Anisotropy decay of fluorescence as an experimental approach to protein dynamics

This minireview makes an initial assessment of the progress made using anisotropy decay measurements for investigating the conformational changes and molecular dynamics in soluble systems. A critical analysis of available data is presented. The anisotropy decays of the tryptophan fluorescence of staphylococcal nuclease, adrenocorticotropin, melittin and of labeled transfer RNA were studied for investigating the functional conformational changes of these systems. The emissions of variously labeled immunoglobulins have been used to elucidate the conformations of these proteins before and after the binding of specific antibodies. Labeled myosin and its fragments have given information on the functional motions of the protein domains. The anisotropy decays of labeled and natural hemoglobin systems have been utilized for exploring the allosteric behavior of these molecules. The data suggest a wide applicability of this technique to the study of protein dynamics and conformational changes of macromolecules.     

Hemoglobin Dallas (α97(G4)Asn→Lys):functional characterization of a high oxygen affinity natural mutant

Hemoglobin Dallas, an α-chain variant with a substitution of lysine for asparagine at position 97(G4), was found to have increased oxygen affinity ($\text{p}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1}\text{mmHg at pH}\text{7.3}\text{and}\text{20°C), diminished cooperativity (0n,}\text{the Hill coefficient}\text{= 1.7}$) and reduced Bohr effect (about 50%). Addition of allosteric effectors (such as 2,3-diphosphoglycerate, inositol hexakisphosphate and bezafibrate) led to a decrease in oxygen affinity and increase in cooperative energy. Kinetic studies at pH 7.0 and 20°C revealed that (i), the overall rate of oxygen dissociation is 1.4-fold slower than that for HbA and (ii), the carbon monoxide dissociation rate is unaffected. The abnormal properties of this hemoglobin variant can be atttributed to a more ‘relaxed’ T-state.     

Allosteric Modulation of Flunitrazepam Binding to Rat Brain Benzodiazepine Receptors by Methyl β-Carboline-3-Carboxylate

The inhibition of flunitrazepam (FNP) binding to rat brain benzodiazepine (BZ) receptors by methyl beta-carboline-3-carboxylate (MCC) was studied. Biphasic dissociation was observed for [3H]FNP and [3H]MCC in cerebral cortex, cerebellum, and hippocampus, although the dissociation of [3H]MCC was much faster. The dissociation rate of [3H]FNP was increased by MCC in the cerebellum, but was not altered in cerebral cortex or hippocampus. [3H]FNP binding stimulated by gamma-aminobutyric acid was enhanced in the presence of MCC in all three regions examined. These results indicate that MCC exerts these effects by interacting with allosteric sites that are different from the FNP recognition sites on the BZ receptors.     

Tryptamine, a Substrate for the Serotonin Transporter in Human Platelets, Modifies the Dissociation Kinetics of [3H]Imipramine Binding: Possible Allosteric Interaction

Tricyclic antidepressants and nontricyclic serotonin (5-hydroxytryptamine) uptake blockers monophasically inhibit [3H]imipramine binding in human platelets. Similarly, serotonin and tryptamine inhibit the binding of [3H]imipramine in the low micromolar range and with a pseudo-Hill coefficient near unity. Dissociation of the [3H]imipramine receptor complex in the presence of uptake inhibitors follows first-order kinetics with a half-life of approximately 60 min. Although serotonin and tryptamine do not decrease [3H]imipramine binding when added under equilibrium conditions, simultaneous addition of serotonin or tryptamine with serotonin uptake inhibitors decreases the rate of ligand-receptor dissociation in a concentration-dependent manner. These data suggest a common site of action for serotonin, which is the substrate of the transporter system, and of tryptamine, its nonhydroxylated analog. This hypothesis is supported by the identification of a high-affinity (Km = 0.55 microM), saturable, and temperature-dependent uptake of [3H]tryptamine in human platelets. Uptake of [3H]tryptamine was inhibited potently by imipramine and nontricyclic serotonin uptake inhibitors with a potency similar to that observed for [3H]serotonin uptake. These data support the hypothesis that in platelets, [3H]imipramine, tricyclic, and nontricyclic serotonin uptake inhibitors bind to a common recognition site that is associated with the serotonin transporter but that differs from the substrate recognition site of the carrier through which serotonin and tryptamine exert a heterotropic allosteric modulation on [3H]imipramine binding.     

Phosphorus nutrition of barley, buckwheat and rape seedlings. II. Influx and efflux of phosphorous by intact roots of different P status

