bluuebunny/crossref_metadata_2025 huggingface dataset:
Gangliosides of Calf Thymus and of Normal and Leukemic Bovine Lymphocytes
Authors: DYATLOVITSKAYA Ella V. , ZABLOTSKAYA Alla E. , AZIZOV Yuri M. , BERGELSON Lev D.
Published: March 2005

The gangliosides of calf thymus and of lymphocytes from blood, lymph nodes, lymph and spleen of normal and leukemic cows were investigated in an attempt to determine whether there exists a relation between the ganglioside composition and the maturity of the lymphocytes. With all normal peripheral lymphocytes studied the largely prevailing (up to 97 %) ganglioside component was found to be N‐glycoloylneuraminosyllactosylceramide. The ganglioside spectrum of calf thymus was much more complex and included at least six different components. By thin‐layer chromatography, methylation analysis and neuraminidase treatment they were identified as N‐glycoloylneuraminosyl and N‐acetylneuraminosyl lactosylceramides, N‐glycoloylneuraminosylneolactotetraosylceramide, di‐(N‐glycoloylneuraminosyl), di‐(N‐acetylneuraminosyl) lactosylceramides and N‐glycoloylneuraminosyl‐N‐acetylneuraminosyllactosylceramide. The ganglioside spectra of leukemic peripheral lymphocytes were different from those of normal peripheral lymphocytes and resembled the ganglioside profile of the thymus. The data obtained indicate that, in the normal animal, the development of thymocytes into mature peripheral lymphocytes is accompanied by loss of disialosylgangliosides and sialosylneolactotetraosylceramide. It is concluded that both the structure and the composition of the lymphocyte gangliosides become more simple during maturation and ageing of the lymphocyte.
On the Renaturation of Ribosomal Protein L11
Authors: KIME Matthew I. , RATCLIFFE R. George , MOORE Peter B. , WILLIAMS Robert J. P.
Published: March 2005

When urea‐denatured preparations of protein L11 from the ribosome of Escherichia coli are introduced into physiological buffers, two completely different configurations can be obtained. One form, by NMR criteria, shows little evidence of stable tertiary interactions; the other shows strong indications of a distinctive folding pattern. The configuration obtained depends on minor details of the method used for returning samples to non‐denaturing conditions.
Binding of Cibacron Blue F3GA to the Flavin and NADH Sites in Cytochrome b5 Reductase
Authors: POMPON Denis , GUIARD Bernard , LEDERER Florence
Published: March 2005

The behaviour of cytochrome b5 reductase holoenzyme and apoenzyme toward blue‐dextran–Sepharose has been studied. Holoenzyme was adsorbed at low ionic strength and could be eluted with 100 μM NADH or NAD+. Flavin‐free enzyme was even more strongly bound and could be eluted with 1 M NaCl, or 100 μM NADH + 10 μM FAD. Separately the cofactors were without effect. FMN was less effective than FAD. ADP and AMP eluted nothing.

Cibacron blue F3GA was found to exert a mixed inhibition on NADH oxidation. Dye binding to holoenzyme elicited a characteristic red shift in its spectrum. Comparison of the difference spectrum amplitudes at 680 and 585 nm showed the presence of a second binding mode at higher dye concentrations.

These results point to the existence for cytochrome b5 reductase of two binding sites with high affinity for blue‐dextran‐Sepharose: the NADH binding site and the flavin binding site. For the latter it is clear that the isoalloxazine pocket must play a role in dye binding. Cytochrome b5 reductase is the second flavoenzyme which has been shown to have affinity for immobilized dye at the flavin site, the first one being flavocytochrome b2, an FMN‐dependent enzyme [D. Pompon and F. Lederer (1978) Eur, J. Biochem. 90, 563–569].
A Photochemical Crosslinkinig Study of the Subunit Structure of Membrane‐Associated Spectrin
Authors: MIDDAUGH C. Russell , JI Tae H.
Published: March 2005

The subunit structure of membrane‐associated spectrin was investigated by crosslinking human erythrocyte membranes with a variety of cleavable photosensitive heterobifunctional reagents by flash photolysis millisecond crosslinking. Crosslinked complexes were analyzed on agarose‐polyacrylamide gels whose high exclusion limit permits the resolution of molecules with Mr larger than 106.

Crosslinking of membrane ghosts produced a series of new bands with apparent molecular weights of approximately 420000, 710000 and 910000, as well as a band at the top of the gels. No significant bands were detectable between the 910000‐Mr band and the material at the gel top. The molecular weights of the three bands correspond to spectrin dimer, trimer, and tetramer, suggesting a limiting tetrameric stoichiometry for membrane‐associated spectrin.

The rate of formation of spectrin oligomers was examined by increasing crosslinking time. Dimers accumulate before trimer and tetramer, and as tetramer increased, dimer diminished. A simple kinetic model is employed which is also consistent with spectrin tetramer existing on the cytoplasmic surface of the erythrocyte membrane as the basic structural unit.
Functional Recognition of Phage RNA by 30‐S Ribosomal Subunits in the Absence of Initiator tRNA
Authors: VAN DUIN Jan , OVERBEEK Gerrit P. , BACKENDORF Claude
Published: March 2005

30‐S ribosomal subunits of Escherichia coli form a stable complex with MS2 RNA or Qβ RNA at 37°C in the absence of initiator tRNA. The complex functions as a precursor of initiation since it can enter the ribosome cycle in the presence of inhibitors of de novo initiation.
Comparative Immunochemical Studies of Cytochrome P‐450CAM of Pseudomonas putida and of Cytochrome P‐450SCC, of Bovine Adrenocortical Mitochondria
Authors: DUS Karl M. , LITCHFIELD William J. , HIPPENMEYER Paul J. , BUMPUS John A. , OBIDOA Onyechi , SPITSBERG Vitaly , JEFCOATE Colin R.
Published: March 2005

Antibodies raised in rabbits against highly purified preparations of two cytochromes (cytochrome P‐450CAM from Pseudomonas putida and cytochrome P‐450scc from bovine adrenocortical mitochondria) were found to be specific for their respective antigens in Ouchterlony doublediffusion assays. In each case a single sharp curve was observed and no response was seen with other P‐450 hemeproteins.

