The effect of age on enolase turnover in the free-living nematode, Turbatrix aceti.

The rates of synthesis and degradation of enolase and total soluble proteins slow with age in the free-living nematode, Turbatrix aceti. The half-lives are 73 and 58 h for soluble protein and enolase, respectively, in young organisms (5 days old). The respective figures are 163 and 161 h for old organisms (22–30 days old). Similar slowing of protein turnover occurs when the organisms are aged by a repeated screening procedure which avoids the use of fluorodeoxyuridine, an inhibitor of DNA synthesis normally added to aging cultures to obtain synchrony. The results support the idea that slowed protein turnover may be responsible for the formation of altered enzymes in old organisms.     

Mercury-Induced Inhibition of Photosystem II Activity and Changes in the Emission of Fluorescence from Phycobilisomes in Intact Cells of the Cyanobacterium, Spirulina platensis

Addition of low concentrations of mercury chloride (HgCl₂ tointact cells of the cyanobacterium, Spirulina platensis causedan enhancement in the intensity of fluorescence emitted fromphycocyanin at room temperature and induced blue shifts in theemission peak suggestive of changes in energy transfer withinthe phycobilisomes. HgCl₂ also suppressed the whole-chain electrontransport activity (H₂O methylviologen) at much lower concentrationsthan that required to inhibit Hill activity supported by para-benzoquinone.The extent of inhibition of Hill activity was much higher underhigh-intensity light than that under low-intensity light. Ourresults indicate that mercury ions at low concentrations affectthe transfer of energy within phycobilisomes and at high concentrationsthey inhibit electron transport in this cyanobacterium. (Received February 21, 1989; Accepted October 2, 1989)     

Altered hepatic microsomal function and elevated protooncogene expression as residual effects in rats exposed to delta-9-tetrahydrocannabinol

The microsomal activation of the potent hepatocarcinogen aflatoxin B1 (AFB1) and the expression of selected protooncogenes were investigated in the livers of rats exposed to delta 9-tetrahydrocannabinol (THC). At equimolar levels of cytochrome P-450, the microsome-mediated binding of AFB1 to DNA was significantly lower (56% of the controls) in preparations from drug exposed rats. Hepatic expression of the c-k-ras protooncogene was 3-fold higher in THC exposed animals. These results suggest the possible occurrence of long lasting residual effects in the rats exposed to THC.     

The nonenzymic hydrolysis of Δ-thiazoline-2-carboxylate: The product of the suspected physiological reaction catalyzed by -amino acid oxidase

Δ²-Thiazoline-2-carboxylate, the product of the suspected physiological reaction catalyzed by -amino acid oxidase, is stable to hydrolysis at 37°C and pH 7 or above, but it hydrolyzes readily at pH 5 or below to give a mixture of N- and S-oxalylcysteamines; the N-oxalyl derivative predominates at pH's above 1 while the S-oxyalyl compound is the major product at high acidities. The pH-rate profile looks like the superposition of two bell-shaped curves. The initial increase in the rate as the pH is lowered is controlled by a pK_a of 3.95 and from pH 1 to 3 the rate is relatively constant (k = 6.7 × 10⁻⁴s⁻¹ at 37°C and ionic strength 0.5 ). Below pH 1 the rate increases again to a maximum in 1 HCl and then decreases in more highly acidic solutions. The rate of conversion of S-oxalylcysteamine to N-oxalylcysteamine is inversely proportional to the hydrogen ion concentration from pH 3 to 5 but becomes largely independent of pH from pH 1 to 2. In the pH-independent region the rate is comparable with that observed by others for S-acetylcysteamine but in the pH-dependent region the rate is 20 to 25 times faster for the oxalyl derivative than for the acetyl compound. At pH 1, N-oxalylcysteamine is partially converted to the S-oxalyl derivative but the rate of hydrolysis (k = 1.0 × 10⁻⁵s⁻¹ at 37°C) to cysteamine and oxalate of this partially equilibrated system occurs at a comparable rate. The results of this investigation are rationalized in terms of what is known about other thiazoline hydrolyses and intramolecular S to N acyl migrations. The main differences in the present case are presumably due to the fact that thiazoline-2-carboxylate can undergo hydrolysis by two reaction manifolds, one with the carboxyl unprotonated and the other with it protonated. The relevance of these results to possible reactions of thiazoline-2-carboxylate in vivo is briefly considered.     

Crystal structure of muconolactone isomerase at 3.3 A resolution

The crystal structure of muconolactone isomerase from Pseudomonas putida, a unique molecule with ten 96 amino acid subunits and 5-fold, and 2-fold symmetries, has been solved at 3.3 A resolution. The non-crystallographic symmetries were used to refine the initial single isomorphous replacement phases and produce an interpretable 10-fold averaged map. The backbone trace is complete and confirmed by the amino acid sequence fit. Each subunit is composed of a body with two alpha-helices and an antiparallel twisted beta-sheet of four strands, and an extended arm. The helices and the sheet fold to form a two-layered structure with an enclosed hydrophobic core and a partially formed putative active site pocket. The C-terminal arm of another subunit related by a local dyad symmetry extends over the core to complete this pocket. The decameric protein is almost spherical, with the helices forming the external coat. There is a large hydrophilic cavity in the center with open ends along the 5-fold axis. Molecular interactions between subunits are extensive. Each subunit contacts four neighbors and loses nearly 40% of its solvent contact area on oligomerization.     

