bluuebunny/crossref_metadata_2025 huggingface dataset:
Fluorescence study of the conformational properties of myoglobin structure
Authors: POSTNIKOVA Galina B. , KOMAROV Yuri E. , YUMAKOVA Elena M.
Published: March 2005

Tryptophanyl fluorescence of high‐spin and low‐spin complexes of sperm whale ferric‐ and ferrousmyoglobins, met‐, azide‐ and cyanomyoglobins and deoxy‐, oxy‐ and carboxymyoglobins has been studied in the pH range 2.5–13. The pH‐dependent fluorescence of sperm whale metmyoglobin acylated at the N‐terminal α‐amino group by methylisothiocyanate and of bovine metmyoglobin, which contains invariant Trp7 and Trp14 but lacks Tyr151, have also been examined. Drastic changes in the fluorescence were registered in the acidic and alkaline pH ranges which are due to denaturation of Mb. Fluorescent and CD data indicate that at pH < 4.5 and pH > 11.5 the unique spatial structure of the protein is destroyed whereas the secondary structure and integrity are essentially preserved. In all sperm whale and bovine myoglobins studied a local conformational change in the surroundings of Trp is observed which precedes alkaline denaturation. It seems to be due to deprotonation of lysine residues and breakage of the salt bridges essential for the maintenance of the native conformation of the N‐terminal and the adjacent region. The parameters of this conformational transition are found to correlate with the spin state of the heme complex. However, analysis of the fluorescence behaviour of different ligand derivatives of myoglobin in the whole pH range studied enables one to conclude that the exact protein conformation depends not only on the spin state of the Fe atom but, to a greater extent, probably on the chemical nature of the ligand and its interaction with the protein groups in the heme cavity. Local conformational changes induced by the replacement of the sixth ligand or by varying pH seem to involve the same region of contacts between the A helix and GH fragment (or between the AE and GH helical complexes) though the extent of the changes may be different.
Fluorescence study of the conformational properties of myoglobin structure
Authors: POSTNIKOVA Galina B. , YUMAKOVA Elena M.
Published: March 2005

The porphyrin and tryptophan fluorescence of sperm whale apomyoglobin complexed with protoporphyrin IX has been studied in the pH range 2–13. It has been shown that the fluorescence and absorption spectra of protoporphyrin incorporated into the heme crevice remain constant in the pH range 5.5–10.8 but change significantly at pH <5.5 and pH >10.8, due to the acid and alkaline denaturation, respectively, of the complex accompanied by dissociation of protoporphyrin IX. At the same pH ranges, the quantum yield of tryptophanyl fluorescence increases sharply as a result of removal of protoporphyrin, acting as a quencher, from the complex. Other parameters of tryptophanyl fluorescence (maximum position, halfwidth and spectrum shape) change in the alkaline region as well. In the acidic pH range, these parameters change only at pH <4.3, indicating that the Trp surroundings are more stable to denaturation than the heme crevice region. Between pH 5.5 and 10.9, where the complex of apomyoglobin with protoporphyrin IX is in its native state, the main parameters of tryptophan fluorescence remain unchanged except for the ratio I325/I350 which diminishes at pH >9.5. Its alteration precedes the alkaline denaturation of the complex and can be explained by a local conformational change induced by the break of the ‘salt bridges’ essential for the maintaince of the native Mb structure in the N‐terminal region. The fluorescence data obtained for apomyoglobin, myoglobin and the complex between protoporphyrin IX and apomyoglobin enable one to compare their structures and to evaluate the role of the porphyrin macrocycle and the iron atom in the formation of the native myoglobin structure and its functioning.
Stimulation of non‐selective cation channels providing Ca2+ influx into platelets by platelet‐activating factor and other aggregation inducers
Authors: AVDONIN Pavel V. , CHEGLAKOV Ivan B. , TKACHUK Vsevolod A.
Published: March 2005

To elucidate the mechanism of the receptor‐stimulated Ca2+ entry into human platelets, the influence of Ca2+‐mobilizing agonists on plasma membrane potential (Em) has been studied. Em changes were registered using potentiometric probe 3,3′‐dipropyl‐2,2′‐thiadicarbocyanine iodide. The agonist effect on Em varied from hyperpolarization to slight and slow rise. On the contrary, after loading of platelets with intracellular Ca2+ indicator quin2, platelet‐activating factor (PAF), thrombin, vasopressin, ADP and thromboxane‐A2‐mimetic U46619 cause substantial transient membrane depolarization. Similar effects were observed after platelet loading with other Ca2+ chelators fura‐2 and indo‐1. Agonist‐induced depolarization considerably reduced if quin2‐loaded platelets were suspended in isoosmotic choline‐containing medium. Using Ba2+ as a substitute of Ca2+, we have demonstrated that in choline‐containing medium PAF‐induced Ba2+ entry into platelets results in membrane depolarization. Dependence on Ba2+ concentration and depolarization kinetics correlates with the dose dependence and kinetics of Ba2+ entry detected by quin2 fluorescence. The agonists also stimulate considerable Na+, Li+ and Cs+ inward currents into platelets. Na+‐dependent depolarization is 2–5‐fold suppressed by extracellular Ca2+ [median inhibitory concentration (IC50) approximately 0.3 mM]. Ni2+ and Cd2+ at similar concentrations block Ca2+ entry and agonist‐induced Na2+ current (IC50 for both cations approximately 50 μM). Agonist‐induced depolarization is blocked by the adenylate cyclase stimulator prostaglandin E1 and the protein kinase C stimulator phorbol ester. It is concluded that agonists stimulate Ca2+ entry into human platelets via receptor‐operated channels which are not strictly selective toward divalent cations and are permeable to Na+, Li+ and Cs+.
Purification, characterization and some properties of diacetyl(acetoin) reductase from Enterobacter aerogenes
Authors: CARBALLO Javier , MARTIN Roberto , BERNARDO Ana , GONZALEZ Josefa
Published: March 2005

