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PURIfICATION AND PROPERTIES Of THE LATENT f1-ATPASE Of MICROCOCCUS-LYSODEIKTICUS

BIOCHIMICA ET BIOPHYSICA ACTA 1979年第2期547卷 230-240页

作者： HUBERMAN, M SALTON, MRJ NYU HOSP SCH MED DEPT MICROBIOL NEW YORK NY 10016 USA

The latent coupling factor (f1)-ATPase of M. lysodeikticus [luteus] was purified to homogeneity as determined by a number of criteria including non-denaturing polyacrylamide gel electrophoresis, crossed immunoelectrophoresis and analytical ultracentrifugation. By inclusion of 1 mM phenylmethyl sulfonyl fluoride, a serine protease inhibitor, in the shock-wash step of release of f1 from the membranes, the spontaneous activation of both crude and purified ATPase by endogenous membrane protease(s) could be prevented, thereby yielding a highly latent ATPase preparation. Equilibrium ultracentrifugation of the latent ATPase gave a MW of 400,000. The ATPase contained 5 different subunits .alpha., .beta., .gamma., .delta. and .epsilon. and their MW determined by SDS[sodium dodecyl sulfate]-polyacrylamide gel electrophoresis were 60,000, 54,000, 37,000, 27,000 and 9000, respectively. The subunit composition was determined with 14C-labeled, f1-ATPase prepared from cells grown on medium containing [U-14C]-labeled algal protein hydrolysate. Within the limitations of this method the results tentatively suggest a subunit composition of 3:3:1:1:3.

关键词： f 1 -ATPase Coupling factor (Micrococcus lysodeikticus)

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STABILIZATION Of RAT-LIVER MITOCHONDRIAL f1-ADENOSINE TRIPHOSPHATASE DURING CHLOROfORM-INDUCED SOLUBILIZATION

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BIOCHIMICA ET BIOPHYSICA ACTA 1979年第2期547卷 177-187页

作者： KOPECKY, J KUZELA, S KRAML, J DRAHOTA, Z SLOVAK ACAD SCI CANC RES INST CS-80936 BRATISLAVA CZECHOSLOVAKIA CHARLES UNIV INST MED CHEM 1 PRAGUE CZECHOSLOVAKIA

Isolation of ATPase from rat liver submitochondrial particles by chloroform treatment requires the presence of ATP or ADP during enzyme solubilization. In the absence of adenine nucleotides the enzyme activity is very low although all protein components of f1-ATPase are released. The low concentrations of ATP or ADP required (5 .mu.M) indicate that the high affinity nucleotide-binding sites are involved in enzyme stabilization. Other nucleotides tested (ITP, GTP, UTP, CTP) were less effective. Polyacrylamide gel electrophoresis and immunodiffusion in agar plates revealed that in the absence of adenine nucleotides a fraction of f1-ATPase released by chloroform treatment is split into fragments. The part of the dissociated enzyme molecule has a MW identical with that of a .beta.-subunit of f1-ATPase. Dissociation of the f1-ATPase molecule was prevented by aurovertin. Crude f1-ATPase solubilized by chloroform treatment was further purified by Sepharose 6B gel filtration. Specific ATPase activity of the purified enzyme was 90 .*** Pi/min per mg protein and the enzyme was composed of 5 protein subunits (.alpha., .beta., .gamma., .delta., .epsilon.) and MW 58,000, 55,000, 28,000, 13,000 and 8000, respectively. Chloroform-released f1-ATPase from rat liver mitochondria displayed immunochemical cross-reactivity with that isolated from beef heart mitochondria.

