bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phosphorylation of a 51‐kDa envelope membrane polypeptide involved in protein translocation into chloroplasts
Authors: HINZ Giselbert , FLÜGGE Ulf Ingo 
Published: March 2005

In this report we demonstrate that a 51‐kDa outer‐envelope membrane protein (P51) is involved in protein translocation into chloroplasts. Furthermore it is shown that phosphorylation of P51 is functionally related to the process of binding and/or importing precursor proteins into chloroplasts. Several lines of evidence have been obtained supporting this suggestion. First, protein import into chloroplasts was inhibited by the membrane‐impermeable agent pyridoxal 5′‐phosphate, which has been shown to react with a component of the protein‐import apparatus. Phosphorylation of envelope membrane polypeptides using [γ‐32P]ATP in the presence of pyridoxal 5′‐phosphate resulted in an increased incorporation of 32P radiolabel into a 51‐kDa membrane polypeptide (P51). A close correlation between the inhibition of protein import and the increase in the phosphorylation state of P51, both as a function of PLP concentration, was observed. Second, binding of purified precursor proteins to chloroplasts resulted in a specific increase in the phosphorylation state of P51. This effect was not exerted by the mature form of the precursor protein lacking the presequence. Third, internally generated ATP was able to compete specifically with externally added [γ‐32P]ATP for the phosphorylation of P51. Fourth, digestion of the outer‐envelope membrane with low amounts of thermolysin resulted in a loss of protein import activity, which was associated with the removal of the phosphorylation site of P51.Phosphorylation of P51 proceeds with an apparent Km (ATP) of about 5 μM, which is much lower than the ATP concentration required for the protein translocation itself. We suggest that two different ATP‐dependent processes are involved in protein translocation into chloroplasts. P51 represent presumably a regulatory component of the protein‐import apparatus or the protein receptor itself.
Partial purification and characterization of cytosolic Tyr‐protein kinase(s) from human erythrocytes
Authors: CLARI Giulio , BRUNATI Anna Maria , MORET Vittorio 
Published: March 2005

Tyrosine‐protein kinase, phosphorylating tyrosine residues of transmembrane band 3 protein, has been partially purified from human erythrocyte cytosol by DEAE‐Sepharose chromatography followed by heparin‐Sepharose chromatography.Such a Tyr‐protein kinase (36 kDa), as distinct from the Ser/Thre‐protein kinases (casein kinase S and TS), appears to display a broader site specificity than does the previously described human erythrocyte P‐Tyr‐protein phosphatase, dephosphorylating band 3 protein. That is, it is able to phosphorylate not only the highly acidic copolymer poly(Glu–Tyr)4:1 but also angiotensin II, lacking an acidic amino acid sequence around the target Tyr residue. Moreover, the phosphorylation of these two substrates exhibits a different pH dependence and a different response to NaCl and 2,3‐bisphosphoglycerate.These results suggest that in intact erythrocytes the cytosolic Tyr‐protein kinase might phosphorylate band 3 not only on Tyr‐8, surrounded by several acidic side‐chains (as demonstrated preferentially to occur in isolated ghosts), but also on other Tyr residues surrounded by other amino acid sequences.
Bovine endothelial cell plasminogen activator inhibitor
Authors: KATAGIRI Kazuya , OKADA Kenji , HATTORI Hiroyuki , YANO Milsuo 
Published: March 2005

A plasminogen activator inhibitor (PAI) was purified from bovine endothelial cell conditioned medium by a simple procedure in the absence of protein denaturant The yield was 2.2 mg from 1.6 1 conditioned medium in a typical experiment. The purified inhibitor showed a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis and reverse fibrin autography with an apparent molecular mass of 45 kDa. The amino‐terminal 40‐amino‐acid sequence was determined and found to be 70% similar to the reported corresponding sequence of human PAI‐1. The amino acid composition also revealed a close relationship between bovine PAI and human PAI‐1.The purified PAI was substantially inactive (570 U/mg) but it could be activated by treatment with protein denaturants such as 1% SDS (1.8 × 105 U/mg) and 4 M guanidine‐HCl (l.5 × 105 U/mg). A more effective activation of this latent PAI was achieved by heat treatment at 100°C for 2.S min, generating the specific activity of 1.0 × 106 U/mg. The heat‐activated PAI lost its activity during incubation at 56°C for 30 min. but repeated heat at 100°C for 2.5 min could regenerate about 70% of the initial activity. Treatment at 37°C, 56°C and 80°C. however, failed to activate the latent PAI at all. These findings suggest that the buried reactive site of the latent PAI is exposed as a result of a heat‐induced, specific conformational change, but tends to be masked again during renaturalion under mild conditions, i.e. the PAI protein takes on preferentially a latent form.
Enzymes as biosensors
Authors: SOULIE Jean‐Michel , RIVIERE Mireille , RICARD Jacques 
Published: March 2005