A new method, faster, milder and more efficient than the one previously described [Bryn, K., Hetland, O. & Stormer, F. C. (1971) Eur. J. Biochem. 18, 116–119], for purification of diacetyl(acetoin) reductase from Enterobacter aerogenes is proposed. The experiments carried out with the electrophoretically pure preparations obtained by this procedure show that the enzyme (a) produces l‐glycols from the corresponding l‐α‐hydroxycarbonyls by reversible reduction of their oxo groups and also reduces the oxo group of uncharged α‐dicarbonyls converting them into l‐α‐hydroxycarbonyls, and (b) is specific for NAD. This is a new enzyme for which we suggest the systematic name of l‐glycol:NAD+ oxidoreductase and the recommended name of l‐glycol dehydrogenase(NAD).

The molecular mass, pI, affinity for substrates and pH profiles of this enzyme are also described.
Reaction mechanism of the reconstituted oxoglutarate carrier from bovine heart mitochondria
Authors: INDIVERI Cesare , DIERKS Thomas , KRÄMER Reinhard , PALMIERI Ferdinando
Published: March 2005

The transport mechanism of the reconstituted oxoglutarate carrier, purified from bovine heart mitochondria, was studied kinetically. A complete set of half‐saturation constants (Km) was established for the two different substrates oxoglutarate and malate on both the external and the internal sides of the membrane. The internal affinities for oxoglutarate (Km 0.17 mM) and malate (Km 0.7 mM) were higher than the corresponding external affinities (Km 0.3 mM and 1.4 mM, respectively). The exclusive presence of a single transport affinity for each substrate on one side of the membrane indicated a unidirectional insertion of the oxoglutarate carrier into the liposomal membrane. The Km values and also the maximum exchange rates (8–11 μmol · min−1· mg protein−1) for oxoglutarate and malate were independent of the nature of the counter substrate on the other side of the membrane. Under these defined conditions we analyzed the antiport mechanism in two‐reactant initial velocity studies varying both the internal and external substrate concentrations. From the kinetic patterns obtained, a sequential type of mechanism became evident, implying that one internal and one external substrate molecule form a ternary complex with the carrier before transport occurs. A quantitative analysis of substrate interaction with the unloaded or single‐substrate‐occupied carrier revealed that rapid‐equilibrium random conditions were fulfilled, characterized by a fast and independent binding of internal and external substrate. This kinetic mechanism agrees with previous results obtained in intact mitochondria. Considering also the data available for other mitochondrial carriers, a common kinetic mechanism (sequential type) for this carrier family is suggested.
Purification and characterization of the oxidase from the marine bacterium Pseudomonas nautica 617
Authors: ARNAUD Sylvain , MALATESTA Francesco , GUIGLIARELLI Bruno , GAYDA Jean‐Pierre , BERTRAND Patrick , MIRAGLIO Roger , DENIS Michel
Published: March 2005

The aerobic respiratory system of the hydrocarbonoclastic marine bacterium Pseudomonas nautica 617 ends with a single terminal oxidase. It is a heme‐containing membranous protein which has been demonstrated only to reduce molecular oxygen to hydrogen peroxide [Denis, M., Arnaud S. & Malatesta, F. (1989) FEBS Lett. 247, 475–479]. The purification of this oxidase was achieved in a single step through by DEAE‐Trisacryl chromatography. SDS/PAGE showed the presence of four subunits. The pI was found to be 4.45 and a Mr of 130000 was determined by gel filtration.

The amino acid composition of the purified terminal oxidase has been determined. About 52% of the residues are hydrophobic, strengthening the membranous nature of this bacterial oxidase. Room temperature optical spectra are typical of heme b with a 560‐nm band for the reduced form in the α range. The prosthetic group is made of two hemes b, one high‐spin (S= 5/2, g⊥= 5.9, g∥∼ 2.0), the other low‐spin (S= 1/2, gz= 2.94, gy= 2.27). No other metal centre was detected by EPR. The two hemes remained unresolved in optical spectra, even at low temperature, and throughout redox titration. They behaved potentiometrically like a one‐electron, single redox couple, with Em= 87 ± 10 mV at pH 7.2 and 293 K. The purified oxidase did not oxidize ferrocytochrome c, but displayed quinol oxidase activity both with the native quinone (2419 nmol O2· min−1· mg protein−1 and commercially available coenzyme (101.74 nmol O2· min−1· mg protein−1). Exposure of the reduced enzyme to CO induced the collapse of α and β bands as occurred during reoxidation. In contrast, NaCN and NaN3 fully inhibited the oxidase activity. Results are discussed with respect to other purified quinol oxidases.
N‐(Phosphonomethyl)glycine (glyphosate) tolerance in Euglena gracilis acquired by either overproduced or resistant 5‐enolpyruvylshikimate‐3‐phosphate synthase
Authors: REINBOTHE Steffen , NELLES Axel , PARTHIER Benno
Published: March 2005

Photoautotrophic cells of Euglena gracilis can be adapted to N‐(phosphonomethyl)glycine (glyphosate) by cultivation in media with progressively higher concentrations of the herbicide. Two different mechanisms of tolerance to the herbicide were observed. One is characterized by the overproduction and 40‐fold accumulation of the target enzyme, 5‐enolpyruvylshikimate‐3‐phosphate synthase, in cells adapted to 6 mM N‐(phosphonomethyl)glycine. The other is connected with a herbicide‐insensitive enzyme. No evidence was obtained for the involvement of the putative multifunctional arom protein previously reported to be involved in the biosynthesis of aromatic amino acids in Euglena. Cells adapted to N‐(phosphonomethyl)glycine excreted shikimate and shikimate 3‐phosphate into the medium; the amounts depended on the actual concentration of the herbicide. Two‐dimensional gel electrophoresis and determination of 5‐enolpyruvylshikimate‐3‐phosphate synthase activity in crude extracts, as well as after separation by non‐denaturing gel electrophoresis, revealed that the overproduction of the enzyme in adapted cells correlates with the accumulation of a 59‐kDa protein. Overproduction of this 59‐kDa protein resulted from a selectively increased level of a mRNA coding for a 64.5‐kDa polypeptide which appeared in adapted cells, as shown by cell‐free translation in the wheat germ system.

