bluuebunny/crossref_metadata_2025 huggingface dataset:
Localization of the tight ADP‐binding site on the membrane‐bound chloroplast coupling factor one
Authors: CZARNECKI Joseph J. , ABBOTT Marilyn S. , SELMAN Bruce R.
Published: March 2005

The photoaffinity analog 2‐azido‐ADP (2‐azidoadenosine 5′‐diphosphate) was used as a probe of the spinach chloroplast ATP synthase. The analog acted as a substrate for photophosphorylation. Several observations suggested that 2‐azido‐ADP and ADP bound to the same class of tight nucleotide binding sites: (a) 2‐azido‐ADP competitively inhibited ADP tight binding (Ki= 1.4 μM); (b) the concentration giving 50% maximum binding, K0.5 for analog tight binding (1 μM) was similar to that observed for ADP (2 μM); (c) nucleotide tight binding required prior membrane energization and was completely reversed by re‐energization; (d) the tight binding of 2‐azido‐[β‐32P]ADP was completely prevented by ADP; (e) the analog inhibited the light‐triggered ATPase activity at micromolar concentrations.

Ultraviolet irradiation of washed thylakoid membranes containing tightly bound 2‐azido‐[β‐32P]ADP resulted in the covalent incorporation of the label into the membranes. Denaturing polyacrylamide gel electrophoresis of the labeled membranes demonstrated that the β subunit of the coupling factor one complex was the only polypeptide in the thylakoid membranes which was labeled. These results identify the β subunit of the coupling factor as the location of the tightly bound ADP on the thylakoid membranes.
Changes in the lateral distribution of the light‐harvesting chlorophyll‐a/b—protein complex induced by its phosphorylation
Authors: LARSSON Ulla K. , JERGIL Bengt , ANDERSSON Bertil
Published: March 2005

The effect of protein phosphorylation on the distribution of chlorophyll‐protein complexes between appressed and non‐appressed thylakoid regions of spinach chloroplasts has been investigated. Stroma lamellae vesicles and inside‐out vesicles, representative of non‐appressed and appressed thylakoids, respectively, were isolated from thylakoid membranes before and after phosphorylation. The fractions were analyzed with respect to the yield of vesicles, incorporation of [32P]phosphate into the light‐harvesting chlorophyll‐a / b—protein complex, chlorophyll composition, and the relative content of the main chlorophyll‐protein complexes. The yield of inside‐out vesicles from phosphorylated thylakoids was 20% lower than that from control thylakoids, indicating a partial destacking. The specific incorporation of [32P]phosphate into the light‐harvesting chlorphyll‐a / b—protein complex was at least four times higher in stroma lamellae vesicles than in inside‐out vesicles. The proportion of the light‐harvesting chlorophyll‐a / b—protein complex in stroma lamellae vesicles increased from 13% to 21% of their total chlorphyll after phosphorylation of the thylakoids. There was also a corresponding increase in the level of chlorophyll b. These observations strongly suggest that phosphorylated light‐harvesting complexes migrate from appressed thylakoids rich in photosystem 2 to non‐appressed thylakoids rich in photosystem 1. In contrast, there was no evidence for a lateral migration of the chlorophyll‐a—protein complex of photosystem 2 after phosphorylation. Our results indicate that a lateral migration of phosphorylated light‐harvesting complexes in combination with a lateral separation of photosystem 1 and 2 to the different thylakoid regions, is a basis for the regulation of excitation energy between the two photosystems.
Purification of stalk‐cell‐inducing morphogens from Dictyostelium discoideum
Authors: KAY Robert R. , DHOKIA Babu , JERMYN Keith A.
Published: March 2005

We have shown previously that developing amoebae of Dictyostelium discoideum release one or more low‐Mr factors, which can induce isolated cells to differentiate into stalk cells in the presence of cyclic AMP [Town, C. D., Gross, J. D. and Kay, R. R. (1976) Nature (Lond.) 262, 717–719; Town, C. D. and Stanford, E. (1979) Proc. Natl Acad. Sci. USA, 76, 308–312]. These differentiation‐inducing factors (DIF) have now been purified by a procedure involving binding to and elution from XAD‐2 resin, extraction into hexane and two steps of reverse‐phase high‐pressure liquid chromatography (HPLC). Our results show the following.

HPLC resolves a major stalk‐cell‐inducing activity (DIF‐1) and at least four minor and more polar activities (DIFs 2–5).

DIF‐1 has been purified at least 3000‐fold over the starting dialysed medium with a recovery of about 2%. This low recovery of DIF‐1 can be explained in part by the loss of non‐specific stimulatory (‘helper’) factors during the purification. A few micrograms purified DIF‐1 were obtained from 1012 cells. This material could induce stalk cell differentiation in the standard assay at less than 0.2 nM.

The biological activity of DIFs 1, 2 and 3 was sensitive to borohydride reduction, suggesting the presence of an essential carbonyl group. DIF‐5 was partially sensitive and DIF‐4 resistant. Other properties of DIF‐1 suggest that it is a non‐polar molecule of Mr < 500, which becomes charged in alkaline solution, and that it is neither a peptide nor has essential sugar moieties.

The purification of DIF should make possible its eventual identification by sensitive physical techniques, such as mass spectroscopy, and will allow further investigation of its biological effects.
The function of cytoplasmic membrane of Paracoccus denitrificans in controlling the rate of reduction of terminal acceptors
Authors: KUČERA Igor , LAUČÍK Jaroslav , DADÁK Vladimír
Published: March 2005

The rate of reduction of terminal acceptors (nitrate, nitrite, and oxygen) in anaerobically grown cells of Paracoccus denitrificans increased on permeabilization of cytoplasmic membrane. It was proved that under aerobic conditions the increase of the rate of nitrate reduction was caused by: (i) the abolishment of the permeability barrier for nitrate, (ii) the enhancement of the influx of redox equivalents to the respiratory chain due to the stimulation of succinate dehydrogenase reaction, and (iii) the inhibition of electron flow to oxygen by endogenously formed nitrite.

