bluuebunny/crossref_metadata_2025 huggingface dataset:
Reconstitution of Na+ transport from purified methylmalonyl‐CoA decarboxylase and phospholipid vesicles
Authors: HILPERT Wilhelm , DIMROTH Peter
Published: March 2005

Methylmalonyl‐CoA decarboxylase from Veillonella alcalescens which was isolated by affinity chromatography on avidin‐Sepharose was incorporated into phospholipid vesicles by the detergent dilution method with octyl glucoside as the detergent. By this procedure the Na+ pump activity was reconstituted. The optimal octyl glucoside concentration for reconstitution was about 2.8%. The activity of reconstituted Na+ transport increased with the amount of enzyme present during reconstitution until a plateau was reached at about 7 μg enzyme/mg phospholipid. All four polypeptides of the decarboxylase were incorporated into the proteoliposomes but the relative amounts of α and γ subunits were considerably reduced.

The reconstitution process was highly asymmetric, since about 80% of the decarboxylase was oriented in the proteoliposomes with the substrate binding site facing to the interior. The orientation was determined from the increase of methylmalonyl‐CoA decarboxylase activity by destruction of the membrane permeability barrier. It was also deduced from the amount of enzyme which was not accessible from the outside to inactivation by avidin.

With these reconstituted vesicles, a steady‐state internal Na+ concentration was established by methylmalonyl‐CoA decarboxylation which under optimized conditions was about 30‐fold higher than in the incubation medium. Sodium ion accumulation in the presence of the Na+ ‐carrying ionophores nigericin or monensin was practically nil. In the presence of valinomycin or carbonylcyanide‐p‐trifluoromethoxy phenylhydrazone the rate of Na+ transport and its steady‐state internal concentration were considerably higher than in the controls which is consistent with the function of methylmalonyl‐CoA decarboxylase as an electrogenic Na+ pump.
An investigation of the mechanism of activation of tryptophan by tryptophanyl‐tRNA synthetase from beef pancreas
Authors: LOWE Gordon , TANSLEY Gaynor
Published: March 2005

Adenosine (S)‐5′‐[α‐17O,18O2]triphosphate has been synthesized and used to investigate the stereochemical course of activation of tryptophan by tryptophanyl‐tRNA synthetase from beef pancreas. It is shown that the reaction proceeds by displacement of Mg pyrophosphate from MgATP with inversion of configuration at Pot. Tryptophanyl‐tRNA synthetase catalyses positional isotope exchange in adenosine 5′‐[β‐18O2]triphosphate in the presence of tryptophan but not in its absence or in the presence of the competitive inhibitors tryptamine and tryptophanol. These observations eliminate a multi‐adenylyl transfer or dissociative mechanism and in combination with the stereochemical study leave a direct associative ‘in line’ displacement at Pα of MgATP as the only tenable mechanism for the activation of tryptophan by tryptophanyl‐tRNA synthetase.
Age‐related growth and protein turnover in the thymus of normal and glucocorticoid‐treated rats
Authors: KELLY Frank James , GOLDSPINK David Frank
Published: March 2005

Age‐related changes in the growth, nucleic acid content and protein turnover of the thymus have been studied in normal male rats.

A rapid and massive atrophy was found at each age 24 h after exposure to cortisone acetate or dexamethasone; the thymus of sexually mature rats being most severely affected.

The steroids decreased (70–90%) total protein synthesis in the thymus more than whole body synthesis (15%), indicating a more pronounced action on the thymus compared with other body tissues.

Initially protein breakdown increased in the steroid‐treated thymus but at longer times this change was reversed.
Isolation and characterization of a precursor form of M collagen from embryonic chicken cartilage
Authors: VON DER MARK Klaus , VAN MENXEL Marion , WIEDEMANN Hanna
Published: March 2005

A disulfide‐cross‐linked collagen has been extracted with neutral salt solutions from organ cultures of embryonic chick sternal cartilage. This collagen, which we term pM collagen, is presumed to be the native extracellular precursor molecule to disulfide‐cross‐linked collagen fragments recently described. Cleavage of pM collagen under native conditions with pepsin gives rise to the collagen fragments M1 and M2, which had also been isolated from pepsin extracts of chick hyaline cartilage [K. von der Mark, M. van Menxel & H. Wiedemann (1982) Eur. J. Biochem. 124, 57–62]. Native pM collagen was purified by DEAE‐cellulose chromatography and agarose gel filtration. On agarose and following polyacrylamide gel electrophoresis, the unreduced molecule migrates with an apparent Mr of 300 000. Reduction of disulfide bridges produces two subunits with Mr 80000 (pMa) and 60 000 (pMb) when compared with collagen standards. Cyanogen bromide cleavage of pMa and pMb, excised from dodecyl sulfate gels, resulted in different peptide maps, indicating that both components are genetically distinct polypeptide chains. The occasional appearance of the unreduced pM collagen as a doublet band on dodecyl sulfate gels and the observation that pMa and pMb occur in non‐stoichiometric ratios suggests that pMa and pMb form separate native molecules, although their incorporation into a single pM molecule cannot be excluded. Native pM collagen was completely digested with bacterial collagenase, and contained hydroxyproline and proline in a ratio of 1.15:1, indicating the absence of significant non‐collagenous domains. Thus it represents, despite several pepsinlabile sites, more likely a largely triplehelical, processed form of collagen rather than a procollagen‐like molecule containing globular domains.