In contrast to this quantitative, adaptive type of tolerance, the second mechanism causing tolerance to N‐(phosphonomethyl)glycine in the Euglena cell line NR 6/50 was probably related to a qualitatively altered 5‐enolpyruvylshikimate‐3‐phosphate synthase, which could not be inhibited by even 2 mM N‐(phosphonomethyl)glycine in vitro. In agreement with this observation, the putatively mutated cell line excreted neither shikimate nor shikimate 3‐phosphate into the growth medium containing N‐(phosphonomethyl)glycine, even if cultivated in the presence of 20 mM or 50 mM N‐(phosphonomethyl)glycine.
Evidence for the localization of the nuclear‐coded 22‐kDa heat‐shock protein in a subfraction of thylakoid membranes
Authors: ADAMSKA Iwona , KLOPPSTECH Klaus
Published: March 2005

The precursor to the nuclear‐coded 22‐kDa heat‐shock protein of chloroplasts (HSP 22) has been transported into isolated intact chloroplasts from heat‐shocked plants. The localization of the mature protein in the chloroplast membrane was investigated. We have shown that the processed HSP 22 of pea was not bound to envelopes and found predominantly in thylakoid membranes. The binding of HSP 22 was stable in the presence of high salt concentrations. Solubilization of thylakoid membranes with Triton X‐100 and phase partitioning with Triton X‐114 indicate an intrinsic localization of HSP 22 or, alternatively, a non‐covalent association with integral membrane protein(s). After fractionation into grana and stroma lamellae, HSP 22 was found mostly in the grana‐membrane subfraction.
Biogenesis of the mitochondrial phosphate carrier
Authors: ZARA Vincenzo , RASSOW Joachim , WACHTER Elmar , TROPSCHUG Maximilian , PALMIERI Ferdinando , NEUPERT Walter , PFANNER Nikolaus
Published: March 2005

The mitochondrial phosphate carrier (PiC) is a member of the family of inner‐membrane carrier proteins which are generally synthesized without a cleavable presequence. Surprisingly, the cDNA sequences of bovine and rat PiC suggested the existence of an amino‐terminal extension sequence in the precursor of PiC. By expressing PiC in vitro, we found that PiC is indeed synthesized as a larger precursor. This precursor was imported and proteolytically processed by mitochondria, whereby the correct amino‐terminus of the mature protein was generated. Import of PiC showed the characteristics of mitochondrial protein uptake, such as dependence on ATP and a membrane potential and involvement of contact sites between mitochondrial outer and inner membranes. The precursor imported in vitro was correctly assembled into the functional form, demonstrating that the authentic import and assembly pathway of PiC was reconstituted when starting with the presequence‐carrying precursor. These results are discussed in connection with the recently postulated role of PiC as an import receptor located in the outer membrane.
The cytosolic and glycosomal glyceraldehyde‐3‐phosphate dehydrogenase from Trypanosoma brucei
Authors: LAMBEIR Anne‐Marie , LOISEAU Ann M. , KUNTZ Douglas A. , VELLIEUX Frederic M. , MICHELS Paul A. M. , OPPERDOES F. R.
Published: March 2005

The protozoan haemoflagellate Trypanosoma brucei has two NAD‐dependent glyceraldehyde‐3‐phosphate dehydrogenase isoenzymes, each with a different localization within the cell. One isoenzyme is found in the cytosol, as in other eukaryotes, while the other is found in the glycosome, a microbody‐like organelle that fulfils an essential role in glycolysis. The kinetic properties of the purified glycosomal and cytosolic isoenzymes were compared with homologous enzymes from other organisms. Both trypanosome enzymes had pH/activity profiles similar to that of other glyceraldehyde‐3‐phosphate dehydrogenases, with optimal activity around pH 8.5–9. Only the yeast enzyme showed its maximal activity at a lower pH. The glycosomal enzyme was more sensitive to changes in ionic strength below 0.1 M, while the cytosolic enzyme resembled more the enzymes from rabbit muscle, human erythrocytes and yeast. The affinity for NAD of the glycosomal enzyme was 5–10‐fold lower than that of the cytosolic, as well as the other enzymes. A similar, but less pronounced, difference was found for its affinity for NADH. These differences are explained by a number of amino acid substitutions in the NAD‐binding domain of the glycosomal isoenzyme.

