bluuebunny/crossref_metadata_2025 huggingface dataset:
Acetylation and methylation sites in histone H4 from Physarum polycephalum
Authors: WATERBORG Jaap H. , FRIED Sharon R. , MATTHEWS Harry R.
Published: March 2005

Histone H4 has been isolated and purified from plasmodia of Physarum polycephalum. The four major fragments produced by hydrolysis of H4 by acetic acid were separated and the complete amino acid sequence of two of them was determined. By analogy with calf H4, these peptides are at the C‐terminus and give the sequence from residue 68 to the C‐terminus (residue 102). In this 35 residue sequence there are two minor differences from calf H4: (i) residue 77 is arginine in Physarum H4 and lysine in calf H4; (ii) lysine‐79 is partially methylated in Physarum. Arginine occurs at position 77 in pea H4 but the occurrence of methylated lysine at position 79 has not been reported for other species. In the N‐terminal region, amino acid compositions of acetic acid, tryptic and chymotryptic peptides indicate that Physarum H4 and calf H4 have identical sequences from the N‐terminus to residue 47. There may be minor differences in the region from residue 46 to residue 67. The sites of acetylation were determined by Edman degradation of acetate‐labelled peptide 4–17 of Physarum H4. Acetylation was observed at positions 5, 8, 12, and 16. The only other labelled peptide was the N‐terminal peptide, which is not susceptible to Edman degradation and is thus probably α‐N‐acetylated as in most other organisms. The results confirm the conservation of H4 sequence and place Physarum H4 in an intermediate position between lower eukaryote H4, such as yeast or Tetrahymena H4, and higher eukaryote H4, such as mammalian H4 or pea H4.
Interaction of mature, unglycosylated and cell‐free synthesized rat α1‐proteinase inhibitor with elastase
Authors: ANDUS Tilo , GROSS Volker , TRAN‐THI Thuy‐Anh , HEINRICH Peter C.
Published: March 2005

Purified rat α1‐proteinase inhibitor (Mr 54 000) and porcine pancreatic elastase (Mr 26 000) formed a complex (Mr 82 000) resistant to heat treatment under denaturing and reducing conditions. A similar α1‐proteinase‐inhibitor‐elastase complex was detected by immunoprecipitation of α1‐proteinase inhibitor from the medium of [35S]methionine‐labeled hepatocyte primary cultures after incubation with elastase. Treatment of hepatocytes with tunicamycin led to the secretion of an unglycosylated α1‐proteinase inhibitor (Mr 41 000) which also formed a complex with elastase (Mr 66 000). Complex formation (Mr 68 000) could also be observed between cell‐free synthesized pre‐α1‐proteinase inhibitor (Mr 43 000) and elastase (Mr 26 000).

The results suggest that neither glycosylation nor removal of the signal peptide are required for the formation of a biologically active conformation of α1‐proteinase inhibitor.
Intracellular forms of transferrin oligosaccharide chains in rat liver
Authors: NAKADA Hiroshi , KOHNO Hirao , KAWASAKI Toshisuke , TASHIRO Yutaka
Published: March 2005

Glycosylation of transferrin was investigated in vivo by using antibody monospecific for rat serum transferrin. Pulse‐chase experiments indicated that, after intravenous injection of [35S]methionine, labeled transferrin appeared in the rough and smooth microsomes and Golgi subfractions in rapid succession in 10 min and that an additional 10 min was required for it to be secreted.

Most of the intracellular transferrin (95%) immunoprecipitated from the total microsome fraction was sensitive to endo‐β‐N‐acetylglucosaminidase H (endo H), whereas serum transferrin was completely resistant to it. Further fractionation of the total microsomes has revealed that the intracellular transferrin immunoprecipitated from the rough and smooth microsomes and GF3 are all endo‐H‐sensitive and most of the endo‐H‐sensitive oligosaccharides were eluted at the position corresponding to Man8GlcNAc on high‐resolution Bio‐Gel chromatography. This finding suggests that the major form of intracellular transferrin oligosaccharide in the course of intracellular transport from the endoplasmic reticulum to the Golgi apparatus is Man8GlcNAc2.

Endo‐H‐resistant forms were first detected in the GF2 but more in GF1, most of which were sensitive to neuraminidase. Since the heavy Golgi subfraction contains mainly cis‐Golgi elements, such as cisternae, and the light subfraction mainly trans‐Golgi elements, such as secretory granules, it is strongly suggested that the processing of these large mannosyloligosaccharide chains and the subsequent addition of terminal sugars to them are performed successively in the trans‐Golgi region just before secretion.
Precise localization of several covalent RNA‐RNA cross‐link in Escherichia coli 16S RNA
Authors: EXPERT‐BEZANCON Alain , MILET Michàle , CARBON Philippe
Published: March 2005

The RNA‐RNA cross‐linking reagent N‐acetyl‐N′‐(p‐glyoxylyl‐benzoyl)cystamine, which reacts via its glyoxal residue with guanines not involved in G · C base pairs, has been used to introduce reversible RNA‐RNA cross‐links into Escherichia coli 16S rRNA. A two‐dimensional gel method has been devised for the separation of the cross‐linked oligonucleotides, and the precise location of guanines involved in four of these cross‐links has been determined by sequencing the oligonucleotides. One cross‐link involves guanosines 1315 and 1360 situated in two hairpin end loops of domain III. The other cross‐links involves pairs of guanosine situated within the same hairpin end loops.
Purification and characterization of two ribosomal proteins of Saccharomyces cerevisiae
Authors: JUAN‐VIDALES Fernando , SÁNCHEZ MADRID Francisco , SAENZ‐ROBLES María Teresa , BALLESTA Juan P. G.
Published: March 2005