Processing of pM collagen to M1 and M2 fragments or other intermediate forms was not observed in cartilage organ culture or in chondrocyte cell cultures within 18h.
The catalytic subunits of protein phosphatase‐1 and protein phosphatase 2A are distinct gene products
Authors: TUNG H. Y. Lim , RESINK Therese J. , HEMMINGS Brian A. , SHENOLIKAR Shirish , COHEN Philip
Published: March 2005

Three forms of protein phosphatase‐1 were isolated from rabbit skeletal muscle that had Mr values of 37000, 34000 and 33000 determined by sodium dodecyl sulphate (SDS) gel electrophoresis. Each species dephosphorylated the β‐subunit of phosphorylase kinase very much faster than the α‐subunit, was inhibited by inhibitors 1 and 2 with equal potency, and was converted to a form dependent on glycogen synthase kinase‐3 and Mg‐ATP for activity by incubation with inhibitor‐2. Digestion with cyanogen bromide or Staphylococcus aureus proteinase followed by SDS gel electrophoresis showed a very similar pattern of cleavage products for all three forms. The Mr‐37000 and Mr‐34000 species were converted to the Mr‐33000 form by incubation with chymotrypsin. It is concluded that the Mr‐33000 and Mr‐34000 forms are derived from the Mr‐37000 component by limited proteolysis. Conversion of the Mr‐37000 to the Mr‐33000 form was accompanied by a two‐fold increase in activity, indicating that an Mr‐4000 fragment at one end of the polypeptide is an inhibitory domain that decreases enzyme activity.

The catalytic subunit of protein phosphatase 2A from rabbit skeletal muscle had an Mr of 36000 determined by SDS gel electrophoresis and its specific activity (3 kU/mg) was much lower than that of the Mr‐37000 (15–20 kU/mg) or Mr‐33/34000 (40–50 kU/mg) forms of protein phosphatase‐1. It dephosphorylated the α‐subunit of phosphorylase kinase 4–5‐fold faster than the β‐subunit, was unaffected by inhibitor‐1 or inhibitor‐2, and preincubation with the latter protein did not result in the production of a glycogen synthase kinase‐3 and Mg‐ATP‐dependent form of the enzyme. Digestion with chymotrypsin did not alter the electrophoretic mobility of protein phosphatase 2A under conditions that caused quantitative conversion of the Mr‐37000 form of protein phosphatase‐1 to the Mr‐33000 species. Digestion with cyanogen bromide or S. aureus proteinase, followed by SDS gel electrophoresis, showed a quite different pattern of cleavage products to those observerd with protein phosphatase 1. Antibody to protein phosphatase‐2A raised in sheep did not cross‐react with any of the forms of protein phosphatase‐1, as judged by immunoelectrophoretic and immunotitration experiments. It is concluded that protein phosphatase‐1 and protein phosphatase‐2A are distinct gene products.
Evidence for the presence of calmodulin in fish mucus
Authors: FLIK Gert , VAN RIJS Jeanne H. , WENDELAAR BONGA Sjoerd E.
Published: March 2005

Partly purified mucus collected from the skin of three species offish contains a protein that, on sodium dodecyl sulphate/polyacrylamide gel electrophoresis, comigrates with bovine brain calmodulin and shows the same calcium‐dependent shift in electrophoretic mobility as calmodulin.

Fish mucus contains a heat‐stable activator of cyclic nucleotide phosphodiesterase; activation is concentration dependent and sensitive to the specific calmodulin inhibitor calmidazolium (R 24571).

The presence of calmodulin in fish mucus is further indicated by means of a specific radioimmunoassay.

A drop in the calcium concentration of the water induces an increase in the immunoassayable calmodulin concentration of mucus, which indicates that the function of calmodulin in mucus is related to control of permeability of the skin epithelium to water and ions.
Changes in form of elongation factor 1 during germination of wheat seeds
Authors: SACCHI Gian Attilio , ZOCCHI Graziano , COCUCCI Sergio
Published: March 2005

Two forms of EF‐1 are present in the high‐speed supernatant fraction from wheat embryo homogenate. In embryos from dry seeds EF‐1H, is 55% of the total amount of EF‐1, while after 40 h of germination this form completely disappears. When germination and protein synthesis are accelerated by means of 6‐benzyladenine, the rate of conversion of EF‐1H is increased. On the other hand, the block of germination and of the evolution of protein synthesis by abscisic acid, block this conversion; the block of water uptake, that stops germination and causes a decrease in protein synthesis, reverses the conversion of EF‐1H to EF‐1L, increasing EF‐1H from 15% to 40% of the total.
The platelet‐fibrinogen interaction
Authors: MARGUERIE Gerard A. , THOMAS‐MAISON Nadine , GINSBERG Mark H. , PLOW Edward F.
Published: March 2005

Fibrinogen participates in platelet aggregation via specific inducible receptors on the cell surface. We have used a photoactivable bifunctional reagent, N‐succinimidyl‐6‐(4′‐azido‐2′‐nitrophenylamino)hexanoate, SANAH, to derivatize 125I‐labeled‐fibrinogen (125I‐Fg) and crosslink it to ADP‐stimulated platelets. Binding experiments established that 125I‐Fg and 125I‐Fg‐SANAH interacted with platelets with the same kinetics and affinity as unlabeled fibrinogen. After photoactivation of the platelet‐bound 125I‐Fg‐SANAH, polyacrylamide gel electrophoresis under reducing conditions revealed formation of a high molecular weight covalent complex with coordinate loss of the Aα chain. 125I‐Fg‐SANAH missing the extreme carboxy‐terminal region of the Aα chain failed to crosslink to the platelets under similar conditions. Crosslinked 125I‐Fg‐SANAH was extracted from the cells in 1% Triton X‐100, and immunoprecipitation with antibodies specific for platelet membrane glycoproteins was used to identify components of the complex. With antibodies to the glycoprotein IIb/III complex (anti‐GP IIb/III), 40 ± 9% of the extracted 125I‐Fg‐SANAH was irnmunoprecipitated. Omission of photoactivation, platelets, or ADP from the reaction or use of unmodified 125I‐Fg resulted in less than 5% immunoprecipitation by the anti‐GP IIb/III. As controls for specificity, anti‐(glycoprotein Ib) or anti‐IgG immunoprecipitated less than 5% of the extracted 125I‐Fg‐SANAH. Under similar conditions, 45% of the GP IIb/III from surface‐labeled platelets was recovered in the anti‐GP IIb/III immunoprecipitate. These results indicate that the Aα chain of fibrinogen comes in close proximity to GP IIb/III when the molecule is bound to its platelet receptor.
Fluorescence‐quenching studies on a conformational transition within a domain of the β2 subunit of Escherichia coli tryptophan synthase
Authors: CHAFFOTTE Alain F. , GOLDBERG Michel E.
Published: March 2005