In enzyme inhibition studies with reconstituted monooxygenase systems both the precipitating antibodies and soluble, monovalent Fab−1 fragments obtained from them by peptic digestion were effective inhibitors. In contrast to the Ouchterlony assay, anti(P‐450CAM) not only stopped camphor hydroxylation completely but also caused measurable inhibition of the cholesterol side‐chain cleavage (desmolase) reaction while anti(P‐450scc) inhibited camphor hydrolxylation significantly in addition to abolishing desmolase activity.

Iodination of cytochromes P‐450CAM and P‐450scc with the help of chloramine‐T produced soluble, radiolabeled preparations with specific activities ranging from 3 × 108 to 1 × 109 counts × min−1×μmol−1 which showed undiminished enzymatic activities and gave a single precipitin curve each in the Ouchterlony assay with anti(P‐450CAM) and anti(P‐450scc), respectively. In the iodinated preparations 60–70% of the tyrosine residues were found to be present as monoiodotyrosines while no other amino acid was labeled.

A sensitive radioimmunoassay was developed, measuring inhibition of binding between 125I‐labeled cytochrome P‐450CAM and its antibodies by cross‐reacting material, which revealed substantial cross‐reactivity of cytochromes P‐45011β (88%), P‐450scc (64%) and P‐450LM‐2 (60%). Conversely, competitive binding o.125I‐labeled cytochrome P‐450sccand unlabeled cross‐reacting antigens to anti(P‐450scc demonstrated cross‐reactivities for cytochromes P‐450sccβ (60%), P‐450CAM (75%) and P‐450LM2 (90%). Incubation of cytochrome P‐450scc with adrenodoxin, and of cytochrome P‐450CAM with putidaredoxin, prior to interaction with anti(P‐450scc) and anti(P‐450CAM), respectively, caused marked decreases in the ability of the corresponding P‐450 hemeproteins to bind to their antibodies.

Even the BrCN‐derived hemepeptides of cytochromes P‐450CAM and P‐450ssc were still able to recognize the antibodies elicited by their parent proteins as evidenced by significant crossreactivities in the radioimmunoassay. It is concluded that the four P‐450 hemeproteins tested in this investigation, cytochromes P‐450CAM, P‐450ssc, P‐45011β, and P‐450LM‐2, have one or more antigenic determinants in common and that a major antibody binding site is associated with the hemepeptide.
Comparison of the Mg2+ and Ca2+ Binding Properties of Troponin Complexes P1‐TI2C and TI2C
Authors: JAHNKE Ulrike , HEILMEYER Ludwig M. G.
Published: March 2005

The phosphoserine present in troponin T of freshly isolated skeletal muscle troponin P1‐TI2C was dephosphorous by alkaline phosphatase and the resulting troponin TI2C characterized by phosphorous content and gel electrophoresis in presence of sodium dodecylsulfate

Both complexes bind Ca2+ in an identical manner with a K0.5 of 5.3 × 10−9 M for the Ca2+/ Mg2+ binding sites and of 1.1 × 10−6 M for the Ca2+‐specific sites. 3.5 mM Mg2+ lower the K0.5 value at the Ca2+/Mg2+ binding sites to 1.3 × 10−7 M in the phospho‐troponin P1‐TI2C and leaves nearly unchanged the value of the dephosphorylated troponin TI2C at 1.2 × 10×8 M. At 10 mM Mg2+ only one dissociation constant of about 1.0 −6 M is determined with both complexes.

In analogy dephosphorylation of troponin P1‐TI2C reduces the affinity for Mg2+ at the Ca2+/Mg2+ binding sites from 6.7 × 10−5 M to 2.0 × 10−3 M. Again the Mg2+‐specific sites are uninfluenced. The possibility is discussed that removal of the phosphate group from troponin T allows the interaction of the N‐terminal domin of troponin T with other amino acid side chains of troponin.
Isolation and Partial Characterization of the Plasma Membrane of Purified Bovine Neutrophils
Authors: MOTTOLA Cristina , GENNARO Renato , MARZULLO Antonio , ROMEO Domenico
Published: March 2005

Large amounts of granulocytes can be isolated from bovine blood by differential centrifugation and hypotonic lysis of erythrocytes, followed by separation of neutrophils and eosinophils by centrifugation through a gradient of colloidal silica.

Careful homogenization of the purified neutrophils and subfractionation of the postnuclear supernatant by centrifugation through a discontinuous sucrose gradient provides a membrane fraction (at the 20/32%, w/w, sucrose interface), which collects about 20–35% of the activity of plasma membrane marker enzymes.

Treatment of the plasma membrane fraction with 0.5 M KCl removes some protein and activity of granule enzymes, leading to an about 20–35‐fold enrichment in specific activity of plasma membrane marker enzymes. In particular, there is a 25‐fold enrichment in a Ca2+‐dependent ATPase, whose half‐maximal reaction velocity is reached at 2.2 × 10‐7 M Ca2+.

High‐resolution sodium dodecyl sulphate/polyacrylamide gel electrophoresis reveals a complex composition of the neutrophil plasma membrane, with about 40 polypeptides stained by Coomassie blue. Ten of these peptides are more intensely stained by the dye; their apparent molecular weight ranges from 81000 to 34000. All these ten polypeptides but one (which is likely to be actin) are markedly enriched in the plasma membrane fraction over the original homogenate.

Since bovine blood can be obtained in unlimited amounts, the procedures here described can be applied to a large‐scale purification of the neutrophil plasma membrane, suitable for biochemical studies.
Spontaneous Dissociation of a Cytochrome Core and a Biglobular Flavoprotein after Mild Trypsinolysis of the Bifunctional Saccharomyces cerevisiae Flavocytochrome b2
Authors: Gervais M. , TEGONI Marietta
Published: March 2005

Saccharomyces cerevisiae flavocytochrome b2 is known as a bifunctional enzyme which behaves as the association of an FMN flavodehydrogenase with its specific acceptor, a b5‐like cytochrome. Mild trypsinolysis gives rise to three complementary fragments (n, X, β′), both prosthetic groups being still bound. After such proteolysis the separation of a biglobular flavoprotein domain (carrying FMN) from a cytochrome domain (with the home) is obtained by molecular sieving under non‐denaturing conditions.

