcinogenic diet as soon as the 12th week of the regimen compared to the patterns of normal and regenerating liver.

New Phosphorylated bands appeared during hepatocarcinogenesis in soluble as well as in particulate fractions. None of these bands was phosphorylatedon tyrosine residues and the global percentage of phosphotyrosine residues among phosphoproteins did not increase during the course of azo‐dye hepatocarcinogenesis.

Electrophoresis of active enzymes showed, from the 12th week of carcinogenic diet, a new band identified as a cAMP‐dependent histone kinase. This form was absent in fetal liver and in regenerating liver following partial hepatectomy. It could be identified as a modified (or a new isozymic) form of protein kinase type II by its chromatographic properties and the nature of its photoaffinity‐labeled regulatory subunits. The ratio of the R1 to R2 regulatory subunits increased in the treated animals, but this phenomenon seemed to be due to cell proliferation rather than to malignancy as it was also observed in regenerating liver following partial hepatectomy. In hepatoma, the increase of the amount of R1 subunits was not associated with a concomitant increase of protein kinase type I activity, which seemed to indicate that, in these tissues, a pool of free R1 subunits could exist.

Finally, combination of DEAE‐cellulose chromatography fractionation and cellulose acetate electrophoresis analysis allowed us to detect in hepatoma a phosvitin kinase band insensitive to cyclic nucleotides and heparin, inactive on casein, which was absent in normal liver. Hepatoma extracts also contained fast‐migrating caseinphosvitin kinase forms which could correspond to modified casein kinase II.
No Arginyl Adenylate Is Detectable as an Intermediate in the Aminoacylation of tRNAArg
Authors: THIEBE Rainer
Published: March 2005

On the supposition that aminoacyl adenylate is a necessary intermediate in the reactions catalyzed by aminoacyl‐tRNA synthetases, six possible reactions requiring this intermediate were tested. With arginyl tRNA synthetase from brewer's yeast they were all negative and with phenylalanyl‐tRNA synthetase they were all positive. Therefore, no evidence for the formation of arginyl adenylate could be provided. This is in contrast to results published elsewhere. It was shown that the reaction proceeds through a quaternary complex. The aminoacylation of the tRNA is followed by a rearrangement of the quaternary complex that also affects the structure of the arginyl‐tRNA.
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.
Interaction of Concanavalin A with Native and Denatured Forms of Jackbean α‐d‐Mannosidase
Authors: BOWLES Dianna J. , CHAPLIN Martin F. , MARCUS Susan E.
Published: March 2005

Tetrameric α‐d‐mannosidase from jackbean is a glycoprotein containing at least one mannosylated oligosaccharide. In the native enzyme, the oliosaccharide is sterically masked from interaction with either endoglucosaminidase H or concanavalin A. Denaturation into subunits permits endoglucosaminidase hydrolysis and removal of the oligosaccharide. The mannosyl residues are attached only to the heavy type of subunit. Removal of the oligosaccharide(s) from the denatured heavy subunit requires the joint action of both α‐d‐mannosidase and N‐acetyl‐β‐d‐glucosaminidase.
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.
Isoleucyl‐tRNA Synthetase from Baker's Yeast
Authors: FREIST Wolfgang , CRAMER Friedrich
Published: March 2005