Oxidized chloroplastic fructose‐bisphosphatase is almost totally inactive at pH 7.5, that is under pH conditions that prevail in the chloroplast stroma. When preincubated for different time periods with fructose 2, 6‐bisphosphate and assayed in the absence of this ligand, it displays an activity which is directly related to the duration of the preincubation phase. This implies that fructose 2, 6‐bisphosphate induces enzymes conformers that appear in sequence and may be competent for catalytic activity. Upon desorption of fructose 2, 6‐bisphosphate the enzyme may retain its active conformation for a time period whose duration depends on magnesium concentration. It thus appears that reduction of the enzyme is not an obligatory prerequisite for its acitivity.Fructose 2, 6‐bisphosphate behaves as a competitive inhibitor of the reduced, active enzyme, with respect to the real substrate. When assayed with the oxidized enzyme, however, its behaves as an acitvator. Moreover, the apparent steady‐state rate that may be measured experimentally depends on both fructose 2, 6‐bisphosphate concentration and the direction of a concentraion change. The reaction velocity experimentally measured is thus a meta‐steady‐state rate and depends on the initial conditions of the system. The fructose‐bisphosphatase system thus displays, with respect to fructose 2, 6‐bisphosphate. a hysteresis loop and may then sense whether the concentration of that ligan is increased ordecreased.A model has been proposed which allows one to explain these results. This model is based on the view that the substrateand fructose 2, 6‐bisphosphate compete for the same site of the enzyme and that this latter ligand stabilizes a conformation competent for enzyme activity. After the ligand has been chased away, the enzyme retains the active conformation for a while and slowly relapses to the initial inactive conformation. The time‐scale of this slow relaxation overlaps that of the steady state of product appearance and this generates meta‐steady‐state kinetic, which is dependent on the initial state and therefore on the history of the system.
Calcium‐dependetn inactivation of the Ca2+‐ATPase from sarcoplasmic reticulum by chemically reactive adenosine triphosphate
Authors: BILL Eberhard , GUTOWSKI Zeynep , BÄUMERT Hans G. 
Published: March 2005

ATP γP‐imidazolidate, synthesized from ATP and carbonyldiimidazole, inhibits the Ca2+‐ATPase of sarcoplasmic reticulum in a biphasic;manne.r A fast first phase is concentration‐dependent while a slower second phase is independent of the inhibitor concentration.
The inhibnition is calcium‐ and magnesium‐dependent. No inhibition occurs in the absence of either cation.
Inhibition of the Ca2+‐ATPase can be prevented by the protection with ATP.
The loss of ATPase activity is pH‐dependent. Maximal inhibition coincides with maximal ATPase activity It indicates a participation of the reacting amino acid side chain in the catalytic cycle.
The incorporation of radioactive inhibitor is reversedin a time‐dependent fashion while the Ca2+ATPase remains inhibited.
We conclude that ATP γP‐imidazolixate initially reacts covalently with an amino acid side chain, probably Asp‐351, but is subsequently expelled by a reaction with a second amino acid.
This two‐step reaction induces an intramolecular cross‐link with can be shown by the creation of a new protein band on SDS‐PAGE which originates in the Ca2+‐ATPase.
Detergentless microemulsions as media for enzymatic reactions
Authors: KHMELNITSKY Yuri L. , HILHORST Riet , VERGER Cees 
Published: March 2005