The marked lack of affinity between the tetrameric flavoprotein (X, β′)4 and the monomeric cytochrome core (n) leads to the hypothesis that the two domains are not tightly associated in the native molecule and might move relative to each other. Their respective mobility is possibly required for the catalytic mechanism. The comparison with previous trypsinolysis studies on the flavocytochrome b2 from Hansenula anomala suggests the presence of two common zones of hypersensitivity to proteases, along the protomeric polypeptide chain, and strongly supports the validity of the triglobular model for both flavocytochromes.
Complete Amino‐Acid Sequence of a Non‐neurotoxic, Non‐enzymatic Phospholipase A2 Homolog from the Venom of the Australian Tiger Snake Notechis scutatus scutatus
Authors: LIND Peter , EAKER David
Published: March 2005

The complete amino acid sequence of Notechis II‐1, a non‐neurotoxic, non‐enzymatic phospholipase A2 homolog from the venom of the Australian tiger snake Notechis s. scutatus has been determined. The protein consists of a single chain of 119 amino acids. The main fragmentation of the reduced and S‐carboxymethylated derivative was accomplished by cleavage with Staphylococcus aureus V8 protease. Tryptic peptides were used to align and complete the sequence, which was determined mainly by automated adman deoradation. Notechis II‐1 contains all of the residues that appear to be invariant in elapid and pancreatic phospholipases A2 except at position 30 in the sequence, where an otherwise invariant glycine is replaced by serine.
Purification of Smooth‐Muscle Desmin and a Protein‐Chemical Comparison of Desmins from Chicken Gizzard and Hog Stomach
Authors: GEISLER Norbert , WEBER Klaus
Published: March 2005

A fast and convenient procedure for the purification of polymerization‐competent smoothmuscle desmin is described. Desmin from chicken gizzard and hog stomach were compared by fingerprint techniques. The two proteins show extensive amino acid sequence homology, although, some clear differences in the peptide patterns are indicated. Comparative amino acid sequence analysis of some of the peptides obtained in pure form directly proves this conclusion.
Isolation and Characterization of the Polypeptide Components from Light‐Harvesting Pigment‐Protein Complex B800–850 of Rhodopseudomonas capsulata
Authors: SHIOZAWA Judith A. , CUENDET Pierre A. , DREWS Gerhart , ZUBER Herbert
Published: March 2005

The light‐harvesting bacteriochlorophyll‐carotenoid‐protein complex B800–850 was been isolated from membranes of the phototroph‐negative mutant strain Y5 of Rhodopseudomonas capsulata. The three polypeptides of the complex have been found to be soluble in chloroform‐methanol (1:1, v/v) in the presence of 0.1 M ammonium acetate. They were extracted from the complex and separated by gel filtration on Sephadex LH‐60 in the same solvent mixture.

Minimum molecular weights based on amino acid composition are 12000, 9300, and 5100. Values previously determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis are 14000, 10000 and 8000. The two smaller polypeptides (polarities 31% and 39%) are completely soluble in chloroform/methanol/ammonium acetate while the largest and most polar (41%) polypeptide is only partially soluble. The largest polypeptide contains no tryptophan. The middle polypeptide contains no cysteine and arginine, while the small polypeptide lacks cysteine. Methionine is shown to be the amino terminus for the small and middle polypeptides by two independent methods (Edman degradation and dansylation). Both methods also indicated that the N terminus of the 14000 polypeptide seems to be blocked. Partial N‐terminal amino acid sequences were obtained for the two smaller polypeptides. No homology between the two proteins was observed.
Citrate Lyase Deacetylase of Rhodopseudomonas gelatinosa. Isolation of the Enzyme and Studies on the Inhibition by l‐Glutamate
Authors: GIFFHORN Friedrich , RODE Hergo , KUHN Anita , GOTTSCHALK Gerhard
Published: March 2005

Citrate lyase deacetylase or acetyl‐S‐(acyl‐carrier protein) enzyme thioester hydrolase (acetate) (EC 3.1.2.‐), was purified 3100‐fold with a yield of 3.8% from cell extracts of Rhodopseudomonas gelatinosa. The final enzyme preparation gave a single protein band upon polyacrylamide‐gel electrophoresis in the absence or in the presence of sodium dodecylsulfate. The molecular weight of the native enzyme was estimated by gel filtration to be 14300 ± 1000. Sodium dodecylsulfate/polyacrylamide gel electrophoresis yielded a molecular weight of 7300 ± 600 indicating that the enzyme consisted of two subunits.

Citrate lyase deacetylase acted as an S‐acetyl enzyme thioesterhydrolase because it catalyzed the conversion of citrate lyase (S‐acetyl form) into citrate lyase (Sulfhydryl form) and acetate.

Citrate lyase deacetylase was strongly inhibited by l‐glutamate. The half‐maximal inhibitor concentration was 7.5 × 10−4 M, and a Ki‐value of 1.2 × 10−4 M was determined. The mode of inhibition appeared to be of the linear mixed type. l‐Glutamate was bound by citrate lyric deacetylase but not by citrate lyase.

The pool concentrations of l‐glutamate inR. gelatinosa were 10 mM when citrate was present in substrate amounts and 2.7 mM after total consumption of citrate. Simulation of conditions in vivo using homogeneous enzyme preparations of citrate lyase and citrate lyase deacetylase, and glutamate concentrations of 10 mM and 2.7 mM respectively, revealed that the rate of citrate lyase inactivation increased from 8% within 15 min during growth on citrate to 50% within 15 min in the absence of citrate.
Properties and Characterization of a Spinach Chloroplast RNA Polymerase Isolated from a Transcriptionally Active DNA‐Protein Complex
Authors: BRIAT Jean‐François , MACHE Régis
Published: March 2005

A chloroplast RNA polymerase has been isolated from a transcriptionally active spinach plastid DNA‐protein complex.

