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PYRIDOXYLATION Of ESSENTIAL LYSINE RESIDUES Of MITOCHONDRIAL ADENOSINE-TRIPHOSPHATASE

BIOCHIMICA ET BIOPHYSICA ACTA 1982年第2期679卷 269-278页

作者： KOGA, PG CROSS, RL SUNY UPSTATE MED CTR DEPT BIOCHEM SYRACUSE NY 13210 USA

Soluble beef-heart mitochondrial ATPase (f1) was incubated with [3H]pyridoxal 5''-phosphate and the Schiff-base complex formed was reduced with sodium borohydride. Spectral measurements indicate that lysin residues are modified and gel electrophoresis in the presence of detergent shows the 3H label to be associated with the 2 largest subunits, .alpha. and .beta.. In the absence of protecting ligands, the loss of ATP hydrolysis activity is linearly dependent on the level of pyridoxylation with complete inactivation correlating to 10 mol pyridoxamine phosphate incorporated per mol enzyme. Partial inactivation of f1 with pyridoxal phosphate has no effect on either the Km for ATP or the ability of bicarbonate to stimulate residual hydrolysis activity, suggesting a mixed population of fully active and fully inactive enzyme. In the presence of excess Mg, the addition of ADP or ATP, but not AMP, decreases the rate and extent of modification of f1 by pyridoxal phosphate. The non-hydrolyzable ATP analog, 5''-adenylyl-.beta.,.gamma.-imidodiphosphate, is particularly effective in protecting f1 against both modification and inactivation. Efrapeptin and Pi have no effect on the modification reaction. Prior modification of f1 with pyridoxal phosphate decreases the number of exchangeable nucleotide binding sites by 1. However, pyridoxylation of f1 is ineffective in displacing endogenous nucleotides bound at non-catalytic sites and does not affect the stoichiometry of Pi binding. The ability of nucleotides to protect against modification and inactivation by pyridoxal phosphate and the loss of 1 exchangeable nucleotide site with the pyridoxylation of f1 suggest the presence of a positively charged lysine residue at the catalytic site of an enzyme that binds 2 negatively charged substrates.

关键词： f 1 -ATPase Pyridoxal phosphate Lysine residue (Bovine heart) f 1 in the isolated soluble form used in these studies, the mitochondrial enzyme is referred to as an ATPase. Efrapeptin, also referred to in the literature as efrastatin and A23871 Hepes N -2-hydroxyethylpiperazine- N ′-2-ethanesulfonic acid Ado PP [NH] P 5′-adenylyl-β, γ-imidodiphosphate fDNB 1-fluoro-2,4-dinitrobenzene DNP 2,4-dinitrophenyl

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ON THE NATURE Of THE fLUORESCENCE DECREASE DUE TO PHOSPHORYLATION Of CHLOROPLAST MEMBRANE-PROTEINS

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BIOCHIMICA ET BIOPHYSICA ACTA 1982年第1期680卷 22-27页

作者： HORTON, P BLACK, MT UNIV SHEFFIELD ARC PHOTOSYNTH GRP SHEFFIELD S10 2TN S YORKSHIRE ENGLAND

Phosphorylation of chloroplast membranes [from pea and spinach] by illumination in the presence of ATP results in a 15-20% increase in the rate of photosystem I electron trasnfer at low light intensity. Phosphorylated membranes when depleted of Mg2+ and resuspended in a low salt medium still show a 17% lower yield of photosystem II fluorescence than do unphosphorylated membranes. A 31% difference occurs after restoration of the maximal yield by addition of Mg2+. The concentration of Mg2+ required to induce a half-maximal increase in fluorescence is 0.9 mM for control and 1.8 mM for phosphorylated chloroplasts. Phosphorylation at 1 mM Mg2+ can cause more than double the amount of decrease in fluorescence yield from photosystem II compared to phosphorylation at 5 mM. The mechanism of the ATP-induced fluorescence changes is discussed. The apparent interaction between phosphorylation and Mg2+ concentration may be a physiologically important phenomenon.