The aminoacylation of three modified tRNAIle species with isoleucine and with valine by isoleucyl‐tRNA synthetase has been investigated by initial rate kinetics. For aminoacylation of tRNAIle‐C‐C‐3′dA with isoleucine, a bi‐bi uni‐uni ping‐pong mechanism has been found by bisubstrate kinetics and inhibition by products and by 3′dATP; for aminoacylation with valine a bi‐uni uni‐bi ping‐pong mechanism. For isoleucylation of tRNAIle‐C‐C‐A(3′NH2) bisubstrate kinetics, inhibition by products and by isoleucinol show a random uni‐bi uni‐uni‐uni ping‐pong mechanism; for valylation of this tRNA a bi‐bi uni‐uni ping‐pong mechanism is observed by bisubstrate kinetics and product inhibition. tRNAIle‐C‐C‐2′dA was aminoacylated under modified conditions with isoleucine in a bi‐bi uni‐uni ping‐pong mechanism with a rapid equilibrium segment as observed by bisubstrate kinetics, inhibition by AMP, by P[NH]P as product analog and by isoleucinol. Aminoacylation with valine is achieved in a rapid equilibrium sequential random AB, ordered C mechanism indicated by bisubstrate kinetics and inhibition by 3′dATP and valinol. All six reactions exhibit orders of substrate addition and product release which are different from those observed in aminoacylation of the natural tRNAIle‐C‐C‐A. The Km values of the three substrates and the kcat values of the six reactions are given. For aminoacylation at the terminal 2′OH group of the tRNA differences of 13.38 and 13.17 kJ in binding energies between valine and isoleucine have been calculated which result in discrimination factors of 181 and 167. For aminoacylation at the terminal 3′‐OH group a differnce of only 4.43 kJ and a low discrimination factor of only 6 is observed. Thus maximal discrimination between the cognate and the noncognate amino acid is only achieved in aminoacylation at the 2′‐OH group and conclusions drawn from experiments with modified tRNAs concerning 2′,3′‐specificity have led to correct results in spite of different catalytic cycles in aminoacylation of the natural and the modified tRNA. The stability of Ile‐tRNAIle‐C‐C‐2′dA and Val‐tRNAIle‐C‐C‐2′dA, the lesser stability of Val‐tRNAVal‐C‐C‐2′dA and the instability of Thr‐tRNAVal‐C‐C‐2′dA are consistent with postulations for a ‘pre‐transfer′ proofreading step for isoleucyl‐tRNA synthetase and a ‘post‐transfer’ hydrolytic editing step for valyl‐tRNA synthetase at the terminal 3′OH group of the tRNA.
Interaction of ADP and Magnesium with the Active Site of Myosin Subfragment‐1 Observed by Reactivity Changes of the Critical Thiols and by Direct Binding Methods at Low and High Ionic Strength
Authors: WATTERSON John G. , FOLETTA Daniela , KUNZ Peter A. , SCHAUB Marcus C.
Published: March 2005

Comprehensive binding studies using direct and indirect methods yield stoichiometry and affinities for the binding of Mg · ADP and uncomplexed ADP to the active site of myosin subfragment‐1. Additionally, the binding parameters for Mg2+ in the ternary complex protein · Mg · ADP are presented for the first time. The indirect method makes use of reactivity changes of the critical thiol‐1 and thiol‐2 groups, which occur upon the binding of the ligand at the active site. The affinity constants derived by this method are corroborated by two independent direct methods, equilibrium dialysis and centrifugation transport. For Mg2+, ADP and Mg · ADP just one mole of ligand binds/mole subfragment‐1. The affinity of Mg · ADP at low ionic strength is 2.1 × 106 M‐1 and only five‐times lower in the absence of Mg2+ has a low affinity of 4.1 × 104 M‐1. At high ionic strength the uncomplexed ADP binds with a 43‐times‐lower affinity than Mg · ADP, whose affinity is 6.9 × 105 M−1. In this case Mg2+ interacts in the ternary complex with the higher affinity of 3.2 × 105 M−1, implying that at high salt concentration it plays a more prominent role in anchoring ADP at the active site.
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.
The active Site of Ribonuclease A from the Crystallographic Studies of Ribonuclease‐A‐Inhibitor Complexes
Authors: BORKAKOTI Nivedita
Published: March 2005

The results of X‐ray analyses of three ribonuclase‐A‐nucleotide complexes, at 2.3 Å, are reproted, A modified purine mononucleotide, 8‐oxo‐guanosine 2′‐phosphate in a synconformation binds at the pyrimidine‐binding site of the catalytic cleft. Solvent molecules are expelled from the active site due to inhibitor binding. The positions of the side‐chains of the active‐site residues Gln‐11, His‐12 and Thr‐45 are well defined and do not alter on inhibitor binding. The mobility of Lys‐41 is greatly reduced in protein‐nucleotde complexes and the terminal amino group interacts directly, with the 2′‐phosphate group of the nucleotides. In the complex of the enzyme with a modified pyrimidine, cytidine‐N(3)‐oxide 2′‐phosphate, His‐119 is stabilised in the minor site of the native protein [Borkakoti, N., M, D. S. and Palmer, R. A. (1982) Acta Crystallogr. B38, 2210–2217], while in the protein‐purine derivative the imidazole group is located in the major site. Inhibitor binding induces movements in the site‐chains of Lys‐7 and Lys‐66 which also modify the conformation of the active‐site cleft of ribonuclease A.
The Stereochemical Course of Nucleotidyl Transfer Catalysed by NAD Pyrophosphorylase
Authors: LOWE Gordon , TANSLEY Gaynor
Published: March 2005