Nitrite inhibits oxygen reduction by its interaction with the terminal part of the respiratory chain (I50= 15 μM) localized at the inner aspect of the cytoplasmic membrane. The distribution of nitrite between intact cells and the suspension medium follows the Nernst equation for monovalent anion. The possible physiological consequences of the low intracellular nitrite concentration are discussed.
Signal sequences, secondary modification and the turnover of miscompartmentalized secretory proteins in Xenopus oocytes
Authors: LANE Charles D. , CHAMPION Janet , CRAIG Roger
Published: March 2005

The cytoplasm of the Xenopus oocyte can be altered by the microinjection of proteins and the regulatory responses to such perturbations can then be studied. We have investigated proteolytic systems within the oocyte which may be involved in the maintenance of the integrity of the different subcellular compartments. Thus primary translation products, made in the wheat germ system under the direction of frog liver, chicken oviduct, rat liver rapidly sedimenting endoplasmic reticulum, rat seminal vesicle, guinea pig mammary gland or honey been venom gland RNA, were injected into oocytes. Their stability in the frog cell cytosol was in general low compared to that of their processed counterparts. The latter were usually obtained by collecting the heterologous proteins exported by RNA‐injected oocytes. Electrophoretic analysis of oocytes injected with particular primary and processed polypeptides permitted measurement of the stabilities of proteins differing only by the presence or absence of a detachable signal sequence, or by the presence of a specific secondary modification. The effect of the latter on protein stability appears slight. However, the presence of a detachable signal sequence destabilizes those miscompartmentalized secretory proteins which are otherwise stable. Indeed all other results are consistent with this concept for they show that primary translation products are in general much less and are never more stable than their processed counterparts. Thus we provide evidence that errors of compartmentation can be corrected in living cells and that this process is often facilitated by the properties conferred on a protein by a detachable signal sequence.
High‐resolution proton NMR as indicator of a silent mutation in the haem cavity of a monomeric allosteric haemoglobin
Authors: LA MAR Gerd N. , ANDERSON Richard R. , CHACKO Vappuram P. , GERSONDE Klaus
Published: March 2005

Comparison of the high‐field 1H‐NMR spectra of the met‐cyano complexes of allosteric haemoglobins III and IV of the insect larva Chironomus thummi thummi (CTT‐III and CTT‐IV) shows that, in addition to the molecular heterogeneity previously described for both Hbs as due to haem orientational disorder [La Mar et al. (1980) J. Biol. Chem. 255, 66–70], CTT‐III but not CTT‐IV exhibits a second heterogeneity as evidenced by splitting of numerous signals. Reconstitution of CTT‐III with modified haems alters the populations of the two haem rotational isomers but leaves the secondary heterogeneity unaffected. This argues directly for locating this secondary heterogeneity solely within the polypeptide chain rather than the result of protein‐haem interaction. We assign this secondary heterogeneity found solely in Hb‐III to a point mutation in the haem cavity, 57E6 (Ile/Thr); CTT‐IV is chemically homogeneous. The observation of significant hyperfine shift differences for the alternative substitution at 57E6, particularly for non‐haem single‐proton resonances thought to arise from distal residues, indicates some structural consequences in the haem cavity due to the point mutation. While a difference in the allosteric properties cannot be detected in that the point mutation appears to leave the pK for the allosteric transition unaltered, subtle influences on the function of the protein in both affinity states cannot be ruled out at this time.
Influence of species and sex on cholesterol 7α‐hydroxylase activity in experimental animals
Authors: BOSISIO Enrica , GALLI Giovanni , GALLI KIENLE Marzia
Published: March 2005

Cholesterol 7α‐hydroxylase activity was evaluated measuring by selected ion monitoring the amount of 7α‐hydroxycholesterol formed from endogenous microsomal cholesterol and from exogenously added [4‐14C]cholesterol in incubations of liver microsomes.

The enzyme activity was determined in female rats, hamsters and guinea pigs and results were compared with those obtained in male rats.

The results suggest that there is no difference between male and females as far as the activity of the enzyme from endogenous cholesterol is concerned at least in rat liver. Moreover, saturation of the enzyme by the endogenous substrate was observed in all female animals in contrast to the male rat for which the enzyme was found to be not saturated, as already reported by others.

From molar radioactivity of 7α‐hydroxycholesterol obtained during incubations with [4‐14C]cholesterol, differences were also evidenced in the equilibration of the substrate with the endogenous pool of cholesterol. In the male rat, equilibration depended upon the concentration of radioactive cholesterol added to the medium and reached completion at 60 μM concentration. In female animals, molar radioactivity of 7α‐hydroxycholesterol was always linearly correlated with that of cholesterol in the medium but it was always lower than that one. This indicates that in the tested female animals only part of exogenously added cholesterol is accessible to the substrate pool of the enzyme.
Primary structure of protamine from the Northern pike Esox lucius
Authors: SPECKERT Wally , KENNEDY Brian , DAISLEY St Laurent , DAVIES Peter
Published: March 2005

The basic nuclear protein in the sperm of the Northern pike is a protamine, 32‐residues long, which behaved as a single component during ion‐exchange chromatography and gel electrophoresis. Amino acid analysis gave close to molar ratios for the eight different residues with no evidence of microheterogeneity. However, the presence of sequence variants was revealed following a combination of automated protein sequencing and cleavage of the protamine by CNBr, endoproteinase Lys‐C and thermolysin.