关键词： Chloroplast membrane phosphorylation Chlorophyll fluorescence Mg 2+ effect Electron transfer (Pea, Spinach chloroplast) PS II Photosystem II PS I Photosystem I f m maximum fluorescence level when all PS II traps are closed f 0 minimum fluorescence level when all PS II traps are open f v = f m - f 0 DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea Hepes N -2-hydroxyethylpiperazine- N ′-2-ethanesulphonic acid LHCP light-harvesting chlorophyll protein Tricine N -tris(hydroxymethyl)methylglycine Chl chlorophyll

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RNA-POLYMERASE ACTIVITIES Of CALLIPHORA-VICINA LARVAE IN RESPONSE TO ECDYSTEROIDS

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INSECT BIOCHEMISTRY 1982年第5期12卷 523-530页

作者： SCHENKEL, H SCHELLER, K UNIV WURZBURG INST ZOOL D-8700 WURZBURG FED REP GER

The effect of ecdysterone on RNA polymerase activities, partially purified from fat body nuclei of C. vicina 3rd instar larvae, was investigated. Three different forms of DNA-dependent RNA polymerase activities were isolated and resolved on the basis of chromatographic behavior and .alpha.-amanitin sensitivity. RNA polymerase I and II are similar to the analogous polymerases from other eukaryotic organisms in their enzymatic properties, such as salt and metal-ion optima and in their DNA requirements. The activities of polymerase I and II were correlated with the endogenous ecdysteroid concentrations of 3rd instars. The enzyme activities increase 2-fold in white puparia compared to 5-day-old larvae. After injection of ecdysterone in 5-day-old larvae the enzyme activities are stimulated within 3 h to the same amount as in white puparia. The enzyme stimulation coincides with the increase in RNA synthesis. A hormone dependent mechanism exists for regulating the intracellular activities of RNA polymerases I and II.

关键词： Calliphora vicina fat-body DNA-dependent RNA polymerases ecdysterone four-day-old larvae WP white puparia PhMeSO 2 f Phenyl-methyl-sulphonyl-fluoride TCA trichloroacetic acid PPi tetra-sodium-pyrophosphate SDS sodium-dodecyl-sulphate

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INVITRO RESPONSE Of ATPASE ACTIVITIES IN TISSUE SUB-CELLULAR PARTICLE PREPARATIONS TO A SERIES Of MONOUNSATURATED C-18 fATTY-ACIDS

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BIOCHEMICAL PHARMACOLOGY 1982年第5期31卷 867-872页

作者： KOCH, RB MISSISSIPPI STATE UNIV DEPT BIOCHEM STATE COLL MS 39762 USA

The positions of the double bond and the cis/trans configurations of 6 mono-unsaturated C18 fatty acids (fA) showed selectivity for inhibition and stimulation of ATPase activities of tissue homogenate fractions. The 13,000 g and 100,000 g sediments (fragments B and C respectively) of tissue homogenates were used as sources of Na+-K+ ATPase and of oligomycin-sensitive (OS) and -insensitive (OIS) Mg2+ ATPase activities. Tissue sources included bovine brain and rat brain, kidney, heart and liver. Cis mono-unsaturated C18 fA caused an apparent uncoupling of mitochondrial coupling factor f1 (Mg2+ ATPase). This was indicated by the loss of oligomycin and 1,1,1-trichloro-2,2 bis (p-chlorophenyl) ethane (DDT) sensitivity in the presence of 50 .mu.M fA (12-C18:1). Thus, a precipitous decrease in OS-Mg2+ ATPase activity. This was especially apparent in a heart tissue preparation. The uncoupling action of the fA was not observed with the trans mono-unsaturated C18 fA. Na+-K+ ATPase activity from a bovine brain nerve ending particle (B) fraction was also sensitive to 12-C18:1fA. However, inhibitory responses of Na+-K+ ATPase activity were different for OS-Mg2+ ATPAse activity. The latter (OS) was not sensitive to the trans 12-C18:1, while the former (Na+-K+) was sensitive to both cis and trans forms of 12-C18:1 and inhibition appeared to be dependent on the position of the double bond.