The fluorescence quenching by acrylamide of the single tryptophan residue in the β2 subunit of tryptophan synthase from Escherichia coli K12 is studied for different states of the protein: the native apo‐enzyme and holo‐enzyme, the nicked apo‐protein and holo‐protein and the isolated proteolytic fragment F1 corresponding to the N‐terminal two thirds of β2. The quenching constants measured are used to estimate the accessibility of the tryptophan residue in these different forms. The results are discussed in terms of conformational transition within the F1 domain, occurring in the presence of the cofactor, pyridoxal 5′‐phosphate, in the native enzyme. The proteolytic cleavage of the native enzyme is shown to render the nicked protein unable to undergo this conformational change.
Characterization of phospholipases A2 of Naja melanoleuca snake venom modified at the N‐terminal region
Authors: van EIJK Jan H. , VERHEIJ Hubertus M. , DE HAAS Gerard H.
Published: March 2005

In phospholipase A2 from Naja melanoleuca snake venom all four lysines were converted into the ɛ‐amidinated derivatives without reaction of the α‐amino group. The amidinated phospholipase (AMPA) showed high enzymatic activity.

Starting from AMPA, chemical modification reactions were carried out at the α‐amino function. This group was blocked with a tert‐butyloxycarbonyl or a phenylthiocarbamyl group. Furthermore the polypeptide chain was shortened by one residue by removing the N‐terminal asparagine, resulting in the formation of des‐Asn1‐AMPA. The native enzyme was shortened by eight residues by cyanogen bromide cleavage at the single methionine8 residue.

Although all modified proteins show a reduced affinity for monomeric lipids, they are easily saturated with micellar substrate analogs. Whereas the removal of the N‐terminal octapeptide abolished all enzymatic activity the other modified enzymes possess a low (1%), but measurable enzymatic activity. It is concluded that chemical modifications in the N‐terminal region give rise to a distortion of the active site, thus reducing the activity of the lipid‐bound enzyme.
Primary structure of flavocytochrome b2 from baker's yeast
Authors: GHRIR Rachid , BECAM Anne‐Marie , LEDERER Florence
Published: March 2005

Reverse‐phase high‐pressure liquid chromatography has been used for the purification of some large cyanogen bromide peptides from flavocytochrome b2 fragment α. Acetonitrile gradients at acid and/or neutral pH using μBondapak C18 columns were useful for the smaller peptides (43 and 67 residues). The two larger ones, αCB1 and αCB2, could only be separated from each other by trifluoroacetic acid/1‐propanol gradients on μBondapak‐CN columns. The various systems tested are presented and compared.

The elucidation of the amino acid sequence of αCB2 (95 residues), αCB3 (67 residues) and αCB4 (43 residues) is described. The fragments were digested with trypsin, chymotrypsin and Staphylococcus aureus V8 protease as necessary. Fragment αCB2 was also cleaved at the unique tryptophanyl bond with cyanogen bromide. Peptides were fractionated by Sephadex chromatography, thin‐layer finger‐printing and/or high‐pressure liquid chromatography. Peptides were sequenced mostly in the liquid phase sequenator.

The cyanogen bromide peptides could be ordered using information obtained previously, as well as additional data obtained in this work. Together with the previous elucidation of cytochrome b2 core sequence and of the hinge region [Guiard, B. and Lederer, F. (1976) Biochimie (Paris) 58, 305–316; Ghrir, R. and Lederer, F. (1981) Eur. J. Biochem. 120 279–287], the present results enable us to present the complete sequence of fragment α (314 residues) with only three overlaps missing between cyanogen bromide peptides. Sequence comparisons with other known flavoproteins do not indicate any noticeable similarity. Structural predictions indicate an alternation of α helices and β structure. The possibility that the non‐heme‐binding portion of fragment α could constitute a flavin‐binding domain is discussed.
Metalloproteinase inhibitors from bovine cartilage and body fluids
Authors: BUNNING Rowena A. D. , MURPHY Gillian , KUMAR Shant , PHILLIPS Paul , REYNOLDS John J.
Published: March 2005

Inhibitors of the mammalian metalloproteinases, collagenase, proteoglycanase and gelatinase were isolated from bovine cartilage (extracts and culture medium) and bovine amniotic fluid and serum. These inhibitors either bind or do not bind to concanavalin‐A – Sepharose, with Mr (gel filtration) of about 30000 and 20000, respectively. Cartilage and chondrocyte culture media contained only concanavalin‐A‐binding inhibitors whereas cartilage extracts contained only a non‐binding inhibitor: serum and amniotic fluid contained both forms of inhibitory activities. In most biochemical respects, particularly in their abilities to inhibit metalloproteinases, all of the inhibitors were found to be similar. It is concluded that the forms of the inhibitors that differ in Mr may be closely related to the tissue inhibitor of metalloproteinases (TIMP) previously purified from rabbit and human sources. These findings help to clarify other studies on collagenase inhibitors and support the concept that TIMP‐like inhibitors may be important in the control of connective tissue degradation.
Presence of poly (ADP‐ribose) polymerase and poly (ADP‐ribose) glycohydrolase in the dinoflagellate Crypthecodinium cohnii
Authors: WERNER Edgar , SOHST Silvia , GROPP Felix , SIMON Dietrich , WAGNER Herbert , KRÖGER Hans
Published: March 2005

