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fATTY-ACID DESATURASE ACTIVITIES ARE MODULATED BY PHYTOSTEROL INCORPORATION IN MICROSOMES

BIOCHIMICA ET BIOPHYSICA ACTA 1989年第2期1005卷 187-191页

作者： LEIKIN, AI BRENNER, RR NATL UNIV LA PLATA CONSEJO NACL INVEST CIENT & TECN FAC CIENCIAS MED RA-1900 LA PLATA ARGENTINA

The effect of phytosterol-rich diets (3% .beta.-sitosterol+2% campesterol) on rat liver microsomal fatty acid desaturases, membrane dynamics and lipid composition was investigated. After a 21 day period, phytosterol was incorporated into microsomes and the membrane fluidity decreased. There were no changes in either the phospholipid composition or in the total sterol content. However, the phytosterol/cholesterol ratio increased. In the animals fed phytosterols, the .delta.5-, .DELTA.6- and .DELTA.9-fatty acid desaturases were significantly more active than in control animals. The changes in the lipid fatty acid composition were consistent with those of the desaturase activities. Hence, it is suggested that: (1) dietary phytosterol modulates desaturase activities;(2) phytosterols make the membrane more rigid but do not induce changes in the relative phospholipid composition;(3) .DELTA.9-, .DELTA.5- and .DELTA.6-desaturase activities increase when the membrane becomes more rigid without changes in the phospholipid composition.

关键词： fatty acid desaturase fatty acid composition Lipid composition Phytosterol Membrane fluidity (Rat liver microsome) C cholesterol P total phosphorus f phytosterol PC phosphatidylcholine PE phosphatidylethanolamine PI phosphatidylinositol SM sphingomyelin DPH 1,6-diphenyl-1,3,5-hexatriene

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INTERSPECIES HOMOLOGY Of CYTOCHROME-P-450 - TOXICOLOGICAL SIGNIfICANCE Of CYTOCHROME-P-450 CROSS-REACTIVE WITH ANTI-RAT P-448-H ANTIBODIES IN LIVER-MICROSOMES fROM DOGS, MONKEYS AND HUMANS

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MUTATION RESEARCH 1989年第3期226卷 163-167页

作者： OHTA, K KITADA, M OHI, H KOMORI, M NAGASHIMA, K SATO, N MUROYA, K KODAMA, T NAGAO, M KAMATAKI, T HOKKAIDO UNIV FAC PHARMACEUT SCI DEPT ANALYT BIOCHEM SAPPORO HOKKAIDO 060 JAPAN HOSP HOKKAIDO ASSOC MED SERV WORKERS SAPPORO 065 JAPAN HOKKAIDO UNIV SCH MED DEPT PATHOL SAPPORO HOKKAIDO 060 JAPAN SAPPORO MED COLL DEPT PATHOL SAPPORO HOKKAIDO 060 JAPAN NATL CANC CTR RES INST TOKYO 104 JAPAN

The mutagenic activation of various promutagens by liver microsomes from dogs, monkeys and humans was investigated. Dog liver microsomes efficiently catalyzed the mutagenic ativation of Trp-P-2 and Glu-P-1 followed by IQ and AAf. Monkey liver microsomes were most active in the activation of IQ followed by Glu-P-1, AAf and Trp-P-2. Although there were remarkable individual differences, human liver microsomes were found to be most active in the mutagenic activation of IQ followed by Trp-P-2, Glu-P-1 and AAf. Antibodies against rat P-448-H inhibited the mutagenic activation of Glu-P-1, Trp-P-2 and IQ in rat and dog liver microsomes, and Glu-P-1 and Trp-P-2 in monkey liver microsomes. The activation of Glu-P-1 and IQ in human liver microsomes was also strongly inhibited by anti-P-448-H antibodies. The amounts of cytochrome P-450 cross-reactive with anti-P-448-H antibodies in human liver microsomes highly correlated with the capacity to active Glu-P-1, Trp-P-2 and IQ but not AAf.