NAD pyrophosphorylase catalyes nucleotidyl trasfer from adensone (R′‐[α‐17O]triphosphate to nicotinamide monocucledotide with inversion of configuration at the α‐P giving (S‐[17O]NAD+. The simplest interpretation of the observation is that the adenyly group is trasferred directly from ATP to the co‐substrate by an ‘in line’ mechanism. It is also shown that snake venom phosphodiesterase hydrolyses NAD+ regio‐specifically at the adenylyl terminus of the pyrophosphate bond.
Structure‐Activity Relationships for Binding and Inactivation of Rabbit Reticulocyte Ribosomes by Quassinoid Antineoplastic Agents
Authors: CONSIDINE R. Tobias , WILLINGHAM Walker , CHANEY Stephen G. , WYRICK Steve , HALL Iris H. , LEE Kuo‐Hsiung
Published: March 2005

A series of quassinoid compounds was investigated to determine the structure‐activity relationships for the binding and inactivation steps associated with the inhibition of protein synthesis by these compounds in a rabbit reticulocyte system. The binding of these compounds to rabbit reticulocyte run‐off ribosomes was measured in a competition‐binding assay using [14C]trichodermin as the competing ligand. Previous studies have shown that trichodermin and quassionoids compete for a single binding site on yeast ribosomes. Our experiments confirmed this behavior with rabbit reticulocyte ribosomes and ind9icated that binding equlibrium could be reached in 2.5 min at 30°C. The comparative binding data indicated the following: (a) The nature of the side‐chain at position 15 has very little effect on the association of these compounds with rabbit reticulocyte ribosomes. There is one exception to this general rule, however. saturation of the double bond in brusaatol (compound II) to give dihydrobrusatol (compound IV) increases the d form 1.6μM to 8.7 μM. (b) Easy access to the A ring is required for optimal binding. The addition of any bulky hydrophobic group to the A ring increased the Kd significantly. However, the double bond in the A ring does not appear to be required for binding since the Kd values for dihydrobrusatol (compound IV) and tetrahydrobrusatol (compound VI) to the corresponding lactol (compound X) has only a minor effect on Kd. In order to determine whether inactivation of rabbit reticulocyte ribosomes had any structural requirements which differed from those required for simple binding, we also compared the concentrations of these compounds required to give 50% inhibition of protein synthesis (ID50) in rabbit reticulocye lysates. Previous studies had indicated that binding of quassinoids to the peptidyl transferase center and inhibition of peptide bond formation was probably the only mode of action of these compounds. Our experiments indicated that the ID50 values for several of thse compounds were the same in whole reticulocytes and reticulocyte lysates, suggesting that transport and/or metabolism of these compounds was probably not limiting for their inhibitory effects. The inhibition studies indicated that (a) The nature of the side‐chain at position 15 had no detectable effect on the inhibition: (b) the double bonds in the A ring and lactone ring are probably involved in the inactivation of rabbit reticulocyte ribosomes.
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.
Interaction of Phospolipase A2 form Naja melanoleuca Sanke Venom with Meoomeric Substrate Analogs
Authors: van EIJK Jan H. , VERHEIJ Hubertus M. , DIJKMAN Ruud , de HAAS Gerared H.
Published: March 2005

Unlike porcine pancreatic phospholipase A2, the enzyme from Naja melanoleuca does not display bisphaisc kinetic behaviour at substrate concentrations around the critical concentration. This snake venom enzyme was further investigateds around the critical micelle cnentration. This snake vemom enyme was futher investigated by dirct biding studes using n‐tridecylphosphochline. Binding of this substrate analog to the enzyme was monitored by using equilibrium gel filtration, equilibrium dialyssis and ultraviolet difference spectroswcopy. It is concluded that, in the presence of submicellar conenraitons of n‐tridecylphosphocholin, a lipid‐pritien complex is formed cosisting of about 4 protein and 36 lipid molecules. Ca2+ ions are required for the formation of this complex. A model is proposed which describes the formation of this type of complex. Thsse lipidfomramtion of this complez. A model is proposed which describes th formation of this type of compelex. These lipid protein aggregates are held responsible for the non‐hyperbolic kinetic behaviour of the skanke venom enzyme towards monomeric substrates.
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.
Magnetic and Spectral Properties of Carp Carbonmonoxyhemoglobin. Competitive Effects of Chloride Ions and Inositol Hexakisphosphate
Authors: CERDONIO Massimo , MORANTE Silvia , VITALE Stefano , DALVIT Claudio , RUSSU Irina M. , HO Chien , De YOUNG Alice , NOBLE Robert W.
Published: March 2005

