bluuebunny/crossref_metadata_2025 huggingface dataset:
Fcγ‐receptor‐mediated changes in the plasma membrane potential induce prostaglandin release from human fibroblasts
Authors: FREY Jürgen , JANES Michael , ENGELHARDT Waltraud , AFTING E.‐G. , GEERDS Christina , Möller Beate
Published: March 2005

Binding of aggregated human immunoglobulin G (IgG) on diploid human fibroblasts leads to a rapid depolarization of the cells within 1‐2 min. We resolved this membrane potential change into its plasma membrane and mitochondrial membrane components by measuring the transmembrane distribution of the lipophilic tritiumlabelled cation tetraphenylphosphonium, [3H]Ph4P+. The responsibility of the plasma membrane for the membrane potential change, induced by binding of IgGs, is demonstrated. The IgG‐induced membrane depolarization leads to the induction of prostaglandin E2 synthesis. Aggregated immunoglobulins (IgG) are specifically bound via the Fc portion because only binding of Fc fragments, in contrast to (Fab′)2 fragments, leads to a stimulation of prostaglandin E2 synthesis comparable to that mediated by IgGs. Depolarization of the plasma membrane by short incubation of the fibroblasts in high‐K+ buffer (5 min) results in a stimulation of prostaglandin E2 synthesis comparable to that mediated by either aggregated human IgGs or Fc fragments. Our previous results on Fcγ‐receptor‐mediated antigen‐IgG‐antibody complex internalization showed that a maximum uptake of these complexes could be detected 60–90 min after binding. Therefore, we conclude that not internalisation but binding of aggregated IgGs to the Fcγ receptors on human fibroblasts is the stimulus for plasma membrane depolarization leading to an enhanced prostaglandin E2 release.
Processing of follitropin and its receptor by cultured pig Sertoli cells
Authors: SAEZ José M. , JAILLARD Christine
Published: March 2005

Immature pig Sertoli cells, cultured in a chemically defined medium, are able to maintain many of their functional characteristics for at least two weeks. This model was used to investigate the binding, internalization and degradation of 125I‐labelled human follitropin (hFSH) and the effects of pig FSH (pFSH) on its own receptors. The binding of 125I‐labelled hFSH was dependent on time, temperature and concentration. At 4°C, the apparent steady state was reached in 8‐12 h and remained constant for at least 24 h, whereas at 33°C the apparent equilibrium was reached in 4–6 h. Thereafter the total binding declined and by 24 h it was less than 50% of the maximum binding. At 33°C the binding for the hormone to its surface receptor was followed by internalization of the hormone (half‐life ∼ 1 h) and its degradation (half‐life ∼ 3 h). The receptor‐mediated internalization of hFSH was blocked by phenylarsine oxide. In the presence of the ionophore monensin (20 μM) the rates of binding and internalization were not modified but the degradation rate was much lower (half‐life ∼ 18 h). Thus, in the presence of monensin, maximum binding increased twofold, and remained constant for 24 h. This increase was mainly due to an increase of the internalized hormone. When Sertoli cells were exposed to pFSH there was a loss of its own receptor, which was both dose‐dependent (ED50= 250 ng/ml) and time‐dependent (t1/2= 14 h). Cycloheximide did not modify the FSH‐induced down‐regulation, whereas monensin enhanced the down‐regulation process. These results show that FSH, like other ligands, is internalized and degraded by its target cells and indicate that the hormone‐mediated down‐regulation is related to the internalization process. However, the discrepancy between the rate of internalization and of hormone‐induced down‐regulation, suggests that some of the internalized receptors are recycled.
Phosphorylase kinase from chicken skeletal muscle
Authors: ANDREEVA Iraida E. , LIVANOVA Nataliya B. , ERONINA Tatyana B. , SILONOVA Galina V. , POGLAZOV Boris F.
Published: March 2005

Phosphorylase kinase has been purified from white and red chicken skeletal muscle to near homogeneity, as judged by sodium dodecyl sulphate (SDS) gel electrophoresis. The molecular mass of the native enzyme, estimated by chromatography on Sepharose 4B, is similar to that of rabbit skeletal muscle phosphorylase kinase, i.e. 1320 kDa. The purified enzyme both from white and red muscles showed four subunits upon polyacrylamide gel electrophoresis in the presence of SDS, corresponding to α′, β, γ′ and δ with molecular mass of 1320 kDa for the native enzyme and on the molar ratio of subunits as estimated from densitometric tracings of the polyacrylamide gels, a subunit formula (α′βγ′δ)4 has been proposed. The antiserum against the mixture of the α′ and β subunits of chicken phosphorylase kinase gave a single precipitin line with the chicken enzyme but did not cross‐react with the rabbit skeletal muscle phosphorylase kinase. The pH 6.8/8.2 activity ratio of phosphorylase kinase from chicken skeletal muscle varied from 0.3 to 0.5 for different preparations of the enzyme. Chicken phosphorylase kinase could utilize rabbit phosphorylase b as a substrate with an apparent Km value of 0.02 mM at pH 8.2. The apparent V (18 μmol min−1 mg−1) and Km values for ATP at pH 8.2 (0.20 mM) were of the same order of magnitude as that of the purified rabbit phosphorylase kinase b. The activity of chicken phosphorylase kinase was largely dependent on Ca2+. The chicken enzyme was activated 2–4‐fold by calmodulin and troponin C, with concentrations for halfmaximal activation of 2 nM and 0.1 μM respectively. Phosphorylation with the catalytic subunit of cAMP‐dependent protein kinase (up to 2 mol 32P/mol αβγδ monomer) and autophosphorylation (up to 8 mol 32P/mol αβγδ monomer) increased the activity 1.5‐fold and 2‐fold respectively. Limited tryptic and chymotryptic hydrolysis of chicken phosphorylase kinase stimulated its activity 2‐fold. Electrophoretic analysis of the products of proteolytic attack suggests some differences in the structure of the rabbit and chicken γ subunits and some similarities in the structure of the rabbit red muscle and chicken α′.
The isolation, purification and amino‐acid sequence of insulin from the teleost fish Cottus scorpius (daddy sculpin)
Authors: CUTFIELD John F. , CUTFIELD Susan M. , CARNE Alan , EMDIN Stefan O. , FALKMER Sture
Published: March 2005