Poly(ADP‐ribose) polymerase and poly(ADP‐ribose) glycohydrolase have been detected in chromatin extracts from the dinoflagellate Crypthecodinium cohnii. Poly(ADP‐ribose) glycohydrolase was detected by the liberation of ADP‐ribose from poly(ADP‐ribose). Poly(ADP‐ribose) polymerase was proved by (a) demonstration of phosphoribosyl‐AMP in the phosphodiesterase digest of the reaction product, (b) demonstratioin of ADP‐ribose oligomers by fractionation of the reaction product on DEAE‐Sephadex. The (ADP‐ribose)‐protein transfer is dependent on DNA; it is inhibited by nicotinamide, thymidine, theophylline and benzamide. The protein‐(ADP‐ribose bond is susceptible to 0.1 M NaOH (70 %) and 0.4 M NH2OH (33 %). Dinoflagellates, nucleated protists, are unique in that their chromatin lacks histones and shows a conformation like bacterial chromatin [Loeblich, A. R., III (1976) J. Protozool. 23, 13–281; poly(ADP‐ribose) polymerase, however, has been found only in eucaryotes. Thus our results suggest that histones were not relevant to the establishment of poly(ADP‐ribose) during evolution.
Solubilization of mannosyltransferase activities for the biosynthesis of mammary glycoproteins
Authors: PRAKASH Chandra , KATIAL Albine , KANG Mohinder S. , VIJAY Inder K.
Published: March 2005

The microsomal preparation from the lactating bovine mammary tissue was solubilized by treatment with nonionic detergent, NP‐40, at a protein/detergent ratio of 1.5:1 and a detergent concentration of 0.5%. Following centrifugation at 147000 ×g for 120 min, the supernatant fraction was incubated with labeled sugar nucleotides, GDP‐Man and UDP‐GlcNAc. It was found to synthesize a series of lipid‐linked saccharides up to (Man)5‐ (GlcNAc)2. The solubilized glycosyltransferases retained up to about 60% of the activity after two weeks of storage at 4°C. The biosynthesis of glycolipids was stimulated by a mixture of lipids obtained by extracting the mammary microsomes with CHCl3/CH3OH (2:1).

A labeled lipid‐linked tetrasaccharide of the structure Manα1 → 3Manβ→ GlcNAcβ→ GlcNAc was isolated by labeling baby hamster kidney cells with [2‐3H]mannose under conditions of glucose starvation followed by extraction of the cells with CHCl3/CH3OH (2:1) and separation of the lipids by high‐performance liquid chromatography. When this lipid‐linked tetrasaccharide was incubated with the solubilized bovine mammary microsomes and GDP‐Man, it was elongated to a lipid‐linked heptasaccharide having the structure Manα1 → 2Manα1 → 2Manα1 → 3(Manα1 → 6)Manβ→ GlcNAcβ→ GlcNAc. The kinetics of the elongation reaction also revealed the intermediary formation of smaller amounts of lipid‐linked pentasaccharide and hexasaccharide. The elongation reaction did not require any divalent metal ion and had a broad pH optimum between 6.8 and 7.6. The lack of inhibition of the elongation reaction by EDTA or amphomycin support earlier studies that GDP‐Man rather than mannosylphosphoryldolichol, is the direct donor of mannosyl residues for the biosynthesis of glycolipids up to (Man)5(GlcNAc)2. Mannosylphosphorylretinol was ineffective as mannosyl donor for the elongation reaction.
Short‐term control of mitochondrial adenine nucleotide translocator by thyroid hormone
Authors: MOWBRAY John , CORRIGALL Jane
Published: March 2005

1. An improved simple technique for measuring adenine nucleotide translocator activity at low medium substrate concentrations is described.

2. Confirming previous reports, thyroidectomy was shown to lead to lowered translocator activity in rat liver mitochondria. The rapidly exchangeable portion of the matrix nucleotide also decreased in hypothyroid preparations even though the total nucleotides increased substantially.

3. The apparent Km of translocator for ADP increased from 2.8 to 6.2 μM in hypothyroid preparations: Mg2+ ions raised this to about 20μM.

4. All of these changes in adenine nucleotide translocation were entirely reversed by 15 min after a single intravenous near‐physiological dose of triiodothyronine.
Adenine nucleotides affect the binding of 3‐phosphoglycerate to pig muscle 3‐phosphoglycerate kinase
Authors: VAS Mária , BATKE József
Published: March 2005

Pig muscle 3‐phosphoglycerate kinase was complexed with 1‐anilino‐8‐naphthalenesulfonate (ANS) in order to monitor the binding of substrates to the enzyme. The enzyme‐dye interaction did not influence the enzymic activity under the experimental conditions used.

By measuring the substrate‐dependent change in the fluorescence emission of ANS molecules tightly bound to the enzyme (Kd≤ 0.05 mM), fluorimetric titrations were carried out in 0.1 M Tris/HCl buffer pH 7.5, containing 5 mM mercaptoethanol, at 20°C. The dissociation constants obtained for the separate bindings of 3‐phosphoglycerate, MgATP, 1,3‐bisphosphoglycerate and MgADP were 0.03 ± 0.01 mM. 0.15 ± 0.1 0mM, 0.00005 ± 0.00001 mM and 0.15 ± 0.10mM respectively.

Binding of 3‐phosphoglycerate is weakened when MgATP is also bound to the enzyme: the dissociation constant of 3‐phosphoglycerate in this ternary complex (0.25 ± 0.08 mM) is comparable to its Km value (0.38 ± 0.10mM). The same weakening can be observed in the non‐productive ternary complexes where MgATP is replaced by MgADP (Kd= 0.20 ± 0.10 mM) or AMP (Kd= 0.12 ± 0.05 mM), whereas adenosine has no such effect. This indicates the importance of the negatively charged phosphate(s) of nucleotides in influencing the binding of 3‐hosphoglycerate.

