Ionic-strength-dependence of the oxidation of native and pyridoxal 5''-phosphate-modified cytochromes c by cytochrome c oxidase.

The ionic-strength-dependences of the rate constants (log k plotted versus square root of 1) for oxidation of native and pyridoxal 5'-phosphate-modified cytochromes c by three different preparations of cytochrome c oxidase have complex non-linear character, which may be explained on the basis of present knowledge of the structure of the oxidase and the monomer-dimer equilibrium of the enzyme. The wave-type curve (with a minimum and a maximum) for oxidation of native cytochrome c by purified cytochrome c oxidase depleted of phospholipids may reflect consecutively inhibition of oxidase monomers (initial descending part), competition between this inhibition and dimer formation, resulting in increased activity (second part with positive slope), and finally inhibition of oxidase dimers (last descending part of the curve). The dependence of oxidation of native cytochrome c by cytochrome c oxidase reconstituted into phospholipid vesicles is a curve with a maximum, without the initial descending part described above. This may reflect the lack of pure monomers in the vesicles, where equilibrium is shifted to dimers even at low ionic strength. Subunit-III-depleted cytochrome c oxidase does not exhibit the maximum seen with the other two enzyme preparations. This may mean that removal of subunit III hinders dimer formation. The charge interactions of each of the cytochromes c (native or modified) with the three cytochrome c oxidase preparations are similar, as judged by the similar slopes of the linear dependences at I values above the optimal one. This shows that subunit III and the phospholipid membrane do not seem to be involved in the specific charge interaction of cytochrome c oxidase with cytochrome c.     

A novel a-type terminal oxidase from Sulfolobus acidocaldarius with cytochrome c oxidase activity

Cytochrome C oxidase was solubilized with a nonionic detergent n-decanoyl-N-methyl glucamide from the membranes of Sulfolobus acidocaldarius, a thermoacidophilic archaebacterium, and was purified. The enzyme oxidized horse heart cytochrome C with a Vmax of 63 mumols/min/mg at 50 degrees C. The activity was sensitive to cyanide. The enzyme also catalyzed oxygen uptake detergent on N, N, N', N'-tetramethyl p-phenylene diamine. An apparent molecular mass was estimated to be 150 kDa. The enzyme is composed of three subunits of 37, 23 and 14 kDa. Spectral characteristics were similar to typical bacterial aa3 except for the presence of a novel 583 nm peak observed in reduced minus oxidized difference spectrum.     

Chemometric Tools to Highlight the Variability of the Chemical Composition and Yield of Lebanese Origanum syriacum L. Essential Oil

This study deals with the variation in the yield and composition of Lebanese Origanum syriacum L. essential oil (EO) according to harvesting time, drying methods used, and geographical location. Plant material was harvested twice a month all over 2013 and 2014 from Qartaba and Achkout located at high altitude and from Byblos at low altitude. EOs of the aerial parts were obtained by hydrodistillation. The highest yields were obtained at full flowering stage and slightly reduced after flowering. The GC/MS analysis revealed the presence of 50 components representing 90.49 – 99.82%, 88.79 – 100%, and 95.28 – 100% of the total oil extracted from plants harvested from Qartaba, Achkout, and Byblos, respectively. The major components in the oils were: carvacrol (2.1 – 79.8%), thymol (0.3 – 83.7%), p‐cymene (2.8 – 43.8%), thymoquinone (0.4 – 27.7%), γ‐terpinene (0.4 – 10.0%), octan‐3‐ol (0.3 – 4.9%), caryophyllene oxide (0.2 – 4.7%), oct‐1‐en‐3‐ol (0.3 – 3.7%), β‐caryophyllene (0.7 – 3.2%), cis‐sabinene hydrate (0.1 – 2.8%), terpinen‐4‐ol (0.1 – 2.8%), and α‐terpinene (0.2 – 2.2%). Independent components analysis (ICA) revealed that two groups were discriminated, reflecting compositional differences in the EOs profiles of the Lebanese oregano samples: O. syriacum grown in Qartaba and Achkout belongs to carvacrol chemotype, while O. syriacum grown in Byblos belongs to thymol chemotype. The flowering phase was the most productive period in terms of yield, bringing marked changes in the EO composition by increasing the amounts of carvacrol or thymol, and decreasing those of thymoquinone and p‐cymene.     