In addition, the effects of suramin, gossypol, agaricic acid and pentalenolactone on the trypanosome enzymes were studied. The trypanocidal drug suramin inhibited both enzymes, but in a different manner. Inhibition of the cytosolic enzyme was competitive with NAD, while in the case of the glycosomal isoenzyme, with NAD as substrate, the drug had an effect both on Km and Vmax. The most potent inhibitor was pentalenolactone, which at micromolar concentrations inhibited the glycosomal enzyme and the enzymes from yeast and Bacillus stearothermophilus in a reversible manner, while the rabbit muscle enzyme was irreversibly inhibited.
Purification and biochemical characterization of a soluble mouse interferon‐γ receptor produced in insect cells
Authors: FOUNTOULAKIS Michael , SCHLAEGER Ernst‐Juergen , GENTZ Reiner , JURANVILLE Jean‐Francois , MANNEBERG Michael , OZMEN Laurence , GAROTTA Gianni
Published: March 2005

The extracellular domain of the mouse interferon γ receptor comprising amino acids 17–243 of the protein was produced in Spodoptera frugiperda cells infected with a recombinant baculovirus. The receptor was mainly secreted into the culture medium and was purified to homogeneity in several hundred milligram amounts. The purification procedure involved four chromatography steps and delivered a soluble and active receptor with an overall recovery of 30%. From each purification run, two pools of soluble receptor with the same interferon γ binding capacity were isolated. Under reducing electrophoretic conditions the protein of pool I migrates as two bands of molecular masses 32 and 34 kDa and of pool II as two bands of 30 and 32 kDa. The soluble receptor of both pools carries a heterogeneous glycosylation. After deglycosylation it appears as one protein band of 27 kDa. N‐linked carbohydrates contribute about 6 kDa and O‐linked carbohydrates 1 kDa to its molecular mass. The nonreduced protein specifically binds interferon γ on ligand blots and in a solid‐phase binding system and competes for the binding of radiolabeled interferon γ to the cell surface receptor. The soluble mouse interferon γ receptor exists as a monomer in physiological buffer and binds interferon γ in its dimeric form. It is stable at room temperature and against tryptic digestion, but is very sensitive to proteinase K digestion. The soluble mouse interferon γ receptor produced in the insect/baculovirus expression system may prove useful to study the function of interferon γ receptor as an antagonist of endogenous interferon γ in the treatment of immunological and inflammatory disorders.
Aminopeptidase P from rat brain
Authors: HARBECK Hans‐Thomas , MENTLEIN Rolf
Published: March 2005

Aminopeptidase P (EC 3.4.11.9) was purified from rat brain cytosol. A subunit Mr of 71000 was determined for the reduced, denaturated protein whereas an Mr of 143 000 was determined for the native enzyme. The purified aminopeptidase P selectively liberated all unblocked, preferentially basic or hydrophobic ultimate amino acids from di‐, tri‐ and oligopeptides with N‐terminal Xaa‐Pro‐ sequences. Corresponding peptides with penultimate Ala instead of Pro were cleaved with much lower rates; oligopeptides with residues other than Pro or Ala in the penultimate position appeared not to be substrates for the enzyme. Several bioactive peptides with Xaa‐Pro sequences, especially bradykinin, substance P, corticotropin‐like intermediate lobe peptide, casomorphin and [Tyr]melanostatin were shortened by the N‐terminal amino acid by aminopeptidase P action. Rat brain aminopeptidase P was optimally active at pH 7.6–8.0 in the presence of Mn2 +. Chelating agents and SH‐reacting reagents inhibited the enzyme, but common inhibitors of aminopeptidases, like amastatin or bestatin, of prolidase or of dipeptidyl peptidases II and IV, like N‐benzoyloxycarbonyl‐proline or ɛ‐benzyl‐oxycarbonyl‐lysyl‐proline, as well as antibiotics like β‐lactam ones, bacitracin or puromycin, had little or no effect.
Characterisation of the specific p‐nitrophenylphosphatase gene and protein of Schizosaccharomyces pombe
Authors: YANG Jiewu , DHAMIJA Sunder S. , SCHWEINGRUBER M. Ernst
Published: March 2005

Cloning and sequencing of the pho2 gene which codes for a specific p‐nitrophenylphosphatase from Schizosaccharomyces pombe is described. The gene has an open contiguous reading frame of 269 amino acids corresponding to a protein with a molecular mass of 29.5 kDa and a calculated pI of 6.6. The sequence reveals four regions that share significant sequence similarity with the corresponding gene PHO13 of Saccharomyces cerevisiae. Purification of the enzyme to apparent homogeneity is reported. The amino acid composition of the purified protein matches well the values predicted from the nucleotide sequence. On SDS/polyacrylamide gels, the enzyme runs as a protein with a molecular mass of 33 kDa, and by Sephadex chromatography under non‐denaturing conditions as 70 kDa. This indicates that the enzyme is a homodimer in its native form. The enzyme is not glycosylated. Its activity is stimulated by Mg2+ and inhibited by Zn2+. The available data on p‐nitrophenylphosphatase do not give any clues to its biological role and its physiological substrates.
Characterization and primary structure of a second thioredoxin from the green alga, Chlamydomonas reinhardtii
Authors: DECOTTIGNIES Paulette , SCHMITTER Jean‐Marie , DUTKA Sophie , JACQUOT Jean‐Pierre , MIGINIAC‐MASLOW Myroslawa
Published: March 2005

A second thioredoxin, Ch1, distinct from the one recently reported [Decottignies, P., Schmitter, J. M., Jacquot, J. P., Dutka, S., Picaud, A. & Gadal, P. (1990) Arch. Biochem. Biophys. 280, 112–121] has been purified from the green alga, Chlamydomonas reinhardtii, and its functional and structural properties investigated. Its activity in various enzymatic assays has been compared with the activities of different plant thioredoxins (Ch2 from C. reinhardtii and spinach m and f). Ch1 cannot serve as a substrate for Escherichia coli thioredoxin reductase, but can be reduced by spinach ferredoxin‐thioredoxin reductase. It is less efficient than its spinach counterpart in the activation of corn leaf NADP‐dependent malate dehydrogenase by light or dithiothreitol, and it only activates spinach fructose‐1,6‐bisphosphatase at very high concentrations. The complete primary structure of C. reinhardtii thioredoxin Ch1 was determined by automated Edman degradation of the intact protein and of peptides derived from trypsin, chymotrypsin and Staphylococcus aureus V8 protease digestions. When needed, peptide masses were verified by plasma desorption mass spectrometry. Ch1 consists of a polypeptide of 111 amino acids (11 634 Da) and contains the well‐conserved active site sequence Trp‐Cys‐Gly‐Pro‐Cys. Compared to thioredoxins from other sources, the algal thioredoxin Ch1 displays few sequence similarities with all the thioredoxins sequenced so far. Preliminary evidence indicates that Ch1 may be an h‐type thioredoxin.
N‐linked glycosylation affects the processing of mouse submaxillary gland prorenin in transfected AtT20 cells
Authors: LADENHEIM Ruth Graciela , SEIDAH Nabil G. , ROUGEON François
Published: March 2005