Catalytic acitivity and stability of cholesterol oxidase dissolved in ternary systems composed on n‐hexane, isopropanol, and water were studied. The dependence of catalytic acitivity on the composition of the system revealed two maxima, in contrast to the behaviour of previously studied enzymes where a single maximum has been observed. The stability profile of cholesterol oxidase showed a single sharp maximum coinciding with the microemulsion region of the phase diagram. Both catalytic activity and the first‐order inactivation rate constant of cholesterol oxidase dissolved in n‐hexane/isopropanol/water ternary systems were found to decrease with decreasing temperature. This decrease was more rapid for the inactivation rate constant than for catalytic activity, the activation energies being 200 and 60 kJ mol−1, respectively.Preparative conversion of cholesterol to cholestenone catalyzed by cholesteroloxidase in n‐hexane/isopropanol/water ternary systems was carried out with 100% yield. Decreased temperature and the presence of catalase were required to achieve high degrees of cholesterol conversion. A simple procedure suitable for rapid separation of the reaction product and recovery of the enzyme was developed.
5′‐Methylbenzimidazolyl‐cobamides are the corrinoids from some sulfate‐reducing and sulfur‐metabolizing bacteria
Authors: KRÄUTLER Bernhard , KOHLER Hans‐Peter E. , STUPPERICH Erhard 
Published: March 2005

The sulfate‐reducing bacteria Desulfobacterium autotrophicum, Desulfobulbus propionicus and Archaeoglobus fulgidus (VC‐16) and the sulfur‐metabolizing archaebacteria Desulfurolobus ambivalens and Thermoplasma acidophilum were found to contain considerable amounts of corrinoids, that were isolated and crystallized in their Coβ‐cyano form. In three other sulfur‐metabolizing archaebacteria, Thermoproteus neutrophilus, Pyrodictium occultum and Staphylothermus marinus significant amounts of corrinoids were not detected under the isolation methods used.The samples from the three sulfate‐reducers were identified with Coα‐[α‐(5′‐methylbenzimidazolyl)]‐Coβ‐cyanocobamide. This corrinoid was also obtained from a 5‐methylbenzimidazole‐supplemented Propionibacterium fermentation and was structurally characterized by ultraviolet/visible, CD, fast‐atom‐bombardment MS, 1H‐ and 13C‐NMR spectroscopy. Also the major corrinoid from T. acidophilum was (tentatively) analyzed as a 5′‐methylbenzimidazolyl‐cobamide, whereas the main corrinoid from D. ambivalens was indicated to be vitamin B12 (a 5′,6′‐dimethylbenzimidazolyl‐cobamide).The 5′‐methylbenzimidazolylcobamides are found here as the common corrins of some sulfate‐reducing and sulfur‐metabolizing bacteria. The structural diversity due to the differing nucleotide bases of the corrins examined here and in methanogenic and acetogenic bacteria appears not to correlate to the biological function(s) of the corrins, but rather to be determined by biosynthetic properties of these organisms under natural growth conditions.
Analysis of the activation of pepsinogen in the presence of protein substrates and estimation of the intrinsic proteolytic activity of pepsinogen
Authors: KAGEYAMA Takashi 
Published: March 2005

Monkey pepsinogen A, monkey progastricsin, and porcine pepsinogen A were activated in the presence of two different protein substrates, namely, reduced and carboxymethylated lysozyme and hemoglobin. In each case, an extensive delay in activation was observed. The intermolecular activation reaction required for the generation of pepsin or gastricsin was strongly inhibited and this inhibition was essentially responsible for the delay. However, the intramolecular reaction required for the generation of the intermediate forms of the proenzymes was scarcely affected. The delay was longer at pH 3.0 than at pH 2.0.Irrespective of the delay in activation of pepsinogen, the digestion of substrates proceeded rapidly, evidence of the significant proteolytic activity of pepsinogen itself. Kinetic experiments demonstrated that pepsinogen changed from an enzymatically inactive species to an active species before the release of the activation segment. The proteolytic activity of the active pepsinogen was highest at pH 2.0, at 37°C and the activity under these conditions was comparable to that of pepsin.
Microtubule‐assembly inhibitor protein
Authors: KOTANI Susumu , IKAI Atsushi , KAWAI Gota , MAEKAWA Shohei , YOKOYAMA Shigeyuki , SAKAI Hikoichi 
Published: March 2005