Insulin from the principal islets of the teleost fish, Cottus scorpius (daddy sculpin), has been isolated and sequenced. Purification involved acid/alcohol extraction, gel filtration, and reverse‐phase high‐performance liquid chromatography to yield nearly 1 mg pure insulin/g wet weight islet tissue. Biological potency was estimated as 40% compared to porcine insulin. The sculpin insulin crystallised in the absence of zinc ions although zinc is known to be present in the islets in significant amounts. Two other hormones, glucagon and pancreatic polypeptide, were copurified with the insulin, and an N‐terminal sequence for pancreatic polypeptide was determined.

The primary structure of sculpin insulin shows a number of sequence changes unique so far amongst teleost fish. These changes occur at A14 (Arg), A15 (Val), and B2 (Asp). The B chain contains 29 amino acids and there is no N‐terminal extension as seen with several other fish. Presumably as a result of the amino acid substitutions, sculpin insulin does not redily form crystals containing zinc‐insulin hexamers, despite the presence of the coordinating B10 His.
Insulin receptors of chicken liver and brain
Authors: SIMON Jean , LEROITH Derek
Published: March 2005

Receptors on membranes of chicken liver and brain bound porcine 125I‐insulin in a specific and temperature‐dependent manner. Competition with unlabeled insulin derivatives exhibited typical insulin potency ratios, i.e. chicken > porcine insulin > human proinsulin (2.1/1/0.02). Apparent binding affinity was higher in brain with a 50% inhibition of tracer binding of 1.3 ± 0.2 nM porcine insulin as compared to 2.8 ± 0.3 nM in liver. The apparent molecular mass of the 125I‐insulin cross‐linked α subunit of the insulin receptor was 139 ± 2 kDa for chicken liver and 127 ± 2kDa for chicken brain. These molecular masses were similar to those of rat liver and brain insulin receptors. Neuraminidase treatment of the cross‐linked insulin receptor increased the mobility of the α subunit from liver but did not affect that from brain, suggesting a difference in the glycosylation of the chicken brain α subunit as previously described in the rat. Despite this change, both receptors could be purified on wheat germ agglutinin (WGA) chromatography after Triton solubilization. In the presence of CTP and vanadate (phosphatase inhibitors) insulin‐stimulatable tyrosine‐specific phosphorylation of exogenous substrates was demonstrated with chicken liver and brain receptors. The reaction was dependent on Mg2+ and Mn2+. As noted with other insulin receptors, the best artificial substrate for phosphorylation was poly(Glu, Tyr)4: 1. In both chicken liver and brain the smallest effective insulin dose as well as maximal stimulation of phosphorylation of the substrate was similar to that seen with rat liver, and in all three tissues chicken insulin was more potent than porcine insulin. In chicken liver an active ATP hydrolytic activity copurified with the insulin receptors during WGA chromatography. Further purification using S‐300 Sephacryl filtration or affinity (insulin‐biotin‐avidin) chromatography could dissociate the phosphorylation and the hydrolytic activities. Gel electrophoresis, under reducing conditions revealed β subunits with apparent Mr of 97–99 kDa in chicken liver and brain, which were phosphorylated in the presence of insulin. Similar apparent molecular masses have been described for the β subunit of rat liver receptors. These studies suggest that both chicken brain and liver insulin receptors exhibit coupling of α and β subunits with fully active tyrosine kinase and that the structural difference of the brain insulin receptor is widespread and phylogenetically old.
Synaptosomal bioenergetics
Authors: KAUPPINEN Risto A. , NICHOLLS David G.
Published: March 2005

The bioenergetic interaction between glycolysis and oxidative phosphorylation in isolated nerve terminals (synaptosomes) from guinea‐pig cerebral cortex is characterized.

Essentially all synaptosomes contain functioning mitochondria.

There is a tight coupling between glycolytic rate and respiration: uncoupler causes a tenfold increase in glycolysis and a sixfold increase in respiration.

Synaptosomes contain little endogenous glycolytic substrate and glycolysis is dependent on external glucose.

In glucose‐free media, or following addition of iodoacetate, synaptosomes continue to respire and to maintain high ATP/ADP ratios.

In contrast to glucose, the endogenous substrate can neither maintain high respiration in the presence of uncoupler nor generate ATP in the presence of cyanide.

Pyruvate, but not succinate, is an excellent substrate for intact synaptosomes.

The in‐situ mitochondrial membrane potential (ΔΨm) is highly dependent upon the availability of glycolytic or exogenous pyruvate; glucose deprivation causes a 20‐mV depolarization, while added pyruvate causes a 6‐mV hyperpolarization even in the presence of glucose.

Inhibition of pyruvate dehydrogenase by arsenite or pyruvate transport by α‐cyano‐4‐hydroxycinnamate has little effect on ATP/ADP ratios; however respiratory capacity is severely restricted.