At position 28 there is an equal probability of having serine or glycine. At position 9 glycine is found more frequently than serine. The reciprocal nature of the substitutions results in glycine and serine contents which are close to a 4:2 ratio. Pike protamines are homologous to those of the trout but show less sequence variation between components.
Isolation and structural characterization of five major sialyloligosaccharides and a sialylglycopeptide from normal human urine
Authors: PARKKINEN Jaakko , FINNE Jukka
Published: March 2005

Five major sialyloligosaccharides and a sialylglycopeptide have been isolated from normal human urine by charcoal adsorption, gel filtration, ion‐exchange chromatography, and paper chromatography. Structural studies including gas‐liquid chromatography of monosaccharide and disaccharide derivatives, methylation analysis, glycosidase treatments, and CrO3 oxidation indicated the following structures for the compounds: 1, NeuAc(α2‐6)Gal(β1‐4)Glc; 2, NeuAc(α2‐6)Gal(β1‐4)GlcNAc; 3, NeuAc(α2‐3)Gal(β1‐4)Glc; 4, NeuAc(α2‐3)Gal(β1‐4)GlcNAc; 5, NeuAc(α2‐3)Gal(β1‐3) [NeuAc(α2‐6)]GalNAc; and 6, NeuAc(α2‐3)Gal(β1‐3) [NeuAc(α2‐6)]GalNAc(α1‐O)Ser. Compounds 4, 5, and 6 have not been described in a free form before.

The presence of compound 5 in urine may suggest that it derives from glycoproteins through a catabolic pathway involving cleavage of the carbohydrate‐peptide linkage by an endo‐N‐acetylgalactosaminidase. The predominating sialyloligosaccharides in urine were compounds 3 and 4. The predominance of the compounds with the sialyl(α2‐3) linkage is of interest in view of the recent discovery of uropathogenic Escherichia coli strains with binding specificity for sialyl(α2‐3)galactosides.
DNA topoisomerase I from calf thymus is inhibited in vitro by poly(ADP‐ribosylation)
Authors: JONGSTRA‐BILEN Jenny , ITTEL Marie‐Elisabeth , NIEDERGANG Claude , VOSBERG Hans‐Peter , MANDEL Paul
Published: March 2005

A slight DNA topoisomerase I activity was detected in highly purified poly(ADP‐Rib)polymerase prepared from calf thymus. This copurified activity was found to be suppressed under conditions where the poly(ADP‐ribosylation) reaction occurs in the presence of NAD.

Purified topoisomerase I from calf thymus was shown to be ADP‐ribosylated by poly(ADP‐Rib) polymerase purified from the same tissue. Poly(ADP‐ribosylation) of topoisomerase I produces an inhibition of the enzymatic activity in parallel to the extent of ADP‐ribosylation.

The fact that a slight poly(ADP‐Rib) polymerase activity was also found to copurify with a topoisomerase I preparation and that toposiomerase I activity can be modified by ADP‐ribosylation, may suggest a spacial and functional correlation of these two enzymes in chromatin.
The effect of heart and skeletal muscle troponin complexes and calmodulin on the Ca2+‐dependent reactions of phosphorylase kinase isoenzymes
Authors: YOSHIKAWA Kazunori , USUI Hirofumi , IMAZU Michinori , TAKEDA Masao , EBASHI Setsuro
Published: March 2005

The dephosphorylated form of phosphorylase kinase was purified 700‐fold from rabbit heart extract. The purified enzyme had a pH 6.8/pH 8.2 activity ratio of 0.04–0.08 and was completely dependent on Ca2+ with an apparent Ka value for Ca2+ of 2.59 μM at pH 6.8. At free Ca2+ concentrations between 0.057 μM and 400 μM, 1.5 μM rabbit heart troponin complex had no significant effect on the reaction. However, 1.5 μM rabbit skeletal muscle troponin complex stimulated the reaction 1.5–2‐fold with a concomitant decrease in the Ka value for Ca2+ to 1.40 μM. No differences in the effects of these troponin complexes were observed when heart‐type and skeletal muscle‐type phosphorylase b isoenzymes from either rabbit or pig were used as substrate. Similar effects of heart and skeletal muscle troponin complexes were observed on the Ca2+‐dependent reaction of the dephosphorylated form of phosphorylase kinase partially purified from rabbit skeletal muscle. A saturating concentration (1.36 μM) of bovine brain calmodulin stimulated 2–5‐fold the Ca2+‐dependent reaction of skeletal muscle phosphorylase kinase, but not the reaction of heart phosphorylase kinase. Heart troponin complex (12 μM) suppressed 80–100% the stimulatory effect of skeletal muscle troponin complex on the reactions of phosphorylase kinase isoenzymes, but had no significant effect on the stimulation by calmodulin of skeletal muscle phosphorylase kinase reaction.
Hepatocyte heterogeneity in glutamate uptake by isolated perfused rat liver
Authors: HÄUSSINGER Dieter , GEROK Wolfgang
Published: March 2005

Glutamate is simultaneously taken up and released by perfused rat liver, as shown by 14CO2 production from [1‐14C]glutamate in the presence of a net glutamate release by the liver, turning to a net glutamate uptake at portal glutamate concentrations above 0.3 mM.

14CO2 production from portal [1‐14C]glutamate is decreased by about 60% in the presence of ammonium ions. This effect is not observed during inhibition of glutamine synthetase by methionine sulfoximine. 14CO2 production from [1‐14C]glutamate is not influenced by glutamine. Also, when glutamate accumulates intracellularly during the metabolism of glutamine (added at high concentrations, 5 mM), 14CO2 production from [1‐14C]glutamate is not affected.

