bluuebunny/crossref_metadata_2025 huggingface dataset:
Cold‐Sensitive Ribosome Assembly in an Esclzerichia coli Mutant Lacking a Single Methyl Group in Ribosomal Protein L3
Authors: LHOEST Jacques , COLSON Charles
Published: March 2005

Ribosomal protein methylation has been well documented but its function remains unclear. We have examined this phenomenon using an Escherichia coli mutant (prmB2), which fails to methylate glutamine residue number 150 of ribosomal protein L3. This mutant exhibits a cold‐sensitive phenotype: its growth rate at 22°C is abnormally low in complete medium. In addition, strains with this mutation accumulate abnormal and unstable ribosomal particles; 50‐S and 30‐S subunits are formed, but at a lower rate. Once assembled, ribosomes with unmethylated L3 are fully active by several criteria. (a) Protein synthesis in vitro with purified 70‐S prmB2 ribosomes is as active as wild‐type using either a natural (R17) or an artificial [poly(U)] messenger. (b) The induction of β‐galactosidase in vivo exhibits normal kinetics and the enzyme has a normal rate of thermal denaturation. (c) These ribosomes are standard when exposed in vitro to a low magnesium concentration or increasing molarities of LiCl.

Efficient methylation of L3 in vitro requires either unfolded ribosomes or a mixture of ribosomal protein and RNA. We suggest that the L3‐specific methyltransferase may qualify as one of the postulated ‘assembly factors’ of the E. coli ribosome.
Structural and Serological Analysis of Citrobacter‐036‐Specific Polvsaccharide, the Homopolymer of (βl→ 2)‐Linked 4‐Deoxy‐d‐arabinohexopyranosyl Units
Authors: ROMANOWSKA Elżbieta , ROMANOWSKA Anna , ŁUGOWSKI Czeslaw , KATZENELLENBOGEN Ewa
Published: March 2005

The chemical structure of the O‐specific polysaccharide of Citrobacter 036 lipopolysaccharide has been estab‐ lished by methylation analysis using gas‐liquid chromatography and mass spectrometry. This polysaccharide is a linear homopolymer composed of (β1 →2)‐linked 4‐deoxy‐d‐arahinohexopyranosyl residues.
Properties of Native and Nicked Elongation Factor Tu from Thermus thermophilus HB 8
Authors: GULEWICZ Krzysztof , FAULHAMMER Heinz Georg , SPRINZL Mathias
Published: March 2005

Two alternative procedures for the isolation of the elongation factor Tu from Thermus thermophilus were compared and the properties of a specifically nicked EF‐Tu · GDP were examined in detail. Although the native elongation factor possessed similar catalytic activities in all reactions investigated as the protein isolated by Arai et al. [Eur. J. Biochem. 92, 509–519 and 521–531 (1978)] it could not be crystallized. The nicked EF‐Tu, consisting of two associated fragments with molecular weights of 41000 and 8000 respectively, was active in binding GDP, GTP and in the formation of Phe‐tRNAPhe. EF‐Tu · GTP ternary complex. However, it did not promote poly(U)‐dependent synthesis of polyphenylalanine on Escherichia coli ribosomes. The isolated fragment of a molecular weight of about 41 000 did not bind GDP. This activity could be reconstituted with the supplement of the small 8000‐M, fragment. It is demonstrated that, in contrast to the native EF‐Tu, the nicked EF‐Tu forms high‐molecular‐weight aggregates. Cleavage of the polypeptide chain of EF‐Tu from T. therrnoplzilus stimulates the crystallization of this protein.
Disulfide Bonds in Egg‐White Riboflavin‐Binding
Authors: KOZIK Andrzej
Published: March 2005

All eight disulfide bonds in the apo–form of egg white riboflavin–binding protein were easily reduced by 2–mercaptoethanol and dithiothreitol. These bonds exhibited nearly the same reactivity, thus they appeared to be exposed in the native structure, or ‘superficial’. The cleavage of protein disulfides resulted in a loss of riboflavin–binding capacity. A correlation between these two related processes, analysed by kinetic and statistical methods, suggested a single bond to be essential for binding of riboflavin by the apoprotein.

In the riboflavin–apoprotein complex the disulfides were rather poorly reducible but they still constituted a single reactivity class. The essential bond was not protected against modification, suggesting it was located out of the riboflavin–binding site.

A postulated subunit structure of riboflavin–binding protein was not confirmed. The cleavage of disulfides caused some aggregation of the protein molecules. Only dimers and high polymers were formed, the former being relatively stable. Hydrophobic forces were probably involved in the formation of dimers.
Microana1ytical Partition of Rat‐Liver Homogenates by Poly(ethy1ene glycol)‐Dextran Counter‐current Distribution
Authors: MORRIS Wesley B. , PETERS Timothy J.
Published: March 2005

The principal organelles from rat liver homogenates were fractionated by counter‐current partition between solutions of dextran T500 and poly(ethylene glycol) 6000 in 0.25 M sucrose containing 10 mM sodium phosphate/phosphoric acid buffer, pH 7.4.

A small‐volume (1.3‐ml sample well) counter‐current partition apparatus capable of 17 transfers, in which separation of the phases was enhanced by low‐speed centrifugation, was used.

Variations in distribution of organelles was observed with increasing concentration of dextran and/or poly(ethylene glycol). At a constant polymer composition of 5.0% (w/w) dextran and 4.0% (w/w) poly(ethylene glycol), increasing concentrations of sodium chloride (2.5 mM, 5 mM and 10 mM) also altered the partition of the organelles.