Two non‐acidic proteins, extracted from the ribosomes of Saccharomyces cerevisiae using 1 M ammonium chloride in the presence of 50% ethanol, have been purified and characterized. Similar proteins are present in other eukaryotic ribosomes tested, as determined by two‐dimensional gel electrophoresis and cross‐reaction with antisera. One of the two yeast proteins, protein YL23, seems to be very well preserved during evolution, since antisera specific for YL23 cross‐react with protein EC L11 from Escherichia coli. The structural similarity between these two proteins parallels a functional equivalence shown by the ability of the bacterial protein to reconstitute the activity of protein‐deficient core particles from yeast. However, in contrast to protein EC L11, protein YL23 interacts with the yeast acidic proteins, forming a complex probably similar to the one made by bacterial protein EC L10 with proteins EC L7 and EC L12 in the E. coli ribosome. Protein YL23 might play similar roles to those of proteins EC L10 and EC L11 in bacteria.
Fluorescence and chemical modification of tryptophan as probes of structure of GTP:AMP phosphotransferase from beef heart mitochondria
Authors: TOMASSELLI Alfredo G. , DOUGHERTY John J. , NODA Lafayette H.
Published: March 2005

Selective modification of the two Trp residues of GTP:AMP phosphotransferase from beef heart mitochondria (Mr 26000; MgGTP + AMP ⇆ MgGDP + ADP) has been attained by treatment of the enzyme with N‐bromosuccinimide at pH 4.0. Almost complete loss of activity is observed when one Trp is oxidized. Fluorescence emission spectra (λexc 295 nm) were recorded over the pH range 1.9–12.2. Quenching constants, K, with acrylamide were 4.9, 3.4, 3.1, 2.4, 9.2 and 9.4 M−1 at respective pH values of 11.1, 7.5, 5.5, 4.0, 1.9 and 7.5 with 6 M guanidine/HCl. Over the pH range 8.0–5.5 the fluorescence peak has a constant height with maximum at 333–334 nm, which can be segregated by acrylamide quenching into a peak with maximum at 338 nm and another with maximum at 330 nm. Dropping the pH from 5.5 to 4.0 results in the fluorescence at 338 nm decreasing to 335 nm (indicative of less exposure of the Trp) while that at 330 nm remains constant. Thus the limitation of reactivity to N‐bromosuccinimide to pH 4.0 or lower cannot be accounted for by increased exposure of the Trp residues but rather must be explained by a change in the microenvironment of each Trp. As shown by K values above, at pH 2.0 Trp residues are exposed to the solvent, as in the case of treatment with 6 M guanidine hydrochloride.

In raising the pH from 8.0 to 12.0 a number of changes occur: (a) the λmax of emission shifts from 333–334 nm to 343 nm; (b) residue(s) become(s) more available to acrylamide quenching; (c) fluorescence decreases and enzymatic activity increases, both with a midpoint at about 10.6; (d) absorption difference spectra show a maximum at 295 nm typical of Tyr ionization. These data are consistent with conformational change as the pH becomes more alkaline making the Trp residue(s) more exposed to the solvent and/or to non‐radiative energy transfer to tyrosinate.
Stereochemistry of the rearrangement of 2‐aminoethanol by ethanolamine ammonia‐lyase
Authors: GANI David , WALLIS O. Caryl , YOUNG Douglas W.
Published: March 2005

(1R)‐2‐Amino[1‐2H1]ethanol and (1S,2RS)‐2‐amino[1,2‐2H2]ethanols have been synthesised by decarboxylation of (2S,3R)‐[3‐2H1]serine and (2S,3S)‐[2,3‐2H2]serine respectively.

The stereochemical integrity of these labelled 2‐aminoethanols has been ascertained from the 1H‐NMR spectra of their N,O‐dicamphanoyl derivatives.

This assay has also been used to confirm that samples of (2R)‐ and (2S)‐2‐amino[2‐2H1]ethanols prepared from (2R)‐ and (2S)‐[2‐2H1]glycines are stereochemically pure.

Ethanolamine ammonia‐lyase rearranges (1R)‐2‐amino[1‐2H1]ethanol to acetaldehyde at approximately the same rate as it rearranges unlabelled 2‐aminoethanol, whilst (1S,2RS)‐2‐amino[1,2‐2H2]ethanol is rearranged at the same rate as the (1,1‐2H2)‐labelled substrate. The isotope effect is approximately kH/k2H= 8.

The 2H‐NMR spectra of the 3,5‐dinitrobenzoates of the ethanol produced by reduction in situ of the acetaldehyde formed in the rearrangements show that the 1‐2H1 label migrates in (1S,2RS)‐2‐amino‐[1,2‐2H2]ethanol and 2‐amino[1,1‐2H2]ethanol but not in (1R)‐2‐amino[1‐2H1]ethanol.

The above results indicate that the adenosylcobalamin‐dependent ethanolamine ammonia‐lyase catalyses the rearrangement of 2‐aminoethanol with migration of the 1‐pro‐S‐hydrogen atom.
The effect of dibutyryladenosine 3′,5′‐monophosphate on the synthesis of bile salts in isolated hepatocytes from rat
Authors: BOTHAM Kathleen M. , BOYD George S.
Published: March 2005