If labeled glutamate is generated intracellularly from added [U‐14C]proline, stimulation of glutamine synthesis by ammonium ions did not affect 14CO2 production from [U‐14C]proline.

After induction of a perivenous liver cell necrosis by CCl4, i.e. conditions associated with an almost complete loss of perivenous glutamine synthesis but no effect on periportal urea synthesis, 14CO2 production from [1‐14C]glutamate is decreased by about 70%.

The results are explained by hepatocyte heterogeneity in glutamate metabolism and indicate a predominant uptake of glutamate (that reaches the liver by the vena portae) by the small perivenous population of glutamine‐synthesizing hepatocytes, whereas glutamate production from glutamine or proline is predominantly periportal. In view of the size of the glutamine synthetase‐containing hepatocyte pool [Gebhardt, R. and Mecke, D. (1983) EMBO J. 2, 567–570], glutamate transport capacity of these hepatocytes would be about 20‐fold higher as compared to other hepatocytes.
Purification, characterisation and reconstitution of glutaconyl‐CoA decarboxylase, a biotin‐dependent sodium pump from anaerobic bacteria
Authors: BUCKEL Wolfgang , SEMMLER Roswitha
Published: March 2005

Glutaconyl‐CoA decarboxylase from Acidaminococcus fermentans is a biotin enzyme, which is integrated into membranes. It is activated by Triton X‐100 and inhibited by avidin. The results obtained by a combination of both agents indicate that biotin and the substrate‐binding site are located on the same side of the membrane.

The decarboxylase was solubilized with Triton X‐100 and purified by affinity chromatography on monomeric avidin‐Sepharose. The enzyme is composed of three types of polypeptides: the group of α chains (Mr 120000–140000) containing the biotin, the β chain (60000) and an apparently hydrophobic γ chain (35000). Sodium ions specifically protected the latter chain from tryptic digestion. It was supposed, therefore, that this chain might function as the Na+ channel. The β and γ chains but not the α chain could be labelled by N‐ethyl‐[14C]maleimide. Similar decarboxylases but with much smaller biotin peptides (Mr 15000–20000) were isolated from Peptococcus aerogenes and Clostridium symbiosum.

The decarboxylases from all three organisms could be reconstituted to active sodium pumps by incubation with phospholipid vesicles and octylglucoside followed by dilution. The Na+ uptake catalysed by the enzyme from A. fermentans was completely inhibited by monensin and activated twofold by valinomycin/K+ indicating an electrogenic Na+ pump. The coupling between Na+ transport and decarboxylation was not tight. During the reaction the ratio decreased from initially 1 to 0.2.

The three organisms mentioned above and Clostridium tetanomorphum without glutaconyl‐CoA decarboxylase are able to ferment glutamate and require 10 mM Na+ for rapid growth. There is no correlation between the concentration of monensin necessary to inhibit growth and the presence of decarboxylase in these organisms.
Purification and characterization of proteases from the polychaete annelid Sabellaria alveolata (L.)
Authors: PEAUCELLIER Gérard
Published: March 2005

Eleven proteases have been purified to electrophoretic homogeneity from crude digestive fluid of polychaete annelids, Sabellaria alveolata. Purification steps were Sephadex G‐100 gel filtration, benzamidine‐cellulose and SBTI‐Sepharose (SBTI = soybean trypsin inhibitor) affinity chromatography, CM‐Sepharose and DEAE‐Sepharose ion‐exchange chromatography.

Nine proteases have been purified in sufficient quantities for characterization. All are active at basic pH and are probably serine proteases, since they are inhibited by phenylmethylsulfonyl fluoride, specific chloromethyl ketone amino acids derivatives, but not by EDTA and p‐chloromercuribenzoate. They do not hydrolyse exopeptidase substrates.

From their properties, they can be divided into five classes.

A trypsin‐like protease, which hydrolyses only trypsin substrates and is inhibited by N‐tosyl‐l‐lysine chloromethyl ketone (TosLysCH2Cl), leupeptin and antipain. It differs from bovine trypsin by its very acidic isoelectric point (below 3.3) and its higher Mr (35000).

A chymotrypsin‐like protease which hydrolyses only chymotrypsin substrates and is inhibited by TosPheCH2Cl, Z‐PheCH2Cl, chymostatin but only slightly by leupeptin and antipain. Its isoelectric point is below 3.3 and its Mr 31000.

Two minor chymotrypsin‐like proteases with slightly broader specificity, since they hydrolyse trypsin substrates significantly and are much more inhibited by leupeptin. They have acidic isoelectric points (3.3 and 3.5) and slightly lower Mr (27000).

Four proteases hydrolyse trypsin and chymotrypsin substrates equally well. Their chymotryptic character is, however, predominant since they are inhibited by TosPheCH2Cl and Z‐PheCH2Cl but not TosLysCH2Cl. They have similar Mr (27000) but isoelectric points ranging from 4.0 to above 9.1.

The last one is very similar but has lower esterolytic activities.