Microtubule‐assembly inhibitor protein (MIP) is an acidic protein with Mr 33000 which inhibits microtubule assembly in vitro [Kotani, S., Murofiushi, H., Nishida, E. & Sakai, H. (1984) J. Biochem. (Tokyo) 96, 959‐969]. Anti‐MIP antibody was affinity‐purified from rabbit anti‐MIP sera raised against chemically modified MIP. MIP was localized in the nucleus in interphase culture cells as revealed by immunofluorescent light microscopy.Immunoblotting experiments showed that MIP exists in a variety of mammalian cells and tissues. Kidney appeared to be a better source of MIP than brain, the original source. Kidney MIP was isolated by the same procedure as for brain MIP and proved to be indistinguishable from brain MIP in the inhibitory acitivity of microtubule assembly, molecular mass, immunoreactivity, and one‐dimensional peptide mapping. Physicochemical characteristics of MIP were studied using the kideny protein. It contained 20% aspartic acid and 25% glutamic acid, accounting for its acidic nature. Hydrodynamically, MIP was a monomer with s20, w= 1.9 S and Mr= 30 000. The frictional ratio, f f0= 1.7, indicated that MIP is not a glubular molecule but has either an elongated or an expanded structure. Circular dichroic results showed a low content of α‐helix or β‐sheet structure for MIP. Proton nuclear magnetic resonance analysis provided evidence that MIP consists mainly of very flexible structures (random‐coli‐like structures), but still contains a hydrophobic core structure below 60°C.
The arrangement of intra‐ and intermolecular disulfide bonds in the carboxyterminal, non‐collagenous aggregation and cross‐linking domain of basement‐membrane type IV collagen
Authors: SIEBOLD Bernhard , DEUTZMANN Rainer , KÜHN Klaus 
Published: March 2005

The hexameric complex of globular domains of type IV collagen was isolated after collagenase digestion of human placenta and the different monomers and dimers present were chromatographically separated. The ratio of α1(IV)NC1 to α2(IV)NC1 was 2:1. About 50% of the NC1 domains were connected to dimers. Predominantly α1‐α1 dimers were found. Only 12% were α2‐α2 dimers and no α1‐α2 dimers could be detected. The majority (88%) of the intermolecular bonds was found to be disulfide bridges. The remainder could not be cleaved by reduction.To elucidate the arrangement of the disulfide bonds, the unreduced α(IV)NC1 monomers were treated with cyanogen bromide, the disulfide‐bridged peptides isolated and characterized by Edman degradation. Each of the two homologous subdomains within a monomer is stabilized by an identical set of three disulfide bonds. In subdomain I, cysteines at positions 20 and 53 are connected with the C‐terminal cysteine pair 108 and 111. Thus formed, the disulfide knot stabilizes two interconnected loops of 32 and 54 residues, respectively. A smaller loop of five residues occurs due to a disulfide bond between the cysteines 65 and 71. A similar disulfide arrangement is indicated for subdomain II which is separated from subdomain I by a segment of 20 amino acid residues. The same arrangement of disulfide bonds has been strongly suggested for the α2(IV)NC1 monomer by the isolation and characterization of its disulfide‐bridged tryptic fragments.Similar investigations on the dimeric α1(IV)NC1 domain established the arrangement of the intermolecular disulfide bonds. They are formed by a complete disulfide exchange between corresponding disulfide knots of two monomeric NC1 domains.
Methoxyretinals in bacteriorhodopsin
Authors: GÄRTNER Wolfgang , OESTERHELT Dieter 
Published: March 2005

Analogue bacteriorhodopsins (BRs) were reconstituted from bacterioopsin and 9‐, 11‐, or 13‐methoxyretinals or their demethyl derivatives, respectively. In organic solvents the retinals occur as cis isomers of the respective double bonds carrying the methoxy group. 9‐Methoxyretinal, present as the 9‐cis isomer, does not form an analogue BR with bacterioopsin in the dark. Upon illumination, a BR is produced with an absorbance maximum at 560 nm. This compound is thermally unstable, and converts back into the 9‐cis‐containing complex (λmax= 410 nm) in the dark. Removal of the 13‐methyl group from this compound (= 9‐methoxy 13‐demethyl retinal) does not change the 9‐cis configuration of the free retinal, but allows the reconstitution of a thermally stable chromoprotein absorbing around 500 nm with a proton translocation rate of about 10% of the BR value, comparable to the 13‐demethyl BR value [Gärtner, W., Towner, P., Hopf, H. & Oesterhelt, D. (1983) Biochemistry 22, 2637‐2644]. 11‐Methoxy BRs (13‐demethyl and 9,13‐didemethyl) absorb around 530 nm and are inactive. 13‐Methoxy retinal (13‐cis isomer) reconstitutes a chromoprotein with an absorbance maximum at 515 nm, which can be photoconverted to a thermostable 460‐nm‐absorbing complex. For the 515‐nm‐absorbing species of 13‐methoxy BR a light‐induced proton translocation was not detected in measurements with cell vesicles (detection of pH changes in the vesicle preparation). Only by photocurrent measurements in a bilayer experiment could a very diminished photocurrent be detected, about 1 ‐ 2% of BR, [Fendler et al. (1987) Biochim. Biophys. Acta 893, 60‐68]. The reconstitution rate of 13‐methoxy BR from 13‐methoxy retinal and bacterioopsin is slower by a factor of 40 compared to 13‐ethyl BR. although both substituents are of similar size.The position 13 of retinal was found to be most sensitive for regulation of the absorption maximum and the formation and stability of the all‐trans isomer, which is the active form for light‐induced proton translocation. The results suggest that an electronic interaction with a charged residue of the binding site exists around position 13 of retinal, which is disturbed when a methoxy group replaces the methyl or ethyl group at that position. This electronic interaction is essential for maintaining the active all‐trans configuration of retinal.
Amino‐acid sequence of ribonuclease T2 from Aspergillus oryzae
Authors: KAWATA Yasushi , SAKIYAMA Fumio , TAMAOKI Hidetsune 
Published: March 2005

