The Pharmacia fast protein liquid chromatography system was employed to fractionate a purified preparation of human LH (hLH) on the anion exchanger Mono Q at pH 7 X 8 into 14 sub-fractions. Each of ...the sub-fractions was characterized by its behaviour on polyacrylamide gel electrophoresis, sodium dodecyl sulphate (SDS) gel electrophoresis, LH receptor binding activity and sialic acid content. All sub-fractions contained sialic acid, were active in binding to LH receptors, and exhibited components typical of hLH subunits on SDS gel electrophoresis. None of the subfractions was homogeneous with respect to charge. There is evidence that part of the heterogeneity results from the presence in some molecules of an internal proteolytic cleavage within the beta-subunit, and fractions enriched in species containing such cleavages were prepared by this method.
YACs and the C. elegans genome Coulson, A; Kozono, Y; Lutterbach, B ...
BioEssays,
August 1991, Letnik:
13, Številka:
8
Journal Article
Recenzirano
During the past decade, it has become apparent that it is within our grasp to understand fully the development and functioning of complex organisms. It is widely accepted that this undertaking must ...include the elucidation of the genetic blueprint - the genome sequence - of a number of model organisms. As a prelude to the determination of these sequences, clone-based physical maps of the genomes of a number of multicellular animals and plants are being constructed. Yeast artificial chromosome (YAC) vectors, by virtue of their relatively unbiased cloning capabilities and capacity to carry large inserts, have come to play a central role in the construction of these maps. The application of YACs to the physical map of the Caenorhabditis elegans genome has enabled cosmid clone 'islands' to be linked together in an efficient manner. The long-range continuity has improved the linkage between the genetic and physical maps, greatly increasing its utility. Since the genome can be represented by a relatively small number of YACs, it has been possible to make replica filters of genomically ordered YACs available to the community at large.
Radioimmunoassays utilizing antisera specific for the carboxyl-terminal portion of human chorionic gonadotrophin (hCG) beta-subunit were used to measure the concentration in human pituitary extracts ...of an immunoactive hCG factor (hCG') which was different from human LH (hLH). The content of hCG' from different human pituitary pools collected between 1966 and 1979 was relatively constant at 0.5-1.1 microgram per gland. The hCG' concentrations observed in acetone-dried powder of individual human pituitary glands (0.4-26 ng/mg) were close to those reported for full-term placental powder. After separation and partial purification of human pituitary glycoprotein hormones, pituitary hCG' was found mainly in the crude human FSH (hFSH) fraction. It was separated from hFSH by diethylaminoethyl-cellulose chromatography at pH 7 and by gel filtration on Sephadex G-100. On gel filtration its molecular size was larger than that of hLH or hFSH and it was strongly bound to Concanavalin A-Sepharose. The most active preparations of pituitary hCG' obtained by these procedures were approximately 5 per cent as potent by specific radioimmunoassay as hCG purified from pregnancy urine. Although the hCG' content in individual pituitary glands was more variable than the hLH content, on average pituitary hCG' was estimated to be around 25- to 50-fold less than the content of hLH, hFSH or human TSH in the human pituitary gland.
This report describes some of the properties of a clinical-grade preparation of human growth hormone (hGH) extracted from acetone-preserved autopsy human pituitary glands and used in Great Britain ...from 1967 to 1980. Gel filtration of this hGH on Sephadex G-100 yielded (on a weight basis) an average of 48% of a high molecular weight fraction, 10% of an intermediate fraction expected to contain dimeric forms of the hormone and 33% of a fraction considered to be the hGH monomer. The immunoassay potency of the monomer fraction was twice that of the clinical-grade preparation and the amino-acid composition of the monomer fraction agreed well with that obtained from published hGH sequence data. The results of polyacrylamide-gel electrophoresis (under reducing and dissociating conditions) and amino-acid analysis of the high molecular weight fraction suggest that it contains around 30% of aggregated hGH as well as other material not separated from hGH by the purification procedure.
Highly purified human pituitary FSH was partially dissociated by treatment with 8M-urea, and alpha- and beta-subunits were isolated by ion-exchange chromatography and gel filtration. Tests of ...biological activity by in-vivo assays and in-vitro radioreceptor assays were in good agreement and showed that preparations of isolated alpha-subunit had less than 1%, and beta-subunit from 2 to 10% of the FSH activity of the intact hormone. In contrast to results reported elsewhere, most of the subunit preparations reassociated with counterpart subunit to regain biological activity equal to that of intact FSH (around 160 mg NIH-FSH-S1/mg). The intact FSH recovered as a by-product after isolation of subunits was of high biological activity, and its LH contamination was reduced by more than 90% when compared with thepurified FSH starting material. The subunits are relatively inactive in a radioimmunoassay specific for intact FSH. Sialic acid and tryptophan determinations indicated that both subunits contain sialic acid and that tryptophan is present only in the beta-subunit.
The actions of human follicle-stimulating hormone (hFSH) and its beta-subunit were examined in several assays in reptiles, including effects on lizard testicular activity (growth and androgen ...production) in vivo, and stimulation of androgen production by snake testes and competition for binding of 125I-labeled hFSH in lizards and snakes in vitro. Binding was also examined with mammalian tissues. The hFSH was highly steroidogenic in the snake and lizard; otherwise results were similar to those observed in mammals. In all cases, the potency of the beta-subunit was only a few per cent of the intact hormone. The potency of hFSH in vivo compared with NIH-FSH ovine standards was several 100 times greater than in vitro. Results for stimulation of androgen production in vivo closely paralleled those for binding assays in both reptiles and mammals. In contrast to previous results for ovine FSH beta-subunit, human FSH beta-subunit has little if any FSH biological activity in reptiles.
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