bluuebunny/crossref_metadata_2025 huggingface dataset:
(R)‐Lactyl‐CoA dehydratase from Clostridium propionicum
Authors: HOFMEISTER Antje E. M. , BUCKEL Wolfgang
Published: March 2005

A new two‐step method for purifying component EII of lactyl‐CoA dehydratase was developed. The source fo the enzyme was Clostridium propionicum grown on either d,l‐alanine or l‐threonine. No difference in these preparations was observed whether during purification or by SDS/PAGE of the pure enzymes. Bothe preparations exhibited similar activities towards (R)‐lactyl‐CoA as well as towards (R)‐2‐hydroxbutyryl‐CoA, the latter being the superior substrate.

Three species of (2R)‐2‐hydroxybutyrate labelled with 3H at C3 were prepared containing 96%, 37% and 63% of the 3H in the 3S‐position. By incubation of these species with acetyl‐CoA, propionate CoA‐transferse and lactyl‐CoA dehydratase 104%, 32% and 70% of the 3H, respectively, was released as 3HOH. The data indicate the stereospecific abstraction of the 3Si hydrogin of (2R)‐2‐hydroxybutyryl‐CoA during the dehydration.

The identity of the product of the dehydration as crotonyl‐CoA was established by the combined action of the enzymes crotonase and (S)‐3‐hydroxybutyryl‐CoA dehydrogenase. The results indicate that the elimination of water from (R)‐2‐hydroxybutyryl‐CoA occurs in a syn mode.

All enzyme activities necessary for the conversion of l‐threonine via (R)‐2‐hydroxybutyryl‐CoA to butyrate were detected in cell‐free extracts of C.propionicum.

A new mechanism for the dehydration of lactyl‐CoA is proposed.
Monoclonal antibodies against Pseudomonas aeruginosa elastase: A neutralizing antibody which recognizes a conformational epitope related to an active site of elastase
Authors: YOKOTA Shin‐ichi , OHTSUKA Hiroshi , NOGUCHI Hiroshi
Published: March 2005

We have established seven murine hybridoma cell lines which produce monoclonal antibodies (mAbs) against Pseudomonas aeruginosa elastase. The seven mAbs recognized at least six different epitopes on the elastase molecule. All mAbs inhibited both enzymatic activities of elastase and protease, in which elastin fluorescein and hide powder azure were used as substrates, respectively. One of them, mAb E‐4D3, strongly neutralized enzymatic activities of peptidase in which furylacryloyl‐glycyl‐leucinamide was used as a substrate, as well as of elastase and protease. In contrast, the other six mAbs did not neutralize peptidase activity at all. The Ki value for furylacryloylglycyl‐leucinamide of E‐4D3, as well as its Fab fragment, was comparable to those for metalloprotease inhibitors such as phosphoramidon and Zincov inhibitor. The binding of mAb E‐4D3 was inhibited by phosphoramidon and Zincov inhibitor, but not by metal chelators such as EDTA and o‐phenanthroline. A line of evidence suggests that mAb E‐4D3 directly interacts with active site and highly neutralizes enzymatic activity of P. aeruginosa elastase. Data of Western blotting and ELISA suggest that mAb E‐4D3 is likely to recognize an elastase molecule in a conformation‐dependent manner as an epitope. In contrast, the neutralizing activity of the other mAbs against elastase and protease seems to be caused by a low acessibility of an enzyme to insoluble and high‐molecular‐mass substrates through the binding and steric hindrance of the mAbs to an enzyme.
Characterization of the rat pulmonary surfactant protein A promoter
Authors: LACAZE‐MASMONTEIL Thierry , FRASLON Caroline , BOURBON Jacques , RAYMONDJEAN Michel , KAHN Axel
Published: March 2005

The expression of the pulmonary surfactant protein A (SP‐A) is developmentally regulated and controlled by several hormones. In an attempt to characterize cis‐acting elements involved in the regulation of SP‐A expression, we have cloned the 5′ flanking sequence of the rat SP‐A gene. The promoter region contains a TATA box but no CAAT box. The transcription start site has been identified by anchored polymerase chain reaction and S1 nuclease mapping of the mature and precursor transcripts. S1 mapping of precursor transcripts has confirmed the stimulating effect of glucocorticoids on SP‐A rat gene transcription in vivo. This hormonal effect may be mediated by a putative glucocorticoid responsive element located 140 bp upstream from the initiation site and protected against DNase 1 digestion in footprinting experiments. In vitro transcription of a G‐free reporter cassette linked to the 212‐bp 5′ flanking DNA fragment has established that this putative promoter region is functional. Efficient transcription of the G‐free reporter cassette was obtained with cell‐free fetal lung extracts, whereas no transcript was detectable with cell‐free liver extracts. Comparative analysis of the human and rat 5′ flanking sequences shows the presence of strongly conserved motifs, unrelated to previously known consensus sequences. Some of these motifs, specifically protected in DNase 1 footprinting studies, could therefore be involved in the regulation of SP‐A gene expression.
The plasma membrane of yeast acquires a novel heat‐shock protein (hsp30) and displays a decline in proton‐pumping ATPase levels in response to both heat shock and the entry to stationary phase
Authors: PANARETOU Barry , PIPER Peter W.
Published: March 2005

Recent studies have revealed that the action of the proton‐translocating ATPase of the plasma membrane of yeast is an important determinant of several stress tolerances and affects the capacity of cells to synthesise heat shock proteins in response to heat shock [Panaretou, B. & Piper, P. W. (1990) J. Gen. Microbiol. 136, 1763–1770; Coote, P. J., Cole, M. B. & Jones, M. V. (1991) J. Gen. Microbiol. 137, 1701–1708]. This study investigated the changes to the protein composition of the Saccharomyces cerevisiae plasma membrane that result from a heat shock to dividing cultures and the entry to stationary growth caused by carbon source limitation. Plasma membranes were prepared from exponential, heat‐shocked and stationary yeast cultures. The proteins of these membrane preparations were then analysed by polyacrylamide gel electrophoresis and immunoblot measurement of ATPase levels.