These proteases of broad specificity do not resemble any known serine protease since they differ from subtilisins by their sensitivity to TosPheCH2Cl.
Association of a polyuridylate‐specific endoribonuclease with small nuclear ribonucleo‐proteins which had been isolated by affinity chromatography using antibodies from a patient with systemic lupus erythematosus
Authors: BACHMANN Michael , TRAUTMANN Frank , MESSER Rudolf , ZAHN Rudolf K. , MEYER ZUM BÜSCHENFELDE Karl.‐H. , MÜLLER Werner E. G.
Published: March 2005

Immunoglobulins, containing antibodies against U1‐snRNP, have been prepared from a patient with systemic lupus erythematosus. After coupling these antibodies to a Sepharose matrix, U‐snRNPs have been isolated and purified from rat liver nuclei by use of immunoaffinity chromatography. The resulting RNPs had the typical protein pattern of U‐snRNPs and a sedimentation coefficient of 12 S. The U‐snRNP preparation was associated with an endoribonuclease which required Mg2+ for optimal activity. The enzyme, with an pH optimum of 6.2, degraded only poly(U). Other single‐stranded polyribo‐ and polydeoxyribonucleotides, tRNA, as well as double‐stranded RNA and DNA were not digested. The products of a terminal digestion are (U)6–12 with 3′‐OH and 5′‐P termini. The possible involvement of this endoribonuclease in the splicing of hnRNA is discussed.
Repair of O6‐ethylguanine DNA lesions in isolated cell nuclei
Authors: RENARD André , VERLY Walter G.
Published: March 2005

Cell nuclei prepared from rat liver were alkylated in vitro with ethylnitrosourea; the nuclear DNA was found to lose O6‐ethylguanine and 7‐ethylguanine during a subsequent incubation at 37°C. The rate of O6‐ethylguanine loss is comparable to that observed in vivo, indicating that no cytoplasmic component is needed for the repair; no free O6‐ethylguanine was found in the incubation medium of the ethylated nuclei. The rate of 7‐ethylguanine loss is higher than the spontaneous depurination in vitro and an amount of free 7‐ethylguanine equivalent to that lost by the nuclear DNA was found in the incubation medium; these results suggest that this DNA lesion is excised by a DNA glycosylase.

The proteins of the chromatin prepared from the isolated nuclei induced the disappearance of O6‐ethylguanine from an added ethylated DNA. No free O6‐ethylguanine was released indicating that the repair is not catalyzed by a DNA glycosylase; no oligonucleotides enriched in O6‐ethylguanine were released either, indicating that the disappearance of O6‐ethylguanine from DNA is not the result of the cooperative action of a specific endonuclease and an exonuclease.

Activities capable of removing O6‐ethylguanine from DNA were found in other cell compartments; most of it, however, is in the nucleus where the main location is chromatin. A pretreatment of the rats with daily low doses of diethylnitrosamine during 3 or 4 weeks increased 2–3‐times the repair activity of the chromatin proteins.
Actions of glucagon on flux rates in perfused rat liver
Authors: KIMMIG Rainer , MAUCH Teri Jo , SCHOLZ Roland
Published: March 2005

The relationship between inhibition of glycolysis and stimulation of oxygen consumption by glucagon was studied in perfused rat livers. The two effects exhibit similar kinetics and dose‐response curves; they are slower and less sensitive to the glucagon concentration than the stimulatory effect on glycogenolysis. A stoichiometry of 1 mol extra oxygen consumed/1.8 mol of diminished lactate plus pyruvate production was found. Under conditions where glucagon did not cause a marked inhibition of glycolysis (i.e. low glycolytic flux rates in the fasted state or in the presence of ethanol), oxygen consumption was also not markedly increased. These findings provide evidence that the major portion of glucagon‐induced stimulation of hepatic respiration in the fed state is due to an enhanced demand for mitochondrial oxidative phosphorylation to compensate for the diminished extramitochondrial ATP production following inhibition of glycolysis by glucagon.
Stimulation of mixed‐function oxidation of 7‐ethoxycoumarin in periportal and pericentral regions of the perfused rat liver by xylitol
Authors: BELINSKY Steven A. , KAUFFMAN Frederick C. , JI Sungchul , LEMASTERS John J. , THURMAN Ronald G.
Published: March 2005

Rates of O‐deethylation of 7‐ethoxycoumarin by perfused livers from fasted, phenobarbital‐treated rats were 3.7 μmol × g−1× h−1. Approximately 50% of the product was conjugated. When rates of 7‐ethoxycoumarin O‐deethylation were varied by infusing different concentrations of substrate, a good correlation (r= 0.91) was found between rates of O‐deethylation of 7‐ethoxycoumarin and fluorescence of 7‐hydroxycoumarin detected from the liver surface. Micro‐light guides (tip diameter 170 μm) placed on periportal and pericentral regions on the liver surface were used to monitor the conversion of nonfluorescent 7‐ethoxycoumarin to fluorescent 7‐hydroxycoumarin. The O‐deethylation of 7‐ethoxycoumarin to 7‐hydroxycoumarin increased fluorescence 64% and 28% in pericentral and periportal regions of the liver lobule, respectively. Rates of 7‐ethoxycoumarin O‐deethylation estimated from these increases in fluorescence were 5.2 μmol × g−1× h−1 in pericentral and 2.2 μmol × g−1× h−1 in periportal regions of the liver. During mixed‐function oxidation of 7‐ethoxycoumarin, the oxidation:reduction state of NADP(H) was similar in both regions of the liver lobule. Xylitol (2 mM) decreased the NADP+/NADPH ratio and stimulated rates of drug metabolism in both regions of the liver lobule. This indicates that conditions exist where the supply of NADPH is an important rate‐determining factor for 7‐ethoxycoumarin metabolism in both periportal and pericentral regions of the liver lobule.
Microtubule assembly using the microtubule‐associated protein MAP‐2 prepared in defined states of phosphorylation with protein kinase and phosphatase
Authors: MURTHY Adavi S. N. , FLAVIN Martin
Published: March 2005