Chemistry in environmentally benign media Part 1. Synthesis and characterization of 1,2-bis[bis(hydroxymethyl)phosphino]ethane (‘HMPE’). X-ray structure of [Pt{(HOH2C)2PCH2CH2P(CH2OH)2}2](Cl)2

The water-soluble bisphosphine, 1,2-bis(bis(hydroxymethyl)phosphino)ethane (1), was synthesized in near quantitative yield by the reaction of bisphosphine, H₂PCH₂CH₂PH₂, with an aqueous formaldehyde in the presence of K₂PtCl₄. The reaction of this water-soluble bisphosphine 1 with cis-Pt(COD)Cl₂ affords the mononuclear bischelate complex, [Pt{(HOH₂C)₂PCH₂CH₂P(CH₂OH)₂}₂](Cl)₂ (2), in near quantitative yield. The new ligand and complex have been characterized spectroscopically and the structure of the metal complex, 2, was determined by X-ray crystallography. The Pt(II) complex 2 crystallizes in the orthorhombic space group Pbca(a=14.623(1), B=16.216(2), C=9.319(4) Å) with Z=4. The final R value is 0.024.     

Crystal structure of thioltransferase at 2.2 A resolution.

We report here the first three-dimensional structure of a mammalian thioltransferase as determined by single crystal X-ray crystallography at 2.2 A resolution. The protein is known for its thiol-redox properties and dehydroascorbate reductase activity. Recombinant pig liver thioltransferase expressed in Escherichia coli was crystallized in its oxidized form by vapor diffusion technique. The structure was determined by multiple isomorphous replacement method using four heavy-atom derivatives. The protein folds into an alpha/beta structure with a four-stranded mixed beta-sheet in the core, flanked on either side by helices. The fold is similar to that found in other thiol-redox proteins, viz. E. coli thioredoxin and bacteriophage T4 glutaredoxin, and thus seems to be conserved in these functionally related proteins. The active site disulfide (Cys 22-Cys 25) is located on a protrusion on the molecular surface. Cys 22, which is known to have an abnormally low pKa of 3.8, is accessible from the exterior of the molecule. Pro 70, which is in close proximity to the disulfide bridge, assumes a conserved cis-peptide configuration. Mutational data available on the protein are in agreement with the three-dimensional structure.     

Analysis of dark-relaxation kinetics of variable fluorescence in intact leaves

The dark-relaxation kinetics of variable fluorescence, F_v, in intact green leaves of Pisum stativum L. and Dolichos lablab L. were analyzed using modulated fluorometers. Fast (t_1/2 = 1 s) and slow (t_1/2 = 7–8 s) phases in f_v dark-decay kinetics were observed; the rate and the relative contribution of each phase in total relaxation depended upon the fluence rate of the actinic light and the point in the induction curve at which the actinic light was switched off. The rate of the slow phase was accelerated markedly by illumination with far-red light; the slow phase was abolished by methyl viologen. The halftime of the fast phase of F_v dark decay decreased from 250 ms in dark-adapted leaves to 12–15 ms upon adaptation to red light which is absorbed by PSII. The analysis of the effect of far-red light, which is absorbed mainly by PSI, on F_v dark decay indicates that the slow phase develops when a fraction of Q_A ^– (the primary stable electron acceptor of PSII) cannot transfer electrons to PSI because of limitation on the availability of P700⁺ (the primary electron donor of PSI). After prolonged illumination of dark-adapted leaves in red (PSII-absorbed) light, a transient. F_v rise appears which is prevented by far-red (PSI-absorbed) light. This transient f_v rise reflects the accumulation of Q_A ^– in the dark. The observation of this transient F_v rise even in the presence of the uncoupler carbonylcyanide m-chlorophenyl hydrazone (CCCP) indicates that a mechanism other than ATP-driven back-transfer of electrons to QA may be responsible for the phenomenon. It is suggested that the fast phase in F_v dark-decay kinetics represents the reoxidation of Q_A ^– by the electron-transport chain to PSI, whereas the slow phase is likely to be related to the interaction of Q_A ^– with the donor side of PSII.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - F_O initial fluorescence level - F_v variable fluorescence - P700 primary electron donor of PSI - PSI, II photosystem I, II - Q_A (Q_A ^–) Q_B (Q_B ^–) primary and secondary stable electron acceptor of PSII in oxidized (reduced) state Supported by grant B6.1/88 DST, Govt. of India.     

Prediction of the profiles of the elution bands of proteins in preparative liquid chromatography

A semiequilibrium model of chromatography has been developed to account for the elution profile of high concentration bands. The use of this model requires the prior determination of the equilibrium isotherms of the studied compounds in the chromatographic phase system. The model is applied here to a study of the isocratic elution bands of chicken albumin and conalbumin on a weak anion exchanger in aqueous buffered solutions. Satisfactory agreement is observed between the experimental elution profiles of large-sample-size bands of chicken albumin and conalbumin.