In contrast to 3‐phosphoglycerate, the binding of the substrate analogue, glycerol 3‐phosphate is practically not affected by the presence of MgATP: the dissociation constant to the free enzyme (0.40 ± 0.10 mM) is comparable to its inhibitory constant (0.70 ± 0.20 mM). This finding and the similarity of the dissociation constant of glycerol 3‐phosphate binding (0.40 ± 0.10mM) and the Km value of 3‐phosphoglycerate (0.38 ± 0.10mM) suggest that, during the enzymic reaction, binding of 3‐phosphoglycerate occurs probably without involvement of the carboxyl group.
Inactivation of pig muscle 3‐phosphoglycerate kinase by thiol modification depends on reagent size
Authors: DÉKÁNY Katalin , VAS Mária
Published: March 2005

Two thiol groups (out of the total seven) of pig muscle 3‐phosphoglycerate kinase can be alkylated in 0.1 M Tris/HCl buffer, pH 7.5, at 20°C either with methyl iodide or with iodoacetamide in a second‐order reaction with rate constants 0.05 ± 0.02 M−1 s−1 and 0.23 ± 0.05 M−1 s−1 respectively. The slow reaction of the remaining five thiols with Ellman's reagent (Nbs2), which requires the unfolding of the protein, is not affected by the nature of the alkylating reagent.

While methylation of the two reactive thiols does not affect either the specific activity of the enzyme or the Km values of the substrates, carboxamidomethylation is accompanied by the loss of enzymic activity.

Both the methylated and the carboxamidomethylated enzymes bind 3‐phosphoglycerate or MgATP practically with the same binding constants as the native enzyme, as detected by fluorimetric titration of enzymes complexed with 1‐anilinonaphthalenesulfonate (ANS). Thus, inactivation during carboxamidomethylation cannot be due to changes in the substrate‐binding ability of the enzyme.

The substrate‐caused changes in the fluorescence emission spectrum of ANS bound to the carboxamidomethylated enzyme are different from the changes observed with the native enzyme. The fluorescence properties of the methylated enzyme do not differ from those of the native enzyme. These differences may reflect the different mode of substrate binding to the carboxamidomethylated enzyme as compared to the native or the methylated one. Thus, the special steric requirements of the enzymic reaction are possibly not fulfilled after carboxamidomethylation.

The presence of the equilibrium mixture of substrates lessens the reactivity of the fast‐reacting thiols towards both alkylating agents, a finding that indicates the effects of substrates on the local conformation around these groups.
Ornithine‐containing lipids in Thiobacillus A2 and Achromobacter sp.
Authors: THIELE Otto Wolfgang , OULEVEY Jacques , HUNNEMAN Donald H.
Published: March 2005

20 bacterial strains (corresponding to 16 species) were screened for ornithine lipids. Only two species (Thiobacillus A2 and Achromobacter sp.) turned out to contain ornithine lipids (2.71 mmol/100 g and 0.38 mmol/100 g bacterial dry weight, respectively). In both ornithine lipids, a 3‐hydroxy fatty acid was amide‐linked to the α‐amino group of ornithine, a normal fatty acid was ester‐linked to the 3‐hydroxy group of the former. The predominant fatty acids were 18:1(11) and 3‐hydroxy‐20:1(13) in Thiobacillus A2, 16:0 and 3‐hydroxy‐18:1(11) in Achromobacter sp. All monounsaturated fatty acids (with one exception) belonged to the (n‐7) family. 11, 12‐Epoxy octadecanoic acid was identified among the ester‐linked fatty acids of Thiobacillus A2. Phosphatidyl‐choline was the principal phospholipid in both bacterial species.
Identification and properties of the 38000‐M, poly(A)‐binding protein of non‐polysomal messenger ribonucleoproteins of cryptobiotic gastrulae of Artemia salina
Authors: DE HERDT Erik , TIHOEN Chris , VAN HOVE Lauris , ROGGEN Erwin , PIOT Etienne , SLEGERS Herman
Published: March 2005

The Mr‐38000 poly(A)‐binding protein interacts with synthetic and natural RNA. A sequence‐independent stoichiometry of one protein per 8–12 nucleotides is measured by filter binding and sucrose gradient centrifugation. Specificity for the poly(A) sequence is demonstrated from poly(A)/RNA mixing experiments. The poly(A)‐binding protein has been identified as the helix‐destabilizing protein HD40 [Marvil, D. K., Nowak, L. and Szer, W. (1980) J. Biol. Chem. 255, 6466–6472] and is characterized by the existence of at least seven ionic species with a pl ranging from 9.2 to 6.6. Acidic ionic species are generated by phosphorylation with mRNP‐associated protein kinase. Different ionic species are present on free mRNP and ribosomes‐mRNP preinitiation complexes. The poly(A)‐binding protein affects mRNA translation and (A)4 polyadenylation. The multifunctionality of the protein is discussed.
Isolation of a novel antibody, which precipitates ribonucleoprotein complex containing threonine tRNA from a patient with polymyositis
Authors: OKADA Norihiro , MUKAI Ryozaburo , HARADA Fumio , KABASHIMA Teizo , NAKAO Yasuro , YAMANE Kazuhide , OHSHIMA Yasumi , SAKAMOTO Kazuichi , ITOH Michiyasu , KASHIWAGI Heihachiro , HAMAGUCHI Hideo
Published: March 2005

We have isolated and characterized a novel antibody from a patient with polymyositis, which precipitates threonine tRNA and an unknown small RNA molecule from 32P‐labeled HeLa extract. Fingerprint analysis showed that the human threonine tRNA precipitated was nearly identical to the murine tRNA described by Harada [(1978) Seikagaku 50, 397–411]. Nucleotide analysis suggested that the other small RNA molecule might be transfer RNA. Since phenol extraction resulted in the loss of immunoprecipitability, the protein portion was presumed to be involved in the recognition of the antigen. Immunofluorescence staining of HeLa cells with the antibody clearly demonstrated that the antigen was located predominantly in the cytoplasm.
The 5S RNA binding protein from yeast (Saccharomyces cerevisiae) ribosomes
Authors: YAGUCHI Makoto , ROLLIN C. Fernand , ROY Camille , NAZAR Ross N.
Published: March 2005