The effect of dibutyryladenosine 3′,5′‐monophosphate (Bt2cAMP) on the synthesis of conjugated cholic, chenodeoxycholic and β‐muricholic acids has been investigated. Hepatocytes were incubated with 1 mM Bt2cAMP for 3 h at 37°C. In cells from rats with a basal rate of bile salt synthesis (soft‐diet‐fed rats) production of conjugated cholic acid was increased about two fold, synthesis of conjugated chenodeoxycholic acid was increased 10–20‐fold but formation of its metabolite, conjugated β‐muricholic acid, was decreased by 30–50% in the presence of the cyclic nucleotide. The sum of the amounts of the three bile salts produced (total bile salt synthesis) was increased 30–50% by Bt2cAMP. When hepatocytes were prepared from rats in which bile salt synthesis had been stimulated by feeding the bile salt sequestrant, cholestyramine, Bt2cAMP had no effect on conjugated cholic acid synthesis, increased conjugated chenodeoxycholic acid production 3–5‐fold and decreased conjugated β‐muricholic acid synthesis by about 50 %. Total bile salt synthesis was unchanged. The ratio of the amount of conjugated cholic acid to conjugated chenodeoxycholic acid + conjugated β‐muricholic acid produced, an indication of the activity of 7 α‐hydroxycholest‐4‐en‐3‐one 12α‐hydroxylase, was raised by Bt2cAMP in hepatocytes from soft‐diet‐fed but not in those from cholestyramine‐fed rats. The effects of the cyclic nucleotide on the synthesis of the three bile salts in hepatocytes from soft‐diet‐fed rats were found to be saturable at a concentration of about 2 mM. Responses were half‐maximal at concentrations of Bt2cAMP varying between 0.5 and 1.5 mM. These results suggest that in hepatocytes from rats with a basal rate of bile salt synthesis Bt2cAMP has effects at three different stages in the pathway, at the level of cholesterol 7α‐hydroxylase, 7α‐hydroxycholest‐4‐en‐3‐one 12α‐hydroxylase and chenodeoxycholine acid 6β‐hydroxylase. In cells from rats in which bile salt synthesis has been stimulated only the effect at the chenodeoxycholic acid 6β‐hydroxylase level is apparent. Bt2cGMP and Bt2cCMP had no effect on the synthesis of any of the bile salts measure, showing that the effects are specific for Bt2cAMP. The ratio of the amounts of the three bile salts found inside the cells to those found in the medium was decreased by about 90 % when Bt2cAMP was present in the hepatocyte incubations. This effect was mimicked by Bt2cGMP and to a lesser exent by Bt2cCMP. These results indicate that Bt2cAMP has an effect on the uptake and/or secretion of bile salts in the hepatocytes.
Precursor nucleotides at the 5′ end are not required for processing by RNase E at the 3′ end of 5‐S rRNA
Authors: SZEBERÉNYI József , ROY Monoj K. , APIRION David
Published: March 2005

7‐S RNA, a single‐site substrate for the processing enzyme RNase E of Escherichia coli, consists of p5 rRNA (the precursors of 5‐S RNA) and the 3′‐end region of the rRNA transcript which is mainly a termination stem and loop. The 7‐S RNA studied here was derived mainly from the rRNA gene cluster rrnD, carried in a multicopy plasmid. It contains four different populations of molecules that differ from each other at their 5′ ends only: the shortest species has the 5′ end of the mature 5‐S rRNA, while the others are one, two and three nucleotides longer. The four different 5′‐end forms were separated in a long sequencing gel. Processing of these forms and unfractionated 7‐S RNA by RNase E in vitro, showed that all forms, even the shortest one, can be processed to p5 rRNA. Since the extra nucleotides at the 5′ end of the molecule could be base‐paired with the region of 7‐S RNA where RNase E cuts, it is concluded that this double‐stranded structure is not required for the action of RNase E in separating the 3′ end of p5 rRNA from the termination stem.
Evidence that 2‐allyl‐2‐isopropylacetamide, phenobarbital and 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine induce the same cytochrome P450 mRNA in chick embryo liver
Authors: BROOKER John D. , SRIVASTAVA Gopesh , BORTHWICK Iain A. , MAY Brian K. , ELLIOTT William H.
Published: March 2005

The induction of cytochrome P450 in chick embryo liver has been studied using three different porphyrinogenic drugs, 2‐allyl‐2‐isopropylacetamide, 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine and phenobarbital. Pulse‐labelling studies have shown that for each drug the cytochrome P450 synthesised either in ovo or in a wheat germ translation system reacted immunologically with antibody raised against the purified 2‐allyl‐2‐isopropylacetamide‐induced enzyme (Mr= 50 000).

To investigate whether this is due to the three drugs inducing the same protein or different proteins with common immunological determinants, nucleic acid hybridization studies have been carried out using a recently characterised 2‐allyl‐2‐isopropylacetamide‐induced cytochrome P450 cloned cDNA probe [Brooker, J. D. et al. (1982) Eur. J. Biochem. 129, 325–333]. It has been shown that the mRNA induced by each drug hybridizes with this probe and all are of similar size. The melting profile of the mRNA · cDNA hybrids indicates that the mRNAs induced by the three drugs have at least 98% homology with the cDNA probe.

Restriction endonuclease digestions of total chick embryo genomal DNA and a chick cytochrome P450 genomal clone indicates that the cytochrome P450 gene homologous with the cDNA probe is represented in the genome only once.

These results strongly suggest that the three drugs cause increased levels of the same cytochrome P450 mRNA, possibly due to enhanced expression of the same gene.

Results are also presented which show that other cytochrome‐P450‐inducing drugs, 3‐methylcholanthrene, β‐naphthoflavone or pregnenolone‐16 α‐carbonitrile do not increase the level of the 2‐allyl‐2‐isopropylacetamide‐inducible mRNA but rather reduce it to a level which was lower than that of the untreated controls.
p‐HMW‐Collagen, a minor collagen obtained from chick embryo cartilage without proteolytic treatment of the tissue
Authors: BRUCKNER Peter , MAYNE Richard , TUDERMAN Leena
Published: March 2005

The fragments of minor collagens of cartilages, called HMW and LMW, were isolated after pepsin treatment of sternal cartilages of young chickens and were shown to be entirely triple‐helical molecules as judged by their circular dichroic spectra. Studies on renaturation kinetics of HMW suggested that the interchain disulfide bonds in HMW reside at one of the ends of the so‐called long arm.

Polyclonal antibodies against HMW were raised and affinity purified. These antibodies did not cross‐react with type II collagen nor with other minor collagens such as LMW and 1α, 2α, 3α collagen in native or denatured structure.