关键词： fA, fatty acids DDT, 1,1,1-trichloro-2,2-bis( p -chlorophenyl)ethane f 1 , water-soluble Mg 2+ ATPase OS, oligomycin sensitive OIS, oligomycin insensitive and Ouab-Ins, ouabain-insensitive

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THE GUINEA-PIG MODEL Of DIISOCYANATE SENSITIZATION .2. PHYSIOLOGIC STUDIES

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JOURNAL Of ALLERGY AND CLINICAL IMMUNOLOGY 1982年第5期70卷 393-398页

作者： BERNSTEIN, IL SPLANSKY, GL CHEN, SE VINEGAR, A UNIV CINCINNATI MED CTR DEPT MED DIV IMMUNOL CINCINNATI OH 45267 USA UNIV CINCINNATI MED CTR DEPT ENVIRONM HLTH CINCINNATI OH 45267 USA

The qualitative and quantitative properties of diisocyanate-specific IgE antisera derived from an IgE-susceptible strain of guinea pigs were investigated by passive sensitization of immunologically virgin recipient animals. These animals received infusions of antisera 48 h before i.v. challenge with specific ligands conjugated to an unrelated carrier protein, human transferrin. The pulmonary response was sublethal and could be measured by physiologic methods. Various measurements were obtained in anesthetized guinea pigs placed in a whole-body pressure plethysmograph. Tidal volume (Vt), dynamic compliance (CL dyn) and pulmonary conductance (GL) were calculated from simultaneous recordings of transpulmonary pressure, Vt and flow. frequency at 1 and 2 min after i.v. challenge were also measured. Mean respiratory frequency was significantly increased in experimental animals 1 min after challenge with homologous hapten-protein conjugates, while maximum changes of Vt, CL dyn and GL occurred 2 min after i.v. challenges. A preponderant number of physiologic abnormalities occurred in animals challenged with homologous hapten-protein conjugates. The mean percent change of Vt and CL dyn from baseline levels was significantly decreased after i.v. challenges with homologous ligands in animals passively sensitized with both hexamethylene diisocyanate-(HDI) and toluene diisocyanate-specific antisera. Decrease of mean GL after challenge with homologous hapten-protein conjugates was significant only in animals passively sensitized with HDI-specific antisera. When the number of positive postchallenge pulmonary function responses was compared with the mean geometric titers of IgE antisera, moderate or severe physiologic responses were associated with infusions of IgE antisera having titers of 160 or greater. Antibody concentration may be a critical determinant in distinguishing between clinical, subclinical and nonreactive states induced by diisocyanate sensitization.

关键词： TDI Toluene diisocyanate HDI Hexamethylene diisocyanate HSA Human serum albumin PCA Passive cutaneous anaphylaxis TR Human transferrin f 1 Respiratory frequency at 1 min after challenge f 2 Respiratory frequency at 2 min after challenge V t Tidal volume C L dyn Dynamic compliance G L Pulmonary conductance

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CHEMICAL-STRUCTURE Of RAT-LIVER CYTOCHROME B5 - ISOLATION Of PEPTIDES BY HIGH-PRESSURE LIQUID-CHROMATOGRAPHY

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BIOCHIMICA ET BIOPHYSICA ACTA 1982年第1期704卷 163-173页

作者： OZOLS, J HEINEMANN, fS UNIV CONNECTICUT CTR HLTH DEPT BIOCHEM FARMINGTON CT 06032 USA

The complete amino acid sequence of rat cytochrome b5 was determined. Isolation of this species of cytochrome b5 in its native form from microsomes by means of detergent solubilization required the inclusion of the protease inhibitor, phenylmethylsulfonyl fluoride, throughout the isolation steps. Omission of the protease inhibitor yielded a 97-residue heme-containing peptide without the membranous segment. The primary structure of the intact molecule was deduced from automated sequence analysis of peptides generated by proteolytic or chemical cleavage and isolated exclusively by reversed-phase HPLC [high pressure liquid chromatography]. The blocked amino terminus of cytochrome b5 was identified as N-acetylalanine. The hexosamine content of the cytochrome preparation was less than 0.1 mol/mol protein, indicating an absence of asparaginyl linked oligosaccharide.