Useful analytical resolution of the various organelles was achieved. In particular, separation of endoplasmic reticulum from plasma membrane components was obtained.
Isolation, Purification, and Chemical Analysis of the Lipopolysaccharide Analysis of the Lipopolysaccharide and Lipid A of Acinetobacter calcoaceticus NCTC 10305
Authors: BRADE Helmut , GALANOS Chris
Published: March 2005

The lipopolysaccharide of Acinetobacter calcoaceticus NCTC 10305 (London) was obtained by a modified phenol/chloroform/light petroleum method from the bacterial cells and from the culture medium in yields of 1.6 % and 2.2%, respectively (based on the bacterial dry weight). On chemical analysis, both preparations proved to be identical. The lipopolysaccharide obtained from the cells was purified by repeated ultracentrifugation, electrodialysis, and precipitation with sodium chloride. It was free of nucleic acids, proteins, and glycans. In the analytical ultracentrifuge, the triethylamine and sodium salt forms of the lipopolysaccliaride showed a s20 value of 8.9 S and 51 S, respectively. The lipopolysaccharide consisted of glucosamine, 3‐deoxy‐D‐manno‐octulosonic acid, D‐glucose, fatty acids and phosphate in a molar ratio of 2: 1: 7: 6: 4. The fatty acids were predominantly lauric acid, 2‐hydroxy, and 3‐hydroxylauric acid in a molar ratio of 1: 1: 2. Only 3‐hydroxylauric acid was found in amide linkage. On mild acid hydrolysis of the lipopolysaccharide, 65 % lipid A were obtained, to which glucosamine was retained quantitatively. It still contained 50% of the original glucose, while one third (15%) of the liberated glucose was in monomeric form.
Conformational Studies of G‐Actin Containing Bound Lanthanide
Authors: CURMI Paul M. G. , BARDEN Julian A. , DOS REMEDIOS Cristobal G.
Published: March 2005

The presence of bound Ln ions gives rise to changes in the ultraviolet absorbance of G‐actin. Previously, Ln ions have been shown to mobilize the adenosine moiety of ATP bound to the actin monomer. The changes in ultraviolet absorbance appear to be fully attributable to this mobilization. Any structural changes induced in G‐actin by the substitution of Ln ions for Ca2+ must be small enough so as not to perturb the environment of any aromatic chromophores other than those exposed by the mobility of the nucleotide. This conclusion is supported by measurements of the rates of proteolytic digestion of actin with and without bound Ln ions. The effect of Ln ions on the actin‐bound nucleotide is not simply due to the increase in positive charge on the actin monomer. This is demonstrated by the absence of any ultraviolet spectral changes on the addition of a fivefold excess of Ca2+. Analysis of the ultraviolet difference spectrum obtained when Ln ions bind to G‐actin indicates that at least one tryptophan residue is partially exposed to the solvent coincident with mobilization of the nucleotide. Tb3+ luminescence enhancement studies indicate that there are no Trp, Tyr or Phe residues in the vicinity of the high‐affinity cation site. These results suggest that the exposed Trp may be close to the nucleotide.
Conformational Changes in Subunit A of Cholera Toxin Following the Binding of Ganglioside to Subunit B
Authors: VAN HEYNINGEN Simon
Published: March 2005

Cholera toxin has been labelled with the fluorescent probe 4‐chloro‐7‐nitrobenzofuran (Nbf‐C1) in both subunits, and the labelled subunits separated by gel‐permeation. They retained their biological activities.

Addition of ganglioside GMI (which binds to subunit B only) to either labelled subunit did not alter the fluorescence of the Nbf probe.

Whole toxin was reconstituted using labelled subunit A and unlabelled subunit B. Addition of ganglioside GM1 to the reconstituted toxin enhanced the fluorescence by about 90%, but did not change the wavelength. This enhancement reached a maximum when the ganglioside to toxin ratio was about 1 to 1. Ganglioside GD1b (which does not bind) did not affect the fluorescence.

These results suggest that the binding of ganglioside to subunit B alters the environment of the Nbf probe bound to subunit A, presumably by a conformational change.
Adenosine Deaminase Covalently Linked to Soluble Dextran
Authors: ROSFMEYER Helmut , KÖRNIG Elke , SEELA Frank
Published: March 2005

Dextran‐linked adenosine deaminase (EC 3.5.4.4) has been prepared. The polymer‐linked enzyme possesses an optimal cnzymatic activity of 27 units/mg immobilized protein (non‐bound enzyme: 200 units/mg protein). Support‐bound adenosine deaminase (4.5 pg protein/mg dextran) shows an enhanced heat stability, a moderately ‐increased Km and a decreased V value compared to those of the free enzyme. The pH dependences of V and PKm values of dextran‐linked adenosine deaminase show only two inflection points compared to three for the free enzyme, which are equivalent to the PK values of the enzyme. Since the missing third inflection point (pH 9.8) can be assigned to the pK value of the ɛ‐amino group of lysyl residues, it can be concluded, that immobilization of adenosine deaminase on cyanogen‐bromide‐activated dextran took place via these lysyl residues. The remaining pK values found from tlie other inflection points are moderately shifted owing to the altered secondary structure. From the temperature dependence of the enzymatic activity, a 40% decrease of the activation energy of the support‐bound enzyme was found, indicating diffusion controlled deamination. The immobilization of adenosine deaminase results in a fluorescence quenching of 80%, without shifting the ultraviolet maximum of the emission spectrum. As already shown for unmodified dextran, the matrix of polymer‐linked adenosinc deamniase is degradable by a bacterial endodextranase (EC 3.2.1.11).
Glycogen Synthase from Rabbit Skeletal Muscle
Authors: PARKER Peter J. , EMBI Noor , CAUDWELL F. Barry , COHEN Philip
Published: March 2005

Glycogen synthase has been shown to be phosphorylated in vitro by five protein kinases, cyclic‐AMP‐dependent protein kinase (sites la, 1b and 2). phosphorylase kinase (site 2), glycogen synthase kinase 3 (sites 3a,3b and 3c), glycogen synthase kinase 4 (site 2) and glycogen synthase kinase 5 (site 5) [Cohen,P. et al. (1982)Eur. J. Biochem. 124, 21–35; Picton, C. et al. (1982) Eur. J. Biocliem. 124, 37–45]. All seven serine residues have now been found to be phosphorylated in vivo.