The carboxyl‐terminal half (CN2 fragment) of the yeast 5S RNA binding protein (YL3) retains an ability to form homogeneous ribonucleoprotein complexes with RNA although the N‐terminal half (CN1) appears to confer specificity for the 5S RNA molecule [Nazar, R. N., Yaguchi, M., Willick, G. E., Rollin, C. F. and Roy, C. (1979) Eur. J. Biochem. 102, 573–582]. The nucleic acid binding site in this fragment was more clearly delineated by cleaving the CN2 fragment with a variety of enzymatic and chemical reagents and further examining the ability of the products to form RNA‐peptide complexes. Hot acetic acid treatment produced a 47‐residue subfragment (CN2‐A1) which originated from the C terminus and continued to form stable ribonucleopeptide complexes. The amino acid sequence of this subfragment was determined to be: ‐Pro‐Ala‐Phe‐Lys‐Pro‐Thr‐Glu‐Lys50‐Phe‐Thr‐Lys‐Glu‐Gln‐Tyr‐Ala‐Ala‐Glu60‐Ser‐Lys‐Lys‐Tyr‐Arg‐Gln‐Thr‐Lys‐Leu‐Ser70 ‐Lys‐Gln‐Gln‐Arg‐Ala‐Ala‐Arg‐Val‐Ala‐Ala80‐Lys‐Ile‐Ala‐Ala‐Leu‐Ala‐Gly‐Gln‐Gln‐COOH, with 12 of the 16 basic residues in the CN2 fragment being present in this binding site. The amino acid sequence of the CN2‐A1 fragment bears a limited homology in both amino acid and charge distribution with histone 2B from mammals and with one of the 5S RNA binding proteins (EL25) from Escherichia coli. The results suggest that many protein binding sites for nucleic acids may share common structural features and further support the notion that the single large eukaryotic 5S RNA protein may have evolved through a fusion of genes for the multiple 5S RNA binding proteins in prokaryotes.
Homology between the primary structures of the major bovine β‐crystallin chains
Authors: BERBERS Guy A. M. , HOEKMAN Willeke A. , BLOEMENDAL Hans , de JONG Wilfried W. , KLEINSCHMIDT Traute , BRAUNITZER Gerhard
Published: March 2005

Partial amino acid sequences of six major subunits of bovine β‐crystallin have been determined by automatic liquid‐phase Edman degradation and the dansyl‐Edman procedure, complemented by amino acid analyses of peptides. The results show that, including the previously established βBp sequence [H. P. C. Driessen et al. (1981) Eur. J. Biochem. 121, 83–91], there exist at least seven primary gene products in bovine β‐crystallin, which exhibit 40% or more sequence homology. Two of the gene products are completely identical except for the presence in one of them of 17 additional residues at the N terminus, possibly caused by differential splicing of the same primary RNA transcript. The rate of evolutionary change of the β chains (4% sequence change per 100 × 106 years) is about equally slow as that of α‐crystallin, and the gene duplications giving rise to the different chains must have occurred very early in vertebrate evolution. The β chains can be divided into two groups, according to sequence homology and presence of deletions/insertions and C‐terminal extension, on which basis a new, rational nomenclature for the β subunits is introduced. The N‐terminal extensions of all β chains are very different in length and sequence, even between homologous β chains in different species. Possible explanations for this finding are discussed.
Specific changes in lactate levels, lactate dehydrogenase patterns and cytochrome b559 in Dictyostelium discoideum caused by queuine
Authors: SCHACHNER Elfriede , ASCHHOFF Hans‐Jürgen , KERSTEN Helga
Published: March 2005

Higher eukaryotes contain tRNA transglycosylases that incorporate the guanine derivative queuine from the nutritional environment into specific tRNAs by exchange with guanine at position 34. Alterations in the queuosine content of specific tRNAs are suggested to be involved in regulatory mechanisms of major routes of metabolism during differentiation.

Dictyostelium discoideum has been applied as a model to investigate the function of queuine or queuine‐containing tRNAs. Axenic strains are supplied with queuine by peptone, but they grow equally well in a defined queuine‐free medium. Queuine‐lacking amoebae, starved in suspension culture for 24 h, lose their ability to differentiate into stalk cells and spores, whereas amoebae sufficiently supplied with queuine will overcome this metabolic stress and undergo further development when plated on agar.

The results presented here show that d(–)‐lactate occurs in the slime mould in millimolar amounts and that its level is remarkably decreased in queuine‐lacking cells after 24 h of starvation in suspension culture. On isoelectric‐focusing polyacrylamide gels, nine different forms of NAD‐dependent d(–)‐lactate dehydrogenase can be separated from extracts of vegetative cells, and six forms from extracts of the starved cells. Under queuine limitation, one form is missing in the starved cells. Low amounts of l(+)‐lactate are usually found in vegetative amoebae but significantly less in queuine‐lacking cells. Five forms of NAD‐dependent l(+)‐lactate dehydrogenase are detectable in extracts from vegetative, queuine‐treated cells, and slight alterations occur in queuine‐deficient amoebae. In the starved cells only one form of l(+)‐lactate dehydrogenase is found, irrespective of the supply of queuine to the cells. A cytochrome of type b with an absorption maximum at 559 nm accumulates during starvation only in queuine‐lacking cells; it might be a component of an NAD‐independent lactic acid oxidoreductase as is cytochrome b557 in yeast and be responsible for the reduced level of lactate in cells lacking queuine in tRNA.
Isolation and characterization of a precursor form of lysosomal α‐glucosidase from human urine
Authors: OUDE ELFERINK Ronald P. J. , BROUWER‐KELDER Elisabeth M. , SURYA Inge , STRIJLAND Anneke , KROOS Marian , REUSER Arnold J. J. , TAGER Joseph M.
Published: March 2005

1. A two‐step procedure is described for the isolation of lysosomal α‐glucosidase from human urine. In the second step, affinity chromatography on Sephadex G‐100, two fractions with acid α‐glucosidase activity were obtained.