关键词： HPLC Sequence analysis Hydrophobic peptide Cytochrome b 5 (Microsomal membrane) f 7 BtOH anhydrous heptafluorobutyric acid TPCK Tos-Phe-CH 2 C1 PMSf phenylmethylsulfonyl fluoride HfBA heptafluorobutyric acid

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MUTAGENICITY Of NIfURPIRINOL (P-7138) IN ESCHERICHIA-COLI WP2 AND SALMONELLA-TYPHIMURIUM TA100

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MUTATION RESEARCH 1982年第1-3期104卷 61-66页

作者： NAKAMURA, Y fUKUSHIMA, H TOMITA, I KIMURA, I SHIZUOKA COLL PHARM HLTH SCI LAB SHIZUOKA 422 JAPAN AICHI CANC CTR VIRAL ONCOL LAB NAGOYA 464 JAPAN

Nifurpirinol, 6-hydroxymethyl-2-[2-(5-nitro-2-furyl)vinyl]pyridine, which was widely used as an antibacterial drug against diseases of fish, was a potent mutagen. The mutagenic effect was dose-related, and the potencies were calculated to be 1.49 .times. 103, 7.24 .times. 104 and 8.49 .times. 105 revertants per 1 .times. 108 survivors at the concentrations of 1 .mu.g/ml to E. coli B/r WP2, E. coli B/r WP2 hcr- and S. typhimurium TA100, respectively, without metabolic activation. Nifurpirinol is susceptible to photodegradation, and its mutagenic activity to S. typhimurium TA100 was decreased to about 1/1000 when the solution of nifurpirinol was exposed to sunlight at room temperature for 4 h.

关键词： NP (Nifurpirinol, P-7138) 6-hydroxymethyl-2-[2-(5-nitro-2-furyl)vinyl]pyridine Af 2 (furyl-furamide) 2-(2-furyl-3-(5-nitro-2-furyl)acrylamide MNNG, N -methyl- N ′-nitro- N -nitrosoguanidine 4-NQO, 4-nitroquinoline-1-oxide MeIQ, 2-amino-3,4-dimethylimidazo[4,5- f ]quinoline

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OPTIMAL CONDITIONS fOR THE ASSAY Of LIPOAMIDE DEHYDROGENASE IN HOMOGENIZED HUMAN-PLATELETS

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CLINICA CHIMICA ACTA 1982年第1期121卷 1-9页

作者： BECKER, DM KNIPPRATH, WG PERLMAN, SL NISSENSON, C KARK, RAP UNIV CALIF LOS ANGELES REED NEUROL RES CTR SCH MED LOS ANGELES CA 90024 USA UNIV CALIF LOS ANGELES CTR ATAXIA DEPT NEUROL LOS ANGELES CA 90024 USA UNIV CALIF LOS ANGELES JERRY LEWIS NEUROMUSC & MENTAL RETARD RES CTR LOS ANGELES CA 90024 USA

Widely different methods have been used to assay lipoamide dehydrogenase in tissues from patients with neurological diseases. Assay conditions were reexamined in homogenized human platelets in the light of results of optimal and inhibitory conditions others have found for the purified pig and rat liver enzymes. Optimal conditions in homogenized platelets for the forward, physiological direction were pH 8.0, 2-4 mmol/l dihydrolipoamide and 1.6-2 mmol/l NAD+ and for the reverse reaction, pH 7.3 1.2-2 mmol/l lipoamide and 0.125-0.2 mmol/l NADH. Km values by the Lineweaver-Burke method were approximately 420 .***/l dihydrolipoamide, 180 .***/l NAD+, 600 .***/l lipoamide and 27 .***/l NADH. The optimal conditions and Km values are similar to those reported for the purified pig and rat enzymes. Assays by these methods should reflect the activity of lipoamide dehydrogenase and not the effects of substrate or cofactor inhibition nor the effects of other interfering enzyme activities.

关键词： NAD + nicotinamide adenine dinucleotide NADH nicotinamide adenine dinucleotide, reduced form LAD lipoamide dehydrogenase EDTA sodium ethylenediamine tetraacetate LA lipoamide thioctic acid amide, α-lipoic acid amide H 2 LA dihydrolipoamide (reduced form of lipoamide) TLC thin layer chromatography R f relative mobility K K Michaelis-Menten kinetic constant V velocity of reaction (μmol×min −1 ) S concentration of substrate

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IMMUNOLOGICAL STUDIES ON BOVINE-MILK LIPOPROTEIN-LIPASE - EffECTS Of fAB fRAGMENTS ON ENZYME-ACTIVITY

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BIOCHIMICA ET BIOPHYSICA ACTA 1982年第1期712卷 10-20页