Glycogen synthase purified from rabbits that had been injected with l‐propranolol contained 0.33 mol of phosphate/mol subunit in site la, 0.38 in site Ib, 0.39 in site 2, 1.27 in sites 3a+3b+3c and 0.65 in site 5, totalling 3.0 mol/mol subunit. The activity ratio (∓ glucose‐6 P) for these preparations was 0.21 and their Ka for glucose‐6‐P was 1.4mM.

Glycogen synthase purified from animals that had been injected with I‐adrenaline contained 0.59 mol phosphate/mol subunit in site la, 0.65 in site lb, 1.03 in site 2, 2.43 in sites 3a + 3b + 3c and 0.65 in site 5, totalling 5.3 mol/mol subunit. The activity ratio of these preparations was 0.04 and their Ka for glucose‐6‐P was 6.0mM.

The results show that cyclic‐AMP‐dependent protein kinase, glycogen synthase kinase 3 and glycogen synthase kinase 5 act as glycogen synthase kinases in vivo. Glycogen synthase kinase 4 may be largely responsible for the phosphorylation of site 2 in propranolol‐treated animals, and cyclic‐AMP‐dependent protein kinase and phos‐ phorylase kinase for the increased phosphorylation of site 2 in response to adrenaline.

The activity of glycogen synthase in vivo in propranolol‐treated animals is largely determined by the state of phosphorylation of sites 3a, 3b and 3c, while the inactivation in response to adrenaline is due to increased phosphorylation at site 2 as well as at sites 3a, 3b and 3c. The finding that the phosphorylation of sites 3a, 3b and 3c is increased from 1.27 mol/mol subunit to 2.43 mol/mol subunit by adrenaline was unexpected, since this enzyme is not regulated by cyclic AMP. Possible explanations for this result are discussed.

Phosphofructokinase was isolated as a byproduct of the purification of glycogen synthase. The phosphate content of this enzyme did not differ significantly between l−propranolol‐treated (0.15 mol/mol subunit) and adrenaline‐treated (0.18 mol/mol subunit) animals. These results indicate that muscle phosphofructokinase is not a substrate for cyclic‐AMP‐dependent protein kinase in vivo.
Control of the ppp(A2′p)nA System in HeLa Cells
Authors: SILVERMAN Robert H. , CAYLEY P. Jane , KNIGHT Matilda , GILBERT Chiristopher S. , KERR Ian M.
Published: March 2005

HeLa cells have an unusually high level of ppp(A2′p)nA synthetase(n = 2 to ≥4) even in the absence of interferon treatment. In accord with this ppp(A2′p)nA and ppp(A2′p)nA‐mediated ribosomal RNA cleavage occur naturally in response to encephalomyocarditis virus infection in control as well as in intcrleron‐treated cells. Despite this, in the absence of interferon treatment, encephalomyocarditis virus grows well In these cells. A possible explanation for this paradox is that the ppp(A2′p)nA dependent RNase is lost or inactivated at later times post‐infection in control but not in interferon‐treated cells. It appears, therefore, to bc the prevention by interferon of the virus‐mediated inhibition of the ppp(A2′p)n‐dependent nuclease rather than the absolute level or induction of the ppp(A2′p)nA synthetase which is crucial for the activity of the ppp(A2′p)nA system in HeLa cells. These results provide evidence for a further level of control in the ppp(A2′p)nA system and show that limited ppp(A2′p)nA‐mediated ribosomal RNA cleavage alone is not sufficient to cause;in inhibition of virus growth.
Peroxisomal and Mitochondrial Carnitine Acetyltransferases in Alkane‐Grown Yeast Candida tropicalis
Authors: UEDA Mitsuyoshi , TANAKA Atsuo , FUKUl Saburo
Published: March 2005

Two types of carnitine acetyltransferases (EC 2.3.1.7) were first isolated from a microorganism, alkane‐grown yeast Candidu tropicalis. Carnitine acetyltransferase activity was induced in the alkane‐grown cells, reaching about twenty times higher than that in the glucose‐grown cells. Localization of the enzyme activity was demon‐ strated, at least, in peroxisomes (microbodies), profusely occurred in the alkane‐grown cells. and in mitochondria. Peroxisomal and mitochondrial carnitine acetyltransferases could be separated using the method of DEAE‐ Sephacel column chromatography and both types were found to exist in the alkane‐grown cells of C. tropicalis.

Each carnitine acetyltransferase was purified using Sephadex G‐200, Sepharose 6B, DEAE.‐Sephacel and Blue‐ Sepharose CL‐6B. In DEAE‐Sephacel chromatography, peroxisomal carnitine acetyltransferase was eluted below 0.15 M KCI concentration and mitochondrial carnitine acetyltransferase above 0.15 M KCI concentration. Except for the localization, little difference was observed in their kinetic properties, substrate specificity and so on. These two carnitine acetyltransferase preparations were only specific to acetyl and propion I groups, the hubstrate specificity not being so broad as that of carnitine acetyltransferase obtained from maminalian tissues.

Roles of these carnitine acetyltransferases in alkane metabolism in yeast are also discussed.
Regulation of the Biosynthesis of CoA at the Level of Pantothenate Kinase
Authors: HALVORSEN Ola , SKREDE Sverre
Published: March 2005

Pantothenate kinase, which is present in cytosol, was studied in preparations from livers of rats fed normal or clofibrate‐enriched diets. Effects of CoA, dephospho‐CoA and different acyl‐CoA derivatives on this enzyme activity were examined in vitro.

With partially purified pantothenate kinase or crude particle‐free supernatant from the liver of normal or clofibrate‐treated rats, Km for pantothenic acid was 0.016 mmol/l at the pH optimum 6.1

Acetyl‐CoA, propionyl‐CoA, malonyl‐CoA and other short‐chain acyl‐CoA derivatives were strong inhibitors of pantothenate kinase, with Ki sin the range 0.001 ‐0.003 mmol/l. The mechanism of inhibition appeared to be of an uncompetitive type.