We have extended our studies on the magnetic properties of carp carbonmonoxyhemoglobin and the dependence of these properties upon solution variables. Using an improved version of the superconducting magnetometer, we have found that the magnetic susceptibility of carp carbonomonxythemoglobin is sensitive to both inositol hexakisphosphate and chloride ion. The dependence upon chloride ion concentration is complex. At relatively low concentrations this anion reverses the effect of inositol hexakisphosphate, restoring paramagnetism. At higher chloride concentrations the protein is converted to a roughly diamagnetic state in the absence of inositol hexakisphosphate. Along with these suceptibility studies, we have examined the effects of these anions on other properties of carp carbonmonoxyhemoglobin. The positions of the Soret bands of human asd carp methemoglobin derivatives are correlated with spin state; changes in the magnetic suceptibility of carbobmonoxyhemoglobi are similarly associated with alterations in this spectral band. We have also examined the effects of these anions on the proton nuclear magnetic resonance specturm of carp carbonomonoxyhemoglobin. Both chloride and inositol hexakisphosphate alter the position of the position of the proton resonances in the ring‐current‐shifted region of the spectrum.
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.
Metal‐Ion‐Promoted Dephosphorylation of the 5′ ‐Triphosphates of Uridine and Thymidine, and a Comparison with the Reactivity in the Corresponding Cytidine and Adenosine Nucleotide Systems
Authors: SIGEL Helmut , HOFSTETTER Fritz
Published: March 2005

First‐order rate constants (50°C; I= 0.1 M, NaClO4) for the dephosphorylation of UTP and TTP (1mM) in the pH range 2—10 are compared with those of ATP and CTP; they all show the same properties indicating that the nucleic base has no influence on the rate. In the presence of Cu2+ or Zn2+(NTP:M2+= 1:1) this changes drastically: ATP‐M2+≫ UTP‐M2+∼ TTP=M2+°CTP‐M2+ > NTP, the Cu2+ ystems being always more reactive than the Zn2+ systems, and these more than the Ni2+ systems. An interafction between the nucleic base and metal ion is important for the Cu2+ ‐ATP and Zn2+ ‐ATP systems, but not for the pyrimidine‐nucleotide systms (these behave like methyltriphosphate). Accordingly, prevention of the Cu2+ ‐purine interaction by the addition of one equivalent of 2,2′ ‐bipyridyl, leading to Cu((Bpy) (NTP)2−, strongly reduces the activity and all four ternary Cu2+ systems now show the same dephosphorylation rate. Addition of a second equivalent of Cu2+ to the Cu2+ ‐UTP 1:1 system enhances the dephosphorylation rate significantly and Job's method provides evidence that a 2:1 complex is the most reactive intermediate. The relation between the initial rate, vo= d[PO43−].dt, and the concentration of Cu2+ ‐UTP in 1:1 and 2:1 systems was determined. The results suggest that the reactive complex with pyrimidine nucleuotides is a monomeric, dinuclear species of the type M2(NTP)(OH)− (its formation is inhibited by ligands like tryptophanate), while with M2+ ‐ATP the reactive complex is a dimer. The connection between the indicated dephosphorylations in vitro, i.e. trans‐phosphorylatins to H2O, and related reactions in vivo are discusse.
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.
Purification and Properties of Aryol Acylamidase from Pseudomonas fluorescens ATCC 39004
Authors: HAMMOND Peter M. , PRICE Christopher P. , SCAWEN Michael D.
Published: March 2005

Aryl acytlamidase has been purified from a strain of Pseudomonas fluorescens ATCC 39004, selected from soil on the basis of its ability to utilise acylanilide compounds as a sole source of carbon. The enzyme was purified to homogeneity by a combination of ion‐exchange, hydrophobic and gel‐permeation chromatography. A relative molecular mass of about 52500 was estimated by gel filtration. The native enzyme was shown to be a monomeric protein by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The enzyme was maximally active at a pH of 806 and at a temperature of 45°C. The enzyme shows Michaelis‐Menten kinetics; Km vlaues for nitroacetanilide (69μM) and hydroxyacetanilide (601μM) were low, indicating that the enzyme has a very high affinity for both substrates.
Control of the Activity of Phosphoertolpyruvate Carboxykinase and the Level of ItsmRNA in Livers of Newborn Rats
Authors: BENVENISTY Nissim , SIMCHON Edit Ben , COHEN Hannah , MENCHER David , MEYUHAS Oded , RESHEF Lea
Published: March 2005

Streptozotocin treatment produces a typical experimental diabetes in neonates exhibiting hyperglycemia, glucosuria, ketonemia and increased level of free fatty acids in the blood. The liver is affected as well, with reduced activity of glycogen synthase and a corresponding decrease in the content of liver glycogen.