The properties of the complex and of the reconstituted system have been compared. The crude enzyme, is at least sevenfold less active when compared with the complex. RNA synthesis by the reconstituted system is sensitive to high ionic strength and heparin, contrarily to RNA synthesis by the chloroplast DNA‐protein complex. On the other hand, rifampicin has no inhibitory effect whatever on the transcriptional system used. The RNA polymerase isolated is more efficient with denatured DNA than with double‐stranded DNA and the best template is chloroplast DNA.

The crude RNA polymerase isolated migrates in a peak of 11 S in glycerol gradient centrifugation and is located in a single band in non‐denaturing polyacrylamide gel electrophoresis.

About 30 polypeptides (Mr 15000–180000) are part of the complex and only eight of them are found in the RNA polymerase preparation. Only five polypeptides are always present with the same yield. They are probably the subunits of the RNA polymerase. The molecular weight of these subunits ranged from 15000–69000, even if the isolation of the enzyme was performed in the presence of protease inhibitors.
Reconstitution of Photosynthetic Energy Conservation
Authors: HAUSKA Gunter , SAMORAY Dietrich , ORLICH Gabriele , NELSON Nathan
Published: March 2005

Photophosphorylation has been reconstituted in a liposomal system containing reaction centers of photosystem I and coupling‐factor complex, both highly purified from spinach chloroplasts. This energy‐converting model system was put together by diluting the preparation of the coupling‐factor complex with an aqueous suspension of proteolipid vesicles, preformed from photosystem‐I reaction centers and soybean phospholipids by sonication. In the presence of reduced N‐methylphenazonium methosulfate the system catalyzed photophosphorylation with rates up to 50 μmol ATP formed × mg chlorophyll−1× h−1, which was sensitive to uncouplers and to N, N′‐dicyclohexylcarbodiimide. The properties of the system in comparison to chloroplasts is discussed.
NADP‐Dependent Isocitrate Dehydrogenase from the Mussel Mytilus edulis L.
Authors: HEAD Erica J. H.
Published: March 2005

A purification scheme is described for NADP‐dependent isocitrate dehydrogenase front the digestive gland of Mytilus edulis. The scheme incorporates three chromatographic steps: hydroxyapatite adsorption, blue‐Sepharose affinity chromatography and DEAE‐Sephacel ion‐exchange chromatography. The subunit molecular weight of the enzyme was 45000 (± 5000) by sodium dodecylsulfate gel electrophoresis. The purified enzyme was homogeneous on dodecylsulfate gels and had a specific activity of 30–50 U/mg protein.
NADP‐Dependent Isocitrate Dehydrogenase from the Mussel Mytilus edulis L.
Authors: HEAD Erica J. H.
Published: March 2005

The kinetic mechanism for NADP‐dependent isocitrate dehydrogenase from tae digestive gland of the mussel Mytilus edulis has been investigated. Initial‐rate studies and substrate‐analogue and product‐inhibition patterns are consistent with a rapid‐equilibrium random‐ordered reaction mechanism, both with respect to substrate addition and product release. Product inhibition by NADP is non‐competitive versus d‐isocitrate at sub‐saturating concentrations of NADP, and competitive with respect to NADP at all d‐isocitrate concentrations. 2‐Oxoglutarate inhibition is competitive versus d‐isicitrate at all NADP concentrations. The inhibition by 2‐oxoglutarate versus NADP is either non‐competitive or uncompetitive. Tricarballylic acid (a substrate analogue of d‐isocitric acid) gives competitive inhibition versus d‐isocitrate at all NADP levels and mixed inhibition versus NADP, at sub‐saturating levels of d‐isocitrate.
Correlation of Enzymatic Activity and Anticoagulant Properties of Phospholipase A2
Authors: VERHEIJ Hubertus M. , BOFFA Marie‐Claire , ROTHEN Christophe , BRYCKAERT Marie‐Claude , VERGER Robert , de HAAS Gerard H.
Published: March 2005

Some highly purified phospholipases A from the venom of viperidae, crotalidae and elapidae were found to have anticoagulant properties. All phospholipases which exhibited anticoagulant properties are characterized by a high isoelectric point, but not all strongly basic phospholipases are anticoagulant. Anticoagulant phospholipases hydrolyse highly packed monomolecular films of phospholipids without any lag time while non‐anticoagulant phospholipases present considerable induction times indicative of a low penetrating power.

When the ester linkages in the procoagulant lipids were replaced by the non‐hydrolysable ether bonds, the mixture retained its clotting ability even in the presence of phospholipases, thus suggesting that anticoagulant phospholipases prevent clot formation by hydrolysis of phospholipids. This was confirmed by chemical modification of phospholipases, viz. alkylation of the activecentre histidine with 1‐bromo‐octan‐2‐one. This modification yielded proteins which had lost their anticoagulant properties but which retained a high affinity for phospholipids.
The Inhibition and Activation of Ca2+‐Dependent Phosphatidylinositol Phosphodiesterase by Phospholipids and Blood Plasma
Authors: DAWSON Rex M. C. , HEMINGTON Norma , IRVINE Robin F.
Published: March 2005

Phosphatidylinositol‐phosphodiesterase (EC 3.1.4.10) was strongly inhibited when ovophosphatidylcholine and saturated phosphatidylcholines with acyl chain lengths of more than eight carbon atoms were mixed with its substrate. In contrast, dihexanoylglycerophosphocholine produces a marked activation attended by a breakdown of the bilayer structure of the substrate.

C12, C14 and C16 lysophosphatidylcholines gave a progressive inhibition of the enzyme with increasing chain length; C10 lysophosphatidylcholine activated the reaction.

The enzyme was strongly inhibited by blood plasma and serum and by blood lipoproteins.