The amino acid sequence of ribonuclease T2 (RNase T2) from Aspergillus oryzae has been determined. This has been achieved by analyzing peptides obtained by digestions with Achromobacter lyticus protease I, Staphylococcus aureus V8 protease, and α‐chymotrypsin of two large cyanogen bromide peptides derived from the reduced and S‐carboxymethylated or S‐aminoethylated protein. Digestion with A. lyticus protease I was successfully used to degrade the N‐terminal half of the S‐aminoethylated protein at cysteine residues.RNase T2 is a glycoprotein consisting of 239 amino acid residues with a relative molecular mass of 29155. The sugarcontent is 7.9% (by mass). Three glycosylation sites were determined at Asns 15, 76 and 239. Apparently RNase T2 has a very low degree of sequence similarity with RNase T1, but a considerable similarity is observed around the amino acid residues involved in substrate recognition and binding in RNase T1. These similar residues may be important for the catalytic activity of RNase T2.
Molecular characteristics and evidence for internalization of vasoactive‐intestinal‐peptide (VIP) receptors in the tumoral rat‐pancreatic acinar cell line AR 4‐2 J
Authors: SVOBODA Michal , DE NEEF Philippe , TASTENOY Michèle , CHRISTOPHE Jean 
Published: March 2005

Vasoactive intestinal peptide (VIP) receptors were investigated in the tumoral acinar cell line AR 4‐2 J derived from rat pancreas [125I]Iodo‐VIP binding to cell membranes showed the following IC50 values for unlabeled peptides: VIP, 0.3 nM; peptide His‐IleNH2, 2 nM; helodermin, 30 nM; secretin, 100 nM. After incubation with 20 nM dexamethasone, the binding capacity increased twofold but affinities were unchanged. External [125I]iodo‐VIP binding to intact cells reached steady state after 5 min at 37°C, while the sequestration‐internalization of the [125I]iodo‐VIP ‐receptor complex (tested by cold acid washing) increased progressively, reaching 75% of total binding after 1 h. This phenomenon was blocked at 4°C. Further data with dexamethasone, tunicamycin, cycloheximide, low temperature, and/or phenylarsine oxide, suggested a half‐life of 2 days for VIP receptors and the necessity of N‐glycosylation for proper translocation.
For chemical [125I]iodo‐VIP cross‐linking bis[2‐(succinimidooxycarbonyloxy)ethyl]sulfone gave the best yield when compared with five other bifunctional reagents. In membranes, the main specifically cross‐linked peptide had MT 66000 under nonreducing conditions, and migrated with lower velocity (–5%) under reducing conditions. Cross‐linking was suppressed by VIP, peptide His‐IleNH2 and helodermin (competitively) and also by GTP. In intact cells, the Mr of [125I]iodo‐VIP‐cross‐linked peptides depended on the mode of cell solubilization. After direct solubilization, the major cross‐linked radioactivity migrated as a smear of Mr 130000–180000 but an MT‐66000 peptide was also detectable. In contrast, the solubilization of cross‐linked cells detached by mild trypsinisation gave mainly the MT‐66000 labeled peptide. This suggests that most VIP receptors in intact, attached cells were in a high‐MT complex and that mild cell treatment was sufficient to disrupt this complex.