The antibodies were used to identify HMW‐related molecules which were synthesized by embryonic chick cartilages in vitro. Some of these molecules were secreted into the organ culture medium and could be recovered from it by ammonium sulfate precipitation. Polyacrylamide gel electrophoresis of this precipitate gave one band of high molecular weight which could be reduced to two bands migrating slightly faster than the α1(II) chain when identified by immunoblotting. These bands could also be identified among about six radiolabelled polypeptides present in the ammonium sulfate precipitate of medium proteins when analysed by polyacrylamide gel electrophoresis followed by fluorography.

The same polypeptides could be recovered from the medium by immunoprecipitation with anti‐HMW antibodies. Their presence in cartilage tissue was shown by immunoblotting of material extracted from cartilage tissue and separated on polyacrylamide gels. We suggest that the protein containing these polypeptide chains represents the parent molecule of the peptic fragment HMW as it is synthesized in vivo and have designated it p‐HMW‐collagen.
A comparison of the structure and activity of cat and trout muscle pyruvate kinases
Authors: HARKINS Richard N. , NOCTON Jennifer C. , RUSSELL Margaret P. , FOTHERGILL Linda A. , MUIRHEAD Hilary
Published: March 2005

Pyruvate kinase was purified from cat and trout muscle. The enzymes had similar amino acid compositions and subunit molecular weights. In contrast to the mammalian enzyme, the trout muscle pyruvate kinase was activated by fructose 1,6‐bisphosphate. However, unlike the l‐type pyruvate kinase from mammalian liver it was not phosphorylated by cyclic‐AMP‐dependent protein kinase. The purified enzyme from cat muscle was carboxymethylated with iodo[2‐14C]acetic acid under conditions that led to the preferential labelling of one especially reactive thiol group. The labelled enzyme was cleaved with CNBr, and the radioactive fragment purified. Amino acid sequence analysis of the reactive‐thiol‐containing fragment from cat muscle pyruvate kinase showed it had the following sequence: Ile‐Gly‐Arg‐[14C]CmCys‐Asn‐Arg‐Ala‐Gly‐Lys‐Pro‐Val‐Ile‐CmCys‐Ala‐Thr‐Gln‐Hse. The corresponding peptide from trout pyruvate kinase had only one difference in its amino acid composition and the sequence around the reactive thiol was identical.
Effect of troponin‐tropomyosin complex and Ca2+ on conformational changes in F‐actin induced by myosin subfragment‐1
Authors: BOROVIKOV Yurii S. , GUSEV Nikolai B.
Published: March 2005

The interaction of regulated and unregulated actin of myosin‐free ghost single fibre with myosin subfragment‐1 free of 5,5′‐dithiobis(2‐nitrobenzoic acid) light chains was investigated by polarized microphotometry. The anisotropy of intrinsic tryptophan fluorescence of regulated actin is Ca2+‐dependent and has a maximal value at low (pCa ≥ 7) and a minimal value at high (pCa ≤ 6) concentrations of Ca2+. The interaction of myosin subfragment‐1 with actin induces cooperative changes in actin structure, which manifest themselves in a decrease in the anisotropy of tryptophan fluorescence. The cooperativity of conformational changes in actin, induced by myosin subfragment‐1, is high for regulated actin in the absence of Ca2+ and low for unregulated and regulated actin in the presence of Ca2+. The data obtained suggest that the decrease of the flexibility of actin filaments, induced by tropomyosin or by Ca‐unsaturated troponin‐tropomyosin complex, results in increased cooperativity of conformational changes of actin induced by myosin subfragment‐1.
Carnitine palmitoyltransferase I
Authors: SIRE Oliver , MANGENEY Marise , MONTAGNE Jaqueline , NORDMANN Roger , NORDMANN Joseph
Published: March 2005

Palmitate oxidation by liver mitochondria from rats treated with d‐galactosamine (GalN) was markedly inhibited, 3 h after administration.

The mitochondrial defect responsible for this inhibition was shown to be an inhibition of the activity of palmitoylcarnitine transferase I (EC 2.3.1.21).

Apparent Km of the enzyme remained unchanged whereas apparent V was reduced by 30%.

Addition of 10 mM GalN did not impair the activity of palmitoylcarnitine transferase I in mitochondria isolated from normal rats.

Inhibition of palmitoylcarnitine biosynthesis by GalN treatment was completely reversed by phospholipid supply.

At this stage of intoxication, mitochondrial phospholipid content was decreased whereas incorporation of [14C]palmitate into phospholipids in isolated hepatocytes was drastically inhibited; the phosphatidyl‐choline/phosphatidylethanolamine ratio was reduced by 33%.

The results obtained from these studies show that the depletion of the phospholipid membrane content could account for the altered functional activity of palmitoylcarnitine transferase I.
Cholinergic synaptic vesicles from Torpedo marmorata contain an atractyloside‐binding protein related to the mitochondrial ADP/ATP carrier
Authors: STADLER Herbert , FENWICK Elizabeth May
Published: March 2005

Atractyloside is known to bind to the ADP/ATP translocase of the inner mitochondrial membrane, a complex formed by two basic protein subunits of relative molecular mass around 30000. We found that synaptic vesicles from the electric organ of Torpedo marmorata, which store acetylcholine and ATP, bind atractyloside as well. Similarly to mitochondria, a protein‐atractyloside complex could be solubilized from vesicle membranes with Triton X‐100. Characterization of the complex by gel filtration, isoelectric focusing and gel electrophoresis revealed that atractyloside was bound to protein V11, earlier described as a major vesicle membrane component with a relative molecular mass around 34000 and a basic isoelectric point.