It is concluded that synaptosomes are valuable models for studying the control of mitochondrial substrate supply in situ.
Biosynthesis of rat‐liver pI‐5.0 esterases in cell‐free systems and in cultured hepatocytes
Authors: ROBBI Mariette , BEAUFAY Henri
Published: March 2005

Rat liver esterases focusing at pH 5.0 (referred to below as pI‐5.0 esterases) are structurally related glycoproteins which differ slightly in their mobility in sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS‐PAGE). They reside in the lumen of the endoplasmic reticulum. We have studied their biosynthesis in cell‐free systems programmed by total liver RNA, using sheep and rabbit antibodies to isolate the translation products related to these enzymes. Our results show that they are assembled as a precursor polypeptide chain (62 kDa) larger than the mature proteins. The pI‐5.0 esterase mRNA could be extracted from bound but not free polysomes. Reticulocyte lysates supplemented with dog pancreas microsomes produced four esterase‐related components in segregated form (61, 60, 58 and 56 kDa). The largest three correspond in electrophoretic mobility to the mature enzymes. They are glycoproteins that bind to concanavalin A, and can be reduced to the size of the shortest component by endo‐β‐N‐acetylglucosaminidase H (endo‐H). Immunopreciptation after biosynthetic labeling of the proteins in cultured hepatocytes also gave three glycosylated components that had the same mobility in SDS‐PAGE as the mature enzymes. When tunicamycin was present in the culture medium, a single immunoprecipitable form was observed. Its apparent Mr was similar to that of the unglycosylated pI 5.0 esterase form synthesized in vitro in the presence of dog pancreas microsomes. Thus the biosynthesis of these esterases has characteristics in common with that of numerous secretory proteins, except for the rather large difference in size (∼ 6 kDa) resulting from the proteolytic processing of their in‐vitro‐synthesized precursor.
Mechanisms of lipid peroxidation dependent upon cytochrome P‐450 LM2
Authors: EKSTRÖM Gunilla , INGELMAN‐SUNDBERG Magnus
Published: March 2005

A mechanism of lipid peroxidation dependent on the oxidase activity of cytochrome P‐450 LM2 in reconstitued membrane vesicles has been investigated. The rate of lipid peroxidation, determined as the formation of thiobarbituric‐acid‐reactive substances, was inhibited by CO. It increased concomitantly to the production of O2− and H2O2, when cytochrome P‐450 LM2 was incorporated into vesicle containing NADPH — cytochrome‐P‐450 reductase, until a 1:1 molar ratio between the enzymes was reached. Also the formation of lipid hydroperoxides was dependent on the presence of cytochrome P‐450 LM2 in the membranes. This lipid peroxidation was not inhibited by hydroxyl radical scavengers and not specifically inhibited by scavengers of singlet oxygen. By contrast, superoxide dismutase was a very potent scavenger of the lipid peroxidation. A half‐maximal effect at 3 ng/ml enzyme was registered, whereas a 100‐fold higher concentration was necessary in order to inhibit O2− formation as detected by succinylated cytochrome c or pyrogallol. The reason for this difference might be inherent in different types of kinetics in the interaction of O2− with different scavengers or might possibly indicate that SOD scavenges another type of reactive oxygen, different from O2−, generated by cytochrome P‐450 LM2. Iron chelators inhibited the P‐450‐dependent lipid peroxidation, whereas iron chelate interacted with NADPH — cytochrome‐P‐450 reductase in the membranes giving rise to reductase‐dependent lipid peroxidation. Neither superoxide dismutase nor EDTA at high concentrations, inhibited CCl4‐initiated lipid peroxidation, indicating the point of action of these compounds at the initiation step in the cytochrome‐P‐450‐LM2‐dependent lipid peroxidation. Superoxide generated by pyrogallol, in three times the amount produced by P‐450 LM2, could not bring about lipid peroxidation. It is suggested that the cytochrome‐P‐450‐dependent lipid peroxidation mechanism might be of importance for intracellular oxidative damage under certain conditions.
Demonstration of cGMP‐dependent protein kinase and cGMP‐dependent phosphorylation in cell‐free extracts of platelets
Authors: WALDMANN Rainer , BAUER Silvia , GÖBEL Claus , HOFMANN Franz , JAKOBS Karl H. , WALTER Ulrich
Published: March 2005

Homogenates, membranes and cytosol of rat and human platelets were found to contain cGMP‐dependent protein kinase immunoreactivity. Specific cGMP‐dependent protein kinase immunoreactivity was about 1.7 pmol protein kinase/mg protein for homogenates of human platelets and 0.7 pmol/mg for homogenates of rat platelets; the majority appeared to be associated with the membrane fraction.

In membranes of platelets low concentrations of cAMP (0.5–2 μM) stimulated the phosphorylation of five major proteins with apparent relative molecular masses, Mr, of 240000, 130000,50000,42000 and 22000 while low concentrations of cGMP (0.5–2 μM) stimulated the phosphorylation of three major proteins with apparent Mr of 130000, 50000 and 46000. An affinity‐purified antibody against the cGMP‐dependent protein kinase was prepared which specifically inhibited the activity of cGMP‐dependent protein kinase. In membranes of human platelets this affinity‐purified antibody inhibited the cGMP‐stimulated phosphorlation of the three proteins with Mr of 130000, 50000 and 46000 while it had no effect on the cAMP‐dependent and cyclic‐nucleotide‐independent protein phosphorylation.

The results demonstrate that platelets contain a cGMP‐dependent protein kinase and at least three specific substrates for this enzyme. Two of these substrates, the proteins with apparent molecular Mr of 130000 and 50000, are substrates for both cAMP‐ and cGMP‐dependent protein kinase. The protein with apparent Mr of 130000 appears to be closely related to an intrinsic plasma membrane protein of vascular smooth muscle cells which is a substrate for a membrane‐associated cGMP‐dependent protein kinase. Therefore, cGMP‐dependent protein kinase and cGMP‐regulated phosphoproteins may mediate in platelets the intracellular effects of those hormones, vasodilators and drugs which elevate the level of cGMP and inhibit platelet aggregation.
Purification and characterization of N,N‐dimethylformamidase from Pseudomonas DMF 3/3
Authors: SCHÄR Hans‐Peter , HOLZMANN Werner , RAMOS TOMBO Gerardo M. , GHISALBA Oreste
Published: March 2005