关键词： Cytochrome P-450 P-448-H Rats Dogs Monkeys Humans SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis PAP peroxidase anti-peroxidase EDTA ethylenediamine tetraacetic acid Trp-P-2 3-amino-1-methyl-5 H -pyrido[4,3- b ]indole acetate Glu-P-1 2-amino-6-methyl-dipyrido[1,2- a :3′,2′- d ]imidazole acetate IQ 2-amino-3-methyl-imidazo[4,5- f ]quinoline AAf 20acetylaminofluorene

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PROLACTIN REGULATION Of MOUSE MAMMARY-TUMOR VIRUS (MMTV) EXPRESSION IN NORMAL MOUSE MAMMARY EPITHELIUM

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MOLECULAR AND CELLULAR ENDOCRINOLOGY 1989年第1期62卷 23-29页

作者： MUNOZ, B BOLANDER, ff UNIV S CAROLINA DEPT BIOL COLUMBIA SC 29208 USA

The hormonal regulation of mouse mammary tumor virus (MMTV) RNA in normal mouse mammary epithelium was studied in an explant system. In tissue from parous mice physiological concentrations of prolactin stimulated MMTV expression, while only pharmacological concentrations of cortisol were effective. Regulation in explants from virgin mice was similar to that in parous animals except that the former were less sensitive to prolactin;this relative unresponsiveness may explain why uninduced tissue from virgin mice does not express MMTV RNA, while that from parous mice does exhibit some basal production. These results suggest that prolactin plays a major role in MMTV expression in normal mammary epithelium and that glucocorticoids may only have a permissive effect or may act through an indirect mechanism requiring high concentrations. These data also suggest that the greater susceptibility of parous mice to MMTV-induced tumorigenesis may reflect the greater prolactin sensitivity in the glands from these animals.

关键词： Mouse mammary tumor virus Mammary gland Prolactin Glucocorticoid Insulin MMTV mouse mammary tumor virus I insulin f cortisol P prolactin T 3 triiodothyronine

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CONfORMATIONAL-CHANGES Of (CA-2+-MG-2+)-ATPASE Of ERYTHROCYTE PLASMA-MEMBRANE CAUSED BY CALMODULIN AND PHOSPHATIDYLSERINE AS REVEALED BY CIRCULAR-DICHROISM AND fLUORESCENCE STUDIES

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BIOCHIMICA ET BIOPHYSICA ACTA 1989年第2期986卷 263-270页

作者： WRZOSEK, A fAMULSKI, KS LEHOTSKY, J PIKULA, S M NENCKI INST EXPTL BIOL DEPT MUSCLE BIOCHEM PASTEURA 3 PL-02093 WARSAW POLAND M NENCKI INST EXPTL BIOL DEPT CELLULAR BIOCHEM PL-02093 WARSAW POLAND

Two spectroscopic techniques, circular dichroism and steady-state fluorescence, were employed in order to study conformational changes of the purified, detergent-solubilized (Ca2+-Mg2+)-ATPase of porcine erythrocyte ghost membranes. Circular dichroism (CD) spectra in the peptide region were obtained from the purified (Ca2+-Mg2+)-ATPase of porcine erythrocyte ghost membranes with the aim to investigate the secondary structure of the enzyme in the presence of calmodulin (CaM) or phosphatidylserine (PS), as well as in the E1 and E2 states. The E1 conformation was stabilized by 10 .mu.M free Ca2+, while the E2 conformation was stabilized by 0.1 mM ethylene glycol bis(2-aminoethylether)-N,N,N'',N''-tetraacetic acid (EGTA). It was found that the E1 and E2 states of the enzyme strikingly differed in their secondary structure (66% and 46% of calculated .alpha.-helix content, respectively). In the presence of Ca2+, PS decreased the helical content of the ATPase to 61%, while CaM to 55%. Quenching of intrinsic fluorescence of (Ca2+-Mg2+)-ATPase by acrylamide, performed in the presence of Ca2+, gave evidence for a single class of tryptophan residues with Stern-Volmer constant (KSV) of 10 M-1. Accessibility of tryptophan residues varied depending on the conformational status of the enzyme. Addition of PS and CaM decreased the KSV value to 76 M-1 and 8.5 M-1, respectively. In the absence of Ca2+, KSV was 7.0 M-1. KI and CsCl were less effective as quenchers. The fluorescence energy transfer between (Ca2+-Mg2+)-ATPase tryptophan residues and dansyl derivative of covalently labeled CaM occurred in the presence of EGTA, but was further promoted by Ca2+. It is concluded that the interaction of CaM and PS with (Ca2+-Mg2+)-ATPase results in different conformational states of the enzyme. CD and fluorescence spectroscopy allowed to distinguish these states from the E1 and E2 conformational forms of the ATPase.