关键词： Solubilization Stabilization f 1 -ATPase Chloroform ATPase adenosine triphosphatase (EC 3.6.1.3) f 1 -ATPa se soluble ATPase from mitochondria P i inorganic orthophosphate SDS sodium dodecyl sulphate

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f=500m~2钛管加热室的几个技术问题

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中国井矿盐 1979年第1期 26-29页

作者：赵泰亨自贡市大安盐厂

我厂真空制盐车间自建成投产以来,加热管的腐蚀问题一直是影响真空制盐生产的关键。长期以来,我们采取了各种措施保护加热管,如外加电流阴极保护、牺牲阳极,用精锡镀管内外等方法。实践证明是能起到一定作用,但并不理想。从国内外的情况... 详细信息

我厂真空制盐车间自建成投产以来,加热管的腐蚀问题一直是影响真空制盐生产的关键。长期以来,我们采取了各种措施保护加热管,如外加电流阴极保护、牺牲阳极,用精锡镀管内外等方法。实践证明是能起到一定作用,但并不理想。从国内外的情况看,解决加热管的腐蚀,从材质上着手是理想的解决办法。根据我国的资源情况,七八年我

关键词：管板管头 f=500m~2 加热管防露管胀接胀管率 f

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RELATIONSHIP Of RAISED ATHEROSCLEROTIC LESIONS TO fATTY STREAKS IN CORONARY HEART-DISEASE AND HYPERTENSION

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ATHEROSCLEROSIS 1979年第1期33卷 125-140页

作者： TRACY, RE STRONG, JP TOCA, V Department of Pathology Louisiana State University Medical Center New Orleans LA 70112 U.S.A.

The abdominal aortas and right coronary arteries removed during autopsies were gathered from over 18,000 subjects in 19 location-race groups. Sudan-stained intimal surfaces were graded for the percent as raised lesions (R) and fatty streaks (f). The proportions of all types of lesions (ATL = f + R) that were raised (RaL = R .div. ATL) were studied. The difference between the observed RaL (in subjects having atherosclerosis-related conditions) and the expected RaL (in age-, sex- and ATL-matched subjects without such conditions) measured the excess of RaL, which by inference indicated the tendency for an excess of raised lesions to be produced in place of fatty streaks. In cases of coronary heart disease (CHD), a large excess of RaL was found consistently on the average. The coronary arteries and aortas of hypertensive subjects, compared with those of nonhypertensives, had a small and inconsistent excess of RaL. Both CHD and hypertension were characterized by a marked tendency for severe involvement by ATL, including extensive fatty streaks, leaving only small remnants of normal intima. Some factors (Class A) act to form fatty streaks and also to continue advancing them into raised lesions. Once the fatty streaks are formed, some new factors (Class B2) convert them into raised lesions. Apparently hypertension is a Class A type of atherogenic agent, and CHD is promoted by exceptionally strong effects of both A and B2 types of causation. If the assumptions of the model are true, then some of the more important causes of atherosclerosis (Class B causes) begin to act only after the fatty streaks have formed.

关键词： Atherogenic agent Atherosclerosis Coronary heart disease fatty streak Hypertension Raised lesion Percent surfaces involved N no lesions (“normal” intima) f fatty streaks R raised lesions ATL all types of lesions = f + R faf fatty streaks among flat surfaces = f÷(N + f) RaL raised among lesions = R÷(f + R) OE excess RaL observed RaL minus the RaL expected for an average basal case of the same age, sex, and ATL

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COUPLING fACTOR ATPASE COMPLEX Of RHODOSPIRILLUM-RUBRUM PURIfICATION AND CHARACTERIZATION Of AN OLIGOMYCIN AND N,N'-DICYCLOHEXYLCARBODIIMIDE-SENSITIVE (CA2+ + MG2+)-ATPASE

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BIOCHIMICA ET BIOPHYSICA ACTA 1979年第1期548卷 106-118页

作者： OREN, R GROMETELHANAN, Z Department of Biochemistry The Weizmann Institute of Science Rehovot Israel

An ATPase complex sensitive to the energy transfer inhibitors oligomycin, dicyclohexylcarbodiimide and venturicidin was solubilized from R. rubrum chromatophores with Triton X-100 and further purified by centrifugation on a glycerol gradient. The partially purified ***.f1 contains 13 distinct polypeptide subunits, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, including the subunits of the oligomycin-insensitive, water-soluble Rrf1 ATPase. The ATPase activity of ***.f1 as that of the membrane-bound enzyme complex depends on Ca2+ or Mg2+ and detailed kinetic studies showed that the divalent cation-ATP complex is the substrate for both ATPase complexes. free ATP and free Mg2+ act as competitive inhibitors, with Ki values of 1 mM and 7 .mu.M, respectively. The subunit composition of the purified ***.f1 and its similarity to the membrane-bound ATPase with respect to cation dependence and sensitivity to energy transfer inhibitors suggest that it contains all the subunits of the R. rubrum coupling factor-ATPase complex.