Phosphatidic acid, and certain lysophosphatidic acids (oleoyl and palmitoylglycerophosphates) activated the hydrolysis at a physiological pH, whereas decanoylglycerophosphate had little effect.

Phosphatidic acid at a concentration of 1% molar total lipid P produced an enhancement of the hydrolysis of the phosphatidylinositol substrate contained in a lipid environment which approximated to the inner (cytoplasmic) lamella of the plasma membrane bilayer of the liver sinusoidal cell surface.

The possible role of phospholipid molecules adjacent to phosphatidylinositol in controlling the hydrolysis of the latter in the proximity of a cell surface receptor is discussed. It is suggested that phosphatidic acid formed by the action of diacylglycerol kinase could amplify any increased hydrolysis of phosphatidylinositol which results from stimulation of the cell by an agonist.
Maternal Inheritance of Cytochrome f in Interspecific Nicotiana Hybrids
Authors: GRAY John C.
Published: March 2005

Cytochrome f has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis, from the leaves of Nicotiana tabacum, Nicotiana glutinosa and their reciprocal hybrids. The cytochrome was extracted from chloroplast membranes by sonication in 2% Triton X‐100 and 4M urea and was subsequently purified by acetone precipitation and chromatography on Ultrogel AcA 22 in the presence of cholate and on Sephadex G‐200 in the presence of sodium dodecylsulphate. The purified cytochrome had a molecular weight of 32700 determined by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate. A difference in the primary structure of cytochrome f from N. tabacum and N. glutinosa was detected by ion‐exchange chromatography of the products of trypsin hydrolysis. Cytochrome f from N. tabacum contained an additional peptide not present in the cytochrome from N. glutinosa. This additional peptide was present in the cytochrome from N. tabacum ♀× N. glutinosa ♂, but not in the cytochrome from N. glutinosa ♀× N. tabacum ♂, indicating a maternal mode of inheritance of the primary structure of cytochrome f. This suggests an extranuclear, probably chloroplast, location for the genetic information for cytochrome f.
Metabolism of Lipid‐Linked Oligosaccharide Intermediates in Rat Spleen Lymphocytes
Authors: HOFLACK Bernard , CACAN René , VERBERT André
Published: March 2005

Double‐labelling experiments show that intact lymphocytes as well as lymphocyte homogenates can utilize GDP‐[14C]mannose and UDP‐N‐[3H]acetylglucosamine to synthesize lipid‐linked oligosaccharide intermediates. However, the intermediates formed are quantitatively and qualitatively different in the two systems. The amount of dolichyl diphosphate oligosaccharides synthesized in both cases was calculated by using external labelling by sodium born [3H]hydride reduction of the glycan moiety obtained after mild acid treatment of [14C]mannose‐labelled dolichyl diphosphate oligosaccharides. This showed that, due to the liberation of intracellular enzymes, a larger amount of dolichyl diphosphate oligosaccharides was synthesized by homogenate. However, this higher glycosyltransferase activity was not detected by the direct measurement of incorporation of labelled GDP‐[14C]mannose and UDP‐N‐[3H] acetylglucosamine, due to isotopic dilution caused by both endogenous soluble UDP‐N‐acetylglucosamine and membrane‐bound dolichyl phosphate mannose accumulated during the homogenization process. In addition, endogenous UDP‐glucose allowed the formation, by homogenate, of glucosylated delichyl diphosphate oligosaccharides which were not observed with intact cells unless exogenous UDP‐glucose was added. These striking differences between the lipid intermediates synthesized by homogenate or by intact cells exclude the possibility that intracellular glycosyltransferases could account for the glycosyltransferase activities observed with whole lymphocyte suspensions. This allows us to conclude that ectoglycosyltransferases involved in the dolichol cycle are present at the outer surface of lymphocytes.
Subcellular Location of Semicarbazide‐Sensitive Amine Oxidase in Rat Aorta
Authors: WIBO Maurice , DUONG A. Tuyet , GODFRAIND Théophile
Published: March 2005

With tyramine as substrate, a considerable part of the amine oxidase activity of rat aorta was inhibited by 0.1 mM semicarbazide. The residual activity was little affected by 1 mM semiicarbazide. Oxidation of 5‐hydroxytryptamine was not inhibited by 0.1 mM semicarbazide. The subcellular location of the semicarbazide‐sensitive and semicarbazide‐resistant amine oxidases was investigated by analytical density gradient centrifugation. The semicarbazide‐resistant enzyme was identified with the mitochondrial monoamine oxidase, located in the outer envelope of mitochondria. The semicarbazide‐sensitive amine oxidase was ascribed to the plasma membrane because it was distributed like 5′‐nucleotidase and (oligomycin‐insensitive) Mg2+‐ATPase in various fractionation experiments, and markedly shifted by digitonin towards higher equilibrium densities in sucrose gradient.
Purification and Characterization of Nucleoside Diphosphatase from Rat‐Liver Microsomes
Authors: OHKUBO Iwao , ISHIBASHI Teruo , TANIGUCHI Naoyuki , MAKITA Akira
Published: March 2005

Nucleoside diphosphatase was purified from rat liver microsomes more than 3000‐fold with a 16% yield using a procedure including concanavalin‐A‐Sepharose and phenyl‐Sepharose column chromatography. The purified enzyme had a specific activity of 2500 units/mg protein and appeared homogeneous by gel electrophoresis. The enzyme had a sedimentation coefficient of 6.5 S by sucrose‐density gradient centrifugation, and a Stokes' radius of 4.8 nm was estimated by the gel filtration technique. Its molecular weight is 130000, but only one single band of Mr 65000 was detected after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The native enzyme seems thus to be composed of two identical subunits. The purified enzyme was confirmed to be a glycoprotein containing approximately 9% carbohydrates. The enzyme had a pH optimum of 7. 5, an isoelectric point of 4.85 and a Km of 2. 5 mM for UDP.