关键词： Erythrocyte plasma membrane Calcium pump ATPase, Ca 2+ - Conformational state Circular dichroism fluorescence energy transfer fluorescence quenching (Porcine blood) CD circular dichroism CaM calmodulin PS phosphatidylserine PC phosphatidylcholine EGTA ethylene glycol bis(2-aminoethyl ether)- N , N , N ′, N ′-tetraacetic acid DDAO (Ammonyx LO) N , N -dimethyloctadecylamine- N -oxide Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid PhMeSO 2 f phenylmethylsulfonyl fluoride K SV Stern-Volmer constant for quenching of intrinsic fluorescence of tryptophan residue f 0 fluorescence intensity without quencher f fluorescence intensity with quencher

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STIMULATION Of fA AND PHOSPHATASE-1 ACTIVITIES BY INSULIN IN 3T3-L1 CELLS

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fEBS LETTERS 1989年第2期258卷 208-210页

作者： VILLAMORUZZI, E Istituto di Patologia Generale Università di Pisa Italy.

The phosphatase-1 activator fA and phosphatase-1 were assayed in 3T3-L1 cells exposed to insulin. The cytosolic fA activity was transiently stimulated (7-8-fold) 1 and 2 min after exposure to 10(-8) M insulin and returned to control values within 5-10 min. Cytosolic phosphatase-1 (assayed after trypsin treatment) was activated (120-140% of controls) between 2 and 5 min and returned to control values within 10 min. Insulin effects were dose-dependent, with maximum stimulation of both activities at 10(-8) M insulin. The possibility that fA and other kinases mediate phosphatase activation by insulin is discussed.

关键词： Protein phosphatase Phosphorylation Insulin Hormone Pase, protein phosphatase Pase-1, protein phosphatase type-1 1–2, phosphatase inhibitor 2, also called modulator f A , kinase that activates Pase-1, identical to glycogen synthase kinase 3 CK-II, casein kinase II CK-I, casein kinase I PDGf, platelet-derived growth factor EGf, epidermal growth factor IGf-1, insulin-like growth factor 1 IBMX, 3-isobutyl-1-methylxanthine PMSf, phenylmethylsulfonyl fluoride ßME, β-mercaptoethanol BSA, bovine serum albumin

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PROTEINS Of NEWCASTLE-DISEASE VIRUS ENVELOPE - INTERACTION BETWEEN THE OUTER HEMAGGLUTININ-NEURAMINIDASE GLYCOPROTEIN AND THE INNER NON-GLYCOSYLATED MATRIX PROTEIN

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BIOCHIMICA ET BIOPHYSICA ACTA 1989年第2期999卷 171-175页

作者： GARCIASASTRE, A CABEZAS, JA VILLAR, E UNIV SALAMANCA FAC BIOL DEPT BIOQUIM & BIOL MOLEC PL MERCED 1 E-37008 SALAMANCA SPAIN

Using linear sucrose-density ultracentrifugation analysis of Triton-solubilized Newcastle Disease virus envelopes, we have evidenced, for the first time, the existence of interactions between the outer hemagglutinin-neuraminidase transmembrane glycoprotein and the inner non-glycosylated peripheral matrix protein. Such interactions seem to be electrostatic. These conclusions are based on the behavior of both proteins at different ionic strengths. When in low ionic strength buffer, hemagglutinin-neuraminidase and matrix proteins band together in the sucrose gradient, whereas at high ionic strength both proteins band at different rates in the gradient. The behavior of the inner matrix protein in our conditions was the expected one for a peripheral protein. The results of these ''in vitro'' studies are also discussed in terms of the possible ''in vivo'' role of such interactions.