关键词： Chromatophore Bacterial photosynthesis Coupling factor ATPase complex Oligomycin-sensitive f 0 · f 1 (Ca 2+ + Mg 2+ -ATPase (Rhodospirillum rubrum) DCCD N , N ′-dicyclohexylcarbodiimide Cf 0 · f 1 detergent-solubilized ATPase complexes isolated from chloroplasts chromatophores f 0 · f 1 detergent-solubilized ATPase complexes isolated mitochondria chromatophores Rrf 0 · f 1 detergent-solubilized ATPase complexes isolated from Rhodospirillum rubrum chromatophores Cf 1 water-soluble DCCD-insensitive ATPase moieties of Cf 0 · f 1 f 1 water-soluble DCCD-insensitive ATPase moieties of f 0 · f 1 Rrf 1 water-soluble DCCD-insensitive ATPase moieties of Rrf 0 · f 1 Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid SDS sodium dodecyl sulfate Tricine N -tris(hydroxymethyl)methylglycine

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INTERACTIONS Of BEEf-HEART MITOCHONDRIAL ADENOSINE-TRIPHOSPHATASE AND AUROVERTIN

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LIfE SCIENCES 1979年第15期24卷 1407-1417页

作者： ALEKSANDROWICZ, Z SCHUSTER, SM UNIV WISCONSIN INST ENZYME RESMADISONWI 53706

Aurovertin forms a complex with soluble beef heart mitochondrial ATPase (f 1 ) while exhibiting a biphasic fluorence enhancement. The effect of substrate, activators and inhibitors of f 1 1 of the fluorescence of the aurovertin-f 1 complex is reported. The aurovertin-f 1 complex can exist in two different states, one showing low fluorescence and the other with high fluorescence. Transition into the low fluorescence state is induced by various nucleoside triphosphates (ATP ± Mg 2+ , ITP ± Mg 2+ , GIP + Gg 2+ , and AMP-P(NH)P ± Mg 2+ ). The rate and extent of fluorescence decrease caused by nucleotide addition (except that caused by ATP) is dependent on the presence of added Mg 2+ . The inhibitors of ATPase activity (AMP-P(NH)P, GMP-P(NH)P and EDTA) at concentrations that inhibit hydrolysis of ATP did not prevent the ATP induced decrease of aurovertin fluorescence. EDTA at high concentration (>0.4 mM) enhanced the effect of ADP. The complex of aurovertin with f 1 that had previously been treated with butanedione loses sensitivity to ATP. Addition of ADP to the system containing butanedione-treated enzyme caused a 2-fold greater enhancement of fluorescence than the addition of ADP to the control system. In contrast to the butanedione-treated enzyme, the complex of aurovertin with f 1 previously treated at pH 5.6 loses sensitivity to ADP. Addition of ATP to this system lowered the fluorescence as in the system containing native enzyme. On the basis of the analyses of the aurovertin fluorescence changes and hydrolytic activity of f 1 , the existence of several types of ligand binding sties with varying degrees of specificity are proposed. It is further proposed that these sites are important in control of the conformation and the catalytic properties of the ATPase molecule.

