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W K Liu

Publications and source records attributed to W K Liu.

At least 109 records · Page 6Linked to original sources
PubMed1984

Deglycosylated ovine lutropin: preparation and characterization by in vitro binding and steroidogenesis.

Ovine lutropin (oLH) subunits were deglycosylated in anhydrous hydrofluoric acid. All possible combinations of native and deglycosylated alpha- and beta-subunits were prepared and characterized. The two native plus deglycosylated counterpart preparations showed properties intermediate between the native only and the deglycosylated subunit hybrid. For brevity, only the properties of the latter hybrid were described in detail. Deglycosylation did not alter subunit-subunit recombination, protein conformation (circular dichroism) or the peptide chains (N-termini). Deglycosylation enhanced receptor-binding activity, but the carbohydrate moieties are important for maximal steroidogenesis (Leydig cell testosterone production). The deglycosylated hybrid molecule acts as an antagonist or agonist to native hormone action depending on the test system and conditions. The deglycosylated hormone binds to the receptor much more rapidly than the native hormone and binding plateaued at a higher level in the case of the deglycosylated hormone, although cross-labeling of tracers (either native or deglycosylated) indicated that competition was for the same type of receptors. To eliminate internalization of the hormone as a factor in the binding studies we used broken cell (membrane) preparations of Leydig cells. Differences in the preparations are thus attributable to a greater 'on-rate' for the deglycosylated hormone. The 'off-rate' from the receptor was essentially equal for both native and deglycosylated hormones. For this reason the deglycosylated hormone has only limited antagonistic action for native hormone activity.

Animals↗
PubMed1984

Porcine inhibin: initial fractionation as a high molecular weight complex.

A high molecular weight complex or aggregate of inhibition was obtained by chromatography of porcine follicular fluid in Fractogel TSK65F. Recovery of activity was good (usually 80-100%), but only 30-60% was recovered as a high molecular weight complex (greater than 160,000) free of albumin and gamma globulin (the two major proteins in follicular fluid). The balance of the activity was distributed in the gamma globulin-albumin region of the chromatogram (i.e., 160,000 down to 65,000 daltons). Distribution in this region of the chromatogram in part reflected the prior processing of the sample (e.g., it was augmented by ethanol or acetone precipitation prior to chromatography). The utility of Fractogel chromatography lies in its ability to resolve a large portion of the inhibin activity from the major proteins (albumin and gamma globulin), plus an efficient recovery of activity. Maximum purification on the Fractogel chromatograms was approximately 20-fold. This product and the other Fractogel fractions were tested for protease activity by a sensitive slab gel procedure. All fractions contained detectable protease activity that could potentially affect inhibin activity during further fractionations. This was shown with a protease fraction isolated from porcine follicular fluid by affinity chromatography. When added to a partially purified inhibin preparation, this protease fraction destroyed 77% of the inhibin activity.

Animals↗
PubMed1982

Toxic effects of chromic sulphate on the common carp, Cyprinus carpio.

The effects of chromic sulphate, which is commonly used in the tanning of leather, on the common carp, Cyprinus carpio and the bighead, Aristichthys nobilis, were studied. C. carpio was found to be more susceptible than A. nobilis to chromic sulphate at the lowest concentration tested (100 mg/l) whereas results obtained at other concentration were similar for both species. Damage to the gills, liver and intestine was observed by histopathological examination. A wide range of chemicals is used in the different stages of treating leather and a possible treatment of the effluent from the tanneries is suggested.

Animals↗
PubMed1981

Steroid and plasminogen activator production by cultured rat granulosa cells in response to hormone treatment.

Granulosa cells isolated from immature, DES-primed female rats were incubated in medium-199 plus 10% chicken serum with addition of FSH, or testosterone, or both. Cultures were incubated at 37 degree C for 7 days; medium samples were taken daily and analyzed for steroids and plasminogen-activator production. Only cultures containing FSH + testosterone produced significant amounts of both estradiol and progesterone after 2 days of incubation. The rate of estradiol production increased steadily up to the 4th day and then leveled off; the production of progesterone reached a maximum around the 3rd day, and then declined rapidly afterward. FSH alone was able to stimulate plasminogen activator production at the first day. Cultures with FSH + testosterone produced an additional peak of plasminogen activator activity at the 4th day. Plasminogen-activator production is thus not correlated with steroidogenesis in a simple way. We conclude that the granulosa cell require the presence of both FSH and testosterone at the beginning of incubation for normal response. Delayed addition of either hormone, or both, to the culture causes damage to the cells ability to produce normal responses to hormone treatment.

Animals↗
PubMed1979

The effect of digested sludge on the common carp, Cyprinus carpio L.