A microtubule‐associated protein (the 270‐kDa MAP‐2) was prepared in two defined states of phosphorylation by (a) phosphorylation by associated kinase to the extent of 11–14 mol/mol, and (b) removal of 70–80% of this phosphate with a protein phosphatase purified from brain. The newly introduced phosphate was in addition to about 10 mol/mol already present in MAP‐2 as isolated; these phosphates were not appreciably released by the phosphatase and did not exchange with ATP. In microtubules assembled with phosphorylated (24 mol/mol) MAP‐2 the assembly rate was decreased, microtubule length and critical concentration for assembly were unaffected, and rates of loss of subunits were increased from both microtubule ends. Phosphorylation also reduced the binding of MAP‐2 to taxol‐stabilized microtubules. These changes were unequivocally due to phosphorylation, since phosphatase treatment reversed all of them. The brain phosphatase used in these experiments was purified 3000‐fold towards histone, but only 100‐fold towards MAP‐2, suggesting brain may contain another enzyme more specific for MAP‐2. Calcineurin, however, had only a low activity for MAP‐2.
Evidence for the presence of a hole in the capsid of turnip yellow mosaic virus after RNA release by freezing and thawing
Authors: KATOUZIAN‐SAFADI Mehrnaz , BERTHET‐COLOMINAS Carmen
Published: March 2005

Turnip yellow mosaic virus (TYMV) RNA escapes from viral capsids after freezing and thawing the virus, and the remaining capsids look very similar to natural capsids in the electron microscope after negative staining [Katouzian‐Safadi, M., Favre, A., and Haenni, A. L. (1980)Eur. J. Biochem. 112, 478–486]. In order to understand how an RNA of 2 × 106Da (33% virus by weight) can escape from a compact protein shell we have compared artificial capsids formed after freezing TYMV and natural capsids producedin vivoin infected plants. We have used various physicochemical techniques including analytical ultracentrifugation, X‐ray scattering, X‐ray diffraction and orientation in a magnetic field. From the combination of these results we conclude that the escape of the RNA is accompanied by the formation of a hole in the capsid surface. The size of such a hole is estimated to 5–9 coat protein subunits.
Ricin and Ricinus communis agglutinin subunits are all derived from a single‐size polypeptide precursor
Authors: BUTTERWORTH Andrew G. , LORD J. Michael
Published: March 2005

Antibodies have been raised in rabbits against the individually purified A and B subunits of the toxic castor bean lectin, ricin, and against the A′ and B′ subunits of Ricinus communis agglutinin type I. Each of the antisera recognised a single polypeptide species of Mr 60 500 when maturing castor bean endosperm mRNA was translated in vitro in a rabbit‐reticulocyte‐derived system. When dog pancreatic microsomal vesicles were included in the translational system, each subunit antiserum precipitated a group of 66 000–68 000‐Mr core‐glycosylated polypeptides which had been translocated into the lumen of the vesicles. The 60 500‐Mr polypeptide appeared to be a common precursor to all four individual lectin subunits since (a) its glycosylated (66 000 – 68 000‐Mr) forms were readily detected in the endoplasmic reticulum fraction isolated from maturing castor bean endosperm and (b) pulse‐chase studies showed that the glycosylated precursors disappeared from the endoplasmic reticulum fraction with the concomittant appearance of authentic lectin subunits in a soluble protein fraction which included protein body matrix components. Antiserum prepared against whole R. communis agglutinin, type I, also precipitated the 65 000‐Mr precursor in vitro and in vivo, but in addition precipitated a non‐glycosylated 34 000‐Mr polypeptide. This smaller protein is not a lectin subunit precursor, contradicting an earlier suggestion. It is most probably a precursor to the 2‐S albumin storage proteins found in castor bean endosperm protein bodies
Purification and properties of bovine liver plasma membrane 5′ nucleotidase
Authors: HARB Jean , MEFLAH Khaled , DUFLOS Yves , BERNARD Serge
Published: March 2005

5′‐Nucleotidase from bovine liver plasma membranes has been extracted by the zwitterionic detergent sulfobetaine 14, and purified to apparent homogeneity. Two affinity chromatographies on concanavalin‐A‐Ultrogel and 5′AMP‐Sepharose 4B followed by AcA‐54‐Ultrogel filtration resulted in a purification of 16000 times relative to the homogenate. Sodium dodecyl sulphate gel electrophoresis indicates that the apparent molecular weight of the subunit is 70000. Cross‐linking of the native enzyme with dimethylpimelimidate followed by gel electrophoresis shows a band with an apparent molecular weight of 140000 indicating that the enzyme is a dimer. 5′‐Nucleotidase is a glycoprotein and its activity is inhibited to different degrees by various lectins, indicating a direct interaction with the enzyme. The purified enzyme shows a sevenfold greater affinity for AMP than the membrane‐bound enzyme. The optimum activity of the purified enzyme occurs at pH 7.5 while the membrane‐bound enzyme showed a wide range of pH optimum (7.5–8.3). An Arrhenius plot of the membrane‐bound enzyme shows a break at 28°C, which disappears in the purified enzyme. The enzyme was inhibited by EDTA, and this inhibition was reversed by divalent cations. This, as well as other evidence, indicates that the enzyme contains a highly bound metal cation, perhaps Mn2+ or Mg2+.
Purification and molecular properties of mouse alcohol dehydrogenase isozymes
Authors: ALGAR Elizabeth M. , SEELEY Tanya‐Lee , HOLMES Roger S.
Published: March 2005

