Nitrosyl cytochrome c oxidase. Formation and properties of mixed valence enzyme

We report the first resonance Raman scattering studies of NO-bound cytochrome c oxidase. Resonance Raman scattering and optical absorption spectra have been obtained on the fully reduced enzyme (a2+, a2+(3) NO) and the mixed valence enzyme (a3+, a2+(3) NO). Clear vibrational frequency shifts are detected in the lines associated with cytochrome a in comparing the two redox states. With 441.6 nm excitation the fully reduced preparation yields a spectrum similar to that of carbon monoxide-bound cytochrome c oxidase and is dominated by the spectrum of reduced cytochrome a. In contrast, in the mixed valence preparation no contributions from reduced cytochrome a are evident in the spectrum, verifying that this heme is no longer in the Fe2+ state. In the mixed valence NO-bound samples, a line appears at approximately 545 cm-1, a frequency similar to that found in NO-bound hemoglobin and myoglobin and assigned as an Fe-N-O-bending mode in those proteins. We do not detect this line in the spectrum of the fully reduced NO-bound enzyme. The carbonyl line of the cytochrome a3 heme formyl group in the fully reduced NO-bound enzyme appears at approximately equal to 1666 cm-1 in the resonance Raman spectrum. In the mixed valence NO-bound preparation the frequency of the carbonyl line increases by 1.2 cm-1 to approximately equal to 1667 cm-1. Thus, modes in cytochrome a2+(3) NO are sensitive to the redox state of the cytochrome a and/or CuA centers. We propose that the redox sensitivity of the formyl mode and the Fe-N-O mode results from an interaction between cytochrome a2+(3) (NO) and the cytochrome a-CuA pair, and is linked to the cytochrome a3 (NO) by the coupling between CuB and the NO-bound cytochrome a3 heme.     

Differing amounts of genetic polymorphism in testes and male accessory glands ofDrosophila melanogaster andDrosophila simulans

We surveyed genetic polymorphism by two-dimensional gel electrophoresis of male reproductive tract proteins in 20 isofemale lines each ofDrosophila melanogaster andDrosophila simulans. After classifying 244 such proteins ofDrosophila melanogaster and 271 ofDrosophila simulans by their distribution between testes and accessory glands within the reproductive tract, significant correlations were found between genetic polymorphism and tissue distribution. In both species, gland-specific proteins were significantly more polymorphic than testis-specific proteins, as well as those found in both testes and glands. Simultaneously, inDrosophila simulans, proteins found in roughly equivalent relative abundance in both testes and glands were significantly less variable than gland-specific and testis-specific proteins, as well as those with a quantitative difference in relative abundance between testes and glands. These correlations may reflect general differences in variability between extracellular and intracellular proteins and between proteins with broad as opposed to tissue-specific distributions.We thank the Natural Sciences and Engineering Research Council of Canada for financial support (Grant A0235 to R.S.S.).     

A photoreversible conformational change in 124 kDa Avena phytochrome

Tryptophan (Trp) fluorescence quenching of phytochrome has been studied using anionic, cationic and neutral quenchers, I-, Cs+ and acrylamide, respectively, in an effort to understand the molecular differences between the Pr and Pfr forms. The data have been analyzed using both Stern-Volmer and modified Stern-Volmer kinetic treatments. The anionic quencher, I-, was proven to be an ineffective quencher with Stern-Volmer constants, Ksv, of 0.60 and 0.63 M-1, respectively, for the Pr and Pfr forms of phytochrome. The cationic quencher, Cs+, showed about a 2-fold difference in the Ksv of Pr and Pfr, indicating a significant change in the fluorescent Trp environments during the Pr to Pfr phototransformation. However, only 25-37% of the fluorescent Trp residues were accessible to the cationic quencher. Most of the fluorescent Trp residues were accessible to acrylamide, but the quenching by acrylamide was indistinguishable for the Pr and Pfr forms. An additional quenching by acrylamide after a saturated quenching with Cs+ showed more than 40% increase in the Ksv of Pfr over Pr. These observations, along with the finding of two distinct components in the Trp fluorescence lifetime, indicate the existence of Trp residues in at least two different sets of environments in the phytochrome protein. The two components of the fluorescence had lifetimes of 1.1 ns (major) and 4.7 ns (minor) for Pr and 0.9 ns (major) and 4.6 ns (minor) for Pfr. Fluorescence quenching was found to be both static and dynamic as the Stern-Volmer constants for the steady-state fluorescence quenching were higher than for the dynamic fluorescence quenching. Based on the quenching results, in combination with the location of Trp residues in the primary structure, we conclude that the Pr to Pfr phototransformation involves a significant conformation change in the phytochrome molecule, preferentially in the 74 kDa chromophore-bearing domain.     