Most mouse inbred strains carry two renin genes, Ren‐1 and Ren‐2. Renin‐2, the product of the Ren‐2 gene, is highly expressed in the submaxillary gland. It is a renin isoenzyme 96% similar to kidney renin‐1, but unglycosylated. In order to investigate if glycosylation of prorenin affects its processing and/or secretion we have introduced two potential N‐linked glycosylation sites into preprorenin‐2 cDNA using site‐directed mutagenesis. Expression plasmids were derived from wild‐type and mutant renin‐2 cDNA and were transfected into AtT20 cells. Both transfected cells, expressing glycosylated or unglycosylated forms, secreted prorenin and renin by the constitutive and regulated pathways, respectively. Prorenin was correctly processed to active renin but the second maturation site was not cleaved in AtT20 cells. The comparison of glycosylated and unglycosylated renin expression showed a diminished secretion of glycosylated active renin. Prevention of glycosylation with tunicamycin resulted in an improved secretion of active renin. Moreover, the efficiency of the trypsin activation in vitro was reduced for glycosylated prorenin and it was restored when the activation was performed on mutant renin secreted from tunicamycin‐treated cells. It is proposed that the bulky carbohydrates attached to prorenin constitute a steric hindrance to proteolysis by maturation enzymes.
α‐Factor‐leader‐directed secretion of recombinant human‐insulin‐like growth factor I from Saccharomyces cerevisiae
Authors: STEUBE Klaus , CHAUDHURI Bhabatosh , MÄRKI Walter , MERRYWEATHER James P. , HEIM Jutta
Published: March 2005

A synthetic gene coding for human‐insulin‐like growth factor I (IGFI) was fused to the leader sequence of yeast prepro‐α‐factor and expressed in Saccharomyces cerevisiae under the control of a glyceraldehyde‐3‐phosphate dehydrogenase promoter fragment. Recombinant IGFI was found inside yeast cells and secreted into the medium. The secreted IGFI migrated on SDS gels with the same electrophoretic mobility as authentic IGFI, i.e. at about 7.5 kDa. HPLC analysis of secreted IGFI revealed the presence of the correctly folded, genuine molecule as well as an isomeric byproduct of equal molecular mass but with two of the three disulfide bonds interchanged.

Inside exponentially growing cells the 7.5‐kDa IGFI was also found, along with up to four additional IGFI‐related polypeptides of higher molecular mass. By endoglycosidase F treatment the three polypeptides between 19–26 kDa were converted to a single peptide of 17 kDa. Since this peptide also reacted with an anti‐α‐factor antibody, it represents most likely the unglycosylated α‐factor–IGFI fusion precursor.

Pulse‐chase experiments established the precursor nature of the intracellular higher‐molecular‐mass IGFI species. Conversion of the primary translation product to the differently glycosylated IGFI precursor proteins and into the mature form occurred very rapidly, within 2 min. Rapid maturation was, however, not followed by an equally rapid secretion of the mature form into the medium: only after 30–40 min did IGFI appear outside the cells.

We therefore postulate the presence of an as yet undefined Golgi or post‐Golgi bottleneck representing a major obstacle in secretion of recombinant IGFI from S. cerevisiae cells.
The synthesis of polyphenylalanine on ribosomes to which erythromycin is bound
Authors: ODOM Obed W. , PICKING William D. , TSALKOVA Tamara , HARDESTY Boyd
Published: March 2005

Erythromycin binds to the large subunit of Escherichia coli ribosomes at a specific site that is very close to the amino acid of aminoacyl‐tRNA bound into the peptidyltransferase center, and to the site to which puromycin is bound, the P and A sites, respectively, of the classical two‐site model of ribosome function. Both erythromycin and puromycin affect fluorescence from fluorescent derivatives of aminoacyl‐tRNAs, while both puromycin and aminoacyl‐tRNAs affect fluorescence of fluorescent derivatives of erythromycylamine. The results demonstrate unequivocally that erythromycin, deacylated tRNA, a peptidyl‐tRNA analogue and puromycin can be bound simultaneously to the same ribosome. Nascent peptides of more than a few amino acids in length block binding of erythromycin to the ribosomes but, unlike most other peptides, long polyphenylalanine chains can be synthesized on ribosomes to which erythromycin is bound. It is suggested that this refractory synthesis in the presence of erythromycin reflects the atypical physical and structural properties of polyphenylalanine.
Preproabrin: genomic cloning, characterisation and the expression of the A‐chain in Escherichia coli
Authors: WOOD Katherine A. , LORD J. Michael , WAWRZYNCZAK Edward J. , PIATAK Michael
Published: March 2005