The N,N‐dimethylformamide‐hydrolyzing enzyme (DMFase) from Pseudomonas DMF 3/3 has been purified to apparent electrophoretic homogeneity with an overall 49‐fold purification, a 24% yield and a final specific activity of 1.98 μmol N,N‐dimethylformamide (DMF) hydrolyzed min−1 (mg protein)−1. The native DMFase has a relative molecular mass of 250000 and is composed of two light‐chain (Mr= 15000) and two heavy‐chain (Mr= 105000) subunits. The stability of DMFase is optimal at pH values above 7.5 and at temperatures below 20°C. The activity of the enzyme is inhibited by metal‐chelating agents such as EDTA and 2,2′‐dipyridyl. Emission and atomic absorption spectroscopy measurements showed that iron is present in significant amounts in DMFase, indicating that it is an iron‐containing amidohydrolase. In the ultraviolet/visible spectrum prominent bands were observed at 224 nm, 280 nm and 396 nm and shoulders are present at 418 nm and 467 nm.

DMFase from Ps. DMF 3/3 has an isoelectric point of 7.7. The enzyme exhibits optimal activity between pH 5 and 6 and at 40°C. The substrate spectrum is rather narrow. The enzyme hydrolyzes preferentially substituted short‐chain aliphatic amides such as DMF, N‐ethylformamide and N‐methylformamide. N,N‐diethylformamide, N,N‐dimethylacetamide and unsubstituted amides, e.g. formamide, prolinamide, acetamide, acrylamide and butyramide are substrates as well, but are hydrolysed at significantly lower rates. DMFase obeys Michaelis‐Menten kinetics and its Km and Vmax values for DMF are 13.8 mM and 1.89 U/mg, respectively, as determined from a Lineweaver‐Burk plot.
Isolation and characterization of the 10‐kDa and 22‐kDa polypeptides of higher plant photosystem 2
Authors: LJUNGBERG Ulf , ÅKERLUND Hans‐Erik , ANDERSSON Bertil
Published: March 2005

Two polypeptides of 10 kDa and 22 kDa, shown to be components of the higher plant photosystem 2, were purified and examined. A NaCl/Triton X‐100 treatment was designed, which released these two polypeptides from the thylakoid membrane, in concert with the extrinsic 16‐kDa and 23‐kDa proteins, concomitant with a loss in oxygen‐evolution activity. After this treatment the oxygen‐evolving activity of the photosystem 2 membranes devoid of the 10‐kDa and the 22‐kDa polypeptides could be restored with CaCl2, but not by readdition of the purified 23‐kDa protein. This defiency was caused by an inability of the 23‐kDa protein to rebind to the photosystem 2 membranes. In analogy, the oxygen‐evolution activity of a highly purified photosystem 2 core preparation, devoid of the 10‐kDa and 22‐kDa polypeptides, was stimulated by CaCl2, but not by the 23‐kDa protein. We, therefore, suggest that the 10‐kDa or the 22‐kDa polypeptides provide a binding‐site for the extrinsic 23‐kDa protein to the thylakoid membrane.

The 10‐kDa and 22‐kDa polypeptides were isolated through ion‐exchange chromatography in the presence of detergents. They both displayed hydrophobic properties, verified by their low proportion of polar amino acid residues and their partition to the hydrophobic phase during Triton X‐114 fractionation. The purified polypeptides did not contain metallic cofactors or substances with absorption in the visible region of the spectrum.
Intracellular transport and tyrosine sulfation of procollagens V
Authors: FESSLER Liselotte I. , CHAPIN Steven , BROSH Sarah , FESSLER John H.
Published: March 2005

Several tyrosine residues of the extracellular p‐collagens V and collagens V are sulfated [Fessler, L. I., Brosh, S., Chapin, S. and Fessler, J. H. (1986) J. Biol. Chem. 261, 5034–5040]. Here, the sulfation of their intracellular precursors, the procollagens V, was studied. A Golgi‐enriched subcellular fraction of chick embryo tendon catalyzed the sulfation of tyrosine residues in both endogenous and added, unsulfated procollagens V with the sulfate donor 3′‐phosphoadenosine 5′‐[35S]phosphosulfate. Intracellular tyrosine sulfation of procollagen V occurred at a point distal to the cis Golgi compartment as judged by change of the N‐linked carbohydrate of procollagen V from being endoglycosidase‐H‐sensitive to being resistant. The time course of the intracellular modifications of procollagen V was determined by incubating tendons with 3H‐labeled amino acids and with [35S]sulfate. The proα(V) chains were synthesised in about 10 min and then assembled into unsulfated procollagen V molecules. Tyrosine sulfation occurred 50 min after completion of polypeptide synthesis and the molecules were successively sulfated in the order in which they had been synthesized. The antimicrotubular drug Nocodazole, which disrupts the spatial organization of the Golgi, decreased the time interval between synthesis of procollagens V and sulfation. The sulfated procollagens V were soon secreted and cut to sulfated p‐collagens V. Sulfated proα1(V) chains were cleaved faster than sulfated proα1′(V) chains. The relationship of sequential protein modification to spatial cellular organization is discussed.
Oscillating dissipative structures in mitochondrial suspensions
Authors: HOLMUHAMEDOV Ehson L.
Published: March 2005

The occurrence of spatial structures in the unstirred layer of an oscillating mitochondrial suspension is reported. The structures are detected by photo camera by light scattering in the unstirred layer of suspension. The spatial structures observed are shown to oscillate with the same period as that of mitochondrial oscillations in the bulk phase. Patterning is not affected by the layer depth within the range 0.3–3.0 mm. Various types of oscillatory states of mitochondria are characterized by the corresponding patterns. Patterning is effectively suppressed by the inhibitors of the respiratory chain (antimycin A or CN−).
The resolution of heterogeneous fluorescence of multitryptophan‐containing proteins studied by a fluorescence‐quenching method
Authors: STRYJEWSKI Wieslaw , WASYLEWSKI Zygmunt
Published: March 2005