2. Fraction I contained α‐glucosidase of Mr 109000, whereas fraction II contained components of Mr 76000 and 70000.

3. α‐Glucosidase in fraction I had an Mr similar to that of the precursor of α‐glucosidase detected in the medium of fibroblasts after labelling with [14C]leucine. The components in fraction II had Mr identical to those of the mature forms of α‐glucosidase found in placenta or cultured human skin fibroblasts.

4. α‐Glucosidase in fraction I contained mannose 6‐phosphate (3.5 mol/mol polypeptide). No mannose 6‐phosphate was present in the components in fraction II.

5. Fraction I, but not fraction II, was avidly endocytosed by α‐glucosidase‐deficient cultured human skin fibroblasts. Endocytosis of fraction I was inhibited by mannose 6‐phosphate.

6. The pH optimum and Km values for p‐nitrophenyl α‐glucoside, maltose and glycogen of fractions I and II α‐glucosidase were almost identical. However, the activity with glycogen relative to that of either p‐nitrophenyl α‐glucoside or maltose was lower in fraction I than in fraction II.

7. It is concluded that fraction I consists of the precursor form of α‐glucosidase and fraction II of the mature forms of the enzyme.

8. The importance of urine as a source of precursors of lysosomal enzymes is discussed.
Pyruvate carboxylase from Aspergillus nidulans
Authors: OSMANI Stephen A. , MAYER Frank , MARSTON Fiona A. O. , SELMES Ian P. , SCRUTTON Michael C.
Published: March 2005

1. A method is described for purification of pyruvate carboxylase from Aspergillus nidulans by affinity chromatography on monomeric avidin‐Sepharose. The purified enzyme is homogeneous as judged by electrophoretic and immunochemical analysis. The sub‐unit Mr determined by electrophoresis in the presence of sodium dodecyl sulphate is 133000 ± 5000.

2. Electron microscopic analysis of purified A. nidulans pyruvate carboxylase after negative staining with uranyl acetate reveals the presence of molecules showing rhomboid and triangular projections as well as a projection showing two intensity maxima. A cleft running along the longitudinal axis of the sub‐unit is observed in the rhomboid and triangular projections. Interconversion between all three projections can be obtained in a tilt series.

3. Significantly better preservation of molecular structure is obtained if A. nidulans pyruvate carboxylase is prepared for electron microscopy in the presence of acetyl‐CoA, 2‐oxoglutarate or as the enzyme‐avidin complex. l‐Aspartate has no significant effect when added alone but markedly decreases the enhanced preservation given by acetyl‐CoA. No marked alterations in molecular dimensions are caused by any of these additions.

4. l‐Aspartate, but not 2‐oxoglutarate, enhances the rate of inactivation observed on incubation of A. nidulans pyruvate carboxylase at 4°C in the presence of 0.5 M KCl.

5. Addition of l‐aspartate in low concentrations enhances the effectiveness of inhibition by 2‐oxoglutarate by causing a decrease in the value of [I]0.5 without affecting the Hill coefficient h or the extent of activity observed at saturating 2‐oxoglutarate concentrations. Conversely addition of low concentrations of 2‐oxoglutarate decreases the concentration of l‐aspartate required to give 50% inhibition but also causes a fall in h and an absolute increase in the extent of activity observed in the presence of saturating l‐aspartate concentrations.

6. The data are consistent with the proposal that A. nidulans pyruvate carboxylase is a tetrameric molecule in which the four sub‐units are located at the corners of a tetrahedron. Metabolites which regulate the activity of the enzyme do not cause major alterations in this molecular structure but may alter its stability. The data presented also provide further evidence supporting the postulate that l‐aspartate and 2‐oxoglutarate occupy distinct regulatory sites on A. nidulans pyruvate carboxylase.
Catalytic significance of binary enzyme‐aldehyde complexes in the liver alcohol dehydrogenase reaction
Authors: ANDERSSON Pia , KVASSMAN Jan , OLDÉN Bertil , PETTERSSON Gösta
Published: March 2005

1. The interaction of liver alcohol dehydrogenase with NADH and aldehyde substrates has been characterized with respect to ternary‐complex formation by the apparently non‐preferred pathway which involves intermediate formation of binary enzyme · aldehyde complexes. Rate constant estimates are reported for dimethylamino‐cinnamaldehyde (DACA) binding to free enzyme and for NADH binding to the enzyme · DACA complex.

2. The rate of NADH (or NAD+) association to liver alcohol dehydrogenase is not detectably affected by DACA binding to the enzyme, but the NADH dissociation rate decreases approximately by a factor of 6. The NADH‐induced increase in affinity of the enzyme for DACA is similarly attributable to a decreased dissociation rate rather than an increased association rate of the aldehyde. DACA dissociates much more rapidly than coenzyme from the enzyme · NADH · aldehyde complex and shows a higher association rate constant than NADH in its interaction with free enzyme.

3. It is concluded from these results that the enzymic reduction of typical aldehyde substrates will conform to a rate equation which is experimentally indistinguishable from that of a compulsory‐order mechanism with coenzyme binding preceding substrate binding, and that this rate equation will obtain irrespective of which pathway for ternary‐complex formation is actually preferred. Rate equations provide no reliable information about the order of ligand binding in ternary‐complex systems.

4. A flow analysis is presented which indicates that coenzyme and substrate are actually bound in random order to liver alcohol dehydrogenase during the enzymic reduction of aldehydes by NADH. The enzyme · aldehyde pathway for ternary‐complex formation is fully kinetically competent, and reaction flow via this pathway may predominate when aldehyde concentrations exceed those required for half‐saturation of free enzyme. Binary enzyme · aldehyde complexes are seemingly insignificant with respect to the rate behaviour of the enzyme, but may provide most significant and even predominant contributions to the catalytic reaction flow.
Resolution of bovine heart cytochrome c oxidase into smaller complexes by controlled subunit denaturation
Authors: CORBLEY Michael J. , AZZI Angelo
Published: March 2005

Bovine heart cytochrome c oxidase has been partially denaturated under mild conditions with 0.1–0.25% lithium dodecyl sulfate and 0.05% Triton X‐100.