The protein composition of plasma membranes displayed two prominent changes in response to both heat shock and the entry to stationary phase: (a) a reduction in the level of the plasma membrane ATPase; and (b) the acquisition of a previously uncharacterised 30 kDa heat‐shock protein (hsp30). The ATPase decline with heat shock probably exerts an important influence over the ability of the cell to maintain ATPase activity, and therefore intracellular pH, during extended periods of stress. Through in vivo pulse‐labelling of plasma membrane proteins synthesised before and during heat shock, followed by subcellular fractionation, it was shown that hsp30 is the only protein induced by the yeast heat‐shock response that substantially copurifies with plasma membranes. It might therefore exert a stress‐protective function specifically at this membrane.
Production and characteristics of human tissue plasminogen‐activator antibodies with region‐oriented synthetic peptides
Authors: WILCZYNSKA Malgorzata , PONIATOWSKI Jacek , JANECKA Anna , CIERNIEWSKI Czeslaw S.
Published: March 2005

We selected six peptide sequences as belonging to potential epitopes of tissue plasminogen activator (tPA) using, as the main criterion for their choice, the location of the peptide sequences on the surface of the protein molecule. The six peptides (corresponding to amino acids 4–8, 11–16, 96–101, 272–277, 371–376 and 514–519) were synthesized, coupled to carrier proteins and injected into rabbits. All of these peptides elicited antibodies and 15–75% binding of the corresponding iodinated peptide was obtained with a 1:100 dilution of antiserum. Only two anti‐(peptide) sera [anti‐(tPA96–101) and anti‐(tPA272–277)] reacted with intact tPA and its heavy chain in Western immunoblotting analysis. These two peptides sequences and fragment tPA11–16 appear to be involved in the structure of native antigenic epitopes of tPA, since they were recognized and antibodies present in antisera raised against native tPA. There was no interaction between anti‐(tPA4–8) and anti‐(tPA371–376) sera with intact one‐chain or two‐chain tPA. In the case of anti‐(tPA4–8) cleavage of one‐chain tPA to two‐chain tPA and reduction of disulfide bonds exposed this epitope.
Three‐dimensional structure of proteolytic fragment 163–231 of bacterioopsin determined from nuclear magnetic resonance data in solution
Authors: BARSUKOV Igor L. , NOLDE Dmitry E. , LOMIZE Andrey L. , ARSENIEV Alexander S.
Published: March 2005

546 NOESY cross‐peak volumes were measured in the two‐dimensional NOESY spectrum of proteolytic fragment 163–231 of bacterioopsin in organic solution. These data and 42 detected hydrogen bonds were applied for determining the peptide spatial structure. The fold of the polypeptide chain was determined by local structure analysis, a distance geometry approach and systematic search for energetically allowed side‐chain rotamers which are consistent with experimental NOESY cross‐peak volumes. The effective rotational correlation time of 6 ns for the molecule was evaluated from optimization of the local structure to meet NOE data and from the dependence on mixing time of the NiH/CiαH cross‐peak volumes of the residues in α‐helical conformation. The resulting structure has two well defined α‐helical regions, 168–191 and 198–227, with root‐mean‐square deviation 44 pm and 69 pm, respectively, between the backbone atoms in 14 final energy refined conformations. The α‐helices correspond to transmembrane segments F and G of bacteriorhodopsin. The segment F contains proline 186, which introduces a kink of about 25° with a disruption of the hydrogen bond with the NH group of the following residue. The segments are connected by a flexible loop region 192–197. Torsion angles X1 are unequivocally defined for 62% of side chains in the α‐helices but half of them differ from electron cryo‐microscopy (ECM) model of bacteriorhodopsin, apparently because of the low resolution of ECM. Nevertheless, the F and G segments can be packed as in the ECM model and with side‐chain conformations consistent with all NMR data in solution.
Purification and biochemical characterization of a putative [6Fe‐6S] prismane‐cluster‐containing protein from Desulfovibrio vulgaris (Hildenborough)
Authors: PIERIK Antonio J. , WOLBERT Ronnie B. G. , MUTSAERS Peter H. A. , HAGEN Wilfred R. , VEEGER Cees
Published: March 2005

A novel iron‐sulfur protein has been isolated from the sulfate‐reducing bacterium Desulfovibrio vulgaris (Hildenborough). It is a stable monomeric protein, which has a molecular mass of 52 kDa, as determined by sedimentation‐equilibrium centrifugation. Analysis of the metal and acid‐labile sulfur content of the protein revealed the presence of 6.3 ± 0.4 Fe/polypeptide and 6.2 ± 0.7 S2‐/polypeptide. Non‐iron transition metals, heme, flavin and selenium were absent. Combining these data with the observation of a very anisotropic S= 1/2 [6Fe‐6S]3+ prismane‐like EPR signal in the dithionite‐reduced protein, we believe that we have encountered the first example of a prismane‐cluster‐containing protein. The prismane protein has a slightly acidic amino acid composition and isoelectric point (pI= 4.9). The ultraviolet/visible spectrum is relatively featureless (ɛ280= 81 mM−1· cm−1, ɛ400= 25 mM−1· cm−1, ɛ400,red= 14 mM−1· cm−1). The shape of the protein is approximately globular (s20,w= 4.18 S). The N‐terminal amino acid sequence is MFs/cFQs/c QETAKNTG. Polyclonal antibodies against the protein were raised. Cytoplasmic localization was inferred from subcellular fractionation studies. Cross‐reactivity of antibodies against this protein indicated the occurrence of a similar protein in D. vulgaris (Monticello) and Desulfovibrio desulfuricans (ATCC 27774). We have not yet identified a physiological function for the prismane protein despite trials for some relevant enzyme activities.
Phosphatidylinositol‐glycan‐specific phospholipase D is an amphiphilic glycoprotein that in serum is associated with high‐density lipoproteins
Authors: HOENER Marius C. , BRODBECK Urs
Published: March 2005