The effects of sewage sludge on the survival of the common carp, Cyprinus carpio, were investigated. It was noted that, when the concentrations of the sludge used for rearing carp exceeded 0.6%, the fish died within 42 days, whereas those reared in 0.2% sludge remained alive and healthy during the trial period. The body and the gills of the dead fish were covered by a veil-like film which looked like coagulated mucus and was formed by the heavy metal ions from the sludge reacting with some constituents of the mucus secreted by the gill. The histopathological symptoms of the various organs further demonstrated that internal injury in liver and kidney was also an important feature of the intoxicants as well as external damage, especially the particles observed around the gills. It was concluded that the optimum concentration of sludge to any species of fish should be determined by testing a series of concentrations before using it as a supplementary fish feed or fish pond fertilizer.

Animals↗
PubMed1977

Isolation and recombination of ovine lutropin subunits with complete recovery of biological activity.

Based on the results obtained in the previous paper (Liu, W.-K., Ascoli, M., and Ward, D. N. (1977) J. Biol. Chem. 252, 5274-5279) we have devised a method that permits the isolation of the subunits of ovine lutropin (oLH) in their "native" form. The hormone was dissociated by guanidine HCl, and the subunits separated by salt precipitation (Sairam, M. R., and Li, C. H. (1974) Arch. Biochem. Biophys. 165, 709-714). The present method differs significantly from that of Sairam and Li in that all operations are carried out at pH 5.0 or higher, a condition we have shown to be crucial to maintain the integrity of the isolated subunits. The yield of subunits was 75 to 85%. The isolated subunits exceed 95% purity (i.e. contamination with the other subunit or intact lutropin is less than 5%), migrate as single bands in sodium dodecyl sulfate-polyacrylamide gels, and show only the NH2-terminal heterogeneity expected from that of the native hormone. Recombination of the subunits can be accomplished with 75 to 85% yield (with respect to mass) and with full recovery of biological activity.

Amino Acid Sequence↗
PubMed1977

Studies of disulfide bond location in ovine lutropin beta subunit.

By combinations of selective chemical cleavage (cyanogen bromide), selective enzymatic cleavage (trypsin, thermolysin), and random cleavage (partial acid hydrolysis) a series of disulfide-containing peptides have been isolated from ovine lutropin beta subunit. These peptides suggest six disulfide linkages between half-cystine residues in positions 23-72, 26-110, 93-100, 34-88, 9-90, and 38-57. The latter pair was placed by elimination of other possibilities. The first three pairs are in agreement with a report by Chung, D., Sairam, M. R. and Li, C. H. (1975) Int. J. Peptide Protein Res. 7, 487-493; the pair 93-100 has also been detected by Reeve, J. R., Cheng, K. W. and Pierce, J. G. (1975) Biochem. Biophys. Res. Commun. 67, 149-155, using partial reduction and alkylation. In an attempt to improve the efficiency of enzymatic attack, a preliminary partial reduction as per Reeve et al. [16] was done. In this instance a peptide suggesting an additional disulfide linkage between half-cystines 23-26 was obtained as well as peptides consistent with the 23-72 and 26-110 placements. This was interpreted as an artifactual opening and recombining during partial reduction-reoxidation to produce the 23-26 linkage. The placement of three disulfide bonds (34-88, 9-90, and 38-57) is in disagreement with the pairings Chung et al. [15] suggest for these six half-cystine residues. Six reasons for uncertainty in the placement of disulfide bonds are discussed. It is concluded the definitive placement of the disputed three disulfide bonds in ovine lutropin beta subunit remains an open question.

Amino Acid Sequence↗
PubMed1977

Effects of beta subunit acylation on lutropin receptor site binding.

The effects of various modifications on the beta subunit of lutropin have been studied using the binding characteristics of the reconstituted hormone in the rat testicular radioligand assay. Conditions for iodinating lutropin and lutropin derivatives were determined which resulted in 15 per cent specific binding when tested immediately and retention of 6 to 7 per cent specific binding even after storage for 6 months. Acetimidinyl, acetyl, and carbamyl derivatives of the beta subunit were prepared and combined with unmodified alpha subunit to form reconstituted lutropin. Modification of the beta subunit was shown to have no effect on the time course of binding to testicular receptors or, with one exception, on the extent of receptor saturation. Very high concentrations of lutropin reconstituted with acetylated beta subunit showed an anomalous binding behavior. Scatchard plots of the binding data support the view that the native hormone has a unique receptor affinity which is irreversibly disrupted by separation of subunits and that derivatization of the beta subunit does not alter this parameter further. These data also suggest that there are no significant differences in the amino groups modified on the beta subunit. Competition and preincubation tests for receptor sites that reacted only with modified lutropin and not with the native hormone were negative.

Animals↗
PubMed1976

Carbohydrate components of the glycopeptides of ovine lutropin.

The three tryptic glycopeptides from ovine lutropin, in which two were from the alpha-subunit (alpha-56 and alpha-82 glycopeptides) and one from the beta-subunits (beta-13 glycopeptide), have been isolated and their carohydrate compositions analyzed. The results indicate that the alpha-56 glycopeptide has the highest amount of carbohydrate, whereas the beta-13 glycopeptide has the least. In general, each of the glycopeptides has similar distribution of various sugars, i.e. high in mannose and glucosamine and low in fucose, sialic acid, galactose and galactosamine. Within the limit of experimental error, the sum of their carbohydrate composition is in agreement with the published data on the intact hormone or separated subunits.