Calcium diphosphatidate membrane traversal is inhibited by common phospholipids and cholesterol but not by plasmalogen

Phosphatidate-mediated Ca2+ membrane traversal is inhibited by phospholipids (PL) such a phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin and lysoPC, but not by PC-plasmalogen. Kinetics of Ca2+ traversal through a 'passive' bilayer consisting of OH-blocked cholesterol show competition between PC and phosphatidic acid (PA); it appears likely that a Ca(PA.PC) complex is formed which is not a transmembrane ionophore but will reduce the amount of phosphatidic acid available for the formation of the ionophore, Ca(PA)2. PS and PI may inhibit Ca2+-traversal in the same manner by forming Ca(PA.PL) complexes. We suggest that PC-plasmalogen, with one of the Ca2+-chelating ester CO groups missing, cannot engage in calcium cages, i.e., Ca(PA.PL) complexes, and thus does not interfere with Ca(PA)2 formation. Double-reciprocal plotting of Ca2+ traversal rates in cholesterol-containing liposomes vs. calcium concentration suggests that cholesterol inhibits Ca2+ traversal by competing with Ca2+ for PA. The inhibition does not seem to be caused by a restructuring or dehydration of the membrane 'hydrogen belts' affected by cholesterol; most probably, it is due to hydrogen bonding of the cholesterol-OH group to a CO group of PA; this reduces the amount of PA available for the calcium ferry. The inhibition by sphingomyelin and lysoPC may also be explained by their OH group interacting with PA via hydrogen bonding. The pH dependence of Ca2+ traversal suggests that H[Ca(PA)2]- can serve as Ca2+ cross-membrane ferry but that at physiological pH, [Ca(PA)2]2- is the predominant ionophore. In conclusion, the results indicate that Ca2+ traversal is strongly dependent on the structure of the hydrogen belts, i.e., the membrane strata occupied by hydrogen bond acceptors (CO of phospholipids) and donors (OH of cholesterol, sphingosine), and that lipid hydrogen belt structures may regulate storage and passage of Ca2+.     

Degradation of apolipoprotein B-100 in human chylomicrons

The purpose of this study was to investigate the molecular forms of apolipoprotein B (ApoB) in human chylomicrons under well-preserved conditions. To this end, plasma and serum were collected from the same normal subjects after ingestion of a fatty meal. The samples were divided into three or four aliquots before the addition of various preservative mixtures, including antibiotics, antioxidants and proteinase inhibitors. The chylomicrons were isolated immediately, and all steps were carried out at or below 4 degrees C. Changes in the molecular weight of ApoB in chylomicrons were followed by a time study using 3.3% polyacrylamide gel electrophoresis containing SDS. ApoB from chylomicrons analyzed within 5 h of blood collection showed a single band with mobility identical to that of ApoB (ApoB-100) in low-density lipoproteins. When analyzed after 1-2 days, satellite bands smaller than ApoB-100 were observed, and a very faint band with Mr 200,000 appeared, which comigrated with intestinal ApoB (ApoB-48). Upon storage, the molecular weight of ApoB was smaller in chylomicrons subjected to a higher number of reflotations than those in chylomicrons washed less frequently, suggesting that purified chylomicrons degrade faster. A longer storage time at 4 degrees C (i.e., 7 or 14 days) revealed a stepwise degradation of ApoB, yielding Mr 200,000 band as the prominent form. The degradation of ApoB-100 was slower when both proteinase inhibitors, leupeptin and epsilon-amino caproic acid, were employed, and the appearance of Mr 200,000 band was quicker when the chylomicrons were processed at higher temperature (15-25 degrees C) in the absence of a proteinase inhibitor. Immunoblotting shows that the segment removed from ApoB-100 was the carboxyl-terminal portion. These results suggest the possible presence of a proteinase(s), which copurified with chylomicrons, and which converts ApoB-100 from a large to a smaller molecular form. Although the stop codon has been discovered recently in intestinal ApoB mRNA, which explains the mechanism for direct synthesis of ApoB-48, apparently ApoB-100 is also synthesized in the intestine of all eight subjects studied here, and the ApoB-100 degrades to a form which is ApoB-48-like.     

Plant water relations as selection criteria for drought tolerance in mustard

Genotypes of mustard (B. juncea) were evaluated for concurrent changes in leaf water potential (Ψ), leaf osmotic potential (π), leaf turgor potential (P) and leaf relative water content (RWC) during moisture stress at reproductive stage of growth. The slope ‘b’ in the regression between Ψ and π varied from 0.43 to 0.97 and was positively correlated with P and RWC. The genotypes with ‘b’ around 0.7 were able to maintain P of about 0.5 MPa at Ψ of − 2.5 MPa and thus such value of ‘b’ seems to provide enough degree of tolerance against drought.     

Preparation of passive bilayer liposomes

In studies of in-membrane molecular interactions, need may arise for a matrix that cannot itself interact, except hydrophobically, with the reactants. Such a bilayer matrix should, ideally, consist of only a hydrophobic zone without ionic outer layers and without hydrogen belts (the membrane strata containing CO and OH groups). However, because of the necessity of anchoring the bilayer to its aqueous surroundings, there must be polar substituents. Hydrophilic ether groups in the form of polyoxyethylenes can provide nearly sufficient anchoring and yet not confer unwanted reactivity to the membrane since they are only very weak H-bond acceptors. The stability of the bilayer is ensured by the presence of a few percent of an amphiphile (which may be the substrate to be studied, e.g. a phospholipid) or by a free polyethylene hydroxy group far remote from the original hydrogen belt region. Our most impermeable liposomes consisted of O-methylcholesterol/O-methoxyethoxyethoxyethylcholesterol; the most readily prepared liposomes were made from O-methylcholesterol and hydroxy(ethoxy)4dodecane (Brij 30) or Triton.