ISOLATION, PURIFICATION, AND CHARACTERIZATION OF DMSP LYASE (DIMETHYLPROPIOTHETIN DETHIOMETHYLASE (4.4.1.3)) FROM THE RED ALGA POLYSIPHONIA PANICULATA1

Polysiphonia paniculata Montagne is an intertidal red alga known to produce large amounts of the compound dimethylsulfoniopropionate (DMSP). Conversion of this substrate into dimethylsulfide is accomplished in P, paniculata by an enzyme called DMSP lyase (dimethylpropiothetin dethiomethyla.se (4.4.1.3)). DMSP lyase has been purified and characterized from P. paniculata. Enzymie activity is found in two different proteins: the larger with a molecular weight of 9.26 ± 10⁴ daltons and the smaller with a molecular weight of 3.65 ± 10⁴ daltons. Specific activity of the enzyme is 526 μmols min⁻¹mg⁻¹ for the smaller protein a nd 263 μmols min ⁻¹ mg⁻¹ for the la rger protein. The Michaelis-Menten constant (K_m) is 72.8 μM ± 17.15 and the v_max is 1.62 μmols min⁻¹± 0.928 for the 92.6-kDa protein. The p1 of the larger protein is 5.8 and 5.9 for the smaller protein. Interaction with cysteine protease inhibitors L-trans-epoxysuccinyl-leucylamido (4-guanidino)-butane, dithiobis-(2-nitrobenzoate), or N -ethylmaleimide inactivated enzyme activity. The presence of either magnesium or calcium with DMSP lyase enhanced activity al concentrations between 20 and 40 μM but had little effect above these levels. Addition of the divalent chelators ethylenebis(oxyethylenenitrilo) tetraacetic acid and ethylenediaminetetraacetate decreased activity of the enzyme, but activity was restored when either chelator was removed and magnesium or calcium was added to the enzyme .     

Conformational studies of heterochiral peptides with diastereoisomeric residues: Crystal and molecular structures of linear dipeptides derived from leucine,isoleucine, and allo-isoleucine

The x-ray diffraction analyses of three N- and C-terminally blocked L , D dipeptides, namely t-Boc-D -Leu-L -Leu-OMe ( 1 ), t-Boc-L -Ile-D -alle-OMe ( 2 ), and t-Boc-D -aIle-L -Ile-OMe (3) containing enantiomeric or diastereomeric amino acid residues have been carried out. The structures were determined by direct methods and refined anisotropically to final R factors of 0.077. 0.058. and 0.072 for ( 1 ) ( 2 ) and ( 3 ), respectively. Peptides 1–3 all assume a similar U-shaped structure with ? and ψ torsion angles cosrresponding to one of the possible calculated minimum energy regions (regions E and G for L residues, and F*. D* and H* for D residues). The peptide backbones of 1-3 are almost super-imposable [provided that the appropriate inversion of the chiral centers of ( 2 ) is made]. Side-chain conformations of Leu residues in peptide ( 1 ) are g^? (tg^?) for the L -Leu residue and the mirrored g⁺ (tg⁺) for the D -Leu residue; however, in peptides ( 2 ) and ( 3 ) the conformations of the isoconfiguralional side chains of the Ile or allo-Ile residues are (g^?t) t and (tg⁺) tfor the L -Ile and the D -allo-Ile moieties, respectively. In all cases, these conformations correspond to the more populated conformers of β-branched residues statistically found in crystal structures of small peptides. The results seem to indicate that, at least in short peptides with enantiomeric or diastereoisomeric residues, the change in chirality in the main-chain atoms perturbs the backbone conformation to a lesser extent and the side chain conformation to a greater extent. © 1995 John Wiley & Sons, Inc.     

Bioactive peptides: Conformational studies of [TYR4] cyclolinopeptide A

The solid state conformational analysis of [Tyr⁴] cyclolinopeptide A has been carried out by x-ray diffraction studies. The crystal structure of the monoclinic form, grown from a dioxane-water mixture [a = 9.849 (5) Å, b = 20.752 (4) Å, c = 16.728 (5) Å, β = 98.83 (3)°, space group P2₁, Z = 2], shows the presence of five intramolecular N-H? O?C hydrogen bonds, with formation of one C₁₇ ring structure, one α-turn (C₁₃), one inverse γ-turn (C₇), and two β-turns (C₁₀, one of type III and one of type 1). The Pro¹-Pro² peptide unit is cis (ω = 5°) all others are trans. The structure is almost superimposable with that of cyclolinopeptide A. The rms deviation for the atoms of the backbones is on the average 0.33 Å. © 1995 John Wiley & Sons, Inc.     