Since earlier experiments have already shown that uptake of ATP into isolated vesicles in vitro is inhibited by atractyloside, we can conclude now that V11 constitutes the nucleotide carrier of this secretory organelle. The structural and functional relationship of the mitochondrial and vesicular nucleotide translocases suggest a common evolutionary origin.
Subcellular fractionation of porcine neutrophils by nitrogen cavitation and sucrose‐density‐gradient centrifugation
Authors: CHIBBER Ramesh , CASTLE Alan G.
Published: March 2005

Neutrophils, isolated in large quantities from porcine blood were disrupted by nitrogen cavitation and separated by differential centrifugation into a nuclear fraction and a post‐nuclear supernatant. The latter was subfractionated by sucrose density gradient centrifugation into cytosol, a fraction consisting of membrane vesicles and two granule‐rich fractions.

The membrane fraction accounted for 1.9% of the protein in the post‐nuclear supernatant, the light granule fraction for 2.2% and the dense granule fraction for 4.2%.

Catalase, lactate dehydrogenase and malate dehydrogenase were largely confined to the cytosol. The dense granule fraction contained the highest quantities of the hydrolytic enzymes, although the membrane fraction was also rich in alkaline and acid phosphatase and γ‐glutamyl transpeptidase activities.

Electron microscopy of the membrane fraction showed intact membrane vesicles, whereas the granular fractions consisted of electron‐dense, membrane‐bound granules. Two granular fractions were isolated which contained granules of differing size and density.

3H‐labeled wheat germ agglutinin bound to the surface of intact neutrophils and when these were disrupted and fractionated the membrane fraction showed a specific binding activity 16‐times greater than that of the cavitated sample.

The membrane fraction interacted with the detergent digitonin and as a result underwent density perturbation increasing from 1.13 g . cm−3 to 1.18 g . cm−3.

Dodecylsulphate‐polyacrylamide gel electrophoresis showed the membrane fraction to consist of at least 40 protein bands, with relative molecular masses ranging from 200000–16000. The granule fractions contained less protein bands, with a protein composition quite distinct from that of the membrane fraction.
Structure of the Mo‐Fe protein component of Azotobacter vinelandii nitrogenase
Authors: VOORDOUW Gerrit , HAAKER Huub , VAN BREEMEN Jan F. L. , VAN BRUGGEN Ernst F. J. , EADY Robert R.
Published: March 2005

The Mo‐Fe protein of nitrogenase from both Azotobacter vinelandii and Klebsiella pneumoniae (Av1 and Kp1. respectively) consists of four subunits of similar, but not identical, relative molecular mass. The hydrodynamic properties of Av1 (sedimentation and diffusion coefficient) and its total relative molecular mass are very similar to those of Kp1 and catalase from bovine liver, a tetramer of four identical subunits.

By electron microscopy the Av1, Kp1 and catalase tetramers are seen as protein particles of diameter 9.0–10.0 nm; no details of the subunit structure can be observed. Av1 (but not Kp1) forms regular polymers of variable length at low ionic strength in the presence of MgCl2. The structure of these polymers, of diameter 21.2 nm, is complex. Optical diffraction studies give a smallest repeating distance of 8.4 nm (corresponding to the diameter of the Av1 tetramer) and indicate a four‐start helix. The latter structure is incompatible with a flat, square subunit arrangement of the Av1 tetramer as proposed by Stasny et al. [(1974) J. Cell. Biol. 60, 311–316]. We propose, therefore, that the subunit arrangement of the Av1 tetramer is of the tetrahedral type. This has also been proposed for the catalase tetramer from optical diffraction studies of electron micrographs of catalase tubes indicating a 222 symmetry [Kiselev, D. A., De Rosier, N. J. and Klug, A. (1968) J. Mol. Biol. 35, 561–566]. Our proposal is in agreement with the recent finding that Av1 protein crystals belong to the P21 space group [Weiniger, M. S. and Mortenson, L. E. (1982) Proc. Natl Acad Sci. USA, 79, 378–380].
Properties of the chromatin repair activity against O6‐ethylguanine lesions in DNA
Authors: RENARD André , VERLY Walter G. , MEHTA Jitendra R. , LUDLUM David B.
Published: March 2005

Chromatin proteins from rat liver contain a repair activity that removes O6‐ethylguanine from ethylnitrosoureatreated DNA. This activity does not depend on divalent cations and works in the presence of EDTA, but does depend on the presence of free thiol groups. Thus, it is destroyed by N‐ethylmaleimide and is protected by dithiothreitol. The repair activity on single‐stranded DNA is only 20% of what it is on double‐stranded DNA; its half‐life at 35°C is 55 min, but DNA, ethylated or not, affords some protection.

The repair reaction is a transethylation from O6‐ethylguanine in DNA onto two different cysteine residues contained in acceptor proteins. The reaction can be followed by monitoring the appearance of ethylated proteins or by disappearance of O6‐ethylguanine from DNA.
Interaction between photoexcited rhodopsin and peripheral enzymes in frog retinal rods
Authors: PFISTER Claude , KÜHN Hermann , CHABRE Marc
Published: March 2005

The major peripheral and soluble proteins in frog rod outer segment preparations, and their interactions with photoexcited rhodopsin, have been compared to those in cattle rod outer segments and found to be similar in both systems. In particular the GTP‐binding protein (G) has the same subunit composition, the same abundance relative to rhodopsin (1/10) and it undergoes the same light and nucleotide‐dependent interactions with rhodopsin in both preparations.

Previous work on cattle rod outer segments has shown that photoexcited rhodopsin (R*), in a state identified with metarhodopsin II, associates with the G protein as a first step to the light‐activated GDP/GTP exchange on G. The complex R*‐G is stable in absence of GTP, but is rapidly dissociated by GTP owing to the GDP/GTP exchange reaction. Low bleaching extents (< 10% R*) in absence of GTP therefore create predominantly R*‐G complexes, whereas bleaching in presence of GTP creates free R*. We report here that, under conditions of complexed R*, two reactions of R* in frog rod outer segments are highly perturbed as compared to free R*: (a) the spectral decay of metarhodopsin II (MII) into later photoproducts, and (b) the phosphorylation of R* by an ATP‐dependent protein kinase.

a) The spectral measurements have been performed using linear dichroism on oriented frog rod outer segments; this technique allows discrimination between MII and later photoproducts absorbing at the same wavelength. Association of R* with G leads to a strong reduction of the amount of MIII formed and to an acceleration of the decay of MIII. Furthermore, MII is significantly stabilized, in agreement with the hypothesis that MII is the intermediate which binds to G.

b) The phosphorylation of R* is strongly inhibited under conditions of R*‐G complex formation as compared to free R*.