From acrylamide quenching results, analyzed by an itterative non‐linear least‐squares method, we have shown that the fluorescence of multitryptophan‐containing proteins, such as horse‐liver alcohol dehydrogenase, 3‐phosphoglycerate kinase and lysozyme, can be resolved for different segmental contributions, each characterized by collisional (Ki) and static (Vi) quenching constants. The ability to resolve the heterogeneous fluorescence of proteins makes it possible to follow changes in dynamics of the individual residues. In yeast 3‐phosphoglycerate kinase, which contains only two tryptophan residues, three fluorescent fractions, characterized by different accessibility to the quencher, were observed. Two of them are assigned to one of the tryptophan residue. This may be interpreted in terms of conformational fluctuations, which facilitate the access of acrylamide molecules to the buried tryptophan residues.
Hydrolysis of p‐nitrophenyl acetate by the peptide chain fragment (336–449) of porcine pancreatic lipase
Authors: DE CARO Josiane D. , ROUIMI Patrick , ROVERY Mireille
Published: March 2005

The incubation of porcine pancreatic lipase (449 amino acids) with chymotrypsin led to the preferential cleavage of the Phe‐335–Ala‐336 bond [Bousset‐Risso et al. (1985) FEBS Lett. 182, 323–326]. Up to now it has not been possible to isolate the fragment (1–335) whereas fragment (336–449) was easily purified. This fragment does not display any activity towards the specific substrates of lipase, triacylglycerols, either in the aggregate form or monomeric solution, but like lipase it hydrolyzes p‐nitrophenyl acetate. The biphasic kinetics of the release of p‐nitrophenol by the fragment with different concentrations of p‐nitrophenyl acetate ([S] > [E]) are very similar to those of lipase and other esterases. The initial burst is equal to 1 mol p‐nitrophenol/mol fragment (when [S] =∞). Ethoxyformic anhydride only reacts with 1 mol histidine out of the 2 mol that the fragment contained. The activity of the fragment towards p‐nitrophenyl acetate hydrolysis is inhibited after ethoxyformic anhydride reaction as in the case of lipase. The results presented led to the hypothesis that in the area (336–449) a part of the active‐site structure of the lipase molecule is included. It would seem that fragment (336–449) is a functional domain of lipase.
Dimeric ubiquinol:cytochrome c reductase of Neurospora mitochondria contains one cooperative ubiquinone‐reduction centre
Authors: LINKE Petra , BECHMANN Georg , GOTHE Alexander , WEISS Hanns
Published: March 2005

Dimeric ubiquinol:cytochrome c reductase of Neurospora mitochondria was isolated as a protein‐Triton complex and free of ubiquinol (Q). The enzyme was incorporated into phosphatidylcholine membranes together with Q. The effects of varying the molar ratio of Q to enzyme on the electron transfer from duroquinol (DHQ2) to the cytochromes c, c1 and b were studied.

The rate of electron flow from DQH2 to cytochrome c was 15 times increased by Q and was maximal when one molecule of Q was bound to one enzyme dimer. The apparent Km value for DQH2 of the Q‐free enzyme was 5 μM and of the Q‐supplemented enzyme 25 μM. The pre‐steady‐state rate of electron transfer from DQH2 to cytochrome c1 was also 15 times increased by Q and was maximal with one Q molecule bound to one enzyme dimer. This effect of Q was inhibited by antimycin. The pre‐steady‐state rate of electron transfer from DQH2 to cytochrome b was 5 times decreased when Q was bound to the enzyme and this effect of Q was insensitive to myxothiazol. The H+/2 e− stoichiometry with DQH2 as substrate of the Q‐supplemented enzyme was 3.6.

These results are interpreted in accordance with a Q‐cycle mechanism operating in a dimeric cytochrome reductase. Each enzyme monomer catalyses a single electron transfer from the QH2‐oxidation centre to the Q‐reduction centre and the two monomers cooperate in the reduction of Q to QH2 at one Q‐reduction centre. This centre contains two different binding sites for Q. DQH2 does not properly react at the QH2‐oxidation centre. DQH2, however, binds to the loose Q‐binding site of the Q‐reduction centre and reduces the Q bound to the tight Q‐binding site of the centre. The QH2 thus formed at the Q‐reduction centre serves as electron donor for the QH2‐oxidation centre.
The heat‐shock response in Drosophila KC 161 cells
Authors: JACKSON Richard J.
Published: March 2005

The effect of heat shock on protein synthesis in the Drosophila melanogaster KC 161 tissue culture cell line was examined with a view to investigating the mechanism underlying the acute reduction in normal cellular protein synthesis typical of heat‐shocked Drosophila cells. However, at 36–37°C, the optimum temperature for induction of the 70‐kDa heat‐shock protein, this cell line did not show such a response. The synthesis of a very limited number of proteins was abruptly turned off following heat shock in the presence or absence of actinomycin, but the rate of synthesis of the majority of normal cellular proteins declined slowly over a three‐hour period. Incubation of heat‐shocked cells in hypertonic media increased the relative proportion of protein synthesis directed towards heat‐shock proteins (as opposed to normal cellular proteins). Incubation with low concentrations of cycloheximide had the converse effect and resulted in a preferential increase in the size of polysomes translating normal cellular mRNAs, greater than the increase in size of polysomes synthesising heat‐shock proteins. Heat shock also resulted in some mRNAs being almost completely displaced from polysomes into the postribosomal supernatant. These observations suggest that competition between normal cellular mRNAs and increasing amounts of heat‐shock mRNAs with a higher affinity for the translation machinery was the main explanation for the gradual reduction in the synthesis of normal cellular proteins, although a slight reduction in overall translation initiation rates cannot be excluded as a subsidiary cause. The results demonstrate that the acute reduction in normal cellular protein synthesis seen in other Drosophila cell lines is not an integral and necessary feature of the heat‐shock response in this organism, which makes it unlikely that the mechanism of this acute shut‐off is intimately connected with the mechanism of induction of heat‐shock mRNAs.
The position of the oligosaccharide side‐chains of phytohemagglutinin and their accessibility to glycosidases determines their subsequent processing in the Golgi
Authors: FAYE Loic , STURM Arnd , BOLLINI Roberto , VITALE Alessandro , CHRISPEELS Maarten J.
Published: March 2005

