bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isolation and Properties of Chloroplast Coupling Factor from Wheat
Authors: MOASE Elaine H. , GREEN Beverley R. 
Published: March 2005

Wheat chloroplast coupling factor (CF1) was estracted with a modification of the chloroform extraction method of Younis et al. (J. Biol. Chem. 252, 1814‐1818, 1977). A one‐step purification on an 8‐25% sucrose gradient yielded a CF1 which was at least 98% pure as judged by sodium dodecyl sulfate/polyacrylamide gel eletrophoresis.
Inclusion of proteolysis inhibitors during extraction and purification consistently gave a CF1containing all five subunits. Selective loss of the δ and ɛ subunits was observed when proteolysis inhibitors were omitted.
Proteolysis inhibitors prevented the release of wheat CF1 from the thylakoid by the low‐ionic‐strength wash method of Strotmann et al. (Biochim. Biophys. Acta, 314, 202‐210, 1973). The enzyme extracted with chloroform and low ionic strength were compared by electrophoresis and no evidence of a difference in molecular weight of any subunit was observed. This suggests that a proteolytic event is not required for release of wheat CF1 by the low‐ionic‐strength method, even though release is inhibited by proteolysis inhibitors.
The γ subunit of wheat CF1 probably contains at least one internal disulfide bridge, as the electrophoretic mobility of this subunit is lower in the presence of reducing agent than in its absence.
Wheat CF1 was viewed by the electron microscope after negative staining. Discrete particles, many appearing hexagonal, were observed at high magnifications. Markham rotational analysis confirmed that the enzyme has sixfold symmetry in at least one of its orientations.
Correlation between Protein Phosphorylation and Progesterone Synthesis in Bovine Luteal Cells Stimulated by Lutropin
Authors: DARBON Jean‐Marie , URSELY Jsocelyne , LEYMARIE Pierre 
Published: March 2005

The implication of cytosolic protein phosphorylation in the regulation by lutropin of steroidogenesis in luteal tissue was investigated, in vitro by incubating selected small bovien luteal cells in the presence of 32P‐labelled phosphate. The incorporation of 32P into cytosolic proteins was measured after fractionation by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate and autoradiography of the gel. Progeserone synthesis, 32P incorporation into cytosolic proteins, cyclic AMP content and protein kinase activity were measured simultaneously aafter incubation for various times and with increasing doses of lutropin.As previously reporte, the gonadotropin as well as dibutyryladenosine 3′, 5′ monophosphate enchanced by 60–150% the degree of phosphorylation of six cytosolic proteins of molecular weight ranging from 50000–115000.Time‐course studies in the presence of a saturating dose of lutropin demonstrated a close correlation between progesterone synthesis, protein phosphorylation and cyclic AMP production. The three parameters were increased within 5 min, the level of cylci AMP and the extent of protein phosphorylation reaching a maximum value after 10‐20 min.Dose‐response curves for progesterone synthesis and protein phosphorylation were nearly identical with a half‐maximal stimulation occurring for both parameters with a physiological concentration of lutropin (3ng/ml). Conversely dose‐response curves for cyclic AMP and protein kinase activity were clearly shifted, as expected, toward the higher concentration of lutropin (half‐maximal stimulation for 90 ng/ml of lutropin).Further characterization of the phosphorylated proteins using a highly resolutive two‐dimensional eletrophoretic technique demonstrated that the number of proteins phosphrylated under lutropin and the degree of stimulation was distinctly higher than suspeted from monodimensional fractionation technique. The 32P incorporation fro two of the nine proteins phosphorylated by lutropin appeared at least ten‐times higher than in the control.The present result are compatible with the implication of at least some of the observed unduced protein phosphorylations in the steroidogenic effect of lutropin in luteal tissue. They sustain the hypothesis of a cyclic‐AMP‐dependent protein kinase activation as a part of the mechanism of lutropin action.
Interrelationships between 3‐Hydroxy‐3‐Methylglutaryl‐CoA Synthase, Acetoacetyl‐CoA and Ketogenesis
Authors: MENAHAN Lawrence A. , HRON W. Theodore , HINKELMAN Doloris G. , MIZIORKO Henry M. 
Published: March 2005

Kinetic and physical approaches have been employed to investigate the binding of acetoacetyl‐CoA to hydroxymethylgluaryl‐CoA synthase. The enzyme has an apparent Km for acetoacetyl‐CoA (0.35μM) which is more than an order of magnitude lower than the K(6‐10μM) measured for substrate inhibition by this metabolite. Hepatic acetoacetyl‐CoA concentration, as measured by a sensitive and highly specific radioactive assay appears to be in the 1‐ 10 μM range; the concentration decreases during diabetic ketoacidosis. Total hepatic activity of hydroxymethylglutaryl‐CoA synthase and levels of mitochondrial enzyme protein, determined by radioimmunoassay, are not appreciably different in livers from control or ketoacidotic animals.In contrast to the decrease in hepatic acetoacetyl‐CoA concentration observed during ketoacidosis, myocardial acetoaetyl‐CoA levels are increased by aaat least tenfold when compared to controls. Elevated acetoacetyl‐CoA levels may serve to inhibit fatty acid utilization y the heart. Thus, a consideration of the multiple interactions of acetoacetyl‐CoA with the enzymes involved in ketone ody production and utilization may be useful in evaluating the metabolic significance of this intermediate.
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.
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.
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.
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.
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.
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.
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.
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.
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.