Interferences between reactions at the three sites involved in R* are discussed: the retinal binding site in the hydrophobic core is sensitive to the presence of GTP‐binding protein at its binding site on the cytoplasmic surface of R*: the kinase and the GTP‐binding protein compete for access to their respective binding sites, both located on the surface of R*.

We also observed a slow and nucleotide‐dependent light‐induced binding of a protein of molecular weight 50 000, which we consider as the equivalent of the 48 000 Mr light‐dependent protein previously identified in cattle rod outer segments.
The multifunctional polypeptide chain of rabbit mammary fatty acid synthase contains a domain homologous with the acyl carrier protein of Escherichia coli
Authors: McCARTHY Alun D. , AITKEN Alastair , HARDIE D. Grahame
Published: March 2005

The phosphopantetheine thiol of rabbit mammary fatty acid synthase was specifically alkylated using chloro[14C]acetyl‐CoA and a radioactive fragment generated by limited elastase digestion of the modified protein was purified by gel filtration. We have previously mapped this fragment to an internal location in the 250000‐Mr polypeptide adjacent to the thioesterase domain [Eur. J. Biochem. 130, 185–193 (1983)]. The purified fragment had apparent molecular weights of 23 000 by gel filtration and 10 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, while amino acid analysis indicated a minimal molecular weight of 10400. We have determined the amino acid sequence of the first 64 residues of the fragment. The phosphopantetheine moiety is esterified to a serine at residue 38 in the sequence. When the sequences of the rabbit acyl carrier fragment and the 8847‐Mr acyl carrier protein of Escherichia coli are aligned, 17 out of 64 residues are identical. These results suggest that the limited proteolysis delineates an internal acyl carrier domain within the rabbit protein and provide the first clear evidence that multifunctional fatty acid synthases have arisen by fusion of ancestral monofunctional proteins.
Proteins from the sperm of the bivalve mollusc Ensis minor
Authors: GIANCOTTI Vincenzo , RUSSO Elio , GASPARINI Maria , SERRANO Domenico , DEL PIERO Donatella , THORNE Alan W. , CARY Peter D. , CRANE‐ROBINSON Colyn
Published: March 2005

Analysis of the total protein of the mature sperm of the bivalve mollusc Ensis minor (razor shell) using gel electrophoresis, amino acid analysis, nuclear magnetic resonance, circular dichroism and trypsin digestion, show it to contain all five histones plus a protamine‐like protein. The histones H3, H4 and probably H2A are similar to those from calf thymus or sea urchin sperm, but the putative H2B appears to have a very high molecular mass (about 20kDa). The histone H1 molecule is unusual, having little or no proline and 8–10 residues of histidine. The protamine‐like species is rich in both lysine as well as arginine and is of much higher molecular mass than fish sperm protamines. Nucleosomes containing the four core histones have been prepared and the nucleosomal repeat shown to be 200 ± 5 base pairs. Checks for the absence of contaminating cells reinforce the conclusion that a histone‐containing nucleθsomal structure co‐exists with a protamine‐like protein in this sperm chromatin.
Minactivin: A human monocyte product which specifically inactivates urokinase‐type plasminogen activators
Authors: GOLDER Jeffrey P. , STEPHENS Ross W.
Published: March 2005

Culture supernatants from monolayers of human peripheral monocytes strongly inhibited colorimetric assays of urokinase in which plasmin was measured by esterolysis. This inhibitory activity of monocyte culture supernatant was enhanced after culture with muramyl dipeptide. Inhibition was specific for plasminogen activators of Mr 52 000 and 36 000, as shown by three methods: (1) inhibition of plasminogen‐dependent fibrinolysis; (2) inhibition at the level of plasminogen activation in a colorimetric assay; (3) the irreversible loss of plasminogen‐activating activity, as evidenced by electrophoresis, after preincubation with culture media. The factor responsible for this inactivation (which we propose to call minactivin) had an apparent Mr of 66 000 on Sephacryl S 300 gel chromatography and interacted with enzyme in a biphasic manner: a rapid partial inhibition (reversible by sodium dodecyl sulphate) was followed by slow inactivation (irreversible by sodium dodecyl sulphate).

It is proposed that secretion of minactivin by monocytes may contribute to regulation of extracellular proteolysis at sites of tissue injury.
Ornithine‐containing lipid of Bordetella pertussis, a new type of hemagglutinin
Authors: KAWAI Yohko , YANO Ikuya
Published: March 2005

The ornithine‐containing lipids of six strains (phases I–IV) of Bordetella pertussis were prepared from the total extractable cellular lipids by thin‐layer chromatography and treatment with phospholipase A. They were compared with those prepared from two strains each of Bordetella parapertussis and Bordetella bronchiseptica. The structures of the ornithine‐containing lipid of B. pertussis and the other two species were resolved by acid and alkaline hydrolysis, gas‐liquid chromatography, infrared absorption spectroscopy, amino acid analysis and combined gas‐liquid chromatography/mass spectrometry. The main structure of the aminolipid of the three species of Bordetella was 3‐hydroxyhexadecanoic acid, amide‐linked to ornithine and esterified to the second hexadecanoic acid. The aminolipid of B. pertussis Sakurayashiki (phase III) exhibited high hemagglutinating activity for human and rabbit erythrocytes, having a minimum hemagglutinating concentration of 1 γg/ml against 8–16 γg/ml for the other strains of Bordetella.