Free CoA has been held to be the main regulator of pantothenate kinase. We found, however, that free CoASH, dephospho‐CoA and long‐chain acyl‐CoA (with Ki 0.003 ndash; 0.08 mmol/l) were less efficient inhibitors than acetyl‐CoA.

With pantothenate kinase from clofibrate‐treated animals, all inhibitors were less potent. This was most pronounced when the enzyme was assayed in a crude supernatant fraction, possibly because the inhibitors were degraded and/or protein bound. Such a reduction of normal inhibition may contribute to the increased biosynthesis of CoA previously observed during clofibrate treatment.

Fasting or diabetes leads to an increase of long‐chain acyl‐CoA and total CoA in the liver. The increase of CoA has been explained by increased acylation of CoA, and thereby reduced feed‐back inhibition by free CoASH at the pantothenate kinase level. We propose another explanation. In these metabolic states, the cytosolic pool of acetyl‐CoA is decreased. Since pantothenate kinase is present only in the cytosol, its activity will be released and the biosynthesis of CoA will increase.

Acetyl‐CoA is probably a more important physiological regulator of pantothenate kinase activity than is free CoASH.
Identification of Ribophorins in Rough Microsomal Membranes from Different Organs of Several Species
Authors: MARCANTONTO Eugene E. , GREBENAU Ruth C. , SABATINI David D. , KREIBICH Gert
Published: March 2005

Microsomes prepared from several animal sources were analyzed for the presence of proteins corresponding to the ribophorins (I and II) which have been previously characterized in rat liver rough microsomes and appear to be involved in the binding of polysomes to endoplasmic reticulum membranes. In rough microsomal membranes from rat lacrimal gland, rabbit liver, dog and chicken pancreas, and mouse myeloma, ribophorin‐like polypeptides with similar electrophoretic mobilities were detected by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. In all cases the polypeptides remained associated with sedimentable polysomes after solubilization of the microsomal membranes with nonionic detergents. Ribophorin‐like polypeptides were absent from smooth microsomes.

Antibodies raised against each rat liver ribophorin, purified by preparative sodium dodecyl sulfate/poly‐ acrylamide gel electrophoresis, immunoprecipitated only the corresponding polypeptide, indicating no crossreactivity between ribophorins I and II. These antibodies also immunoprecipitated the homologous ribophorins found in microsomal preparations from other organs and species.
Two‐Dimensional NMR Spectroscopy of Siomycin A
Authors: CLAYDEN Nigel J. , INAGAKI Fuyuhiko , WILLIAMS Robert J. P. , MORRIS Gareth A. , TORI Kazuo , TOKURA Katsuya , MIYAZAWA Tatsuo
Published: March 2005

A trial application of a recent two‐dimensional nuclear magnetic resonance experiment to the polypeptide antibiotic siomycin A is described. Proton ‐ Carbon‐13 chemical shift correlation measures the proton and carbon‐13 chemical shift for cach directly bonded CH group in a molecule, in a single experiment. The resultant map of correlated chemical shifts enables the carbon‐13 spectrum to be assigned directly from the known proton shifts, and allows individual proton signals to be identified without problems of overlap. The signal‐to‐noise ratio available from such techniques should enable their application to aqueous protein solutions using currently available high‐field spectrometers.
Temperature Dependence of the Activity of DNA‐Modifying Enzymes: Endonucleases and DNA Ligase
Authors: POHL Fritz M. , THOMAE Ralf , KARST Antje
Published: March 2005

The activities of 17 endonucleases: the restriction endonucleases AvaI, BamHI, EcoRI, HindIII, PstI and SalI, which cleave pBR322 DNA once: aluI, AvaII, CfoI, HaeIII, HhaI, HinfI, HpaII and TaqI, which cut pBR322 DNA several times, and three ‘unspecific’ nucleases (S1 nuclease, staphylococcal nuclease and DNase I from bovine pancreas) were determined between 0° and 65°C. The reaction was followed by the disappearance of covalently closed circular pBR322 DNA, using the alkaline ethidium fluorescence assay of Morgan et al. [Nucleic Acids Res. (1979) 7, 547‐594]; the activity of T4 DNA ligase was similarly measured by the conversion of nicked circular DNA to closed circular DNA. For each enzyme, small aliquots of the same solution were incubated at different temperatures simultaneously in a temperature gradient device, resulting in a high relative precision.

The experimental results are summarized by the simplest possible theoretical description, using linear or exponential kinetics and apparent activation energies Ea for the enzymatic reaction, Ei for the enzyme inactivation and Ti for the inactivation temperature. To a good approximation these three parameters suffice for describing the temperature dependence of the activity of most of the enzymes.
Unique Characteristics of Rat Spleen Lymphocyte, L1210 Lymphoma and HeLa Cells in Glutathione Biosynthesis from Sulfur‐Containing Amino Acids
Authors: BRODIE Ann E. , POTTER Joanne , REED Donald J.
Published: March 2005

Suspensions of rat spleen lymphocyte, murine L1210 lymphoma and HeLa cells were partially depleted of glutathione (GSH) with diethyl maleate and allowed to utilize either [35S]methionine, [35S]cystine or [35S]‐cysteine for GSH synthesis. Lymphocytes preferentially utilize cysteine, compared to cystine, at a ratio of about 30 to 1, which was not related to differences in the extent of amino acid uptake. Only HeLa cells displayed a slight utilization of methionine via the cystathionine pathways for cysteine and GSH biosynthesis. HeLa and L1210 cells readily utilized either cystine or cysteine for GSH synthesis. The three cell types accumulated detectable levels of intracellular cysteine glutathione mixed disulfide when incubated in a medium containing a high concentration of cystine. Various enzyme activities were measured including γ‐glutamyl transpeptidase, GSH S‐transferase and γ‐cystathionase. These results support the concept of a dynamic interorgan relationship of GSH to plasma cyst(e)ine that may have importance for growth of various cell types in vivo.
Characterization and Cloning of the mRNAs Specific for the Lactating Mouse Mammary Gland
Authors: HENNIGHAUSEN Lothar G. , SIPPEL Albrecht E.
Published: March 2005

We have characterized and cloned the lactation‐specific mRNAs of mouse mammary glands. The group of eight milk‐protein‐specific mRNAs were identified (a) by size and antigenic properties of their translation products in vitro and (b) by characterization of their respective cDNA clones.