Synthetic oligonucleotides representing all possible sequences of an N‐terminal and an internal region of the A‐chain of abrin C were used to generate a probe specific for abrin‐related sequences using the polymerase chain reaction on Abrus precatorius genomic DNA. A lambda phage library constructed from genomic DNA isolated from leaf tissue of A. precatorius was screened and positive hybridising clones were characterised by restriction enzyme analysis. The coding regions of unique clones were characterised by DNA sequencing. One clone encodes a preproprotein closely related to abrin C with 83% similarity between the A‐chain sequences. Based on similarity with the ricin toxins and Ricinus communis agglutinin, the preproabrin consists of an A‐chain of 251 amino acids preceded by 34 amino acids containing an N‐terminal signal peptide, followed by a 14‐amino‐acid linker and a B‐chain of 263 amino acids. The mature A‐chain of the preproabrin has been expressed cytoplasmically in Escherichia coli and the soluble recombinant protein was produced at levels exceeding 6% of total cell protein. The recombinant A‐chain has been purified to homogeneity and its ability to depurinate 28S rRNA in rat liver ribosomes has been demonstrated in vitro.
Primary structure of bovine lactoperoxidase, a fourth member of a mammalian heme peroxidase family
Authors: CALS Marie‐Madeleine , MAILLIART Pascal , BRIGNON Ghislaine , ANGLADE Patricia , DUMAS Bruno Ribadeau
Published: March 2005

Much is known about bovine lactoperoxidase but no data are available on its primary structure. In this work its main active fraction was isolated from cow's milk and sequenced using a conventional strategy. A clear similarity was found with human myeloperoxidase, eosinophil peroxidase and thyroperoxidase, the sequences of which were recently elucidated from those of their cDNAs and/or genes. The single peptide chain of bovine lactoperoxidase contains 612 amino acid residues, including 15 half‐cystines and 4 or 5 potential N‐glycosylation sites. The corresponding peptide segments of human myeloperoxidase, eosinophil peroxidase and thyroperoxidase display 55%, 54% and 45% identity with bovine lactoperoxidase, respectively, with 14 out of the 15 half‐cystines present in each of the four enzymes being located in identical positions. The occurrence of an odd number of half‐cystines in bovine lactoperoxidase supports the recent finding of a heme thiol released from this enzyme by a reducing agent, suggesting that the heme is bound to the peptide chain via a disulfide linkage, since the absence of free thiol in the enzyme was reported long ago.
A spectroscopic and conformational study of pertussis toxin
Authors: SEABROOK Richard N. , ATKINSON Tony , IRONS Laurence I.
Published: March 2005

The conformation of native pertussis toxin has been investigated by secondary structure prediction and by circular dichroism, fluorescence and second‐derivative ultraviolet absorption spectroscopy. The far‐ultraviolet circular dichroic spectrum is characteristic of a protein of high β‐sheet and low α‐helix content. This is also shown by an analysis of the circular dichroic spectrum with the Contin programme which indicates that the toxin possesses 53%β‐sheet, 10%α‐helix and 37%β‐turn/loop secondary structure. Second‐derivative ultraviolet absorption spectroscopy suggests that 34 tyrosine residues are solvent‐exposed and quenching of tryptophan fluorescence emission has shown that 4 tryptophan residues are accessible to iodide ions. One of these tryptophans appears to be in close proximity to a positively charged side‐chain, since only 3 tryptophans are accessible to caesium ion fluorescence quenching. When excited at 280 nm, the emission spectrum contains a significant contribution from tyrosine fluorescence, which may be a consequence of the high proportion (55%) of surface‐exposed tyrosines. No changes in the circular dichroic spectra of the toxin were found in the presence of the substrate NAD. However, NAD did quench both tyrosine and tryptophan fluorescence emission but did not change the shape of the emission spectrum, or the accessibility of the tryptophans to either the ionic fluorescence quenchers or the neutral quencher acrylamide.
The complex between a tissue inhibitor of metalloproteinases (TIMP‐2) and 72‐kDa progelatinase is a metalloproteinase inhibitor
Authors: KOLKENBROCK Hansjörg , ORGEL Dagmar , HECKER‐KIA Adelheid , NOACK Wolfgang , ULBRICH Norbert
Published: March 2005

Human rheumatoid synovial cells in culture secrete both 72‐kDa progelatinase and a complex consisting of 72‐kDa progelatinase and a 24‐kDa inhibitor of metalloproteinases, TIMP‐2. In addition, the culture medium contains TIMP‐1, the classical inhibitor of metalloproteinases, with a molecular mass of 30 kDa. TIMP‐1 does not form a complex with free 72‐kDa progelatinase.

Free progelatinase and progelatinase complexed with TIMP‐2 can be activated with the organomercury compound p‐aminophenylmercury acetate. The activated complex shows less than 10% the enzyme activity of activated free gelatinase.

The progelatinase – TIMP‐2 complex could be shown to be an inhibitor for other metalloproteinases, such as gelatinase and collagenase secreted by human rheumatoid synovia fibroblasts, as well as for the corresponding enzymes from human neutrophils.
Orientation of the haems of the ubiquinol oxidase: O2 reductase, cytochrome bo of Escherichia coli
Authors: SALERNO John C. , INGLEDEW W. John
Published: March 2005

The orientation of the two haems of the Escherichia coli ubiquinol oxidase:O2 reductase, cytochrome bo, has been determined by electron paramagnetic resonance studies on oriented multilayer preparations of cytoplasmic membrane fragments. The enzyme contains a low‐spin b‐like haem and a high‐spin b‐like haem, designated cytochromes b and o respectively. Both haems are oriented with their planes perpendicular to the membrane plane, further extending the catalogue of structural and functional similarities between this enzyme and the mammalian cytochrome c oxidase, cytochrome aa3.
Cultured human fibroblasts contain a large pool of precursor β1‐integrin but lack an intracellular pool of mature subunit
Authors: De STROOPER Bart , Van LEUVEN Fred , CARMELIET Geert , Van Den BERGHE Herman , CASSIMAN Jean‐Jacques
Published: March 2005