Phosphatidylinositol (PtdIns)‐glycan‐specific phospholipase D was purified from bovine and human serum by phase separation in Triton X‐114 and by chromatography on DEAE‐cellulose, octyl‐Sepharose, concanavalin‐A–Sepharose, and hydroxyapatite. The purification of the two enzymes was approximately 1200‐fold with a recovery of 3–5%. Bovine serum contained about 40 μ/ml of PtdIns‐glycan‐specific phospholipase D, about 10 times more than the amount determined in human serum. PtdIns‐glycan‐specific phospholipase D is also present in mammalian cerebrospinal fluid and in mammalian milk but to a much lesser extent than in serum. Enzyme from bovine and human serum displayed amphiphilic properties as revealed by sucrose density gradient centrifugation and gel filtration in the absence and presence of detergent. On density gradient centrifugation, both enzymes sedimented with an apparent sedimentation coefficient of about 6.0 S in the presence of 0.1% Triton X‐100, and formed aggregates up to 14.5 S in the absence of detergent. Upon gel filtration, the bovine and human enzymes migrated with a Stokes' radius of 6.5 nm and 6.6 nm, respectively, in the presence of Triton X‐100. In the absence of Triton X‐100, both enzymes gave a Stokes' radius of 8.8 nm. Serial centrifugation of serum at increasing NaBr concentrations revealed that the majority of the enzyme is contained in the high‐density lipoprotein fraction. PtdIns‐glycan‐specific phospholipase D from bovine and human serum contained 27 and 28 N‐acetylglucosamine residues, respectively. Treatment with N‐glycosidase F decreased the apparent molecular mass of the bovine and human enzyme from 115 and 123 kDa to 91 and 87 kDa, respectively. Sequence analysis of peptides derived from PtdIns‐glycan‐specific phospholipase D of bovine serum by CNBr cleavage gave 100% identity to the sequence published for the bovine liver enzyme while there was 83% similarity and 74% identity to the sequence of peptides obtained from the human serum enzyme.
Selenoprotein synthesis in E. coli
Authors: EHRENREICH Armin , FORCHHAMMER Karl , TORMAY Peter , VEPREK Barbara , BÖCK August
Published: March 2005

The product of the selD gene from Escherichia coli catalyses the formation of an activated selenium compound which is required for the synthesis of Sec‐tRNA (Sec, selenocysteine) from Ser‐tRNA and for the formation of the unusual nucleoside 5‐methylaminomethyl‐2‐selenouridine in several tRNA species. selD was overexpressed in a T7 promoter/polymerase system and purified to apparent homogeneity. Purified SELD protein is a monomer of 37 kDa in its native state and catalyses a selenium‐dependent ATP‐cleavage reaction delivering AMP and releasing the β‐phosphate as orthophosphate. The γ‐phosphate group of ATP was not liberated in a form able to form a complex with molybdate. It was precluded that any putative covalent or non‐covalent ligand of SELD not removed during purification participated in the reaction. In a double‐labelling experiment employing [75Se]selenite plus dithiothreitol and [γ‐32P]ATP the 75Se and 32P radioactivities co‐chromatographed on a poly(ethyleneimine)‐cellulose column. No radioactivity originating from ATP eluted in this position when [α‐32P]ATP or [β‐32P]ATP or [14C]ATP were offered as substrates. The results support the speculation that the product of SELD is a phosphoselenoate with the phosphate moiety derived phosphoselenoate from the γ‐phosphate group of ATP. The α,β cleavage of ATP is also supported by the finding that neither adenosine 5′‐[α,β‐methylene]triphosphate nor adenosine 5′‐[β,γ‐methylene]triphosphate served as substrates in the reaction.
The carboxylesterase family exhibits C‐terminal sequence diversity reflecting the presence or absence of endoplasmic‐reticulum‐retention sequences
Authors: MEDDA Sukumar , PROIA Richard L.
Published: March 2005

Resident proteins of the endoplasmic reticulum lumen are continuously retrieved from an early Golgi compartment by a receptor‐mediated mechanism. The sorting or retention sequence on the endoplasmic reticulum proteins is located at the C‐terminus and was initially shown to be the tetrapeptide KDEL in mammalian cells and HDEL in Saccharomyces cerevisiae. The carboxylesterases are a large family of enzymes primarily localized to the lumen of the endoplasmic reticulum. Retention sequences in these proteins have been difficult to identify due to atypical and heterogeneous C‐terminal sequences. Utilizing the polymerase chain reaction with degenerate primers, we have identified and characterized the C‐termini of four members of the carboxylesterase family from rat liver. Three of the carboxylesterases sequences contained C‐terminal sequences (HVEL, HNEL or HTEL) resembling the yeast sorting signal which were reported to be non‐functional in mammalian cells. A fourth carboxylesterase contained a distinct C‐terminal sequence, TEHT. A full‐length esterase cDNA clone, terminating in the sequence HVEL, was isolated and was used to assess the retention capabilities of the various esterase C‐terminal sequences. This esterase was retained in COS‐1 cells, but was secreted when its C‐terminal tetrapeptide, HVEL, was deleted. Addition of C‐terminal sequences containing HNEL and HTEL resulted in efficient retention. However, the C‐terminal sequence containing TEHT was not a functional retention signal. Both HDEL, the authentic yeast retention signal, and KDEL were efficient retention sequences for the esterase. These studies show that some members of the rat liver carboxylesterase family contain novel C‐terminal retention sequences that resemble the yeast signal. At least one member of the family does not contain a C‐terminal retention signal and probably represents a secretory form.
cDNA cloning and deduced amino acid sequence of a major, glycine‐rich cuticular protein from the coleopteran Tenebrio molitor
Authors: CHARLES Jean‐Philippe , BOUHIN Hervé , QUENNEDEY Brigitte , COURRENT Annie , DELACHAMBRE Jean
Published: March 2005