Two α‐caseins (43 kDa and 39 kDa) are encoded by mRNAs of 1600 nucleotides and two β‐caseins (26 kDa) are encoded by mRNAs of 1450 nucleotides in length. Three smaller caseins, γ‐casein (23.7 kDa), δcasein (21 kDa) and £‐casein (14.5 kDa) are synthesized by mRNAs of 880, 1150 and 860 nucleotides. Beside these casein mRNAs a mammary specific 620 nucleotide mRNA codes for a novel acidic whey protein (13.7 kDa).

cDNA clones corresponding to the mRNAs for the lactation‐specific proteins have been isolated from a mammary‐specific cDNA library. Cloned α‐casein cDNA hybridizes to both β‐casein specific mRNAs and cloned β‐casein cDNAs hybridize with both β‐casein specific mRNAs. By RNA blot analysis we show that the cloned β ‐casein cDNAs for mouse α‐casein, β‐casein, γ‐casein and &‐casein and the acidic whey protein cross‐hybridize with mRNAs of the rat, demonstrating partial sequence homology between the corresponding mRNAs of those species.
Effect of Tunicamycin on Thyroglobulin Secretion
Authors: BJORKMAN Ulla , EKHOLM Ragnar
Published: March 2005

Secretion of thyroglobulin was studied by incubating pig thyroid follicles, isolated by collagenase digestion and opened up by trypsinization. When followed over periods of 4 h, the secretion of [14C]leucine‐labeled thyroglobulin into the medium was reduced by 60‐95% in the presence of 1 pg/ml and 5 μg/ml of tunicamycin. These concentrations of the antibiotic reduced incorporation of [3H]mannose into the follicle proteins by 70‐80 %, but did not significantly influence the incorporation of [14C]leucine.

Rat thyroid lobes were labeled with [3H]leucine for 20 min and chase‐incubated for 0‐4 h. In electron microscopic autoradiographs obtained immediately after labeling, the label was restricted to the follicle cells and concentrated over the endoplasmic reticulum both in controls and in specimens exposed to tunicamycin (5 μg/ml). After 4 h chase most radioactivity was located in the follicle lumen in controls whereas in tuni‐ camycin‐exposed lobes almost all labeled material was retained in the follicle cells.

It is concluded that tunicamycin suppresses thyroglobulin secretion and that this is not due to inhibited protein synthesis.
Biosynthesis of Two Structurally Different Pro‐opiomelanocortins in the Pars Intermedia of the Amphibian Pituitary Gland
Authors: MARTENS Gerard J. M. , BIERMANS Peter‐Paul J. , JENKS Bruce G. , VAN OVERBEEKE A. P.
Published: March 2005

This study reports the biosynthesis of two forms of pro‐opiomelanocortin in the pars intermedia of the pituitary gland of the African clawed toad Xenopus laevis. The two forms could be resolved by dodecyl sulphate gel electrophoresis on a 9–16% acrylamide gradient and their molecular weights were 38200 and 37300. Incubation of neurointermediate lobes with [3H]glucosamine followed by tryptic digestion of the newly synthesized glycoproteins, revealed that both prohormones have only one glycosylated site, namely within the region corresponding to γ3‐melanotropin. Biosynthesis of proteins in lobes treated with tunicamycin to prevent glycosylation again resulted in the production of two pro‐opiomelanocortins (Mr 35000 and 34200), indicating that the two forms differ in their primary structure. This notion was corroborated by the results of tryptic mapping of the newly synthesized prohormones. The maps showed that the primary structures of the two forms of pro‐opiomelanocortin differ in at least two parts of the molecules, one part concerning the endorphin region.
Copurification of Citrate Lyase and Citrate Lyase Ligase from Rhodopseudomonas gelatinosa and Subsequent Separation of the Two Enzymes
Authors: ANTRANIKIAN Garabed , GOTTSCHALK Gerhard
Published: March 2005

A procedure has been worked out which allowed the purification and crystallization of a citrate lyase/citrate lyase ligase complex from Rhodopseudomonas gelatinosa. The complex was subsequently separated to yield two homogeneous enzymes.

Citrate lyase ligase was purified 365‐fold with a yield of 3.23%. The molecular weight of the enzyme was estimated to be 39500, the enzyme consisted of one polypeptide chain. The reaction rates for ATP, acetate and citrate lyase (sulfhydryl form) followed Michaelis‐Menten kinetics (Km values: 0.14 mM, 5 mM and 37 nM respectively).