In contrast, the activity of liver cytosolic phosphoenolpyruvate carboxykinase and the level of its mRNA are not affected. Using a cDNA containing P‐pyruvate carboxykinase sequence, the relative abundance of the enzyme mRNA was estimated. The level of the mRNA was readily observed increasing by glucocorticoid treatment or decreasing in response to administered load of glucose. These parallel the changes observed in the activity of the enzyme under these treatments, indicating that the level of P‐pyruvate carboxykinase mRNA actually determines that of the cnzyme. The failure of diabetes to incrcase the level of enzyme mRNA and the limited response to glucose loading strongly suggest that the mechanisms controlling the level of P‐pyruvate carboxykinase mRNA in neonates are relatively resistant to insulin. This is unique to nconates, since in both the adult and the fetal liver, P‐pyruvate carboxykinase readily responds to insulin. The minimal levels of glucocorticoids characteristic of neonates may be associated with this phenomenon.
Purification and Characterization of a Melanoma Cell Plasminogen Activator
Authors: WALLÉN Per , POHL Gunnar , BERGSDORF Nils , RÅNBY Mats , NY Tor , JÖRNVALL Hans
Published: March 2005

The plasminogen activator from a human melanoma cell line was purified with immunoadsorption as a major step. the cells were cultured in the presence of aprotinin in order to avoid proteolysis. A three‐step purification involved adsorption on antibodies to porcine tissue plasminogen activator before chromatographies on arginine‐Sepharose and Sephadex G‐150. All solvents contained Tween‐80 (0.01%) and, except for the last step, aprotinin. The final product had a specific acitivity of about 220000 IU/mg measured against the WHO urokinase standard. The activator obtained has an apparent Mr of 72000 and consists of single‐chain molecules.

Evidence was obtained that four different types of activator variants occur. First and known previously, the one‐chain form can be proteolytically cleaved into a two‐chain form. Secondly, both the one‐chain and two‐chain molecules exhibit two forms with molecular weight differences of about 3000 (possibly due to carbohydrate differences). Thirdly, the one‐chain preparations contain two variants, each constituting about 50% of the material and differing in length by three N‐terminal amino acids. Finally, a possible positional microheterogeneity was detected.

Digestion with plasmin yields the two‐chain form with disulfide‐bonded polypeptide chains, ‘A′ and ‘B′ (from the N‐terminal and C‐terminal parts, respectively). At the same time, the variability of the original N terminus is removed. The A chain keeps the two Mr variants (now about 40000 and 37000, respectively). The B chain (Mr about 33000) contains thee active site of the molecule, as demonstrated by labelling with [3H]diisopropyl phosphofluoridate, and is homologous to the enzymatically active chains of thrombin, plasmin and other serine proteases. In contrast to these enzymes, the plasminogen activator is enzymatically active in the one‐chain form. A speculative explanation for this activity may possibly be the presence of an ɛ‐amino group of a lysine residue at a position close to the bond cleaved in the two‐chain form.
Relationship of Collagen‐Tailed Acetylcholinesterase with Basal Lamina Components
Authors: GRASSI Jacques , MASSOULIÉ Jean , TIMPL Rupert
Published: March 2005

In view of their supposed localization in extracellular structures, such as basal lamina, we have investigated the possible interactions of collagen‐tailed forms of acetylcholinesterase from Electrophorus and bovine superior cervical ganglion with matrix proteins: laminin, fibronectin and types IV and V collagens. Using binding and sedimentation assays, with iodinated or non‐radioactive matrix proteins, we have not observed any significant interaction, in conditions of high or low ionic strength. We also examined whether the collagen tail of acetylcholinesterase asymmetric forms possessed an immunological relationship with known collagen types (I, III, IV, V) from mammalian sources. We found no specific immunoreactivity with any of the 32 sera studied, either with the iodinated Electrophorus or with the native bovine enzyme. We conclude from these negative results that the collagen‐like tail of acetylcholinesterase is clearly distinct from the classical types of collagen and that asymmetric forms of the enzyme do not interact specifically with the matrix proteins studied. This does not exclude the possibility of specific interactions with other components, remaining to be identified.