Phytohemagglutinin (PHA), the glycoprotein lectin of Phaseolus vulgaris has two types of asparagine‐linked oligosaccharides per polypeptide: a high‐mannose chain with the formula (Man)8–9(GlcNAc)2 on Asn12 and a modified chain with fewer mannose residues and additional fucose and xylose residues on Asn60. Glycosylation of PHA is a cotranslational process, which occurs in the endoplasmic reticulum, and newly synthesized PHA has two high‐mannose chains. Transport of PHA to the protein bodies via the Golgi complex is accompanied by the modification of one of the two high‐mannose chains. Why is only one chain modified, while the other remains in the high‐mannose configuration? By determining the effect of digestion with various glycosidases (α‐mannosidase, endo‐β‐N‐acetylglucosaminidase H and endo‐β‐N‐acetylglucosaminidase F) on native and denatured PHA we obtained evidence consistent with the interpretation that the accessibility of oligosaccharide chains to modifying enzymes is of major importance in determining whether a high‐mannose chain becomes modified or not. The high‐mannose chain of mature undenatured PHA is only partially accessible to glycosidases, while PHA obtained from the endoplasmic reticulum has one high‐mannose chain, which is readily accessible to α‐mannosidase and endoglycosidases H and F. We show that this readily accessible chain is in the same position on the polypeptide (Asn60) as the modified oligosaccharide on mature PHA. Thus, accessibility of the oligosaccharide side‐chains to processing enzymes in the Golgi determines whether a particular oligosaccharide side‐chain is processed or not.
The effects of pressure on yeast cytochrome c peroxidase
Authors: KORNBLATT Jack A. , ENGLISH Ann M. , HUI BON HOA Gaston
Published: March 2005

The effects of pressure on cytochrome c peroxidase [CcP(FeIII)], its cyano derivative (CcP · CN) and its enzyme‐substrate complex (ES) have been studied. The effects of pressure on the binding of the substrate analog porphyrin cytochrome c (porphyrin c) to CcP · CN and ES have also been studied.

High pressure causes CcP(FeIII) to undergo a high‐spin to low‐spin transition but has no detectable effect on either CcP · CN, which is already low spin, or on ES. The low‐spin CcP(FeIII) structure at pressure is similar to the low‐spin form at low temperature and the low‐spin form of horseadish peroxidase at high pressure. ΔV° associated with the spin equilibrium is about 30 ml/mol and is independent of temperature. ΔG° is small, 4.7 kj/mol at 0 °C, while ΔH° is 14.2 kJ/mol at 1 bar (100 kPa).

Pressure has no detectable effect on the binding equilibria of mixtures of CcP · CN plus porphyrin c or ES plus porphyrin c. This indicates that the interaction of CcP and porphyrin c results in little or no volume change; the same is true in the case of cytochrome c oxidase and porphyrin c.
Structural analysis of poly(ADP‐ribose) polymerase in higher and lower eukaryotes
Authors: SCOVASSI A. Ivana , IZZO Raffaella , FRANCHI Elisabetta , BERTAZZONI Umberto
Published: March 2005

A phylogenetic survey for the poly(ADP‐ribose)polymerase has been conducted by analyzing enzyme activity in various organisms and determining the structure of the catalytic peptides by renaturation of functional activities of the enzyme in situ after electrophoresis in denaturating conditions (activity gel).

The enzyme is widely distributed in cells from all different classes of vertebrates, from arthropods, mollusks and plant cells but could not be detected in echinoderms, nematodes, platyhelminthes, thallophytes (including yeast) and bacteria. The presence on activity gels of a catalytic peptide with Mr= 115000–120000 was demonstrated in vertebrates, arthropods and mollusks but no activity bands were recovered in many lower eukaryotes, in plant cells and bacteria. By using an immunological procedure that used an antiserum against homogeneous calf thymus poly(ADP‐ribose) polymerase, common immunoreactive peptides were visualized in mammals, avians, reptiles, amphibians and fishes, while lacking in non‐vertebrate organisms.

Our results indicate that the structure of poly(ADP‐ribose) polymerase is conserved down to the mollusks suggesting its important role for DNA metabolism of multicellular organisms.
Interaction of enzymes involved in triosephosphate metabolism
Authors: TOMPA Péter , BÄR Jörg , BATKE József
Published: March 2005

The affinity of baker's yeast (Saccharomyces cerevisiae) fructose‐1, 6‐bisphosphate aldolase towards the metabolically related enzymes phosphofructokinase and glyceraldehyde‐3‐phosphate dehydrogenase was tested by using a fluorescence‐probe technique with fluorescein isothiocyanate attached covalently to the enzymes. The dissociation constants of the enzyme‐enzyme complexes, as well as the rate constants of association and dissociation, were determined. Data were compared with the parameters derived from a mammalian (rabbit muscle) system, known from the literature and determined under the same conditions (pH 7.5 or 8.5 in 0.05 M Tris/HCl buffer at 20°C). The comparison reveals similarities in the supramolecular organization of these cytoplasmic enzymes in phylogenetically distant species. Moreover, the fact that in vitro hybrid complexes are formed of stability comparable to that of non‐hybrid complexes indicates that this ancient characteristic is probably conserved during evolution.