作者： SHIRAI, K WISNER, DA JOHNSON, JD SRIVASTAVA, LS JACKSON, RL UNIV CINCINNATI COLL MED DEPT PHARMACOL & CELL BIOPHYS DIV LIPOPROT RES CINCINNATI OH 45267 USA UNIV CINCINNATI COLL MED DEPT BIOL CHEM CINCINNATI OH 45267 USA UNIV CINCINNATI COLL MED DEPT MED CINCINNATI OH 45267 USA

Rabbit antiserum was prepared against purified bovine milk lipoprotein lipase. Immunoelectrophoresis of lipoprotein lipase gave a single precipitin line against the antibody which was coincident with enzyme activity. The .gamma.-globulin fraction inhibited heparin-releasable lipoprotein lipase activity of bovine arterial intima, heart, muscle and adipose tissue. The antibody also inhibited the lipoprotein lipase activities from adipose tissue of human and pig, but not that of rat and dog. fab fragments were prepared by papain digestion of the .gamma.-globulin fraction. fab fragments inhibited the lipoprotein lipase-catalyzed hydrolysis of dimyristoylphosphatidylcholine vesicles and trioleoylglycerol emulsions to the same extent. The fab fragments also inhibited the lipolysis of human plasma very low density lipoproteins. The change of the kinetic parameters for the lipoprotein lipase-catalyzed hydrolysis of trioleoylglycerol of the fab fragments was accompanied with a 3-fold increase in Km and a 10-fold decrease in Vmax. Preincubation of lipoprotein lipase with apolipoprotein C-II, the activator protein for lipoprotein lipase, did not prevent inhibition of enzyme activity by the fab fragments. Preincubation with dipalmitoylphosphatidylcholine-emulsified trioleoylglycerol or Triton X-100-emulsified trioleoylglycerol had a protective effect (remaining activity 7.0 or 25.8%, respectively, compared to 1.0 or 0.4% with no preincubation). The addition of both apolipoprotein C-II and substrate prior to the incubation with the fab fragments was associated with an increased protective effect against inhibition of enzyme activity;remaining activity with dipalmitoylphosphatidylcholine-emulsified trioleoylglycerol was 40.6% and, with Triton X-100-emulsified trioleoylglycerol, 45.4%. Human plasma very low density lipoproteins also protected against the inhibition of enzyme activity by the fab fragments. The interaction of lipoprotein lipase with apolipoprotein C-II in the presen

关键词： Triacylglycerol hydrolysis Triacylglycerol-rich lipoprotein Antibody f ab fragment γ-Globulin inhibition Lipoprotein lipase (Bovine milk) DPPC dipalmitoylphosphatidylcholine DMPC dimyristoylphosphatidylcholine VLDL very low density lipoproteins ( d < 1.006 g / ml ) DnsPE dansylphosphatidylethanolamine

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BRONCHIAL RESPONSE TO HISTAMINE AfTER INHALED PROPRANOLOL AND ATROPINE IN MONKEYS

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JOURNAL Of ALLERGY AND CLINICAL IMMUNOLOGY 1982年第2期69卷 213-220页

作者： PARE, PD NICHOLLS, I UNIV BRITISH COLUMBIA ST PAULS HOSP PULM RES LAB VANCOUVER V6Z 1Y6 BC CANADA

To investigate the nature of the variable response to inhaled histamine in monkeys, dose-response curves were performed in a group of 10 anesthetized Macaca mulatta monkeys before and after administration of propranolol and atropine in inhaled doses sufficient to produce significant beta-adrenergic and cholinergic blockade of airway smooth muscle. Animals were studied in a volume-displacement body plethysmograph and changes in pulmonary resistance (R1), dynamic compliance (Cdyn), frequency (f) and tidal volume (VT) were plotted against inhaled histamine concentration (0.016-64 mg/ml). Sensitivity to histamine was assessed by the concentrations producing a 50% increase in R1 and a 50% decrease in Cdyn. A wide range of sensitivity (0.44-3.3 mg/ml for R1, 0.47-11.3 mg/ml for Cdyn) was found in this small group of animals, and this was not influenced by prior inhalation of propranolol. Atropine inhalation resulted in a marked decrease in sensitivity in all animals but did not reduce the variability of response.

关键词： P L Transpulmonary pressure V Volume V̇ flow R L Pulmonary resistance Cdyn Dynamic compliance V T Tidal volume f Breathing frequency

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