On the basis of direct measurement of metal content in the native enzyme, the rat liver nucleoside diphosphatase was found to be a metalloenzyme containing 0.9 mol zinc and 0.1 mol manganese/mol 65000‐Mr subunit. Metal‐free nucleoside diphosphatase has been prepared. The activity of the metal‐free enzyme was restored by the addition of several divalent cations, zinc being the most effective.
Purification and Primary Structure Determination of the N‐Terminal Blocked Protein, L11, from Escherichia coli Ribosomes
Authors: DOGNIN Mai J. , WITTMANN‐LIEBOLD Brigitte
Published: March 2005

Protein L11 was isolated from the 50‐S subunit of Escherichia coli ribosomes, using two salt extractions and two chromatographic separations on CM‐cellulose. The unusual behavior of the protein when run on sodium dodecyl sulfate electrophoresis showed multiple bands.

The complete primary structure of protein L11 is presented in detail. Its sequence was derived from peptides obtained by digesting the protein with trypsin, chymotrypsin, thermolysin, Staphylococcus aureus protease and, after modification, with trypsin. Chemical cleavage was performed with cyanogen bromide. Sequencing of the various peptides was achieved by manual micro‐dansyl‐Edman degradations and automatic methods. The N‐terminal residue of the protein is blocked and was not degradable in the liquid‐phase sequenator by the Edman method. It was identified by a combination of enzymatic cleavage and mass spectrometry.

Protein L11 contains three methylated amino acid residues, a Nα‐trimethylalanine, and two residues of Nα‐trimethyllysine. Their behaviour and influence in the sequence elucidation are described. The protein contains 141 amino acid residues and has a molecular weight of 14874. Secondary structure predictions of the protein are given, and its sequence is compared with those of other E. coli ribosomal proteins.
A Critical Period of Ecdysterone Action on Sensitive Clones of Drosophila Cultured in vitro: the Maturation of the Cells
Authors: BEST‐BELPOMME Martin , COURGEON Anne‐Marie
Published: March 2005

Ecdysterone‐sensitive clones cultured in vitro were isolated from established cell lines of Drosophila melanogaster. The clones FC and 89K are ecdysterone‐inducible for two enzymatic activities: acetylcholinesterase and β‐galactosidase. No activity could be detected in untreated cells, whereas after treatment with 50–250 nM ecdysterone, the activity appeared after one day and increased during 3–4 days.

We wanted to modulate the response of the cells by varying the conditions of the hormonal stimulus. Mimicking the physiological situation of Drosophila (the ecdysterone peak corresponding to the molts is preceded by low levels) we pretreated the cells with a subthreshold concentration (1–5 nM) for 2 days and then we added the stimulating concentration of 50–250 nM ecdysterone. The enzymatic activities were then detectable within the following hours and the final level of induction was about twice the one of cells without pretreatment.

Thus, the continuous presence of a subthreshold concentration of ecdysterone provokes the maturation of the cells which become able to respond to the hormonal stimulus by a quicker and higher enzymatic induction.

The cellular maturation seems to be a critical period. It is altosid‐sensitive. Altosid (a juvenile hormone analog) abolishes the effects of the ecdysterone‐induced maturation.
Glucosyltransferase Activities in Liver Mitochondria
Authors: GATEAU Odile , MORELIS Renee , LOUISOT Pierre
Published: March 2005

Mitochondria, and specially outer mitochondrial membranes, incorporate d‐[125C]glucose from UDP‐d‐[14C]glucose into products extracted with organic solvents and into a residual precipitate, with a pH optimum of about 6.5 in (2‐N‐morpholine‐ethane)‐sulfonic acid (MES) buffer.

The chloroform/methanol (2:1, v/v) extract contains two products. The major [14C]glucolipid is stable to mild alkali, but releases [14C]glucose upon mild acid hydrolysis. It is retained on DEAE‐cellulose (acetate form) and is eluted with the same ionic strength as an hexosyldolichyl monophosphate diester. This [14C] glucolipid has the same chromatographic behaviour as dolichyl‐mannosylphosphate in neutral, acidic and basic solvent systems; and its biosynthesis is greatly increased by exogenous dolichylmonophosphate. the other [14C]glucolipid is stable upon mild acid hydrolysis and is not retained on DEAE‐cellulose. On silicic acid it is eluted with acetone. The biosynthesis of this compound is stimulated by exogenous ceramide. This glucolipid has the same chromatographic mobility in different solvent systems as glucosylceramide isolated from the liver of a patient with Gaucher's disease. Biosynthesis of these two glucolipids is inhibited by UDP, but only biosynthesis of dolichylglucosyl monophosphate is reversible with this nucleotide.

The biosynthesis of these different glucosylated derivatives is stimulated by the addition of divalent cations (Mn2+, Mg2+). The effect of these two metal ions on dolichylglucosyl monophosphate and glucosylceramide formation is studied in different conditions.
A γ‐like DNA Polymerase in Spinach Chloroplasts
Authors: SALA Francesco , AMILENI Anna R. , PARISI Bruno , SPADARI Silvio
Published: March 2005

A DNA polymerase has been extracted from spinach chloroplasts and purified by chromatography on DEAE‐cellulose and hydroxyapatite. A great similarity between the purified chioropiast polymerase and the mammalian mitochondrial DNA polymerase γ was found by several criteria: preference for the synthetic primer‐template (dT)12–18· poly(rA), optimal requirement for Mn2+ (0.1–1.0 mM), KCl (100 mM) and pH (8–9), high relative molecular mass (approximately 105000), resistance to aphidicolin and inhibition by N‐ethylmaleimide. Some peculiar features of the chloroplast DNA polymerase have, however, been noticed.

The mammalian DNA polymerase y has been suggested to be responsible for the replication of mitochondrial DNA. Thus, both the presence of a γ‐like DNA polymerase in chloroplasts and the similarities between the chloroplast and the mitochondrial DNA (absence of a nucleosomal structure and presence of displacement loops) lead to the suggestion that chloroplast DNA is also replicated by a γ‐like DNA polymerase and that the γ polymerases present in eukaryotes are, therefore, involved in a strand‐displacement DNA synthesis.