In Coleoptera, the elytra (forewings), with a very hard and thick cuticle, protect the membranous and delicate hindwings against mechanical stress. We have isolated and characterized a cDNA encoding a major cuticle protein in Tenebrio molitor, named ACP‐20. The deduced amino acid sequence is roughly tripartite, with two terminal glycine‐rich domains and a central region showing pronounced similarities with some other hard cuticle proteins. Northern blot and in situ hybridization analyses reveal that ACP‐20 gene expression is developmentally regulated since transcript accumulation occurs only in epidermal regions synthesizing hard cuticle and is restricted to the period of preecdysial adult cuticle deposition. Moreover, application of a juvenile hormone analogue prevents the appearance of the transcript, indicating that juvenile hormone, a key molecule involved in the control of insect metamorphosis, negatively regulates the expression of the ACP‐20 gene.
Stimulation of phospholipase C by guanine‐nucleotide‐binding protein βγ subunits
Authors: CAMPS Montserrat , HOU Cuifen , SIDIROPOULOS Dimitrios , STOCK Jeffry B. , JAKOBS Karl H. , GIERSCHIK Peter
Published: March 2005

We have previously shown that soluble fractions obtained from human HL‐60 granulocytes contain a phospholipase C which is markedly stimulated by the stable GTP analogue guanosine 5′‐[3‐O‐thio]triphosphate (Camps, M., Hou, C., Jakobs, K. H. and Gierschik, P. (1990) Biochem. J. 271, 743–748]. To investigate whether this stimulation was due to a soluble α subunit of a heterotrimeric guanine‐nucleotide‐binding protein or a soluble low‐molecular‐mass GTP‐binding protein, we have examined the effect of purified guanine‐nucleotide‐binding protein βγ dimers on the phospholipase‐C‐mediated formation of inositol phosphates by HL‐60 cytosol. We found that βγ subunits, purified from bovine retinal transducin (βγt), markedly stimulated the hydrolysis of phosphatidylinositol 4,5‐bisphosphate by this phospholipase C preparation. The stimulation of phospholipase C by βγt was not secondary to a phospholipase‐A2‐mediated generation of arachidonic acid, was prevented by the GDP‐liganded transducin α subunit and was additive to activation of phospholipase C by guanosine 5′‐[3‐O‐thio]triphosphate. βγt also stimulated soluble phospholipase C from human and bovine peripheral neutrophils, as well as membrane‐bound, detergent‐solubilized phospholipase C from HL‐60 cells. Stimulation of soluble HL‐60 phospholipase C was not restricted to βγt, but was also observed with highly purified βγ subunits from bovine brain. Fractionation of HL‐60 cytosol by anion‐exchange chromatography revealed the existence of at least two distinct forms of phospholipase C in HL‐60 granulocytes. Only one of these forms was sensitive to stimulation by βγt, demonstrating that stimulation of phospholipase C by βγ subunits is isozyme specific. Taken together, our results suggest that guanine‐nucleotide‐binding protein βγ subunits may play an important and active role in mediating the stimulation of phospholipase C by heterotrimeric guanine‐nucleotide‐binding proteins.
Purification to homogeneity and characterization of a redoxyendonuclease from calf thymus
Authors: HUQ Ikramul , HAUKANES Bjørn‐Ivar , HELLAND Dag E.
Published: March 2005

A redoxyendonuclease from calf thymus was purified to apparent homogeneity. The redoxyendonuclease recognized and induced cleavage of DNA damaged by ultraviolet light. The enzyme preparation produced a single band of a relative molecular mass of approximately 34 kDa upon SDS/PAGE. The apurinic/apyrimidinic endonuclease and the DNA glycosylase activities remained associated in the apparently homogeneous preparation of the enzyme. The redoxyendonuclease activity displayed a broad pH optimum between pH 5.0–8.5 and exhibited no requirement for divalent cations. By application of FPLC columns Mono‐S, Mono‐Q and Mono‐P, the isoelectric point (pI) of the enzyme was found to be approximately 8.0. Using the DNA sequencing procedure of Maxam and Gilbert [Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65, 499–560] the purified enzyme was found to incise ultraviolet‐light‐irradiated DNA at pyrimidine sites as observed previously with a more crude form of the enzyme. While the most frequently cleavaged sites for the crude preparation were at cytosine residues, the apparently homogeneous enzyme preparation frequently induced cleavage sites at both cytosine and guanine residues. Predominant incision induced by the apparently homogeneous preparation was observed at guanine residues when a particular DNA sequence was used as substrate. Furthermore, the 16 N‐terminal amino acid residues of the purified enzyme were identified. The sequence did not show any significant similarity to other known proteins.
Chemical cross‐linking indicates a staggered and antiparallel protofilament of desmin intermediate filaments and characterizes one higher‐level complex between protofilaments
Authors: GEISLER Norbert , SCHÜNEMANN Jürgen , WEBER Klaus
Published: March 2005

