Conversion of type II procollagen to collagen in Vitro: Removal of the carboxy-terminal extension is inhibited by several naturally occurring amino acids,polyamines, and structurally related compounds

Chick embryo sterna, which actively synthesize type II procollagen, were pulse-labeled with radioactive proline; protein synthesis was then inhibited by unlabeled proline and cycloheximide. After the inhibition of protein synthesis, several amino acids, polyamines, or structurally related compounds were added to the incubation medium. The conversion of procollagen, first to two intermediates, pC-collagen and pN-collagen, and then to collagen, was monitored by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The addition of 50 mm β-alanine, arginine, asparagine, glutamine, hydroxylysine, lysine, or ornithine, as well as agmatine, ?-aminocaproic acid, S-2-aminoethylcysteine, cadaverine, canavanine, putrescine, or spermine clearly inhibited the removal of the carboxy-terminal extension and pC-collagen accumulated; the removal of the amino-terminal extension was not affected. The inhibition of the conversion was reversible and unaffected by fetal calf serum. The results suggest that the conversion of type II procollagen to collagen requires at least two separate proteinases for the removal of amino-terminal and carboxy-terminal extensions. The results further suggest that naturally occurring molecules may be used to modulate the rate of conversion of procollagen to collagen, and development of analogs of these compounds may provide the means to interfere with excessive deposition of collagen in diseases with tissue fibrosis.     

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.     

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).     

A Human Active Lower Limb Model for Chinese Pedestrian Safety Evaluation

A subsystem impactor test for pedestrian lower limb injury evaluation has been brought in China New Car Assessment Protocol(CNCAP).Concerning large anthropometr...