Acetyl Coenzyme A: Deacetylvindoline O-Acetyltransferase,A Novel Enzyme from Catharanthus

A new enzyme, Acetyl Coenzyme A: deacetylvindoline 0-acetyl transferase (EC 2.3.1. -) which catalyses the synthesis of vindoline from acetyl coenzyme A and deacetylvindoline was isolated from the soluble protein extract of Catharanthus roseus leaves and purified approximately 365-fold. The enzyme had an apparent pI of 4.6 upon chromatofocusing, an apparent molecular weight of 45,000 daltons and a pH optimum between 8.0 to 9.0. Dithiothreitol was essential to maintain enzyme activity.Substrate saturation studies of this enzyme resulted in Michaelis Menton kinetics giving Km values of 5.4 and 0.7µM respectively for acetyl coenzyme A and deacetylvindoline. Studies of the forward reaction demonstrated an absolute requirement for acetyl coenzyme A and deacetylvindoline derivatives containing a double bond at positions 6, 7, whereas the reverse reaction occurred only in the presence of free coenzyme A and vindoline derivatives containing the same double bond. The forward reaction was subject to product inhibition by coenzyme A with an apparent Ki of 8 µM, but was not inhibited by up to 2 mM vindoline. The rate of reaction could therefore be regulated by the level of free coenzyme A in the cell, unaffected by the accumulation of indole alkaloid product.It was suggested that this enzyme catalyses a late step in the biosynthesis of vindoline.     

Proteoglycan modifications in cultured osteogenesis imperfecta skin fibroblasts

The PGs produced in the growth medium by skin fibroblast cultures from two O.I. affected patients were investigated. After density gradient centrifugation, in the most dense fraction two main families of molecules appeared. The patient with the more severe clinical picture showed a lower content of the PGs with the highest molecular weight. The GAG composition of PGs was different in the two patients. The more severely affected one showed an increase of HS and a decrease of ChS content, in agreement with the lower value of galactosamine to glucosamine ratio in urinary GAGs.     

Dion tomasellii (Zamiaceae), a new species with two varieties from western Mexico

Dion tomasellii sp. nov. occurs with an interrupted distribution from Guerrero to central Sonora. It is characterized by falcate to subfalcate leaflets. The populations from Sonora and northern Sinaloa are segregated asD. tomasellii var.sonorense on the basis of their narrower and glaucous leaflets.     

Temperature-dependent inactivation of nucleic acid binding and aggregation of the 1,25-dihydroxyvitamin D3 receptor

The interaction of the 1α,25-dihydroxyvitamin D₃ receptor with immobilized calf thymus DNA has been compared with its sedimentation properties on hypotonic sucrose gradients. Forty to sixty percent of total hormone:receptor complexes formed at 4 °C were retained by DNA-cellulose and could be eluted by 0.18 to 0.2 m KCl. In contrast, heating preparations to 25 °C rapidly and irreversibly converted receptor to a form which bound hormone and DEAE-cellulose normally, but was unable to associate with DNA. Similarly, the ability of receptor to aggregate to a 6 S species was labile at 25 °C. Stabilization of receptor in the DNA binding aggregating form was accomplished using Ca²⁺, Mg²⁺, Mn²⁺, or Na₂MoO₄ while several protease and phosphatase inhibitors were ineffective. An examination of DNA binding properties of aggregating and nonaggregating receptor forms revealed that only receptor competent to enter into aggregates could bind DNA suggesting that a functional nucleic acid binding site, and, hence, a nucleic acid interaction is necessary for aggregate formation. Consistent with this view, an RNA:receptor interaction appears to be involved in formation of the 6 S complex since removal of RNA by ribonuclease treatment or purification of receptor reduced aggregation, an effect that could be reversed by addition of purified RNA.     

6β-Hydroxyipolamiide,an iridoid glucoside from Stachytarpheta mutabilis

A new iridoid glucoside has been isolated from Stachytarpheta mutabilis and assigned the structure and configuration of 6β-hydroxyipolamiide on the basis of ¹H NMR and ¹³C NMR evidence. The conversion of this compound into penta- acetyllamiol proved the above assignment.     

Interactions between retinyl phosphate and bivalent cations.

In the presence of Mn(II) ions, the u.v. absorption spectrum of retinyl phosphate (Ret-P) solubilized in Triton X-100 micelles, phosphatidylcholine liposomes or rat liver microsomes exhibited a shift from the maximum of 330 nm to 287 nm. The effect of Mn(II) was reversed by adding EDTA or phosphate buffer. The same spectral change was found in the presence of poly-L-lysine in place of Mn(II) ions. The e.s.r. spectrum of Mn(II) in the presence or in the absence of Ret-P clearly showed that approx. 75% of the initial concentration of Mn(II) ions is bound to Ret-P when the molar ratio of Ret-P to Mn(II) ions is 4:1; no such binding occurred in the presence of retinol or retinoic acid. The appearance of two isosbestic points at 303 and 368 nm, in the presence of Mn(II) ions, suggests the existence of an equilibrium between an Mn(II)-bound monomer and an Mn(II)-bound dimer of Ret-P in Triton X-100 micelles. The same effect on the u.v.-absorption spectrum of Ret-P was also induced by Co(II), Cr(II), Zn(II) and Fe(II), but not by Mg2+ or Cu(II). The formation of the 'metachromatic complex' between Ret-P and Mn(II) or Co(II) inhibited the synthesis of retinyl phosphate mannose (Ret-P-Man) from exogenous and endogenous Ret-P and guanosine diphosphate [14C]mannose when bovine serum albumin was added after the metal ion. However, the order of addition did not influence Ret-P-Man synthesis in incubations containing MgCl2, which does not form the metachromatic complex with Ret-P. These results suggest that the bioavailability of proteins, polyamines and metal ions may control the extent to which Ret-P can be mannosylated in the intact membrane.