Alcohol dehydrogenase isozymes from mouse liver (A2 and B2) and stomach (C2) tissues have been purified to homogeneity using triazine‐dye affinity chromatography. The enzymes are dimers with similar but distinct subunit sizes, as determined by SDS/polyacrylamide gel electrophoresis: A, 43000; B, 39000, and C, 47000. Zinc analyses and 1,10‐phenanthroline inhibition studies indicated that the A and C subunits each contained two atoms of zinc, with at least one being involved catalytically, whereas the B subunit probably contained a single non‐catalytic zinc atom. The isozymes exhibited widely divergent kinetic characteristics. A2 exhibited a Km value for ethanol of 0.15 mM and a broad substrate specificity, with Km values decreasing dramatically with an increase in chain length: C2 also exhibited this broad specificity for alcohols but showed a Km value of 232 mM for ethanol. These isozymes also showed broad substrate specificities as aldehyde reductases. In contrast, B2 showed no detectable activity as an aldehyde reductase for the aldehydes examined, and used ethanol as substrate only at very high concentrations (>0.5 M). The isozyme exhibited low Km and high Vmax values, however, with medium‐chain alcohols. Immunological studies showed that A2 was immunologically distinct from the B2 and C2 isozymes. In vitro molecular hybridization studies gave no evidence for association between the alcohol dehydrogenase subunits. The results confirm genetic analyses [Holmes, Albanese, Whitehead and Duley (1981) J. Exp. Zool. 215, 151–157] which are consistent with at least three structural genes encoding alcohol dehydrogenase in the mouse and confirm the role of the major liver isozyme (A2) in ethanol metabolism.
Kinetic and thermodynamic evidence of a molybdate interaction with glucocorticoid receptor in calf thymus
Authors: CARDO Paolo P. , GAMBETTI Maurizio , VIGNALE Bruno , DIVANO Maria Carla
Published: March 2005

The effect of molybdate on the kinetic and thermodynamic properties of the dexamethasone‐receptor interaction was studied in calf thymus cytosol.

In the presence of molybdate both the equilibrium binding studies and the association and dissociation experiments reveal a significantly lower affinity of the receptor for [3H]dexamethasone. At 0°C the equilibrium dissociation constant increases from 0.8 nM to 1.8 nM, the association rate constant shifts from 1.5 × 108 M−1 h−1 to 0.2 × 108 M−1 h−1, whereas the rate of dissociation of the untransformed receptor increases from 0.04 h−1 to 1.1 h−1 in the molybdate‐containing buffer. All these effects appear dependent on the concentration of molybdate but the dissociation of the transformed receptor (0.01 h−1) is unaffected. The enthalpy for the association, ΔH+, increases at least twofold whereas the entropy, both for the association (ΔS*=−25 to +104 J K−1 mol−1) and for the equilibrium (ΔS°=−100 to +38 J K−1 mol−1), is markedly influenced by the presence of molybdate.

Taken all together these data suggest that molybdate interacts with the receptor molecule turning it into a form that displays low affinity for steroid, in addition to the well‐documented incapacity to transform itself. This fact leads us to think that both the binding and the transformation are the expression of conformational modifications involving molybdate‐sensitive groups.
The presence of the iron‐sulfur protein (subunit V) of complex III in mitochondria of heme‐deficient yeast cells lacking iron‐sulfur clusters detectable by electron paramagnetic resonance
Authors: LIN Ching‐I. P. , OHNISHI Tomoko , CLEJAN Liviu , BEATTIE Diana S.
Published: March 2005

The presence of subunit V, the iron‐sulfur protein, of complex III has been demonstrated in mitochondria from a mutant of Saccharomyces cerevisiae which lacks 5‐aminolevulinic acid synthase and, hence, is devoid of heme. The mature form (24 kDa) of the iron‐sulfur protein was observed in equal amounts in the heme‐deficient and heme‐sufficient cells with antiserum against subunit V and either the sensitive immuno‐transfer technique or immunoprecipitation from dodecylsulfate‐solubilized mitochondria. In addition, a slight shoulder with a molecular mass 1.5 kDa larger than the mature form was present in mitochondria from the heme‐deficient cells.

Electron paramagnetic resonance spectroscopy revealed the absence of iron‐sulfur signals due to clusters S‐1, S‐2 and S‐3 of succinate dehydrogenase or to Rieske's iron‐sulfur cluster of complex III in mitochondria from the hemedeficient cells. The lack of iron‐sulfur centers in these cells may be a consequence of the absence of sulfite reductase in the cells without heme.
Brown‐adipose‐tissue mitochondria: the regulation of the 32 000‐Mr uncoupling protein by fatty acids and purine nucleotides
Authors: RIAL Eduardo , POUSTIE Alison , NICHOLLS David G.
Published: March 2005

The increased proton permeability induced by the addition of a synthetic proton translocator to non‐respiring hamster brown‐fat mitochondria is unaffected by purine nucleotide addition. In contrast the permeability induced by fatty acids is inhibited by nucleotide, indicating that fatty acids act at the 32000‐Mr uncoupling protein. Fatty acids lower the affinity of nucleotide binding to the 32000‐Mr protein, but not sufficiently to explain their uncoupling action. The sensitivity of the fatty acid modulation of permeability is dependent on chain length, extent of unsaturation and pH. There is a requirement for an unesterified carboxyl group. In respiring mitochondria fatty acids act in the presence of nucleotide by lowering the ‘break‐point’ potential at which the conductance of the 32000‐Mr protein increases. Fatty acids have no effect on the chloride uniport activity of the 32000‐Mr protein, but decouple the interference between chloride and protons when the simultaneous transport of both ions is attempted.
Casein kinase activity in rat mammary gland Golgi vesicles
Authors: WEST David W. , CLEGG Roger A.
Published: March 2005

A Golgi vesicle preparation isolated from the mammary tissue of rats in mid‐lactation has been shown to contain the caseins of rat milk. These proteins were phosphorylated when the Golgi vesicles were incubated in the presence of [γ‐32P] ATP. Although this phosphorylation occurred when the physical integrity of the vesicles was maintained, it was markedly increased when the membrane structure was disrupted by hypoosmotic conditions or by use of detergents. The kinase responsible has been shown to be responsive to the intravesicular concentration of Ca2+ and to the extravesicular concentration of Mg2+. These results have been interpreted in terms of a model suggesting a transmembrane location for the enzyme with binding sites on the cytosolic membrane face for Mg2+ and possibly also for ATP and on the luminal surface for Ca2+ and the caseins.