Previous work has shown the presence of an important intracellular pool of β1‐integrin subunit in human skin fibroblasts as detected with monoclonal antibody DH12 [De Strooper, B., Van der Schueren, B., Jaspers, M., Saison, M., Spaepen, M., Van Leuven, F., Van den Berghe, H. & Cassiman, J. J. (1989) J. Histochem. Cytochem. 37, 299–307]. To analyze this more quantitatively, a radioimmunoassay with radioiodinated monoclonal antibody was developed. The total amount of specific binding sites for monoclonal antibody DH12 on skin fibroblasts was between 0.8–1.5 × 106/cell. After permeabilizing the cells with digitonin, a threefold increase in specific binding was observed, which suggested that about 60% of the total amount of β1‐subunit was localized intracellularly. From pulse/chase experiments, it was deduced that an important pool of precursor subunit, as defined by its sensitivity to endoglycosidase treatment, existed in fibroblasts. Since in steady‐state‐labeling conditions, at least three to four times more precursor than mature subunit was immunoprecipitated with monoclonal antibody DH12, we suggested that the intracellular pool of β1‐integrin subunit is mainly precursor pool. This precursor pool contains a degradation compartment and a maturation compartment. Other investigators have found evidence for a recirculating pool of mature integrin in Chinese hamster ovary cells. Therefore, the presence of a recirculating pool of integrin in human fibroblasts was also considered. The data obtained with mAb DH12 showed that less than 10% of the surface pool of integrin was internalized by endocytosis. Since, however, cross linking of β1‐integrins with polyclonal antibodies leads to rapid endocytosis of most of the integrin, it remains possible that the quantitatively small effect was actually an artefact induced by the divalent mAb. We conclude that the intracellular pool of β1‐integrins observed in our previous studies consists of precursor and that in skin fibroblasts no mature β1‐integrin is available intracellularly for rapid quantitative modulations at the cell surface.
Core I protein of bovine ubiquinol–cytochrome‐c reductase; an additional member of the mitochondrial‐protein‐processing family
Authors: GENCIC Simonida , SCHÄGGER Hermann , von JAGOW Gebhard
Published: March 2005

Core I and core II proteins are the largest nuclear‐encoded subunits of the mitochondrial ubiquinol–cytochrome‐c reductase (bc1 complex) lacking redox prosthetic groups. cDNA clones of the two bovine core proteins have been isolated by the screening of λ ZAP cDNA libraries either with an oligonucleotide probe based on the sequence of an internal peptide or with a polymerase‐chain‐reaction‐amplified fragment. The core I precursor protein consists of 362 amino acids with a 34‐amino‐acid presequence typical for mitochondrial targeting signals. The mature protein migrates in SDS/polyacrylamide gels with an apparent molecular mass of 47 kDa, which does not correspond to the actual molecular mass of the protein of 35.8 kDa deduced from the cDNA sequence. The core II precursor protein is composed of 453 amino acids having a 14‐amino‐acid presequence as a targeting sequence. Comparison of the core I amino acid sequence with sequences of the newly discovered protein family [Schulte, U., Arretz, M., Schneider, H., Tropschug, M., Wachter, E., Neupert, W. & Weiss, H. (1989) Nature 339, 147–149] comprising the processing enhancing protein (PEP), matrix processing peptidase (MPP), and core I and II proteins from Neurospora crassa and Saccharomyces cerevisiae, revealed a remarkable identity of 39% and a high similarity of 49% to N. crassa PEP, which in this fungus is identical to core I. Core II protein is only a distant relative of this protein family. Based on these sequence comparisons and data obtained by genomic Southern blots, we anticipate that the bovine core I subunit, like the N. crassa core I protein, is bifunctional, being responsible for the maintenance of electron transport and processing of proteins during their import into the mitochondrial matrix. The analysis of the primary structure of the two core proteins completes the set of primary structures of all subunits of bovine ubiquinol–cytochrome‐c reductase.
The unusual action of (R,S)‐2‐hydroxy‐2‐trifluoromethyl‐trans‐n‐octadec‐4‐enoic acid on 5‐lipoxygenase from potato tubers
Authors: BUTOVICH Igor A. , SOLOSHONOK Vadim A. , KUKHAR Valeri P.
Published: March 2005

We found that (R,S)‐2‐hydroxy‐2‐trifluoromethyl‐trans‐n‐octadec‐4‐enoic acid (HTFOA) is a powerful activator of 5‐lipoxygenase from potato tubers. The degree of activation of the enzyme is proportional to the HTFOA concentration and is a maximum at about 0.1 mM independently of initial substrate concentration (25 μM or 0.1 mM). At greater concentrations of HTFOA, enzyme inhibition takes place. Enzyme activation is inversely proportional to the substrate (linoleic acid) concentration. The results may be explained by assuming that a regulatory center exists in the enzyme molecule, which shows affinity to both substances: activator and linoleic acid.
Synergistic effects of Ca2+ and wheat germ agglutinin on the lamellar‐hexagonal (HII) phase transition of glycophorin‐containing egg‐phosphatidylethanolamine membranes
Authors: TAMPÉ Robert , GALLA Hans‐Joachim
Published: March 2005

Glycophorin has been reconstituted into egg phosphatidylethanolamine (egg‐PtdEtn) membranes. Stable vesicles were obtained at a molar ratio of 8 × 10−4 of inserted protein/lipid. This macroscopic change from lipid aggregates to lipid vesicles was followed by density gradient centrifugation. Vesicles formed in the presence of protein enclose the dye calcein and are stable with time and temperature. Membrane aggregation does not occur, as was demonstrated by energy‐transfer experiments. The phase transition from the fluid lamellar Lα phase to the inverted hexagonal HII phase observed at 29°C in pure egg‐PtdEtn membranes is suppressed and finally disappears in the presence of glycophorin. The transition enthalpy decreases linearly from ΔH= 4 kJ/mol in pure lipids to zero at a protein/lipid molar ratio of 1:1000.