Tetrameric rods, protofilaments and assembled filaments of desmin, the intermediate filament protein of muscle, have been chemically cross‐linked with the lysine specific cross‐linkers EGS [ethylene glycol bis(succinimidylsuccinate), 1.61 nm span] and bis(sulfosuccinimidyl) suberate (1.14 nm span). One bis(sulfosuccinimidyl)suberate and two EGS cross‐links were isolated from the rod and characterized. They show that the two coiled coils in the rod tetramer are staggered by approximately 15–20 nm and strongly indicate an antiparallel arrangement in which the inner overlapping part of the rod is formed by the amino‐terminal helices 1A, 1B and 2A. Both EGS cross‐links identified in the rod were also isolated from cross‐linked filaments. The isolated rod, therefore, represents a complex also present in identical, or very similar form in protofilaments and in assembled filaments. Cross‐linked filaments yielded a third EGS cross‐link that must have been formed between neighboring protofilaments. It connects the highly conserved carboxy‐terminus of helix 2B of the first protofilament to the overlap region formed by helices 1A and 2A of the second protofilament. The restrictions posed by these cross‐links on current filament models are discussed.
Mitochondrial dolichyl‐phosphate mannose synthase
Authors: GASNIER Françoise , ROUSSON Robert , LERME Fabienne , VAGANAY Elisabeth , LOUISOT Pierre , GATEAU‐ROESCH Odile
Published: March 2005

Mitochondrial dolichyl‐phosphate mannose synthase has been purified to homogeneity using an original procedure, reconstitution into specific phospholipid vesicles and sedimentation on a sucrose gradient as final step.

The enzyme has an apparent molecular mass of 30 kDa on an SDS/polyacrylamide gel. Increased enzyme activity could be correlated with this polypeptide band.

A specific antibody was raised in rabbits against this transferase. Specific IgG obtained from the immune serum removed enzymatic activity from a detergent extract of mitochondrial outer membrane and reacted specifically with the 30‐kDa band on immunoblots. Furthermore, an immunocytochemical experiment proved the localization of dolichyl‐phosphate mannose synthase on the cytosolic face of the outer membrane of mitochondria.
Two classes of receptor specific for sperm‐activating peptide III in sand‐dollar spermatozoa
Authors: YOSHINO Ken‐ichi , SUZUKI Norio
Published: March 2005

We characterized receptors specific for sperm‐activating peptide III (SAP‐III: DSDSAQNLIQ) in spermatozoa of the sand dollar, Clypeaster japonicus, using both binding and cross‐linking techniques. Analyses of the data obtained from the equilibrium binding of a radiolabeled SAP‐III analogue to C. japonicus spermatozoa, using Klotz, Scatchard and Hill plots, showed the presence of two classes of receptors specific for SAP‐III in the spermatozoa. One of the receptors (high‐affinity) had a Kd of 3.4 nM and 3.4 × 104 binding sites/spermatozoon. The other receptor (low‐affinity) had a Kd of 48 nM, with 6.1 × 104 binding sites/spermatozoon. The Kd of the high‐affinity receptor was comparable to the median effective concentration of the intracellular‐pH‐increasing activity of SAP‐III and that of the low‐affinity receptor was comparable to the median effective concentration of the cellular‐cGMP‐elevating activity of the peptide. In addition, Scatchard and Hill plots of the data suggested the existence of positive cooperativity between the high‐affinity members. Similar results were also obtained from a binding experiment using a sperm‐membrane fraction prepared from C. japonicus spermatozoa. The incubation of intact spermatozoa or sperm plasma membranes with the radioiodinated SAP‐III analogue and a chemical cross‐linking reagent, disuccinimidyl suberate, resulted in the radiolabeling of three proteins with molecular masses of 126, 87 and 64 kDa, estimated by SDS/PAGE under reducing conditions.
Unique structure in the intergenic and 5′ external transcribed spacer of the ribosomal RNA gene from the pea aphid Acyrthosiphon pisum
Authors: KWON O‐Yu , ISHIKAWA Hajime
Published: March 2005

We analyzed the DNA sequence of the 5′ external transcribed spacer (ETS) and part of the intergenic transcribed spacer (IGS) of the aphid ribosomal RNA gene (rDNA). The 5′ ETS of aphid rDNA consists of 843 nucleotides with a G/C content of 69 mol/100 mol, far higher than that of any other known 5′ ETS for insect rDNA. The IGS of aphid rDNA contained a characteristic array of repeated sequences of 247 nucleotides. The repeated sequences were identical. It was shown that the number of the repeating sequence is heterogeneous.
A novel cyclic analog of neuropeptide Y specific for the Y2 receptor
Authors: BECK‐SICKINGER Annette G. , GROUZMANN Eric , HOFFMANN Eike , GAIDA Wolfram , VAN MEIR Erwin G. , WAEBER Bernard , JUNG Günther
Published: March 2005

The low‐molecular‐mass, cyclic analog of neuropeptide Y, [Ahx5–24, γ‐Glu2‐ɛ‐Lys30] NPY (; Ahx, 6‐aminohexanoic acid; NPY, neuropeptide Y), was synthesized and investigated for receptor binding, inhibition of forskolin‐stimulated cAMP accumulation, inhibition of electrically stimulated rat vas deferens contractions and ability to increase blood pressure. Like the linear peptide [Ahx5–24] NPY (YPSK‐Ahx‐RHYINLITRQRY), the more rigid, cyclic analog showed good correlation between receptor binding to rabbit kidney membranes and biological activity in the vas deferens assay. Binding of this peptide to a new Y2‐receptor‐expressing cell line was slightly reduced, compared to the linear peptide [Ahx5–24] NPY, however inhibition of cAMP accumulation was even more efficient.