Anatomical distribution of primary amine oxidase activity in four adipose depots and plasma of severely obese women with or without a dysmetabolic profile

Semicarbazide-sensitive amine oxidase (SSAO), identical to primary amine oxidase or vascular adhesion protein-1, is a membrane enzyme that generates hydrogen peroxide. SSAO is highly expressed at the adipocyte surface, and its plasma levels increase with type 2 diabetes. Since visceral adipose tissue (AT) is more tightly associated with obesity complications than subcutaneous (SC) abdominal fat, we compared SSAO activity in plasma and 4 distinct AT locations in 48 severely obese women (body mass index (BMI), averaging 54 ± 11 kg/m²), with or without a dysmetabolic profile. Higher glucose and triacylglycerol levels vs lower high-density lipoprotein (HDL)-cholesterol characterized dysmetabolic women (DYS; n = 25) from non-dysmetabolic (NDYS; n = 23), age- and weight-matched subjects. SC, mesenteric (ME), omental (OM), and round ligament (RL) fat locations were collected during bariatric surgery. SSAO capacity to oxidize up to 1 mM benzylamine was determined in AT and plasma with radiometric and fluorimetric methods. Plasma SSAO was higher in the DYS group. SSAO activity was higher in fat than in plasma, when expressed as radiolabeled benzaldehyde per milligram of protein. In ATs from DYS women, protein content was 10 % higher, and basal hydrogen peroxide release lower than in NDYS subjects, except for RL location. The SSAO affinity towards benzylamine did not exhibit regional variation and was not altered by a dysmetabolic profile (K _m averaging 184 ± 7 μM; n = 183). Although radiometric and fluorimetric methods gave different estimates of oxidase activity, both indicated that AT SSAO activity did not vary according to anatomical location and/or metabolic status in severely obese women.     

Interaction between the cell-cycle-control proteins p34cdc2 and p9CKShs2. Evidence for two cooperative binding domains in p9CKShs2.

A universal intracellular factor, the 'M-phase-promoting factor' (MPF), displaying histone H1 kinase activity and constituted of at least two subunits, p34cdc2 and cyclin Bcdc13, triggers the G2----M transition of the cell cycle in all organisms. The yeast p13suc1 and p18CKS1 subunits and their functionally interchangeable human homologues, p9CKShs1 and p9CKShs2, directly interact with p34cdc2 and may actually be part of the MPF complex. We have chemically synthesized p9CKShs2 and several of its peptide domains in order to investigate the binding of p9CKShs2 and p34cdc2. Several arguments support the hypothesis that the N-terminal half (peptide B) and the C-terminal half (peptide E) each contain a p34cdc2-binding site and that these two binding domains cooperate in establishing a stable p9CKShs2-p34cdc2 complex: (a) only the combination of peptides B + E, and not B or E alone, is able to elute the cdc2 kinase from p9CKShs1-Sepharose beads; (b) only immobilized peptides B + E, and not immobilized B or E, bind the cdc2 kinase; (c) only the peptides B + E combination, and not B or E alone, can compete with p9CKShs1 for cdc2 kinase binding; (d) only when supplemented with E or B free peptide does the cdc2 kinase bind to B- or E-Sepharose beads, respectively. No binding occurs in the absence of free peptide. This additivity cannot be attributed to the formation of a B-E complex mimicking the full-length p9CKShs2. The cyclin B subunit is not required for the formation of the p9CKShs2-p34cdc2 complex through these two binding domains. The implications of the existence of two cooperative p34cdc2-binding domains in p9CKShs2 on the structure of the active M-phase-specific kinase is discussed.