A possible regulatory mechanism is presented, based on the dynamic competition, with each other, of the enzymes involved in triosephosphate metablism.
The cobalt‐nitrosyl stretching vibration as a sensitive resonance Raman probe for distal histidine‐nitrosyl interaction in monomeric hemoglobins
Authors: YU Nai‐Teng , THOMPSON Helen M. , MIZUKAMI Hiroshi , GERSONDE Klaus
Published: March 2005

The Co‐NO stretching vibration has been assigned in the resonance Raman spectra of various cobaltsubstituted monomeric hemoglobins by employing isotope‐labeling of nitrosyl (14N16O, 15N16O, 14N18O). Monomeric hemoglobins with a distal histidine (sperm whale myoglobin and leghemoglobin) exhibit this vibration at 573–575 cm−1, wherease hemoglobins without distal histidine (elephant myoglobin and insect hemoglobin from Chironomus thummi thummi, CTT III) show this vibration in the range of 553–558 cm−1. The Fe‐NO stretching vibration which occurs in the range of 554–556 cm−1 does not reflect the distal histidine‐ligand interaction. Therefore, the Co‐NO moiety which is isoelectronic with the Fe‐O2 moiety is a good monitor for distal effects on the exogenous ligand of hemoglobins, especially due to the fact that in hemoglobins with distal histidine the Fe‐O2 stretching vibration (567–572 cm−1) is similar to the Co‐NO stretching vibration.
Synthesis of ATP by soluble mitochondrial F1 ATPase and F1–inhibitor‐protein complex in the presence of organic solvents
Authors: GÓMEZ PUYOU Armando , TUENA DE GÓMEZ PUYOU Marietta , de MEIS Leopoldo
Published: March 2005

The F1 and F1–inhibitor‐protein complex synthesized tightly bound ATP from ADP and Pi when the organic solvents dimethylsulfoxide (20–50% v/v), ethyleneglycol (20–60% v/v) or poly(ethyleneglycol) 4000 and 8000 (30–50% w/v) were included in the assay media. There was no synthesis of tightly bound ATP in the absence of organic solvents.

In the presence of 50% dimethylsulfoxide, maximal synthesis of ATP was obtained at pH values between 6.5 and 7.7. In both F1 and F1 ‐inhibitor‐protein there was no synthesis of ATP in the absence of MgCl2. The rate of ATP synthesis became faster as the MgCl2 concentration in the medium was raised from 0.1–10 mM. The Km for Pi of F1 was in the range of 0.8–1.5 mM. The Km for Pi of the F1 ‐inhibitor‐protein was much higher than that of F1 and could not be measured.

In the presence of 10 mM MgCl2 and 2 mM Pi, the rate constants of ATP synthesis by F1 and F1–inhibitor‐protein were 5.2–10.4 h−1 and 3.5–5.9 h−1 respectively. For both enzymes the rate constant of ATP hydrolysis was 0.69 h−1. The tightly bound ATP of F1 and F1‐inhibitor‐protein were hydrolyzed at a much slower rate when either the Pi concentration or the MgCl2 concentration was suddenly decreased.

Both in presence and absence of Mg2+, 40–60% of the radioactive tightly bound ATP synthesized by F1 was hydrolyzed when non‐radioactive ATP was added to the assay medium. This was not observed when F1 ‐inhibitor‐protein was used.
Calcium transport in membrane vesicles ofStreptococcus cremoris
Authors: DRIESSEN Arnold J. M. , KONINGS Wil N.
Published: March 2005

Rightside‐out membrane vesicles ofStreptococcus cremoriswere fused with proteoliposomes containing the light‐driven proton pump bacteriorhodopsin by a low‐pH fusion procedure reported earlier [Driessen, A. J. M., Hellingwerf, K. J. & Konings, W. N. (1985)Biochim. Biophys. Acta 808, 1–12]. In these fused membranes a proton motive force, interior positive and acid, can be generated in the light and this proton motive force can drive the uptake of Ca2+. Collapsing ΔΨ with a concomitant increase in ΔpH stimulates Ca2+uptake while dissipation of the ΔpH results in a reduced rate of Ca2+uptake. Also an artificially generated ΔpH, interior acid, can drive Ca2+uptake inS. cremorismembrane vesicles.

Ca2+uptake depends strongly on the presence of external phosphate while Ca2+‐efflux‐induced proton flux is independent of the presence of external phosphate. Ca2+accumulation is abolished by the divalent cation ionophore A23187.

Calcium extrusion from intact cells is accelerated by lactose. Collapse of the proton motive force by the uncoupler carbonylcyanidep‐trifluoromethoxyphenylhydrazone or inhibition of the membrane‐bound ATPase byN,N′‐dicyclohexylcarbodiimide strongly inhibits Ca2+release. Further studies on Ca2+efflux at different external pH values in the presence of either valinomycin or nigericin suggested that Ca2+exit from intact cells is an electrogenic process. It is concluded that Ca2+efflux inS. cremorisis mediated by a secondary transport system catalyzing exchange of calcium ions and protons.
Isolation and characterization of mutants from Schyzosaccharomyces pombe defective in glycerol catabolism
Authors: GANCEDO Carlos , LLOBELL Antonio , RIBAS Juan‐Carlos , LUCHI Francisca
Published: March 2005