Unlike the linear peptide [Ahx5–24] NPY, the cyclic analog did not induce a blood pressure increase in rats. Reduced binding to Y1 receptor‐expressing SK‐N‐MC cells, as well as the loss of capability of signal transduction, suggest that only Y2‐mediated activity is preserved after cyclization. The selectivity of the cyclic compound for Y2 subtypes of NPY receptors with respect to inhibition of cAMP accumulation is more than fortyfold increased, as compared to the linear NPY‐(13–36) peptide, which has been used to determine Y2 selectivity so far.
The polyanion‐binding domain of cytoplasmic Lys‐tRNA synthetase from Saccharomyces cerevisiae is not essential for cell viability
Authors: MARTINEZ Ricardo , MIRANDE Marc
Published: March 2005

Cytoplasmic Lys‐tRNA synthetase (LysRS) from Saccharomyces cerevisiae is a dimeric enzyme made up of identical subunits of 68 kDa. By limited proteolysis, this enzyme can be converted to a truncated dimer without loss of activity. Whereas the native enzyme strongly interacts with polyanionic carriers, the modified form displays reduced binding properties. KRS1 is the structural gene for yeast cytoplasmic LysRS. It encodes a polypeptide with an amino‐terminal extension composed of about 60–70 amino acid residues, compared to its prokaryotic counterpart. This segment, containing 13 lysine residues, is removed upon proteolytic treatment of the native enzyme. The aim of the present study was to probe in vivo the significance of this amino‐terminal extension. We have constructed derivatives of the KRS1 gene, encoding enzymes lacking 58 or 69 amino‐terminal residues and, by site‐directed mutagenesis, we have changed four or eight lysine residues from the amino‐terminal segment of LysRS into glutamic acids. Engineered proteins were expressed in vivo after replacement of the wild‐type KRS1 allele. The mutant enzymes displayed reduced specific activities (2–100‐fold). A series of carboxy‐terminal deletions, encompassing 3, 10 or 15 amino acids, were introduced into the LysRS mutants with modified amino‐terminal extensions. The removal of three residues led to a 2–7‐fold increase in the specific activity of the mutant enzymes. This partial compensatory effect suggests that interactions between the two extreme regions of yeast LysRS are required for a proper conformation of the native enzyme. All KRS1 derivatives were able to sustain growth of yeast cells, although the mutant cell lines displaying a low LysRS activity grew more slowly. The expression, as single‐copy genes, of mutant enzymes with a complete deletion of the amino‐terminal extension or with four Lys → Glu mutations, that displayed specific activities close to that of the wild‐type LysRS, had no discernable effect on cell growth. We conclude that the polycationic extensions of eukaryotic aminoacyl‐tRNA synthetases are dispensable, in vivo, for aminoacylation activities. The results are discussed in relation to the triggering role in in situ compartmentalization of protein synthesis that has been ascribed to the polypeptide‐chain extensions that characterize most, if not all, eukaryotic aminoacyl‐tRNA synthetases.
DNase‐I hypersensitivity analysis of the L‐type pyruvate kinase gene in rats and transgenic mice
Authors: BOQUET Didier , VAULONT Sophie , TREMP Günter , RIPOCHE Marie‐Anne , DAEGELEN Dominique , JAMI Jacques , KAHN Axel , RAYMONDJEAN Michel
Published: March 2005

The rat L‐type pyruvate kinase gene possesses two promoters located 500 bp apart. The L′ promoter is specific to erythroid cells. The L promoter is specific to liver and is regulated by diet and hormones; positively by glucose and insulin and negatively by glucagon via cAMP. The DNA elements involved in this tissue‐specific and hormone‐regulated gene expression are located within 3.2 kbp of 5′ flanking region as previously demonstrated by transgenic mice analysis [Tremp, G. L., Boquet, D., Ripoche, M. A., Cognet, M., Yu‐Chun, L., Jami, J., Kahn, A. and Daegelen, D. (1989) J. Biol. Chem. 264, 19 904–19 910]. Moreover, we have observed in these mice that gene expression was dependent on the transgene copy number and independent of the integration site. We present here DNase‐I‐hypersensitivity analysis of the endogenous rat L‐type pyruvate kinase gene and of two transgene constructs in relation to development, tissue differentiation, nutritional and hormonal status. In rats, two groups of proximal sites were detected on the endogenous gene; hypersensitive site (HSS) HSS‐1 in adult liver and HSS‐A in fetal liver (a major erythropoietic tissue). Both groups are probably related to the transcriptional initiation complexes at either the L or L′ promoter. Two other distal groups were detected; HSS‐2 at −3 kbp (with respect to the liver‐specific cap site) in adult liver and HSS‐B around −4 kbp in fetal liver. These sites are thought to correspond to activating sequences; in adult liver, deletion of a fragment encompassing HSS‐2 provokes a dramatic reduction of transcription starting at the L promoter of the transgene. In adult liver, HSS‐1 appears to be a transcription‐associated site, being greatly weakened in fasted rats, while HSS‐2 is transcription independent.

The pattern of DNase‐I hypersensitivity is similar for the rat endogenous gene and for the complete rat transgene; the liver‐specific HSS‐1 and HSS‐2 are present and the intensity of the sites is correlated to the number of integrated copies. Interestingly, HSS‐1 is still detectable and its intensity remains proportional to the number of integrated copies in a truncated transgene with HSS‐2 deletion, while this transgene is very weakly (but nevertheless tissue‐specifically) expressed. These results strongly suggest that each transgene copy possesses a complete set of specific nucleoprotein complexes and that, with or without HSS‐2, the DNA is in a potentially active configuration.
Molecular cloning, sequencing and restriction mapping of the genomic sequence encoding human proacrosin
Authors: VAZQUEZ‐LEVIN Monica H. , REVENTOS Jaume , GORDON Jon W.
Published: March 2005