Seedlings of barley (Hordeum vulgare L. cvs Salka and Zita), buckwheat (Fagopyrum esculentum Moench) and rape (Brassica napus L. ssp. napus cv. Line) were raised at 8 or 10 different extenral P concentrations in the range 0–2000 μM. Apart from P, the nutrient solutions were complete. Phosphate influx in roots of different P status was determined by use of a nutrient solution containing 0.1 mM³²P-labelled phosphate. A double labelling technique was used for simultaneous determination of influx (³³P) and efflux (³²P) of phosphorus by roots of barley and rape with three selected P levels. Flux determinations were also done in presence of a metabolic uncoupler (2,4-dinitrophenol) and a protein synthesis inhibitor (cycloheximide). Influx of phosphate was maximal at a certin optimal P level of the roots and decreased at both lower and higher P levels. Maximum phosphate influex [μmol (g root)-^?1 h^?1] were: rape 4,4, buckwheat 2.2, barley cv. Salka 1.6, barley cv. Zita 1.5. Both Hill plots and plots of the untransformed decreasing phosphate influx vs root P concentrations above the optimal were linear and had high correlation coefficients. The Hill coefficient varied between -3.1 and -4.2. The decrease of phosphate influx from the maximum to the lowest value at the highest P concentration of the root was 60–70%. Hence, phosphate influex appeared to be regulated through negative feedback by the internal level of phosphorous in the roots. The regulation mechanism seems bascially similar for the three species and may be of an allosteric type. P efflux from roots of low and optimal (with regard to P influx) P status was 15–20% of the simultaneous P influx. Contary to P influx, P efflux increased at high P status and almost eliminated (barley) or halved (rape) net P uptake. 2,4-Dinitrophenol reduced both P influx and P efflux by low P roots and gave linearly increasing P efflux with increasing root P status. This indicates that P efflux partly occurred by counter transport and ion exchange at the uptake sites, partly by passive P efflux along an electrochemical potential gradient. Phosphate influx was not affected by inhibition of barley root growth with cycloheximide, but P efflux increased considerably.     

枯草杆菌中性蛋白酶天然抑制剂的筛选

用透明因法和福林──酚法筛选了２１种植物中的蛋白酶活性抑制剂,其中来自鼓皮、高粱、玉米和土豆的抑制剂的活性在本研究条件下达５０％以上。进一步研究了麸皮、玉米两种抑制剂在不同温度和ｐＨ值下的稳定性,结果表明,两者的稳定性相差甚远,是两类不同的抑制剂。玉米抑制剂比麸皮抑制剂更稳定。     

Effects of anions, acetazolamide, thiocyanate and amiloride on fluid secretion by the Malpighian tubules of Locusta migratoria L.

The effect of Cl⁻, HCO₃⁻, Br⁻, acetazolamide, thiocyanate and amiloride on urine formation in Locusta migratoria Malpighian tubules have been determined. The rate of fluid secretion depends markedly on the concentration of Cl⁻ in the bathing solution with concentrations less than 90 mM resulting in reduced fluid secretion. Substitution of Br⁻ for Cl⁻ had no significant effect on the rate of the fluid secretion. Replacement of NaHCO₃ with NaCl in Hepes buffered Ringer solution reduced the rate of urine production by 23%. Fluid secretions were reduced in the presence of 10⁻⁴–10⁻² M acetazolamide, a carbonic anhydrase inhibitor. The combined effect of acetazolamide in the absence of HCO₃⁻ appears to be additive. A 1 mM concentration of thiocyanate, an ionic inhibitor, reduced fluid secretion by 35%. Amiloride interferes with the electrogenic entry of Na⁺ into the cell and a 1 mM solution reduced fluid secretion by 94% with secretion completely inhibited in 80% of the tubules tested.     

Inhibition of Glutamate Dehydrogenase in Brain Mitochondria and Synaptosomes by Mg2+ and Polyamines: A Possible Cause for Its Low In Vivo Activity

Abstract: Magnesium and the polyamines putrescine, spermidine, and spermine inhibited the activity of glutamate dehydrogenase in permeabilized rat brain mitochondria in a concentration-dependent manner. The inhibitory effect was observed on both the reductive amination of 2-oxoglutarate and oxidative deamination of glutamate, as well as in the presence and absence of ADP and leucine, the allosteric activators of the enzyme. Kinetic studies at various concentrations of substrates showed that inhibition by magnesium and spermine was very pronounced at 2-oxoglutarate concentrations less than 0.5 m M and NADH levels less than 0.08 m M . The presence of the former compounds also accentuated the inhibitory effect of high concentrations of 2-oxoglutarate (>2.0 m M ) and NADH (>0.32 m M ). Addition of magnesium and spermine to suspensions of synaptosomes decreased the amount of ammonia produced from glutamate. It is suggested that polyamines and magnesium, normal constituents of mammalian brain, are responsible, at least in part, for the low glutamate dehydrogenase activity in vivo.     

Involvement of putrescine in the inductive rooting phase of poplar shoots raised in vitro

Micropropagated poplar shoots rooted 100% on a rooting medium (A) containing NAA, but they did not root in the absence of auxin (NA). Putrescine, but not spermidine and spermine, promoted rooting up to 42% when added to the NA medium. Cyclohexylamine (CHA), an inhibitor of spermine synthase, also promoted (up to 36%) rooting in the absence of auxin. The inhibitors of polyamine biosynthesis DFMA (α-difluoromethylarginine) and DFMO (α-difluoromethylomithine), aminoguanidine (AG) and methylglyoxal-bis-guanylhydrazone (MGBG), inhibited rooting when applied in the presence of auxin and had no effect in its absence.
The rooting inductive phase (in the presence of auxin) was determined by periodical transfer of shoots from A to NA medium, and by changes in peroxidase activity, to be 7 h. Putrescine (not spermidine and spermine) accumulated to a maximum during the inductive phase. Both putrescine and CHA promoted rooting on NA medium when applied during the first 7 h. In contrast DFMA and AG inhibited rooting during this period. The results point to the involvement of putrescine and its Δ¹-pyrroline pathway, in the inductive phase of rooting in poplar shoots.