An α‐like DNA polymerase activity, present and predominant in crude leaf extracts, was practically absent from purified chloroplast preparations.
Improved Procedures for Purification of the Bandeiraea simplicifolia I Isolectins and Bandeiraea simplicifolia II Lectin by Affinity Chromatography
Authors: DELMOTTE Francis M. , GOLDSTEIN Irwin J.
Published: March 2005

Bandeiraea simplicifolia plant seeds contain a family of five α‐d‐gal actopyranosyl‐binding isolectins (BS I‐A4, A3B, A2B2, AB3, B4) and N‐acetyl‐d‐glucosamine‐binding lectin (BS II). After Pi/NaCl extraction and ammonium sulfate fractionation BS II is specifically adsorbed onto p‐aninobenzyl‐1‐thio‐N‐acetyl‐β‐d‐glucosaminide‐succinylaminohexylaminyl—Sepharose‐4B. The BS I isolectins pass through this column and BS II is selectively eluted by Pi/NaCl containing 2 mM N‐acetyl‐d‐glucosamine or by 0.1 M sodium acetate buffer pH 3.6. The material not bound to the column is loaded onto p‐aminophenyl‐β‐d‐galactopyranosyl‐succinylaminohexylaminyl—Sepharose 4B. BS I‐A4 is specifically eluted in a sharp peak with Pi/NaCl containing 1 mM N‐acetyl‐d‐galactosamine. Then, BS I‐A3B, A2B2, AB3 and B4 are selectively eluted, in a single peak for each isolectin, with Pi/NaCl containing 3 mM, 8 mM, 15 mM and 50 mM methyl α‐d‐galactopyranoside, respectively.
Biogenesis of Glyoxysomes
Authors: ZIMMERMANN Richard , NEUPERT Walter
Published: March 2005

Biosynthesis of isocitrate lyase, a tetrameric enzyme of the glyoxysomal matrix, was studied in Neurospora crassa, in which the formation of glyoxysomes was induced by a substitution of sucrose medium by acetate medium.

Translation of Neurospora mRNA in reticulocyte lysates yields a product which has the same apparent molecular weight as the subunit of the functional enzyme. Using N‐formyl[35S]methionyl tRNAMetf as a label, the translation product shows the same apparent size which indicates that the amino terminus has no additional ‘signal’‐type sequence.

Read‐out systems employing free and membrane‐bound polysomes show that only free ribosomes are active in the synthesis of isocitrate lyase.

Isocitrate lyase synthesized in reticulocyte lysate is released into the supernatant and is soluble in a monomeric form. It interacts with Triton X‐100 to form mixed micells in contrast to the functional tetrameric form.

Transfer of isocitrate lyase synthesized in vitro into isolated glyoxysomes is suggested by results of experiments in which supernatants from reticulocyte lysates are incubated with a particle fraction isolated from acetate‐grown cells. No transfer occurs when particles from non‐induced cells are employed. Resistance to added proteinase is used as a criterion for transmembrane transfer.

The data support a post‐translational transfer mechanism for isocitrate lyase. They suggest that isocitrate lyase passes through a cytosolic precurscr pool as a monomer and is transferred into glyoxysomes.
RNA Synthesis in Rabbit Endometrial Nuclei
Authors: MÜLLER Heidrun , BEATO Miguel
Published: March 2005

Endometrical nuclei, prepared front rabbits subjected to different hormonal treatments, were used for the cell‐free synthesis of RNA. Optimal conditions for the incorporation of [3H]UMP into RNA are described, leading to the synthesis of relatively undegraded RNA molecules. Under these conditions there is virtually no initiation of new RNA chains in vitro, and RNA chain elongation is inhibited up to 60% by low concentrations of α‐amanitin and up to 90% by actinomycin D. The synthesis of RNA is slightly inhibited in the presence of Hg‐CTP and monothioglycerol, but newly synthesized mercurated RNA can be efficiently separated from endogenous RNA upon chromatography on sulfhydryl‐Sepharose under stringent conditions. The RNA synthesized in vitro by endometrial nuclei from pseudopregnant rabbits contains RNA sequences transcribed from the uteroglobin gene, as demonstrated by hybridization to an excess of purified preuteroglobin cDNA. In endometrial cells from pseudopregnant animals the number of RNA polymerase II molecules transcribing the uteroglobin gene is 12‐fold higher than in control animals, demonstrating that at least part of the hormoNatly induced accumulation of preuteroglobin mRNA is due to an increased rate of transcription of the uteroglobin gene.
Synthesis in vitro of Keratin Polypeptides Directed by mRNA Isolated from Newborn and Adult Mouse Epidermis
Authors: SCHWEIZER Jürgen , GOERTTLER Klaus
Published: March 2005

A method is described which allows the isolation of undegraded total cellular RNA from living epidermis in a one‐step procedure. Epidermal tissue is homogenized in 4 M guanidinium thiocyanate/2% sarkosyl buffer and centrifuged through a 5.7 M CsCl cushion. Under appropriate density conditions in the homogenate, large amounts of RNA are sedimented free of DNA and protein to the bottom of the tubes. A prerequisite for the applicability of this method is the preparation of pure epidermis under conditions which efficiently inactivate endogenous RNases.

Oligo(dT) affinity chromatography of total RNA yields 3–4% of polyadenylated RNA. Translation of poly(A)‐rich fractions from both newborn and adult mouse epidermis in a cell‐free reticulocyte system leads to the synthesis of proteins with electrophoretic mobilities identical to those of native keratin polypeptides of the corresponding tissue. Similar to native keratin polypeptides, these proteins are highly insoluble and can selectively be enriched by high‐salt extraction of the translation assays. The CNBr cleavage pattern of these purified proteins is identical to that of native keratins. There is no evidence for the synthesis of precursors of these proteins.
Further Electron Microscope Studies on Pyruvate Carboxylase
Authors: MAYER Frank , WALLACE John C. , KEECH D. Bruce
Published: March 2005

Using negative staining and electron microscopic tilting techniques in conjunction with modelling experiments, the fine structure of chicken, sheep and rat pyruvate carboxylases has been studied. The overall configuration appears to be a tetrahedron‐like structure consisting of two pairs of subunits in two different planes orthogonal to each other with the opposing pairs of subunits interacting with each other on their convex surfaces.