From its reactivity towards CO and CN–, an unmasking of the heme a was inferred in this enzyme. The catalytic activity was lost during the denaturation and small spectral differences became visible. Spectra and ligand binding properties of the denatured enzyme were reversed by dilution in 2% Triton X‐100. This suggests that during the denaturation procedure the hemes were not displaced from their original sites.

By gel filtration of the partially denatured enzyme the following complexes of subunits were obtained: I–III, I–II– III, II–IV–V–VI–VII and IV–V–VI–VII. The first three complexes retained almost all the heme, and their spectral characteristics were very similar to those of the partially denatured cytochrome c oxidase. The data, in combination with the information that subunit III does not contain heme [Saraste et al. (1980) FEBS Lett. 114, 35–38], suggest that the hemes are attached to subunit I and II.

After denaturation of cytochrome c oxidase under more drastic conditions some of the heme was also found to be associated with the smaller subunits, but its spectral characteristics were radically altered, becoming almost identical to those of free heme.
Involvement of the 3′ side of the anticodon loop of yeast tRNATyr in messenger‐free binding to ribosomes
Authors: WEYGAND‐DURAŠEVIĆ Ivana , NÖTHIG‐LASLO Vesna , KUĆAN Željko
Published: March 2005

Electron‐spin resonance (ESR) spectra of a nitroxide spin‐label attached to residue i6A‐37 of yeast tRNATyr were measured in complexes of deacylated tRNATyr with Escherichia coli ribosomes. A Scatchard plot, obtained in the absence of mRNA, indicated strong binding with an association constant of 1 × 107l · mol–1, suggesting the P‐site binding. The ESR spectrum of free tRNATyr, characteristic for a rapidly tumbling nitroxide, changes to a spectrum with extensively broadened lines in the ribosome‐tRNA complex. The original spectrum can be restored upon long incubations of the complex with an excess of extraneous tRNA. ESR spectra suggest that the spin‐label motion is drastically perturbed though not completely blocked in the ribosome‐tRNATyr complex. Since ESR spectra of a spin‐label attached to the opposite, i.e. 5′, side of the anticodon loop are only slightly perturbed by the messenger‐free binding to ribosomes [Rodriguez et al. (1980) J. Biol. Chem. 255, 8116–8120], it is concluded that the two sides of the anticodon loop face entirely different environments when bound to the P site, the 3′ side being oriented towards the surface of the ribosome, and the other side towards its environment or a large cavity.
Biosynthesis and compartmentalization of rat‐intestinal vitamin‐D‐dependent calcium‐binding protein
Authors: LEONARD Warren J. , STRAUSS Arnold W. , GO Mae F. , ALPERS David H. , GORDON Jeffrey I.
Published: March 2005

We have purified the primary translation product of rat intestinal vitamin‐D‐dependent calcium‐binding protein mRNA from wheat germ and ascites cell‐free systems. We show that calcium‐binding protein is neither synthesized as a larger precursor nor likely to be exported from the intestinal epithelium. Our conclusions are based on the following observations. (1) The primary translation product, NH2‐terminally labeled with formyl[35S]methionine, comigrates with the mature cytoplasmic protein during electrophoresis through denaturing gels. (2) It does not possess a cleavable signal peptide sequence or internal signal equivalent as judged by co‐ and post‐translational cleavage assays in vitro. (3) The NH2 terminus of the cell‐free product is acetylated. (4) Comparison of the NH2‐terminal amino acid sequences of the primary translation product and cyanogen bromide peptides obtained from the blocked, purified cytoplasmic protein.

The kinetics of calcium‐binding protein mRNA accumulation and decay in rachitic intestinal epithelium after primary and secondary stimulation with 1,25‐dihydroxycholecalciferol (calcitriol) were studied using the cell‐free translation system. The results are reminiscent of other steroid‐hormone‐inducible systems. Both the rate of mRNA accumulation and the peak response were greater after secondary stimulation.
The mechanism of inactivation of lipoxygenases by acetylenic fatty acids
Authors: KÜHN Hartmut , HOLZHÜTTER Hermann‐Georg , SCHEWE Tankred , HIEBSCH Christa , RAPOPORT Samuel M.
Published: March 2005

The inactivation of soybean lipoxygenase by 5,8,11,14‐eicosatetraynoic acid was studied in detail. The inactivation was found to be time‐dependent and irreversible. A kinetic scheme, based on the assumption of a rapid inactivation of the enzyme‐product complex, yielded a Km value for 5,8,11,14‐eicosatetraynoic acid of 1.3 μM, which is about a tenth of that described for arachidonic acid, and a reaction constant k+2 of 0.006s−1, which is four orders of magnitude lower. The reasons for these differences are discussed. Several types of experimental evidence indicate that the first step of the enzyme inactivation is the conversion of 5,8,11,14‐eicosatetraynoic acid via a lipoxygenase reaction: (a) the conversion of radioactively labelled methyl ester of 5,8,11,14‐eicosatetraynoic acid to other products; (b) the oxygen requirement of the inactivation; (c) the competitive protective effect of linoleic acid; (d) the similarity of the activation energy for both the dioxygenation of linoleic acid and the enzyme inactivation by 5,8,11,14‐eicosatetraynoic acid; (e) the formation of one mole methionine sulfoxide/mole enzyme during the reaction with 5,8,11,14‐eicosatetraynoic acid, similar to the suicidal reaction of reticulocyte lipoxygenase with 13Ls‐hydroperoxy‐linoleic acid.

These results, as well as the lack of covalent binding of 14C‐labelled 5,8,11,14‐eicosatetraynoic acid methyl ester, contradict the allene mechanism postulated by others [D. T. Downing, D. G. Ahern, and M. Bachta (1970) Biochem. Biophys. Res. Commun. 40, 218–223; K. H. Gibson (1977) Chem. Soc. Rev. 6, 489–510]. It is assumed that the susceptible methionine is located at the active centre of the enzyme.