All of these aminolipids showed some degree of microheterogeneity. Because the 3‐hydroxyhexadecanoic acid content was especially high in strain Sakurayashiki, it was presumed that the intensity of hemagglutinating activity of the aminolipid was affected by the chain length of the central 3‐hydroxy fatty acid, that is the aminolipid containing 3‐hydroxyhexadecanoic acid had high hemagglutinating activity. The hemagglutination was inhibited by phosphatidylcholine at concentrations of more than 20 γg/ml. Other inhibitory substances were cysteine, sphingomyelin, acidic amino acids, histidine, unsaturated fatty acid and basic amino acids. Furthermore, the divalent cations Ca2+ and Mg2+ inhibited this hemagglutination at a concentration of 1 mM. The O‐deacylated ornithine‐containing lipid that had lost hexadecanoic acid did not have any hemagglutinating activity but did have hemolytic activity. Observation by electron microscopy indicated that erythrocytes were combined by the liposomes of the ornithine‐containing lipids.

On the basis of these results, the proposed mechanism of hemagglutination by the aminolipids is that the liposomes of the aminolipids combine erythrocytes by hydrophobic interaction between the fatty acid moieties of the aminolipid and the lipids of the surface of erythrocytes, and by ionic interaction between the ornithine of the aminolipid and the protein of the surface of the erythrocytes. In addition, the hemagglutinating activity of phosphatidylserine was found to be due to its similar structure to that of the ornithine‐containing lipid and the mechanism was also presumed to be similar. The mechanism of hemagglutination by these aminolipids was distinct from that of lectins.
Kinetic effects of chemical and physical uncoupling on the energy‐transducing ATPase from spinach chloroplasts
Authors: SHERMAN Paula A. , WIMMER Mary J.
Published: March 2005

Ammonium chloride, an uncoupler of photophosphorylation which stimulates the membrane‐bound chloroplast coupling factor ATPase when added after light/dithiothreitol activation, causes a decrease in the number of extra water oxygens incorporated into the phosphate formed during ATP hydrolysis. This observation is in contrast to the long‐reported insensitivity of intermediate Pi:H2O oxygen exchange to uncoupler dinitrophenol in the mitochondrial F1 ATPase system. The effect of ammonium chloride on the CF1‐catalyzed oxygen exchange reaction is consistent with ATPase activity stimulation caused by increased partitioning forward of the enzyme products complex. In line with the oxygen exchange data, ammonium chloride causes an increase in the apparent Km of the enzyme for substrate ATP. The effect of ammonium chloride on the pattern of the intermediate Pi : H2O oxygen exchange is not a threshold phenomenon; the extent of exchange decreases in a continuous fashion, paralleling the stimulation of ATPase activity. The uncoupler CF3OPhzC(CN)2 also decreases the extent of oxygen exchange upon stimulating the membrane‐bound ATPase, while phlorizin, an energy‐transfer inhibitor, has essentially no effect on exchange although it inhibits the ATPase reaction. Similar to the effect of chemical uncoupling on the membrane‐bound enzyme, physical removal of the coupling factor ATPase from the thylakoid membrane also results in an increase in forward partitioning of the enzyme · ADP · Pi complex. The modulation of oxygen exchange observed by altering the degree of coupling is similar to that which accompanies changing ATP concentration in the mitochondrial ATPase system [Russo, J. A., Lamos, C. M. and Mitchell, R. A. (1978) Biochemistry 17, 473–480 and Choate, G. L., Hutton, R. L. and Boyer, P. D. (1979) J. Biol. Chem. 254, 286–290]. However, the uncoupler modulation is not readily correlated with the degree to which multiple catalytic sites are occupied by substrate.
The interaction of nocardicin A with the penicillin‐binding proteins of Bacillus megaterium KM
Authors: TODD John A. , YON Jeff R. , ELLAR David J.
Published: March 2005

The inhibition of elongation of Bacillus megaterium KM growing in the presence of low concentrations of nocardicin A resulted in the production of osmotically stable, actively dividing coccal‐shaped cells. Saturation of penicillin‐binding proteins 3a and 3b with nocardicin A in vivo at these concentrations was correlated with the inhibition of cell elongation. Analysis of the DD‐carboxypeptidase activity of isolated vegetative membranes of B. megaterium KM in vitro indicated that penicillin‐binding protein 4 is not a DD‐carboxypeptidase under the assay conditions used. Penicillin‐binding proteins were analysed by two‐dimensional gel electrophoresis and the suitability of lysozyme treatment of cells as a method of membrane preparation was investigated with regard to the detection of proteins with highly labile penicillin‐binding activities in vitro.
Evidence for an ATP‐driven proton pump in rat thyroid phagolysosomes
Authors: FOUCHIER Francis , DANG Josseline
Published: March 2005

During incubations at 37°C in appropriate media (buffered 0.25 M sucrose) isolated thyroid phagolysosomes degrade the thyroglobulin they contain (labelled with 131I in vivo) giving rise to trichloroacetic‐acid‐soluble radioiodine. Thyroglobulin‐degradation is unaffected by external pH (7 or 8) or by 20–40 mM external NaCl or KCl, while it is strongly inhibited by ionophores and protonophores. As a consequence, thyroglobulin degradation can be used as an index of the intralysosomal pH which appears to be powerfully maintained in basal conditions (no ionophore and no protonophore) by the strong impermeability of the lysosomal membranes to various compounds including ionic species MgATP which does not modify basal proteolysis prevents or minimizes the alkalinizing effects of both ionophores and protonophores. ATP can thus be concluded to promote a protonic flux inward thyroid lysosomes via the activity of a lysosomal ATP‐driven proton pump regulated by the magnitude of the intralysosomal pH.
Mutual conformational changes of tryptophanyl‐tRNA synthetase and tRNATrp in the course of their specific interaction
Authors: BERESTEN Sergey , SCHEINKER Vladimir , FAVOROVA Olga , KISSELEV Lev
Published: March 2005

tRNATrp (beef, yeast) is capable of accelerating limited tryptic hydrolysis of the N‐terminal part in the polypeptide chains of dimeric beef pancreas tryptophanyl‐tRNA synthetase; it can also eliminate the protective effect of tryptophanyl adenylate on the enzyme proteolysis. The effect of tRNA on the proteolysis is manifested even when the 3′‐CCA terminus is removed. It has been concluded that the conformation of the synthetase changes when it forms a complex with tRNATrp. Yeast tRNATrp lacking the 3′‐half of the acceptor stem can still interact with the synthetase and, to certain extent, induces changes in the conformation of the latter.