The predominant form of the enzyme particles mounted and stained in the presence of acetylcoenzyme A consisted of a compact, triangular outline enclosing three readily visible intensity maxima. When samples were mounted in the absence of acetyl‐coenzyme A the molecules were more ‘open’ predominantly rhomboid structures. From tilting experiments it is concluded that the rhomboid images found in the absence of acetyl‐coenzyme A represent partly or wholly flattened forms of the tetrahedron‐like molecule. A feature of the enzyme when mounted in the absence of acetyl‐coenzyme A was the existence of a ‘cleft’ along the longitudinal midline of each subunit, suggesting that the subunits may consist of two distinct domains.
Separation and Characterization of an Autolytic Endo‐β‐glucosaminidase from Bacillus cereus
Authors: KAWAGISHI Shigenori , ARAKI Yoshio , ITO Eiji
Published: March 2005

An autolytic endo‐β‐glucosaminidase, capable of cleaving the glycoside linkages of N‐unsubstituted glucosamine in the glycan moiety of cell wall peptidoglycan, was purified 470‐fold from a salt extract of the 20000 x g precipitate fraction obtained after sonication of a lysozyme‐resistant strain of Bacillus cereus. The properties of this enzyme were studied.

The purified enzyme preparation was also active towards the glycan chain of fully N‐acetylated cell wall peptidoglycan.

The endo‐β‐glucosaminidase was inactive towards the cell wall peptidoglycan unless the peptide portion of this polymer was removed either by the action of N‐acetylmuramyl‐l‐alanine amidase or by the treatment with alkali in aqueous dimethyl sulfoxide.

Studies on the action of this enzyme towards chemically modified glycans revealed that the carboxyl groups of muramic acid residues are indispensable to a substrate for this enzyme.
Fluorescence Probes as Monitors of Surface Membrane Fluidity Gradients in Murine Fibroblasts
Authors: SCHROEDER Friedhelm
Published: March 2005

Fluorescence probe molecules were utilized in conjunction with quenching agents to investigate the possibility that mammalian cell surface membranes may display vertical asymmetry of physico‐chemical properties. Two different approaches indicated that the outer monolayer of a murine fibroblast surface membrane was more fluid than the inner monolayer, First, a fluorescent glucosamine derivative of trans‐parinaric acid did not penetrate intact LM cells (a strain of transformed murine fibroblasts) or phagosomes derived from these cells. This probe molecule was easily accessible to nonpenetrating quenching agents such as trinitrophenyl‐glycine, indicating that it resided in the exposed membrane monolayer. The fluorescence polarization, P, of glucosamine trans‐parinarate in intact LM cells was 0.218 ± 0.009 while in phagocytosed latex bead membranes the polarization was 0.248 0.006. Second, trans‐parinaric acid, 1,6‐diphenyl‐1,3,5‐hexatriene, and N‐phenyl‐l‐naphthylamine were used as fluorescent membrane probes in isolated plasma membrane vesicles. Their fluorescence in the plasma membrane was quenched by either covalently linking trinitrophenyl groups to exposed NH2 constituents on the membrane surface or by adding the water‐soluble non‐penetrating quenching agent, trinitrophenyl‐glycine. Trinitrophenyl‐amino groups have an absorption maximum at 415 nm and can therefore chemically quench the fluorescence of trans‐parinarate, 1,6‐diphenyl‐1,3,5‐hexatriene, and N‐phenyl‐1‐naphthylamine which have fluorescence emission maxima near 415 nm. With both methods, when only the outer monolayer amino groups were trinitrophenylated, the absorption‐corrected fluorescence emission and the relative fluorescence efficiency of trans‐parinarate in the plasma membrane were decreased by 45 ± 5% and 44 ± 6% respectively. Neither quenching method altered the fluorescence lifetime of trans‐parinarate. In contrast, when both sides of the plasma membrane had covalently linked trinitrophenyl groups, these parameters were diminished by 90–95%. Similar results were obtained with 1,6‐diphenyl‐1,3,5‐hexatriene and N‐phenyl‐1‐naphthylamine. Polarization measurements of trans‐parinarate indicated that the inside monolayer of the membrane was more rigid (P= 0.364 ± 0.005) than the whole membrane (P= 0.323 ± 0.005). Similar results were obtained with 1,6‐diphenyl‐l,3,5‐hexatriene. However, N‐phenyl‐1‐naphthylamine which resides near the polar interface of the bilayer showed no difference in polarization upon quenching by either method. Thus, results obtained with glucosamine trans‐parinarate in the absence of quenching agents or with trans‐parinarate and 1,6‐diphenyl‐1,3,5‐hexatriene in the presence of quenching agents indicated that a vertical asymmetry or gradient of certain physicochemical parameters may exist in LM cell plasma membranes. Preliminary data indicated that this asymmetry could be altered by lipid polar head group manipulation.
Activity Determination of 3‐Iodopyridineadenine Dinucleotide and Its Phosphate as Hydride Acceptors in the Presence, of Dehydrogenases Using a Coupled Redox System
Authors: ABDALLAH Mohamed A. , BIELLMANN Jean‐François
Published: March 2005

A new procedure for the activity measurement of NAD(P)+‐dependent dehydrogenases has been devised using an electron‐transferring agent, phenazine methosulfate, and an electron acceptor, 3‐(4,5‐dimethylthiazolyl)‐2,5‐diphenyltetrazolium bromide. The reduction of the latter is determined by an increase in absorbance at 578 nm. 3‐Iodopyridineadenine dinucleotide was found to be active as an hydride acceptor with horse liver alcohol dehydrogenase and lactate dehydrogenase but showed no activity with glyceraldehyde‐3‐phosphate dehydrogenase nor did its phosphate with 3‐phosphogluconate dehydrogenase.