The chemical identity of the immunoreactive LHRH-like peptide biosynthesized in the human placenta

Freshly obtained human placental trophoblasts were minced and pulselabeled for 30 min at 37°C with tritiated L-Tyrosine. After homogenisation, the crude extract was centrifuged and deproteinized with 10% TCA. The supernatant was defatted and the peptides concentrated through hydrophobic binding on ODS-silica cartridges. The bound, crude peptide extract was eluted and subjected to gradient, reverse-phase High Performance Liquid Chromatography. The fractions corresponding to the absorption peak of reference, synthetic LHRH were collected and extensively purified to radioactive homogeneity by further multiple HPLC. After digestion with pyroglutamate aminopeptidase, the resulting nonapeptide was manually sequenced by dansyl-Edman degradation. All the incorporated radioactivity was found to reside exclusively in residue number 4 of the nonapeptide; thus establishing for the first time the primary sequence of biosynthetic placental LHRH as: pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂, identical to its hypothalamic counterpart.     

稀土元素Pr~(3+)、Nd~(3+)对蚕豆(Vicia feba)叶片原生质膜上Ca~(2+)·Mg~(2+)-ATPase活性的影响

本文介绍用二相分配法制备蚕豆叶片原生质膜上的Ca~(2+)·Mg~(2+)-ATPase,用以研究镧系,稀土离子对此酶活性的影响。初步证实Pr~(3+)、Nd~(3+)对依赖于CaM的以及不依赖于CaM的蚕豆叶片原生质膜上Ca~(2+)·Mg~(2+)-ATPase活性的抑制不是CaM专一的。     

Stage-specific cuticular proteins of Ostertagia circumcincta and Ostertagia ostertagi

In this study we have shown that NHS-biotin and I¹²⁵-streptavidin can detect cuticular polypeptides of Ostertagia spp. The labelled polypeptide profile of intact nematodes is simple compared to the profile obtained by labelling homogenates. None of the major internal polypeptides are labelled and the subset of proteins labelled in intact nematodes appears to be mainly surface associated. The results presented here demonstrate that NHS-biotin may be used as a reagent for the analysis of surface polypeptides. The surface polypeptide profiles of the five major developmental stages (L1, L2, L3, L4 and adult) of Ostertagia circumcincta show a series of stage-specific molecules with no polypeptides common to all stages, indicating that the cuticle is a dynamic structure which changes throughout the life cycle. Similarity comparison of Ostertagia ostertagi L3 and L4 stage surface profiles showed that each stage is clearly distinct; comparison of these stages between the two species shows an overall similarity.     

De novo biosynthesis of enkephalins and their homologues in the human placenta

Fresh trophoblastic preparations of two human placentae delivered at term were pulse labelled for 30, 120 and 240 min with tritiated L-tyrosine. After deproteinizing and defatting, the peptide extracts were first concentrated through reversible hydrophobic binding on octadecasilyl-silica particles, prior to further resolution by repetetive high-performance liquid chromatography. Four peptides were isolated and purified to radioactive homogeneity, namely Met-enkephalin, Leu-enkephalin, (Arg⁶)-Leu-enkephalin, and (Arg⁶, Arg⁷)-Leu-enkephalin. Their presence and identity were further confirmed by substractive Edman degradation and by radioimmunoassay. No detectable amounts of radioactive Dynorphin could be trapped, however. Under the incubation conditions used, reference tritiated Leu-enkephalin had a biological half-life of circa 9.5 min.     

The phosphorylation of brain microtubular proteins in situ and in vitro

The phosphorylation of microtubular proteins isolated by reassembly in vitro from slices of guinea-pig cerebral cortex labelled with [32P]orthophosphate was investigated. Under the conditions tested, both and the alpha and beta forms of tubulin contained metabolically-active P which accounted for about one third of the total 32P incorporated into protein; the remaining protein-bound 32P was associated with 3-4 minor high MW components co-purifying with tubulin during two cycles of assembly-disassembly. Microtubular protein prepared in this way contained approx. 0.8 mol of alkalilabile P/mol of tubulin dimer (M.W. 110,000). In vitro studies showed that reassembled microtubular protein preparations catalysed the incorporation of up to 0.55 mol of P/mol of tubulin dimer during incubation with Mg2+ and [gamma 32P]ATP. The reaction was linear during the first 30 min of incubation at 37 degrees C. Cyclic AMP (10 microM, final concentration) caused a transient increase in the initial rates of tubulin phosphorylation. Little label was incorporated into the minor high M.W. components under these conditions. The in vitro phosphorylation of microtubular protein increased in a non-linear manner with respect to protein concentration: this was in contrast to earlier experiments showing linear kinetics when chromatographically isolated tubulin was tested for intrinsic kinase activity. Isolated microtubular protein preparations bound [3H]GTP, [3H]ATP and to a lesser extent, [3H]cyclic AMP, and exhibited Ca(2+)-ATPase activity (up to 60 pmol Pi released min/mg protein at 37 degrees C).