关键词： f 1 , soluble mitochondrial ATPase AMP-P(NH)P, adenylyl imidodiphosphate GMP-P(NH)P, guanylyl imidodiphosphate

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fINE-STRUCTURE Of THE NUCLEAR APPARATUS Of REMANELLA-MULTINUCLEATA KAHL (CILIOPHORA, GYMNOSTOMATA)

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ARCHIV fUR PROTISTENKUNDE 1979年第1-2期121卷 1-19页

作者： RAIKOV, IB ACAD SCI USSR INST CYTOL LENINGRAD USSR

The nuclear apparatus of R. multinucleata Kahl (family Loxodidae) is a longitudinal row of unstable nuclear groups, each consisting of 2 or 3 macronuclei and 1 or 2 micronuclei. Both macro- and micronuclei have a typical nuclear envelope carrying pores and strengthened at the cytoplasmic side by an electron dense sheet and 1-3 layers of flattened cisternae of the smooth endoplasmic reticulum. The chromosomes remain condensed in interphase micronuclei and may fuse with each other into a common spongy mass. Micronuclei lack both granular component (RNP) and nucleoli, but often contain small, apparently proteinaceous nuclear bodies. Macronuclei contain several nucleoli of typical structure (fibrillar core, granular cortex), with a thread-like nucleolar organizer embedded in each. The macronuclear chromatin is partly dispersed, partly condensed into both numerous small bodies and a few large spherical chromocentres which are often vacuolized. In some specimens, the macronuclei contain spheres of medium electron density and finely granular structure, containing RNA and protein.

关键词： AB amorphous body in a micronucleus CB macronuclear chromatin bodies CC macronuclear chromocentre CM micronuclear chromatin mass CS micronuclear chromosomes D dictyosome DL dense layer outside the nuclear envelope f fibrillar zone of a nucleolus fNM fibrils of the macronuclear matrix G granular zone of a nucleolus GER granular endoplasmic reticulum IS inclusion into the macronuclear sphere KL micronuclear karyolymph LP lipid globule MA macronucleus MC mitochondrion MI micronucleus MTB microtubule N nucleolus NE nuclear envelope NO nucleolar organizer PER perinuclear smooth endoplasmic reticulum S macronuclear sphere SER smooth endoplasmic reticulum (except the perinuclear one) V cytoplasmic vacuole VCC vacuole inside a chromocentre VN vacuole inside a nucleolus

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TRYPTIC PROTEOLYSIS Of COUPLING fACTOR-LATENT ATPASE fROM MYCOBACTERIUM-PHLEI - THEORETICAL MODELING Of STRUCTURE-fUNCTION RELATIONSHIPS

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BIOCHIMICA ET BIOPHYSICA ACTA 1979年第1期547卷 1-17页

作者： RITZGOLD, CJ GOLD, CM BRODIE, Af UNIV SO CALIF SCH MED DEPT BIOCHEM LOS ANGELES CA 90033 USA

Trypsin treatment of solubilized coupling factor-latent ATPase from M. phlei alters its subunit structure and functional properties. This coupling factor exhibits ATPase activity following trypsin treatment. Concurrently, both the ability of the enzyme to rebind to membranes depleted of coupling factor and its capacity for coupled phosphorylation are lost. The native .alpha. (64,000 dalton) subunit undergoes limited proteolytic digestion, and the .delta. (14,000 dalton) subunit is partially lost. During the course of tryptic proteolysis, the coupling factor molecule may exist in 1 of 10 unique structural states (e.g., the native, ATPase-inactive molecule exists in the .alpha..alpha..alpha. state). Rigorous analysis of the experimental data by theoretical modeling provided information concerning the intermediate structural states leading to the fully ATPase-activated .alpha.".alpha.".alpha." state under different conditions of trypsin treatment. The theoretical models of structure-function relationships that best represented the experimental data predicted that the native coupling factor molecule contains 3 copies of the .alpha. (64,000 dalton) form of the .alpha. subunit, that the .alpha." (58,000 dalton) .alpha. subunit species contributes maximally and the .alpha.'' (61,000 dalton) form about half-maximally to ATPase activity, that membrane rebinding ability is proportional to the number of native .alpha. subunits in the enzyme, and that at least 1 native .alpha. subunit/molecule is required for full expression of coupled phosphorylation. These results indicate an essential role for the .alpha. subunit in the regulation of ATPase activity and in the ability of the solubilized coupling factor to rebind to depleted membranes.