In the present study, molecular cloning, sequencing and restriction mapping of the genomic sequence encoding human proacrosin is described. The full‐length cDNA encoding human proacrosin was utilized to recover a 17‐kb human genomic clone which was sequenced without further subcloning. The nucleotide sequences of the exons agree with the sequence of the cDNA reported previously. More than 500 bases of the promoter region were sequenced and found to be highly GC rich but devoid of an identifiable TATA box. These findings are generally consistent with a recently published report [Keime, S., Adham, I. M. & Engel, W. (1990) Eur. J. Biochem. 190, 195–200]. However, further sequence analysis revealed discrepancies between our clone and that previously reported. Sequencing of the first intron showed similarity with the published data for 54 bases of the 5′ region, beginning with the donor splice site, and for 114 bases at the 3′ end. However, 500 bases sequenced distal to the initial 54 bases at the 5′ end of intron 1 showed no similarity with the published sequence. In addition, the boundaries of intron 3 differed such that a cytosine residue previously reported to be in exon 3 was found to be the first base of exon 4. Detailed studies were undertaken to confirm that our clone constitutes the authentic sequence of human proacrosin. Cloning and characterization of the human proacrosin gene may allow for informative studies of its regulation, and for a more detailed examination of its role in fertilization.
Characterisation of yeast phosphoglycerate kinase modified by mutagenesis at residue 21
Authors: WALKER Philip A. , JOÂO Heidi C. , LITTLECHILD Jennifer A. , WILLIAMS Robert J. P. , WATSON Herman C.
Published: March 2005

Site‐directed mutagenesis has been used to produce mutant forms of yeast phosphoglycerate kinase in which the conserved active‐site residue, Arg21, has been replaced by a methionine or a lysine. Kinetic results obtained using these mutant enzymes show that their Km for both 3‐phospho‐D‐glycerate and ATP are significantly different from those recorded for the wild‐type enzyme. The Vmax for the lysine mutant is reduced by a factor of two from that of the wild‐type enzyme whereas the Vmax for the methionine mutant is reduced more than sevenfold. A very clean electron‐density‐difference map shows little, if any, evidence of a structural change associated with the C‐terminal domain, although resonances in the NMR spectra associated with the ATP‐binding site (C‐terminal domain) are also affected by the mutation as one might expect from the kinetic results. The NMR data show that binding at both the 3‐phospho‐D‐glycerate and the non‐productive ATP‐binding site (associated with the N‐terminal domain) are affected in the mutant in a way which is different to that associated with the wild‐type enzyme. These results, taken together with the X‐ray and kinetic data, indicate that the non‐productive ATP‐binding site and the activating anion‐binding site are both associated with the basic patch region of yeast phosphoglycerate kinase.
The cholesterol‐side‐chain‐cleaving cytochrome P450 spin‐state equilibrium
Authors: LANGE Reinhard , LARROQUE Christian , ANZENBACHER Pavel
Published: March 2005

We have investigated the spin‐state equilibrium of adrenal mitochondrial P450scc (cholesterol‐side‐chain‐cleaving, CYP11A1) by absorption spectroscopy in the Soret band as a function of pH and temperature. The van't Hoff plot of the high‐spin/low‐spin equilibrium is not linear and is shifted towards high spin by lowering the pH. This non‐linearity resolves clearly into two phases when the temperature range is extended from 37°C to −20°C using ethylene glycol as anti‐freeze cosolvent. This enabled us to measure the enthalpy and entropy changes which are ΔHA= 0.7 kJ · mol−1 and ΔSA= 5 J · K−1· mol−1 at low temperatures and ΔHB=−42 kJ · mol−1 and ΔSB=−152 J · K−1· mol−1 at high temperatures. The transition temperature, Tbreak, between both phases decreases as a function of pH.

The experimental data can be fitted by a minimal reactional model comprising a temperature dependent conformational transition and two ionisation steps (one for each conformation), the pK of which is 1.5 ± 0.5 higher in the low‐temperature conformation. The deduced conformational equilibrium is affected by physiological effectors: Tbreak depends on the nature of the substrate intermediate and on the presence of the physiological electron donor, adrenodoxin.
The cholesterol‐side‐chain‐cleaving cytochrome P450 spin‐state equilibrium
Authors: LANGE Reinhard , PANTALONI Antoine , SALDANA Jean‐Louis
Published: March 2005

We have confirmed and characterised structurally the enzyme conformational changes deduced from the preceding thermodynamic analysis of the adrenal mitochondrial cholesterol‐side‐chain‐cleaving cytochrome P450 spin‐state equilibrium. The spin‐transition kinetics following rapid pH jumps were multiphasic in aqueous buffer and biphasic in the presence of 35% ethylene glycol. The activation energy between −2°C and 30°C of both phases was exceptionally high (Ea= 147 kJ · mol−1), suggesting the involvement of large‐scale conformational changes. The pH and temperature effects on the CD spectrum show that the enzyme is in equilibrium between at least two conformations which are predicted by the thermodynamic model, but which are not directly correlated to the spin state. The CD changes between 260 nm and 280 nm indicate that the conformation prevailing at high temperatures is characterised by a decreased polarity of the tyrosine environments; the changes between 200 nm and 250 nm suggest furthermore a 4% decreased protein helical content.
Purification and characterization of a novel thermostable 4‐α‐glucanotransferase of Thermotoga maritima cloned in Escherichia coli
Authors: LIEBL Wolfgang , FEIL Robert , GABELSBERGER Josef , KELLERMANN Josef , SCHLEIFER Karl‐Heinz
Published: March 2005

Maltodextrin glycoslyltransferase (4‐α‐glucanotransferase) of the extremely thermophilic ancestral bacterium Thermotoga maritima has been purified from an Escherichia coli clone expressing the corresponding T. maritima MSB8 chromosomal gene. T. maritima 4‐α‐glucanotransferase, an approximately 53‐kDa monomeric enzyme, is the most thermophilic glycosyltransferase described to date. It retained more than 90% of its maximum activity at temperatures from 55°C up to 80°C.