关键词： Newcastle Disease Virus Sialidase Hemagglutinin-neuraminidase Protein M HN hemagglutinin-neuraminidase glycoprotein f fusion glycoprotein L large protein M matrix protein NDV Newcastle Disease Virus NP nucleocapsid protein P phosphoprotein SDS sodium dodecyl sulfate

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REVERSIBLE MODULATION Of THE MITOCHONDRIAL ATP SYNTHASE WITH ENERGY DEMAND IN CULTURED RAT CARDIOMYOCYTES

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fEBS LETTERS 1989年第1-2期256卷 97-100页

作者： DAS, AM HARRIS, DA UNIV OXFORD DEPT BIOCHEMS PARKS RDOXFORD OX1 3QUENGLAND

The ATP synthase capacity of rat heart myocytes can be measured in sonicates of cultured cardiomyocytes. In these cells, transitions in ATP synthase capacity occur on changing to the anoxic or uncoupled state (drop in ATP synthase capacity of over 40%) or on electrically stimulating the cells to contract (rise of 70%). These changes occur rapidly (half time less than l min) and are completely reversed on returning to the original conditions. It is proposed that mitochondria in vivo are directly regulated at the level of the ATP synthase. The naturally occurring inhibitor protein from mitochondria may be responsible for this regulation. Mitochondria; Cardiomyocyte; ATP synthase; ATPase inhibitor protein; Metabolic regulation; Anoxia

关键词： f 1 , soluble portion of the mitochondrial ATP synthase complex If 1 , naturally occurring inhibitor protein of f i fCCP, carbonyl cyanide p -trifluoromethoxyphenyl hydrazone Hepes, 4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid

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DCCD SENSITIVITY Of ELECTRON AND PROTON-TRANSfER BY NADH - UBIQUINONE OXIDOREDUCTASE IN BOVINE HEART SUBMITOCHONDRIAL PARTICLES - A THERMODYNAMIC APPROACH

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BIOCHIMICA ET BIOPHYSICA ACTA 1989年第2期974卷 219-222页

作者： VUOKILA, PT HASSINEN, IE UNIV OULU DEPT MED BIOCHEM KAJAANINTIE 52 A SF-90220 OULU FINLAND

The sensitivity of the H+/2e- ratio of the redox-driven proton pumping by the NADH : ubiquinone reductase (complex I) of the submitochondrial particles to dicyclohexylcarbodiimide (DCCD) was studied by a thermodynamic approach, measuring the membrane potential and .*** across the membrane and the redox potential differences across the complex I span of the respiratory chain. The .***/.DELTA.~.mu.H+ ratio did not decrease upon additions of 50 or 100 nmol of DCCD per mg protein in the presence of oligomycin although the H+/2e- ratio has been demonstrated to decrease upon DCCD addition in kinetic experiments with mitochondria. Complex I then becomes reminiscent of the cytochrome bc1 complex, which shows DCCD sensitivity of the kinetically but not thermodynamically determined H+/2e- ratio.

关键词： NADH: ubiquinone oxidoreductase Proton transfer Dicyclohexylcarbodiimide Membrane potential Submitochondrial particle (Bovine heart) CCCP carbonyl cyanide m -chlorophenylhydrazine complex I NADH : ubiquinone oxidoreductase complex III ubiquinol: cytochrome c oxidoreductase complex IV cytochrome c oxidase complex DCCD dicyclohexylcarbodiimide Δ Δ μ ˜ H + electrochemical potential of protons Δψ membrane potential E h actual redox potential E m standard redox potential at pH specified Δ E redox potential difference ETP, bovine heart submitochondrial particles T absolute temperature R the gas constant f the faraday constant ΔG Gibbs free energy Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid MeNH 3 + methylammonium cation TMPD N,N,N′,N′ -tetramethyl- p -phenylenediamine