Mutants unable to grow on glycerol were isolated from the fission yeast Schyzosaccharomyces pombe. Two types of mutants were obtained: one type was able to grow on dihydroxyacetone while the other one did not grow on this compound. The first type of mutants was defective in glycerol dehydrogenase while the second one was affected both in the glycerol dehydrogenase and in dihydroxyacetone kinase. It was found that the second type was defective in the derepression of several enzymes. The mutations wre nuclear and monogenic and defined two complementation groups. Spontaneous revertants, able to grow on glycerol, were obtained from the first type of mutants. They have regained the glycerol dehydrogenase activity. The results presented provide genetic evidence for a pathway of glycerol catabolism in Sch. pombe involving dehydrogenation of glycerol as the first step followed by phosphorylation of the dihydroxyacetone formed.
Sequence‐specific DNA‐binding proteins which interact with (G + C)‐rich sequences flanking the chicken c‐myc gene
Authors: LOBANENKOV Victor V. , NICOLAS Robert H. , PLUMB Mark A. , WRIGHT Carol A. , GOODWIN Graham H.
Published: March 2005

The interaction of nuclear sequence‐specific DNA‐binding proteins from definitive chicken erythrocytes, thymus and proliferating transformed erythroid precursor (HD3) cell with the 700‐base‐pair (700‐bp) DNA 5′‐flanking region of the chicken c‐myc gene was investigated by in vitro footprint analysis. The major HD3 protein‐binding activity binds to a site (site V) 200 bp upstream from the ‘cap’ site but, after further fractionation, a second distinct binding activity is detected to a site (site VIII) which contains both the ‘CAAT’ and ‘SP1‐binding’ consensus sequences. Protein from thymus and erythrocyte cells which express c‐myc at lower levels, bind to seven and eight sites repectively, In common with HD3 cell protein, they both bind to site VIII and, although binding to the sequence at site V is also detected, the footprint protection pattern is sufficiently different (site V′) to suggest the involvement of different proteins in terminally differentiated and proliferating cells. The DNA‐binding activities were partially fractionated by high‐performance liquid chromatography gel filtration and include an erythrocyte‐specific protein which binds to a c‐myc gene poly(dG) homopolymer sequence similar to that found upsteram of the chicken βA‐globin gene.
13C NMR evidence for bacteriochlorophyll c formation by the C5 pathway in green sulfur bacterium, Prosthecochloris
Authors: OH‐HAMA Tamiko , SETO Haruo , MIYACHI Shigetoh
Published: March 2005

The 13C NMR spectra of the pheophorbide of bacteriochlorophyll c, formed in the presence of l‐[1‐13C]glutamate and [2‐13C]glycine and [13C]bicarbonate in Prosthecochloris aestaurii, were analysed. The isotope in the glutamate was specifically incorporated into the eight carbon atoms in the tetrapyrrole macrocycle derived from the C‐5 of 5‐aminolevulinic acid, while no specific enrichment of these eight carbon atoms was observed in the spectrum of the pigment formed in the presence of [2‐13C]glycine. These labelling patterns provide evidence for the operation of the C5 pathway of 5‐aminolevulinic acid synthesis for bacteriochlorophyll c formation in the bacterium.

The labelling of bacteriochlorophyll c by [13C]bicarbonate is consistent with its formation from 5‐[1,4,5‐13C]aminolevulinic acid formed by the C5 pathway from [1,2,5‐13C]glutamic acid. It is proposed that this glutamate is the transamination product of 2‐[1,2,5‐13C]oxoglutaric acid, arising by carboxylation of [1,4‐13C]succinyl‐CoA with 13CO2 catalysed by 2‐oxoglutaric acid synthase activity, and that the labelled succinyl‐CoA is, in turn, derived by the fixation of 13CO2 by the reductive tricarboxylic acid cycle.

The 13C chemical shifts of two sp2 quaternary carbons of bacteriopheophorbide c methyl ester (C‐2 and C‐4) were reassigned.
The mouse myoglobin gene
Authors: BLANCHETOT Alain , PRICE Melanie , JEFFREYS Alec J.
Published: March 2005

Seal myoglobin (Mb) exons 1 and 3 were used as probes to isolate the functional mouse Mb gene. This gene has a very low level of Mb expression in skeletal muscle. Although it is shorter, the mouse Mb gene displays the common organisation found in human and seal Mb genes. In addition, we have defined blocks of conserved sequences in the 5′ flanking region by comparison with other mammalian Mb genes. Moreover, about 103 bases upstream of the cap site we identified a repetitive B1 element directly associated with two overlapping open reading frames, containing a putative polyadenylation signal. A polypyrimidine‐rich region has also been located upstream from the gene.
Aminoacylaminonucleoside inhibitors of protein synthesis
Authors: THEOCHARIS Dimitrios A. , KALPAXIS Dimitrios L. , COUTSOGEORGOPOULOS Charalambos
Published: March 2005

In a model system derived from Escherichia coli, Ac[3H]Phe‐puromycin is produced in a pseudo‐first‐order reaction between the preformed Ac[3H]Phe‐tRNA‐poly(U)‐ribosome complex (complex C) and excess puromycin [Kalpaxis et al. Eur. J. Biochem. 154, 267,1986]. Amicetin and gougerotin inhibit this reaction to various degrees depending on whether or not complex C is allowed to interact with the inhibitor (I) prior to the addition of puromycin (S). The kinetic analysis shows a phase where competitive inhibition can be observed provided that S and I are added simultaneously. After preincubating C with I, the inhibition becomes of the mixed non‐competitive type. The Ki (the dissociation constant of the CI complex), calculated from the competitive plot, is 20.0 μM for amicetin and 15.0 μM for gougerotin. This inhibition constant (Ki) cannot distinguish amicetin from gougerotin. Its acceptance as a criterion of potency does not explain why after preincubation amicetin proves to be a stronger inhibitor than gougerotin. The determination of the apparent catalytic rate constants of peptidyltransferase at various inhibitor concentrations and the appropriate replotting of these rate constants distinguish amicetin from gougerotin. A new approach for evaluating the potency of these inhibitors is proposed. The familiar Ki is supplemented with an apparent kinetic constant obtained from a replot in which the intercepts of the double‐reciprocal plots (1/Kobs versus 1/[S]) are plotted versus the inhibitor concentration.