Citrate lyase ligase exhibited a high substrate specificity and could not react with citrate lyases from non‐phototrophic microorganisms. In contrast to the ligase from Streptococcus diacetilactis, the enzyme from R. gelatinosa was extremely labile; however, it could be stabilized by nucleotides, the most potent stabilizing one being ADP.
Interaction of the hnRNA of Amphibian Oocytes with Fibril‐Forming Proteins
Authors: KLOETZEL Peter‐M. , JOHNSON Rosalyn M. , SOMMERVILLE John
Published: March 2005

A ribonucleoprotein fraction that contains most of the rapidly labelled hnRNA has been isolated from gently ruptured oocytes of Triturus cristatus. This fraction consists of large aggregates of ribonucleoprotein and has a high (30:1) ratio of protein to RNA. The labelled RNA is contained in ribonucleoprotein particles that have a density of 1.27 g/cm3 in Cs2SO4 gradients (1.39 g/cm3 after formaldehyde fixation in CsCl gradients). Evidence is presented that the particles are associated in vivo with a librillar protein network. When the ribonucleoprotein aggregates are treated with ribonuclease, high salt concentration and nonionic detergent, a fibrillar protein residue is produced which contains many species of protein but a few that have electrophoretic characteristics that are identical to major ribonucleoprotein particle proteins. Isolated labelled hnRNA has been shown to bind specifically polypeptides of molecular weight 60000 and 54000 that are found in both particle and fibril preparations. In binding assays in vitro, these polypeptides are found to interact with mRNA to a lesser extent and not with rRNA. The isolated 60000‐Mr and 54000‐Mr proteins have the dual ability of forming ribonucleoprotein ‘particles’ with hnRNA and of polymerizing to generate 10‐nm fibrillar structures in the absence of RNA. The possible cellular functions of these proteins are discussed.
Calcium Uptake in Isolated Hepatic Plasma‐Membrane Vesicles
Authors: KRAUS‐FRIEDMANN Naomi , BIBER Jürg , MURER Heini , CARAFOLI Ernesto
Published: March 2005

A liver plasma‐membrane fraction capable of Ca2+ uptake was isolated. The fraction exhibited high Na+, K+‐ATPase, low glucose‐6 phosphatase activity, and transported alanine in a Na+‐dependent fashion. The uptake of Ca2+ was ATP‐dependent; UTP, GTP, or CTP did not substitute for ATP. The presence of oxalate did not significantly alter the rate of uptake. The pH optimum of the reaction was basic (no uptake was visible at pH 6.8). These properties are at variance with those of the endoplasmic reticulum Ca2+ uptake system, which is oxalate‐dependent, and has an acid pH optimum. The ATP‐dependent Ca2+ uptake has a Km(Ca2+) of 1.4 × 10–8 M and a Vmax of transport of 30 nmol × mg protein–1× min–1. No conclusive results were obtained on the calmodulin‐sensitivity of the process: addition of calmodulin to the vesicles did not stimulate uptake, and the anti‐calmodulin drug trifluoperazine had no inhibitory effect. However, another anti‐calmodulin drug (R 24571) had a limited, but statistically significant, inhibitory action. A partial release of the accumulated Ca2+ from the vesicles could be induced by the addition of Na+, and incubation of the vesicles in a high Na+ medium (as compared to high K+ medium) resulted in lower (about 25%) calcium uptake. Partial release of the accumulated Ca2+ could be induced also by the addition of H+. The releasing effect of H+, taken together with the absence of Ca2+ uptake at acid pH, suggests the possibility of a H+/Ca2+ exchange.
The Slow, Tight‐Binding Inhibition of Cathepsin B by Leupeptin
Authors: BAICI Antonio , GYGER‐MARAZZI Maya
Published: March 2005

Leupeptin was found to be a slow, tight‐binding inhibitor of cathepsin B from human spleen and rabbit liver. During the enzyme‐catalyzed reaction in the presence of inhibitor a concentration‐dependent transient state, lasting several minutes, preceeded the attainment of the steady state and was characterized by a concave upward or a concave downward lag phase depending on whether the enzyme had been preincubated with the inhibitor or not, respectively. From the pre‐steady‐state phase of the curves both kon and koff for the formation of the enzyme‐inhibitor complex could be calculated. Ki, as the ratio koff/kon, was in good agreement with the inhibition constant obtained using a steady‐state treatment. kon was 1.8 × 105 M–1 s–1 and 2.0 × 105 M–1 s–1 for the human and rabbit enzyme, respectively and the slowness of the binding process fitted into the general concept of enzyme hysteresis. The activation of the essential cysteine residue of cathepsin B by dithiothreitol was also a very slow process characterized by a second‐order rate constant of 4.1 M–1 s–1. The kinetic features of leupeptin binding allow the prediction of the possible efficiency of this inhibitor on cathepsin B in vivo. It is shown that in order for leupeptin to be a physiologically significant inhibitor of cathepsin B, its concentration at the target site must exceed 10 μM, at least. This contrasts with the predictions drawn from the value of Ki (∼ 5 nM), which would suggest an effective inhibition of the enzyme already at a concentration of 0.05 μM.
Transient Unfolding of Trypsin‐Digested Chromatin Core Particles
Authors: GRIGORYEV Sergei A. , KRASHENINNIKOV Igor A.
Published: March 2005

Limited digestion of nucleosome core particles with trypsin caused cleavage and removal of N‐terminal histone sequences of 10–30 amino acids. The proteolyzed core particles exhibited salt‐dependent structural transitions revealed by sedimentation, circular dichroism and nuclease‐cutting assays, while the intact nucleosome cores were not affected under the experimental conditions.

The results obtained indicate that the observed transitions correspond to the transient unfolding of terminal segments of core particle nucleoprotein caused by the increase of its net negative charge. The excision of the N‐terminal histone domains therefore leads to partial destabilization but not to irreversible disruption of the compact nucleosome structure.
Distance Measurement by Energy Transfer: The 3′ End of 16‐S RNA and Proteins S4 and S17 of the Ribosome of Escherichia coli
Authors: EPE Bernd , WOOLLEY Paul , STEINHÄUSER Klaus G. , LITTLECHILD Jennifer
Published: March 2005

Escherichia coli ribosomal proteins S4 and S17 were specifically labelled at their thiol groups with the acetylaminoethyl‐dansyl and/or bimane fluorophores. Each formed a complex with 16‐S RNA and, when the other 30‐S ribosomal proteins were added, a complete 30‐S subunit with at least partial activity. If the 3′ end of the RNA was also labelled (with fluorescein) then the distance between the two fluorophores could be measured by Förster‐type energy transfer. The result for S4 was 6.0 nm (60 Å) in the ribonucleoprotein complex and 5.6 nm (56 Å) in the 30‐S subunit, and for S17 6.3 nm (63 Å) in the complex and 6.2 nm (62 Å) in the subunit. There is no evidence for a major change in the relative disposition of the 3′ and 5′ ends of the 16‐S RNA during formation of the 30‐S subunit. Sources of error are discussed, including the question of multiple labelling.