关键词： Tryptic proteolysis f 1 -ATPase Coupling factor (Structure-function relationships, Mycobacterium)

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ALTERATIONS IN SPECIfICITY Of THE GLUCOCORTICOID RECEPTOR WITH TEMPERATURE IN RAT SPLENIC LYMPHOCYTES

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JOURNAL Of STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY 1979年第1期10卷 21-29页

作者： MACDONALD, RG CIDLOWSKI, JA UNIV VERMONT COLL MED DEPT BIOCHEM BURLINGTON VT 05401 USA

Rat splenic lymphocytes contain specific, saturable glucocorticoid receptors. When suspensions of these cells were incubated at ***. C in the presence of [3H]-triamcinolone acetonide ([3H]-TA), 80% of receptor-bound steroid was found associated with the cytoplasm, whereas at ***. C, 80% of [3H]-TA binding was associated with the nucleus. The equilibrium dissociation constant for interaction of [3H]-TA with the cytoplasmic receptor at ***. C is 5.6 nM while that for nuclear binding at ***. C was 0.85 nM, suggesting an increase in receptor affinity for [3H]-TA at physiological temperatures. Specificity studies showed a high degree of glucocorticoid specificity at ***. C in cytoplasm and nucleus, with little competition observed for either progesterone (P) or cortexolone (S). At ***. C, P and S competed with [3H]-TA for receptor binding as effectively as dexamethasone. ***., testosterone, ***.-dihydrotestosterone and cortisone did not compete under any of these conditions. When cells were incubated at ***. C in the presence of 0.02 .mu.M [3H]-TA and concentrations of P and S ranging from 0.04-2.56 .mu.M, [3H]-TA binding was inhibited at steroid concentrations as low as 0.1 .mu.M. If these same cells were then warmed to ***. C [3H]-TA binding in cytoplasm and nucleus was inhibited only at P and S concentrations .gtoreq. 1 .mu.M. There is, concomitant with increasing temperature, an increase in affinity and specificity of the receptor for active glucocorticoids. This specificity change was regarded as evidence for a conformational change in the hormone-receptor complex coincident with activation.

关键词： TA triamcinolone acetonide (9α-fluoro-11β, 16α, 17α, 21-tetrahydroxy-pregna-1,4-diene-3, 20-dione-16, 17-acetonide DEX dexamethasone (9α-fluoro-11β, 17, 21-trihydroxy-16α-methylpregna-1,4-diene-3, 20-dione) f cortisol E cortisone B cortico-sterone S cortexolone P progesterone T testosterone DHT 5α-dihydrotestosterone E 2 estradiol-17β

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INHIBITION Of INSULIN RECEPTOR-BINDING BY DIMETHYL-SULfOXIDE

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BIOCHIMICA ET BIOPHYSICA ACTA 1979年第3期582卷 486-495页

作者： VANOBBERGHEN, E DEMEYTS, P ROTH, J Diabetes Branch National Institute of Arthritis Metabolism and Digestive Diseases National Institutes of Health Bethesda MD 20014 U.S.A.

Little is known of the effects of the solvent on hormone-receptor interactions. In the present study the effect of the polar solvent dimethyl sulfoxide on the binding of insulin to its surface receptors on cultured human lymphocytes of the IM-9 line was investigated. At concentrations exceeding 0.1% (v/v), dimethyl sulfoxide produced a dose-related inhibition of 125 I-labeled insulin binding. Insulin binding was totally abolished in 20% dimethyl sulfoxide. This inhibition was immediately present and was totally reversible. Analysis of the data of binding at steady state indicated that the decrease in binding of 125 I-labeled insulin was due to a reduced affinity of the insulin receptor without noticeable change in the concentration of receptor sites. Kinetic studies showed that the decreased affinity could largely be accounted for by a decreased association rate constant; effects on dissociation and negative cooperativity of the insulin receptor were affected to a much lesser extent.

关键词： Insulin Receptor binding Dimethyl sulfoxide (Negative cooperativity) Me 2 SO, dimethyl sulfoxide, HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid B / f bound/free R 0 total concentration of receptor sites per cell K average affinity K e limiting high affinity state K f limiting low affinity state

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