Amino Acids↗
PubMed1976

Reaction of tetranitromethane with lutropin, oxytocin, and vasopressin.

Tetranitromethane reaction with intact ovine lutropin and its isolated subunits was studied using spectrophotometric measurements, amino acid analysis, and isolation of tyrosyl peptides. Tyrosyl residues in the beta subunit (beta37, beta59) did not react with tetranitromethane in the intact hormone, but were nitrated in the isolated subunit. The sequence and extent of reaction of tetranitromethane with the tyrosyl residues in the alpha subunit was alpha21 = alpha92 = alpha93 (in intact hormone or isolated subunit) greater than alpha 41 (reacted in isolated subunit only) greater than alpha 30 (reacted in isolated subunit in 8 M urea only). Polymerization was observed as a side reaction in agreement with previous studies. The degree of polymerization appeared to be related to both primary sequence and tertiary structure, and for lutropin had the relation: alpha subunit (93% polymerized) greater than intact hormone greater than beta subunit (less than 40%). Polymerization observed with vasopressin was significantly greater than with oxytocin; for these peptides the tyrosine residues in the monomeric product were converted to 3-nitrotyrosine. Neither 3-nitrotyrosine nor tyrosine was detected in the polymerized by-products. In the tetranitromethane reaction with intact ovine lutropin, other reaction products charcterized by absorption spectra were found. Peptides isolated from these products lacked the characteristic 428 nm abosrption maxima of 3-nitrotyrosyl peptides and showed instead absorption in the 310 to 350 nm region. Similar products from tetranitromethane reactions with di- and tripeptides containing tyrosine have been observed previously (Boyd, N.D., and Smith, D.B. (1971) Can. J. Biochem, 49, 154-161), but they have not been studied in proteins. A possible relationship to the polymerization side reaction is suggested.

Amino Acid Sequence↗
PubMed1976

Effect of selective nitration of ovine lutropin on the subunit association and biological activity of the hormone.

Different nitrated lutropin (LH) subunits were tested for their abilities to recombine with their counterpart subunits. (A detailed description of the preparations tested is presented in the preceding paper (Burleigh, B. D., Liu, W.K., and Ward, D.N. (1976) J. Biol. Chem. 251, 308-315).) The results indicated that the fully nitrated beta subunit of ovine LH (oLHBeta) (diatyrosyl oHbeta) can combine with the alpha subunit of ovine LH (oLHalpha) as wen which tyrosine residues alpha21, alpha41, alpha93 are modified) recombined partially with oLHbeta yielding about 40% as recombined product under specified recombination conditions. The fully nitrated oLHalpha (pentaatyrosyl oLH) combined poorly with oLHbeta and only 20 to 25% was obtained as the recombined product. The various nitrated LH and recombined LH derivatives containing separately nitrated subunits were tested for their potency in an in vitro radioligand assay and their in vivo biological potency in the ovarian ascorbic acid depletion assay. In the radioligand assay, the monoatyrosyl oLH and the oLHalpha + diatyrosyl oLHbeta were the most active derivatives and had about the same competitive inhibtion curves as that of oLHalpha + oLHbeta. Diatyrosyl oLH had intermediate native potency and the triatyrosyl oLH, tetraatyrosyl oLH + OLHbeta, and pentaatyrosyl oLHalpha + oLHbeta were the least active derivatives in this series. No tetranitromethane-modified LH derivative in our study was completely devoid of the specific binding activity in the radioligand assay. At a dosage of 1000-fold excess over the 125I-labeled oH, all the preparations tested in this series completely inhibited the specific binding of 125I-labeled oLH to the hormone receptors in rat testis preparations. In the in vivo ovarian ascorbic acid depletion assay, both the monoatyrosyl oLH and oLHalpha + diatyrosyl oLHbeta retained about 50% of its original activity; the diatyrosyl oLH and triatyrosyl oLH had about 10% of the activity and the recombined products of tetraatyrosyl oHalpha + oLHbeta and pentaatyrosyl oLHalpha + oLHbeta showed virtually no activity. Based on the results obtained from this paper and the preceding paper, the role of the individual tyrosine residues in oLH is assessed.

Animals↗
PubMed1975

Preparation of lutropin with acetyl or acetimidinyl substituents on the amino groups of the beta-subunit.

The free amino groups in ovine lutropin beta subunit were acylated with acetic anhydride and methyl acetimidinate-HCl to produce the corresponding acetyl and acetimidinyl ovine lutropin beta derivative. These two derivatives recombined with ovine lutropin alpha as well as native ovine lutropin beta, but produced lutropin derivatives which were 33-50% less active than the ovine lutropin alpha + ovine lutropin beta in biological assays.

Acetylation↗