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REGULATION Of PHOTOSYNTHESIS IN LEAVES Of C-4 PLANTS fOLLOWING A TRANSITION fROM HIGH TO LOW LIGHT

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BIOCHIMICA ET BIOPHYSICA ACTA 1989年第2期973卷 176-184页

作者： DONCASTER, HD ADCOCK, MD LEEGOOD, RC UNIV SHEFFIELD PHOTOSYNTH RES INST SHEFFIELD S10 2TN S YORKSHIRE ENGLAND UNIV SHEFFIELD DEPT PLANT SCI SHEFFIELD S10 2TN S YORKSHIRE ENGLAND

The aim of this work was to investigate the factors which regulate photosynthetic carbon assimilation in leaves of maize (Zea mays) and Amaranthus edulis following a decrease in photon flux density. The principal observations in maize were that, after a decrease in photon flux density;(i) there were a transient inhibition of the rate of carbon assimilation;(ii) the pool of phosphoenol pyruvate rose while the flux decreased, indicating rapid regulation of the activity of phosphoenolpyruvate carboxylase;(iii) the triose-P/glycerate-3-P ratio declined drastically;at the same time the pool of pyruvate increased several-fold;(iv) the activation state of NADP+-malate dehydrogenase declined rapidly, while the activation states of pyruvate, P1 dikinase, fructose-1,6-bisphosphatase and sucrose phosphate synthase changed relatively slowly;(v) there was a rapid decrease in non-photochemical quenching of chlorophyll fluorescence and a rapid oxidation and re-reduction of QA followed by a slow oxidation of QA;(vi) in A. edulis the content of aspartate and of the total pool of C4-cycle intermediates increased. The observations are discussed in terms of the regulation of C4 photosynthesis and in terms of the supply of, and demand for, the products of electron transport by carbon metabolism. It is suggested that the transient inhibition of photosynthetic carbon assimilation in maize leaves is accompanied by a high demand for ATP which is met by cyclic electron transport.

关键词： Carbon assimilation Chlorophyll fluorescence Photosynthesis, C4 (Zea) (Amaranthus) (Leaf) PGA glycerate-3- P RuBP ribulose-l,5-bisphosphate fBP fructose-l,6-bisphosphate PEP phosphor enol pyruvate G6P glucose-6-phosphate f6P fmctose-6-phosphate f o fluorescence level when all Photosystem II centres are open f m fluorescence yield when all centres are closed q Q photochemical fluorescence quenching q NP non-photochemical fluorescence quenching PfD photon flux density

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ALPHA-3-BETA-3 COMPLEX Of THERMOPHILIC ATP SYNTHASE - CATALYSIS WITHOUT THE GAMMA-SUBUNIT

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fEBS LETTERS 1989年第1期249卷 67-69页

作者： KAGAWA, Y OHTA, S OTAWARAHAMAMOTO, Y Department of Biochemistry Jichi Medical School Tochigi-ken Japan.

A complex of the alpha- and beta-subunits of thermophilic ATP synthase showed about 25% of the ATPase activity of the alpha beta gamma complex. The alpha 3 beta 3 hexamer structure was analyzed by sedimentation (11.2 S) and gel filtration (310 kDa). Dilution of the alpha beta complex caused dissociation of the complex and rapid loss of ATPase activity which was restored by addition of the gamma-subunit. A previous method using urea for isolating the subunits resulted in an alpha beta complex with lower activity than that prepared by over-expression of the genes. The alpha beta-ATP complex was formed from the alpha beta complex, ADP and Pi in the presence of dimethyl sulfoxide.

关键词： f 1 -ATPase Oligomer, α 3 β 3 ATPase ATP synthesis (Thermophilic bacteria) f 1 , catalytic portion of ATP synthase Tf 1 , f 1 from thermophilic bacterium PS3 Ef 1 , f 1 from E. coli AMPPNP, adenyl-5′-yl imidodiphosphate DMSO, dimethyl sulfoxide DTT, dithiothreitol Mes, 4-morpholineethanesulfonic acid α, β and γ, subunits of f 1 HPLC, high-performance liquid chromatography

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