Ca2+ ions and wheat germ lectin act synergistically on the phase behavior of vesicles containing glycophorin and phosphatidylethanolamine. Differential scanning calorimetry scans show that the lamellar‐to‐hexagonal phase transition is reinduced. The membranes aggregate and exchange lipid as could be demonstrated by energy transfer experiments. The dye calcein is released but only if the temperature exceeds the lamellar‐to‐hexagonal phase transition temperature of the pure egg‐PtdEtn.
The C‐terminal positively charged region of subunit 8 of yeast mitochondrial ATP synthase is required for efficient assembly of this subunit into the membrane F0sector
Authors: GRASSO David G. , NERO Debra , LAW Ruby H. P. , DEVENISH Rodney J. , NAGLEY Phillip
Published: March 2005

This paper deals with a truncated derivative of subunit 8 of yeast mitochondrial ATP synthase in which a conserved positively charged residue (Lys47) has been removed by site‐directed mutagenesis together with the C‐terminal residue (Leu48). This derivative has been expressed as a chimaeric precursor N9L/Y8‐1(K47‐STP) carrying an N‐terminal cleavable leader sequence (N9L), fused by a short bridging sequence to the truncated subunit‐8 passenger protein. Allotopic expression of N9L/Y8‐1(K47‐STP)in vivoin anaap 1 mit−host yeast strain lacking endogenous subunit 8 leads to partial restoration of bioenergetic function in the transformant strain denoted T475. Import and assembly studies were carried outin vitrousing target mitochondria from strain YGL‐1 partially depleted in subunit 8; such controlled depletion has been previously shown to be required for the efficient assembly (monitored immunochemically) of full‐length subunit 8 importedin vitroas the precursor N9L/Y8‐1. It was found that N9L/Y8‐1(K47‐STP) synthesizedin vitrowas imported successfully into YGL‐1 mitochondria, but no significant assembly of the truncated subunit 8 was observed in these or any other mitochondria tested. The bioenergetic defects in T475 mitochondria are ascribed to the impaired assembly of the subunit‐8 variantin vivo, resulting from the truncation at Lys47. In consequence, T475 mitochondria behave as though partially depleted of subunit 8. This conclusion was supported by the ability of isolated T475 mitochondria to provide a vehicle for the efficient import and assembly of subunit 8 processed form full‐length N9L/Y8‐1. Two related aspects of import and assembly have been addressed as part of the analysis of truncated subunit 8. First, mitochondria from strain T2‐1, anaapl mit−mutant genetically reconstituted by allotopic expression of N9L/Y8‐1, were also found to be effective in thein vitroassembly of subunit 8 derived from imported N9L/Y8‐1. This suggests an intramitochondrial shortage of subunit 8 delivered by allotopic expression of N9L/Y8‐1in vivo, which may underlie the incomplete restoration of energy coupling in T2‐1 mitochondria compared to those of wild‐type yeast. Second, on allotopic expression of N9L/Y8‐2 (containing subunit 8 directly fused to N9L) in theaap 1 mit−host, a rescued transformant strain T10‐1 was generated which displays bioenergetic defects superficially similar to those of T475. Processed subunit 8 clearly assembled into the ATP synthase of isolated YGL‐1 mitochondria, in spite of the relatively weak import of N9L/Y8‐2in vitro. This demonstrates the ability of thein vitroassembly system to distinguish assembly properties of variants of subunit 8, such as N9L/Y8‐1(K47‐STP) and N9L/Y8‐2.
Activation and solubilization of the retinal cGMP‐specific phosphodiesterase by limited proteolysis
Authors: CATTY Patrice , DETERRE Philippe
Published: March 2005

The cGMP‐specific phosphodiesterase (PDE) of vertebrate retinal rod outer segments (ROS) is a peripheral enzyme activated in vivo by transducin. In vitro artificial activation can be achieved using trypsin. This was described as resulting from degradation of the inhibitory γ subunit (2 copies/PDE molecule), leaving intact the αβ catalytic core. It was, however, observed that trypsin could induce the release of PDE (or solubilization) from the ROS membranes before its activation [Wensel, T. G. & Stryer, L. (1986) Proteins Struct. Funct. Genet. 1, 90–99]. Studying the time course of this solubilization, we were able to purify a trypsin‐solubilized PDE still completely inhibited (i.e. with its two γ subunits bound). The tryptic solubilization of PDE is therefore complete before any functional degradation of the γ subunits occurs. It was recently suggested that this solubilization could coincide with the cleavage of a C‐terminal fragment of the α subunit, which can be labeled by methylation of a terminal cysteine residue [Ong, O. C., Ota, I. M., Clarke, S. & Fung, B. K. K. (1989) Proc. Natl Acad. Sci. USA 86, 9238–9242]. We present the following evidence indicating that the C‐terminus of the PDE β subunit is mainly responsible for PDE anchorage to the ROS membrane. (a) The trypsin‐solubilized PDEαβγ2 has intact blocked N‐termini. (b) It is still methylated on PDEα. (c) The C‐terminus of PDEβ can also be labeled by methylation and its tryptic cleavage coincides well with the PDE solubilization. (d) Sequential cleavage of the α and β polypeptides can also be detected by high‐resolution gel electrophoresis: the first cleavage appears on the β subunit and is completed when cleavage of the α subunit begins. The time course for cleavage of the γ subunits appears to be slower than for the β subunit and comparable to that of the α subunit. Upon longer trypsinization, a 70‐kDa polypeptide appears which seems to be a degradation product of PDEβ. Gel‐filtration analysis, however, shows that this 70‐kDa fragment does not dissociate from the catalytic core.