The proposed action modus is the transfer of 1,4‐α‐glucanosyl chains, thus resulting in the disproportionation of 1,4‐α‐glucans. It converted soluble starch, amylopectin, and amylose, thereby changing the iodine staining properties of these substrates. The addition of low‐molecular‐mass malto‐oligosaccharides, which act as glucanosyl acceptor molecules, enhanced the reaction and resulted in the formation of a series of linear maltohomologues from two to more than nine glucose units in size. Use of either of the malto‐oligosaccharides maltotetraose, maltopentaose, maltohexaose, or maltoheptaose as sole substrate also yielded linear maltohomologues. On the other hand, maltose and maltotriose were not disproportionated by 4‐α‐glucanotransferase, although both were good acceptors for glucanosyl transfer. Glucose did not function as an acceptor in transfer reactions. Glucose also never appeared as a reaction product. The chain length of glucanosyl segments transferred ranged from two to probably far more than six glucose residues.

Comparison of the N‐terminal amino acid sequence of 4‐α‐glucanotransferase with other published protein sequences revealed significant similarity to sequences near the N‐termini of various eucaryotic maltases and bacterial cyclodextrin glycosyltransferases, suggesting its relatedness on the molecular level with other starch‐ and maltodextrin‐converting enzymes.
Site‐directed mutagenesis of the Streptomyces R61 DD‐peptidase
Authors: HADONOU Ayaovi M. , WILKIN Jean‐Marc , VARETTO Louis , JORIS Bernard , LAMOTTE‐BRASSEUR Josette , KLEIN Daniel , DUEZ Colette , GHUYSEN Jean‐Marie , FRÈRE Jean‐Marie
Published: March 2005

The importance of various residues in the Streptomyces R61 penicillin‐sensitive DD‐peptidase has been assessed by site‐directed mutagenesis. The replacement of the active Ser62 by a Cys residue yielded an inactive protein which was also unable to recognize penicillin. The activity of the Lys65→Arg mutant with the peptide and thiolester substrates was decreased 100–200‐fold and the rate of penicillin inactivation was decreased 20 000‐fold or more. The mutant thus behaved as a poor, but penicillin‐resistant, DD‐peptidase.

The other studied mutations, the mutations Phe58→Leu, Tyr90→Asn, Thr101→Asn, Phe164→Ala, Asp225→Glu and Asp225→Asn had little influence on the catalytic and penicillin‐binding properties. The Asp225 mutants did not exhibit an increased sensitivity to cefotaxime. The Phe164→Ala mutant was significantly more unstable than the wild‐type enzyme.
A comparison of the catalytic properties of cellobiose: quinone oxidoreductase and cellobiose oxidase from Phanerochaete chrysosporium
Authors: SAMEJIMA Masahiro , ERIKSSON Karl‐Erik L.
Published: March 2005

Several catalytic properties of the FAD enzyme cellobiose:quinone oxidoreductase (CBQ) and the heme/FAD enzyme, cellobiose oxidase (CBO) have been investigated and compared. Dichlorophenolindophenol was found to be a very good electron acceptor for cellobiose oxidation by both enzymes. The optimal pH value for this oxidation with dichlorophenol‐indophenol as a co‐substrate was observed around pH 4 for both enzymes. The turnover numbers of this reaction were also very similar. The Km values for cellobiose oxidation were identical, whereas the Km for CBO with dichlorophenol‐indophenol is lower than that of CBQ.

Atmospheric oxygen is a very poor electron acceptor for both CBO and CBQ, however, CBO can utilize cytochrome c as an effective electron acceptor, while CBQ cannot. The specific activity of CBO for cytochrome c is thus about 200‐times higher than for oxygen. Thus, one way to distinguish the two enzymes is by the cytochrome‐c‐reducing ability of CBO. Therefore, we propose that the nomenclature for CBO is tentatively changed to cellobiose:cytochrome c oxidoreductase until a rational name can be installed.

Both enzymes have radical‐reducing activities. The cation radical, derived from 1,2,4,5‐tetramethoxybenzene, was reduced by both enzymes at almost the same reaction rate. The phenoxyradical produced by lignin peroxidase, catalyzing the oxidation of acetosyringon, was also reduced by both enzymes. The reduction of phenoxyradicals formed by phenoloxidases (lignin peroxidases, as well as laccases) may be important in preventing repolymerization reactions which we suggest would significantly facilitate lignin degradation.
Malonate decarboxylase of Malonomonas rubra, a novel type of biotin‐containing acetyl enzyme
Authors: HILBI Hubert , DEHNING Irmtraut , SCHINK Bernhard , DIMROTH Peter
Published: March 2005

Cell suspensions or crude extracts of Malonomonas rubra grown anaerobically on malonate catalyze the decarboxylation of this substrate at a rate of 1.7–2.5 μmol · min−1· mg protein−1 which is consistent with the malonate degradation rate during growth. After fractionation of the cell extract by ultracentrifugation, neither the soluble nor the particulate fraction alone catalyzed the decarboxylation of malonate, but on recombination of the two fractions 87% of the activity of the unfractionated extract was restored. The decarboxylation pathway did not involve the intermediate formation of malonyl‐CoA, but decarboxylation proceeded directly with free malonate. The catalytic activity of the enzyme was completely abolished on incubation with hydroxylamine or NaSCN. Approximately 50–65% of the original decarboxylase activity was restored by incubation of the extract with ATP in the presence of acetate, and the extent of reactivation increased after incubation with dithioerythritol. Reactivation of the enzyme was also obtained by chemical acetylation with acetic anhydride. These results indicate modification of the decarboxylase by deacetylation leading to inactivation and by acetylation of the inactivated enzyme specimens leading to reactivation. It is suggested that the catalytic mechanism involves exchange of the enzyme‐bound acetyl residues by malonyl residues and subsequent decarboxylation releasing CO2 and rregenerating the acetyl‐enzyme. The decarboxylase was inhibited by avidin but ot by an avidin‐biotin complex indicating that biotin is involved in catalysis. A single biotin‐containing 120‐kDa polypeptide was present in the extract and is a likely component of malonate decarboxylase.