In order to measure more accurately the extent of energy transfer a procedure based upon enzymic digestion was developed and is detailed in this paper.
Signal Peptide Digestion in Escherichia coli
Authors: HUSSAIN Musaddeq , OZAWA Yuji , ICHIHARA Shigeyuki , MIZUSHIMA Shoji
Published: March 2005

Upon incubation of the envelope fraction of Escherichia coli a precursor of the major outer membrane lipoprotein that accumulates in the cytoplasmic membrane of the globomycin‐treated cell is processed to the mature form [Hussain, M., Ichihara, S., and Mizushima, S. (1980) J. Biol. Chem. 255, 3707–3712; (1982) J. Biol. Chem. 257, 5177–5182]. When this precursor‐containing envelope fraction was incubated in the presence of protease inhibitors such as antipain, leupeptin, chymostatin and elastatinal, a new peptide appeared on a polyacrylamide gel at the position where the signal peptide was expected to appear. This was proved to be the signal peptide of the lipoprotein from the following facts: (a) its appearance is in proportion to the appearance of the lipoprotein and disappearance of the precursor; (b) when the cleavage of the signal peptide from the precursor was inhibited by globomycin, the peptide did not appear on the gel; and (c) the results of labeling of the peptide with [3H]leucine, [35S]methionine and [3H]arginine were consistent with the amino acid composition of the signal peptide. The signal peptide thus accumulated in the envelope fraction was hydrolyzed by an enzyme named ‘signal peptide peptidase’ when the envelope fraction was washed to remove the inhibitors. The hydrolysis was inhibited by re‐addition of these inhibitors. The signal peptide peptidase hydrolyzed the signal peptide only after its cleavage from the lipoprotein precursor.
Fatty Acids as Acute Regulators of the Proton Conductance of Hamster Brown‐Fat Mitochondria
Authors: LOCKE Rebecca M. , RIAL Eduardo , SCOTT Ian D. , NICHOLLS David G.
Published: March 2005

Possible mechanisms are evaluated for the acute regulation of the hamster brown‐fat mitochondrial proton‐conductance pathway which is active during non‐shivering thermogenesis. Isolated mitochondria are incubated under conditions designed to approximate to the non‐thermogenic state, and the effect of the steady infusion of fatty acids or acyl derivatives upon respiration, membrane potential and membrane proton conductance is monitored continuously. Fatty acids increase the proton conductance with no detectable threshold concentration, allowing the generated acyl carnitine to be rapidly oxidized. The extent of depolarization and of respiratory increase is a function of the rate of infusion. Immediately infusion is terminated the conductance decreases, the mitochondria repolarize and respiration returns to the initial rate. Infusion of acyl‐CoA and acylearnitine cause only a slight depolarization or respiratory increase after high concentrations of these derivatives have accumulated. Any factor which decreases the rate of conversion of fatty acid to acyl‐CoA potentiates the conductance increase. An effect of acyl‐CoA upon chloride permeability is not specific to brown‐fat mitochondria. Fatty acids infused into rat liver mitochondrial incubations produced a small conductance increase, comparable to that of acyl‐CoA or acylcarnitine. It is concluded that fatty acids are the most plausible acute regulators of the proton conductance. The relation to the brown‐fat‐specific 32000‐Mr protein is discussed.
The Acute Regulation of Mitochondrial Proton Conductance in Cells and Mitochondria from the Brown Fat of Cold‐Adapted and Warm‐Adapted Guinea Pigs
Authors: LOCKE Rebecca M. , RIAL Eduardo , NICHOLLS David G.
Published: March 2005

Cells and mitochondria were prepared from the brown adipose tissue of adult guinea‐pigs adapted to either 4–7°C or 22–25°C. The cold‐adapted cells displayed noradrenaline‐stimulated, propranolol‐sensitive respiration, but noradrenaline failed to increase the respiration of the warm‐adapted cells. Purine‐nucleotide‐sensitive proton conductance was greater in cold‐adapted mitochondria than in warm‐adapted controls. At the same time cold‐adapted mitochondria were extremely sensitive to the uncoupling effect of endogenous and infused fatty acids, and resembled the mitochondria from the brown adipose tissue of cold‐adapted hamsters. Warm‐adapted mitochondria were ninefold less sensitive, and resembled liver mitochondria. With cold‐adapted, but not warm‐adapted mitochondria, respiration increased proportionately to the rate of fatty acid infusion. It is concluded that the presence of the 32000‐Mr proton conductance pathway is necessary for the expression of a high sensitivity to fatty acid uncoupling, suggesting that the fatty acids interact directly with this protein to modulate the proton conductance during the acute regulation of thermogenesis.
Component E of the DNA‐Dependent RNA Polymerase of the Archaebacterium Thermoplasma acidophilum Is Required for the Transcription of Native DNA
Authors: SCHNABEL Ralf , ZILLIG Wolfram , SCHNABEL Heinke
Published: March 2005

The role of component E of the DNA‐dependent RNA polymerase of the archaebacterium Thermoplasma acidophilum in the transcription of Thermoplasma DNA has been analyzed.

Component E (Mr 22000) is released upon formation of the binary complex of the polymerase with the DNA. Enzyme not containing component E is inactive on DNA but active on poly[d(A‐T) · d(A‐T)]. The activity on DNA can be restored by addition of component E.

Two states of the binding complex between RNA polymerase and DNA, differing in their tonic strength stabilities, have been distinguished both in the presence and absence of component E. between the two states is 43°C.