Kinetic changes of αB crystallin expression in neoplastic cells and syngeneic rat fibroblasts at various subculture stages

B crystallin, a structural protein of the mammalian lens essential for the maintenance of lens transparency, is also expressed, at variable levels, in many extraocular tissues where it plays a protective role in stress conditions. In fact, heat or toxic shocks, as well as pathological states, increase B crystallin levels in many cell types. Here we show that B crystallin expression is also modulated in subcultures of rat fibroblasts and Galliera sarcoma cells. Western blots analysis with anti B crystallin antibodies reveals the presence of the protein in both cell populations, although the kinetic pattern of expression is different. Galliera fibroblasts constitutively express the protein up to the 70th subculture and afterwards the synthesis ceases. On the other hand, Galliera sarcoma cells do not contain B crystallin in the early stages of the culture, but there is a progressive increases between the 20th and 40th cell subculture. Differences also exist concerning the intracellular distribution: B crystallin is diffusely localized in the cytoplasm of fibroblasts while in sarcoma cells it localizes mainly to the perinuclear region. B crystallin is totally recovered as soluble protein in the supernatants obtained after low speed centrifugation of fibroblast homogenates, while in sarcoma cells a portion of the protein is also recovered in the insoluble pellet. Intracellular pH measurements show an alkaline cytosol in sarcoma cells compared to fibroblasts. Heat shock treatment of fibroblast subcultures constitutively expressing B crystallin induces an over-expression of the protein, while in fibroblasts whose biosynthetic capacity is lost, heat shock is unable to activate the crystallin gene. Correlation between B crystallin expression and proliferative rate shows that highly proliferating fibroblasts do not express B crystallin, while neoplastic cells do.     

The eects of a ring chromosome on the meiotic segregation of other chromosomes in Saccharomyces cerevisiae

Meiotic chromosome segregation must occur with high fidelity in order to prevent the generation of aneuploid cells. We have previously described the identification and genetic characterization of a yeast mutant with defects in meiotic sister-chromatid segregation. We attributed the phenotype in this mutant to a dominant allele, which we referred to as SID1-1. These mutants appeared to exhibit high levels of nondisjunction and precocious separation of sister-chromatids of chromosome III, as well as precocious separation of sister chromatids of chromosome VIII and a univalent artificial chromosome. We show here that the unusual meiotic behavior of chromosome III in these strains is due to the presence of a ring III chromosome, rather than a mutant gene. Additional experiments demonstrate that a ring III/rod III pair alters the meiotic segregation of a univalent artificial chromosome.     

Proton/electron stoichiometry of mitochondrialbc 1 complex. Influence of pH and transmembrane δpH

The effect of pH and transmembrane pH on the efficiency of the proton pump of the mitochondrialbc ₁ complex bothin situ and in the reconstituted state was studied. In both cases the H⁺/e ^– ratio for vectorial proton translocation by thebc ₁ complex respiring at the steady state, under conditions in which the transmembrane pH difference (pH) represents the only component of the proton motive force (p), was significantly lower than that measured under level flow conditions. The latter amounts, at neutral pH, to 1 (2 including the scalar H⁺ release). In the reconstituted system steady-state pH was modulated by changing the intravesicular buffer as well as the intra/extra-liposomal pH. Under these conditions the H⁺/e^– ratio varied inversely with the pH. The data presented show that pH exerts a critical control on the proton pump of thebc ₁ complex. Increasing the external pH above neutrality caused a decrease of the level flowH ⁺/e ^– ratio. This effect is explained in terms of proton/electron linkage inb cytochromes.     

Genomewide Scan of Multiple Sclerosis in Finnish Multiplex Families

Multiple sclerosis (MS) is a neurological, demyelinating disorder with a putative autoimmune etiology. It is thought to be a multifactorial disease with a complex mode of inheritance. Here we report the results of a two-stage genomewide scan for loci predisposing to MS. The first stage of the screen, with a low-resolution map, was performed in a selection of 16 pedigrees collected from an isolated Finnish population. Multipoint, non-parametric linkage analysis of the 328 markers did not reveal statistically significant results. However, 10 slightly interesting regions (P = .1-.15) emerged, including our previous findings of the HLA complex on 6p21 and a putative locus on 5p14-p12. Eight of these novel regions were further analyzed by use of denser marker maps, in the second stage of the scan. For the chromosomal regions 4cen, 11tel, and 17q, the statistical significance increased, but not conclusively; for 2q32 and 10q21, the statistical significance did not change. Accordingly, genotyping of the high-density markers in these regions was performed, and the data were analyzed by use of two-point, parametric linkage analysis using the complete pedigree information of the 21 Finnish multiplex families. We detected suggestive evidence for a predisposing locus on chromosomal region 17q22-q24. Several markers on 17q22-q24 yielded positive LOD scores, with the maximum LOD score (Zmax) occurring with D17S807 (Zmax = 2.8, theta = .04; dominant model). Interestingly, a suggestive linkage between MS and the markers on 17q22-q24 was also revealed by a recent genomewide scan in MS families from the United Kingdom.