The susceptibility of single‐stranded and double‐stranded regions of tRNATrp to cleavage with endonucleases has been studied, and the results are indicative of the fact that, regardless of considerable differences in the nucleotide sequence of yeast and beef tRNATrp, their three‐dimensional structures are similar. This fact is consistent with the finding that parameters for the interaction of these tRNAsTrp with beef tryptophanyl‐tRNA synthetase are rather close.

The three‐dimensional structure of tRNATrp is altered when the enzyme forms a complex with it, as seen from (a) a change in the circular dichroic spectrum and (b) an elevated susceptibility of the anticodon and, apparently, acceptor stems to cleavage with nuclease.

The conversion of exposed cytidine residues in tRNATrp into uridine residues results in a loss of the acceptor activity; the capability to accelerate limited tryptic hydrolysis of tryptophanyl‐tRNA synthetase is also lost although the enzyme‐substrate complex, as seen from circular dichroic spectra, can still be formed. The conversion of cytosine in the anticodon stem into uracil modifies the conformation of the anticodon stem.

The anticodon arm (including the anticodon) and the acceptor stem play an essential role in the interaction between tRNATrp and tryptophanyl‐tRNA synthetase.
Characterization of phosphodiesterase catalytic sites by means of cyclic nucleotide derivatives
Authors: COUCHIE Dominique , PETRIDIS Georg , JASTORFF Bernd , ERNEUX Christophe
Published: March 2005

Cyclic nucleotide derivatives have been used as a tool to characterize distinct catalytic sites on phosphodiesterase enzyme forms: the cGMP‐stimulated enzyme from rat liver and the calmodulin‐sensitive enzyme from rat or bovine brain. Under appropriate assay conditions, the analogues showed linear competitive inhibition with respect to cAMP (adenosine 3′,5′‐monophosphate) as substrate. The inhibition sequence of the fully activated cGMP‐stimulated phosphodiesterase was identical to the inhibition sequence of the desensitized enzyme, i. e. the enzyme which has lost its ability to be stimulated by cGMP. The inhibition pattern could, therefore, not be attributed to competition with cGMP at an allosteric‐activating site. Also, the inhibition sequence of the calmodulin‐sensitive phosphodiesterase was maintained whether activity was basal or fully stimulated by calmodulin. When cAMP and cGMP, with identical chemical ligands substituted at the same position, were compared as inhibitors of the calmodulin‐sensitive phosphodiesterase, the cGMP analogues were always the more potent suggesting that, for that enzyme, the catalytic site was sensitive to a guanine‐type cyclic nucleotide structure. Comparing the two phosphodiesterases, it was possible to establish both similar and specific inhibitor potencies of cyclic nucleotide derivatives. In particular, the two enzymes exhibited large differences in analogue specificity modified at C‐6, 6‐chloropurine 3′,5′‐monophosphate or purine 3′,5′‐monophosphate.
Interaction of α‐1,2‐mannosidase with anionic phospholipids
Authors: FORSEE W. Thomas , SCHUTZBACH John S.
Published: March 2005

A partially purified microsomal α‐1,2‐mannosidase from rabbit liver binds to vesicles that contain anionic phospholipids. The affinity of the enzyme for the phospholipid vesicles is dependent upon the negative charge density of the vesicles rather than the concentration of the anionic phospholipid or the phospholipid specificity. A phosphatidylinositol/phosphatidylcholine ratio of 1:3 is sufficient to bind all of the enzyme. The association of enzyme with anionic phospholipids involves ionic interactions and can be readily reversed by high ionic strength or changes in pH. The α‐mannosidase is inhibited when bound to the anionic phospholipid vesicles, but the enzyme can be reactivated when released by NaCl or CaCl2. When bound to anionic phospholipid vesicles, the enzyme was also found to undergo a slow inactivation process that was time and temperature dependent and could not be reversed by the addition of CaCl2.
Reversibility characteristics of glucose‐induced trehalase activation associated with the breaking of dormancy in yeast ascospores
Authors: THEVELEIN Johan M. , JONES Kerri‐Ann
Published: March 2005

The breaking of dormancy in yeast ascospores by addition of glucose is associated with a sudden tenfold increase in the activity of trehalase. The rapid activation of trehalase is followed by a slower inactivation process which is greatly retarded in the presence of nitrogen sources and cycloheximide. When glucose is washed away from the spores after some time and the spores resuspended in glucose‐free medium, the trehalase activity decreases sharply. Subsequent addition of new glucose partially reactivates the enzyme. The extent of reactivation decreases further with each subsequent activation/inactivation step. Changing the duration of the inactivation periods has no effect on this diminution of the reversibility. However, prolonging the duration of the activation step speeds up the loss of reversibility. On the other hand, addition of a nitrogen source or cycloheximide completely prevents the loss of reversibility. The results of the reversibility studies are in agreement with the phosphorylation mechanism which has been proposed for the underlying molecular process of trehalase activation. Apparently, they are also in agreement with proteolytic breakdown being responsible for the inactivation of trehalase after its initial activation. However, the effect of cycloheximide and nitrogen sources, at least in ascospores, does not appear to be due to inhibition or repression of protease synthesis, respectively, since the addition in the presence of glucose of a nitrogen source after trehalase inactivation immediately reactivates the enzyme completely.