Component E appears to open productive binding sites in native DNA in addition to the non‐productive strong binding sites available for component‐E‐deficient enzyme.
Effects of Chronic Stimulation at Low Frequency on the Lipid Phase of Sarcoplasmic Reticulum in Rabbit Fast‐Twitch Muscle
Authors: SARZALA M. Gabriela , SZYMAŃSKA Graiyna , WIEHRER Walter , PETTE Dirk
Published: March 2005

The transformation of sarcoplasmic reticulum in fast‐twitch rabbit muscle by low‐frequency indirect stimulation was studied. Peptide pattern analyses and ATPase activities revealed transformation of the sarcoplasmic reticulum into a slow‐twitch muscle type to be completed after 30 days. Concomitant changes in the lipid phase consisted in a decrease of the phospholipid/protein ratio, a decrease in the phosphatidylcholine content and in changes of the saturated fatty acid moiety of the phospholipid fraction. Significant increases were seen in content of lysophosphatidylcholine (lysolecithin) and in nonesterified fatty acid. Parallel increases in lysolecithin acyl‐ transferase activity indicated an enhanced phospholipid turnover. The changes in the phospholipid matrix did not lead to a complete transformation. Moreover, the decrease in Ca2+ uptake and Ca2+‐dependent ATPase activity preceded most of the changes in the phospholipids. It appears thus that these alterations occurred independently.
Purification and Properties of a d‐Galactose/N‐Acetyl‐d‐ galactosamine‐Specific Lectin from Erythrina cristagalli
Authors: IGLESIAS Jose L. , LIS Halina , SHARON Nathan
Published: March 2005

The lectin from the seeds of Erythrina cristagalli has been isolated in high yield (75 %) and homogeneous form by affinity chromatography on a column of D‐galactose‐derivatised Sepharose. It is a glycoprotein with a molecular weight of 56800±900 and s20.w, = 3.9 S, composed of two subunits (apparent molecular weights of 28000 and 26000 respectively) both of which are glycosylated. The total carbohydrate content is 4.5% and it is comprised of mannose, N‐acetylglucosamine, fucose and xylose in amounts corresponding to 7, 4, 2 and 2 mo1/56800 Da respectively. The amino acid composition of the lectin is characterised by a high content of acidic and hydroxy amino acids, low content of methionine and absence of cysteine. Valine is the only N‐terminal amino acid detected. The lectin is a metalloprotein in that it contains 0.093% Mn and 0.13percnt; Ca (1 mol and 1.9 mo1/56 800 Da respectively), both of which are tightly bound to the protein.

E. cristagalli lectin agglutinates untreated human erythrocytes of all blood types, as well as rabbit erythrocytes, at a concentration of 5‐10 μg/ml. It is mitogenic for human peripheral blood T lymphocytes at an optimal concentration of about 100 pμg/ml, but is not mitogenic for mouse thymocytes or splenocytes.

d‐GahtOSe and various d‐galactosides inhibit the hemagglutinating activity of the lectin. N‐Acetyllactosarnine is most potent, completely inhibiting four agglutinating units of the lectin at 0.4 mM concentration. Lactose, N‐acetyl‐d‐galactosamine and d‐galactose are 5, 16 and 35 times less active respectively. Lactose specifically perturbs the ultraviolet spectrum of the lectin in the aromatic region. The difference spectrum obtained upon binding of the disaccharide to the lectin shows maxima at 291 nni and 282‐284 nm, indicating a change in the environment of tryptophan residues of the protein upon binding of sugar.
A Sphingomyelin Transfer Protein in Rat Tumors and Fetal Liver
Authors: DYATLOVITSKAYA Ella V. , TIMOFEEVA Natalya G. , YAKIMENKO Ekaterina F. , BARSUKOV Leonid I. , MUZYA Galina I. , BERGELSON Lev D.
Published: March 2005

Affinity resins containing covalently bound phospholipids were used to compare the affinity of the phosphatidylcholine transfer protein from beef liver and a low‐specificity lipid transfer protein from rat hepatoma 27 to phosphatidylcholine and sphingomyelin. Binding experiments demonstrated that the beef liver protein associates specifically with immobilized phosphatidylcholine whereas the hepatoma protein showed a preference for sphingomyelin.

Purified antiserum raised against the hepatoma sphingomyelin transfer protein was used to determine the presence of that protein in the cytosol of various experimental tumors as well as in those of normal, regenerating and fetal rat liver. The protein was found to be well expressed in all the tumors examined and in fetal liver as determined by immunodiffusion whereas only minute amounts could be detected in normal liver and in 30‐h regenerated liver. The presence of the sphingomyelin transfer protein in cytosol was in parallel with the presence of sphingomyelin in the corresponding mitochondria. It is suggested that the occurrence of sphingomyelin in tumor and fetal liver mitochondria may be due to protein‐catalyzed sphingomyelin transfer from the endoplasmic reticulum.
Identification of Casein Kinase II and Phosphorylated Proteins Associated with Messenger Ribonucleoproteins Particles from Reticulocytes
Authors: RITTSCHOF Daniel , TRAUGH Jolinda A.
Published: March 2005

Messenger ribonucleoprotein (mRNP) particles, isolated from reticulocyte polysomes and purified by buoyant density centrilfugation in metrizamide, contained an endogenous protein kinase activity. The cyclic‐nucleotide‐independent protein kinase phosphorylated casein using either ATP or GTP as the phosphoryl donor and had properties similar to casein kinase II, an enzyme previously purified and characterized from the post‐ribosomal supernate of reticulocytes. Antibody prepared to casein kinase II was shown to inhibit the protein kinase activity in the mRNP particles. The endogenous enzyme phosphorylated four peptides (Mr, 125000, 107000, 76000 and 63000) in the mRNP particle. Three of the four peptides, plus another (Mr, 175000), were phosphorylated by purified casein kinase II while two peptides (Mr, 95000) and Mr, 76000) were phosphorylated with casein kinase I. The mRNP particles were not substrates for the CAMP‐dependent protein kinases.