Others have postulated that the assembly of caseins into micelles occurs in Golgi vesicles and requires both prior phosphorylation of the proteins and the presence of Ca2+. In this investigation we demonstrate that treatments which increase the intravesicular casein phosphorylation also alter the Ca2+ balance within the vesicle lumen. These results are discussed in relation to the ATP‐dependent accumulation of Ca2+ by the mammary gland Golgi vesicles.
Synthesis of photosynthetic membrane proteins directed by RNA from rough thylakoids of Chlamydomonas reinhardtii
Authors: MARGULIES Maurice M.
Published: March 2005

Messenger RNA activities associated with thylakoids of Chlamydomonas reinhardtii were examined by translation of RNA of rough thylakoids (thylakoids with bound ribosomes) in a wheat germ protein‐synthesis system. The RNA fraction, active in translation, did not bind to oligod(T)‐cellulose indicating that the mRNA associated with the rough thylakoids contains little, if any, 3′ polyadenylic acid. Assuming that 1% of the thylakoid RNA was mRNA, it was found to have a specific translation activity between that of globin mRNA and tobacco mosaic virus RNA. Translation products of thylakoid RNA were immunoprecipitated by crossed immunoelectrophoresis, using antisera to two polypeptides synthesized within the chloroplast, one of which specifically precipitates the β subunit of chloroplast coupling factor 1. Each antiserum immunoprecipitated radioactive polypeptide from translates of thylakoid RNA, indicating that the presence of mRNA for both polypeptides is associated with the thylakoids. The results suggest that thylakoid‐bound polyribosomes are involved in the synthesis of thylakoid polypeptides.
A small chromatin‐associated RNA homologous to repetitive DNA sequences
Authors: HOLOUBEK Viktor , DEACON Nicholas J. , BUCKLE David W. , NAORA Hiroto
Published: March 2005

The chromatins of rat liver and chicken reticulocytes contain a small RNA of identical length (29 nucleotides). This RNA species differs from the degradation products of nuclear RNA present in the chomatin in possessing a free 3′‐hydroxyl terminus. It stays associated with chromatin and is not released from the nuclei during the extraction of nuclear ribonucleoprotein particles. This RNA species was purified from the third fraction of non‐histone chromosomal proteins eluted from Sephadex G‐200 (fraction 3 RNA). When fraction 3 RNA, isolated from rat liver, was used to screen a rat genomic library approximately 3% of the phage plaques hybridized with the RNA. DNA isolated from four randomly selected hybridizing phage clones hybridized with purified 29‐nucleotide RNA demonstrating that the sequences present in the four clones represent those of the 29‐nucleotide RNA species and not those of minor contaminants. Each of the four clones contained a different amount of sequences complementary to the RNA species 29‐nucleotides‐long and in each clone the DNA sequences homologous to this RNA were surrounded by different restriction sites. Hybridization of 3′‐32P‐labeled fraction 3 RNA with blots of total genomic DNA established that some sequences homologous to this RNA are dispersed in the DNA and others are present on one EcoRI restriction fragment, which is approximately 6000 base pairs long. Fraction 3 RNA is not a degradation product of ribosomal or transfer RNA, it is transcribed from middle repetitive DNA and probably originates by specific release during the formation and/or processing of high‐molecular‐weight transcripts. In normal liver cells its sequences are not released into the cytoplasm.
Structure determination of the carbohydrate chains of rat α‐fetoprotein
Authors: BAYARD Bernard , KERCKAERT Jean‐Pierre , STRECKER Gérard , DORLAND Lambertus , VAN HALBEEK Herman , VLIEGENTHART Johannes F. G.
Published: March 2005

By affinity chromatography on concanavalin A (ConA) linked to Sepharose, S‐carboxymethylated rat α‐fetoprotein could be separated into two molecular variants: a ConA‐reactive and a ConA‐nonreactive fraction. The carbohydrate chains were quantitatively released from the protein by hydrazinolysis. Based on methylation analysis and high‐resolution 1H‐NMR spectroscopy of the re‐N‐acetylated hydrazinolysates, the carbohydrate structures of the two ConA‐molecular variants of α‐fetoprotein were established. The ConA‐reactive species contains two N‐glycosidic carbohydrate units per molecule, both having the following structure:

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Lysosomes revisited
Authors: de DUVE Christian
Published: March 2005

The first international symposium on lysosomes [1] was held in 1963 at the Ciba Foundation, under the chairmanship of Danielli and with the participation of a prestigious membership. It is interesting to compare our present understanding of the properties and functions of lysosomes with what was known 20 years ago.
Protein conformation and proton nuclear‐magnetic‐resonance chemical shifts
Authors: PARDI Arthur , WAGNER Gerhard , WÜTHRICH Kurt
Published: March 2005

The nuclear magnetic resonance (NMR) chemical shifts of the polypeptide backbone protons in basic pancreatic trypsin inhibitor from bovine organs and the inhibitors E and K from the venom of Dendroaspis polylepis polylepis have been analyzed. Using the corresponding shifts in model peptides, the chemical shifts observed in the proteins were decomposed into random‐coil shifts and conformation‐dependent shifts. Correlations between contributions to the latter term and the polypeptide conformation were investigated by using the crystal structure of the bovine inhibitor. In addition to the well‐known ring‐current effects, a correlation was found between chemical shifts of amide and Cα protons and the length of the hydrogen bonds formed by these protons with nearby oxygen atoms as acceptor groups. There remain sizeable and as yet unexplained residual conformation shifts. Overall, the present treatment provides a satisfactory qualitative explanation for the outstandingly large shifts of backbone hydrogen atoms in these diamagnetic proteins.