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H C Li

Publications and source records attributed to H C Li.

At least 91 records · Page 5Linked to original sources
PubMed1982

Enzymatic properties of human glucuronyltransferase and a sensitive method for its assay in a stable B lymphocyte cell line.

Properties of lymphocyte glucuronyltransferase were studied in homogenates of SN1006 cells. A sensitive assay procedure for lymphocyte glucuronyltransferase was developed utilizing radioactive testosterone as the acceptor substrate and TLC for separation of the metabolite. The method is capable of detecting picomolar quantities of the product. The enzyme activity exhibited a broad pH optimum, and was subject to activation by the detergent Lubrol WX and Mn++ ions. The activity conformed to the Michaelis-Menten kinetics giving apparent Km values of 0.8 mM and 11 microM, for UDPGA and testosterone, respectively. 4-Methylumbelliferone, a-naphthol and p-nitrophenol behaved as competitive inhibitors of testosterone glucuronidation. The results indicate that the method could be used for genetic studies of human lymphocyte glucuronyltransferase, and that the enzyme is of consequence in detoxication of exogenous as well as endogenous substrates.

B-Lymphocytes↗
PubMed1982

On the functional heterogeneity of UDP-glucuronyl transferase of mouse liver microsomes.

Fractionation of sodium cholate solubilized microsomes from mouse liver on Sepharose CL-48 yielded three protein peaks with UDP-glucuronyltransferase activities. Of these three peaks, only peak II contained activities towards all the substrates tested: p-nitrophenol, 1-naphthol, morphine, testosterone and estrone. These glucuronyltransferase activities could not be dissociated by further chromatography on DEAE-Sepharose CL-6B and isoelectric focusing. The results show the presence of a functional form of glucuronyltransferase with a wide substrate specificity, and indicate that in addition, other forms with narrower studied specificities may also be present in mouse liver microsomes.

Animals↗
PubMed1981

Serotyping of Ureaplasma urealyticum by immunoperoxidase assay.

The immunoperoxidase method was applied to the identification of Urea-plasma urealyticum serotypes. The assay used highly diluted antisera and could be run directly on primary plate isolates. It was ideal for detecting and identifying mixed serotypes because stained and unstained colonies could be visualized simultaneously by conventional light microscopy. Antisera run against eight serotypes revealed one-way cross-reactions between serotypes 3 and 5 and antiserum to 2, and between serotype 4 and antiserum to 8, at dilutions of less than 1:150. This cross-reactivity could be diluted out at the optimal antiserum dilution for the immunoperoxidase assay, but not for the growth inhibition assay or immunofluorescence test. The immunoperoxidase assay therefore proved ideal for serotyping U. urealyticum.

Cross Reactions↗
PubMed1981

Substrate specificity of human UDP-glucuronyltransferase in cultured lymphocytes.

1. This study establishes the presence of UDP-glucuronyltransferase activity for non-steroidal as well as steroidal substrates, in cultured human B-lymphocytes. Glucuronidation of alpha-naphthol and testosterone was demonstrated in homogenates of two cell lines, SN1006 and RPMI-1788, and that of phenolphthalein, 4-methylumbelliferone, p-nitrophenol and estradiol in the cell line with the higher glucuronyltransferase activity, SN1006. 2. Kinetic studies of testosterone glucuronidation in homogenates of both cell lines revealed a similarity in the behaviour of glucuronyltransferase of these cells. Thus, comparable apparent Km values for UDPGA (0.63 mM) and for testosterone (14 microgram) were observed, although apparent maximal velocities, Vmax, differed several-fold (3.0 versus 0.55 pmol/10(6) cells per min, in SN1006 and RPMI-1788 cells, respectively). 3. Kinetic studies of glucuronidation of testosterone, estradiol, phenolphthalein, alpha-naphthol, 4-methylumbelliferone, and p-nitrophenol yielded comparable apparent Km values for UDPGA (0.56-0.67 mM), suggesting that the same, or similar, glucuronyltransferase(s) catalyse(s) glucuronidation of this wide range of substrates in lymphocytes. This was reinforced by the observation of competitive inhibition of testosterone glucuronidation by alpha-naphthol (Ki 0.25mM), 4-methylumbelliferone (Ki 0.8mM) and p-nitrophenol (Ki 0.8 mM). Thus, lymphocyte glucuronyltransferase activity with a broad substrate specificity, for steroidal and non-steroidal aglycones, is indicated.

Cell Line↗
PubMed1980

Purification of a high molecular weight kininogen from rat plasma.

A high molecular weight kininogen has been isolated from rat plasma and purified. At each preparative step the kininogen concentration and purity were monitored by assay on the perfused isolated rat uterus in terms of bradykinin equivalents formed per mg protein following incubation of the plasma fractions with rodent acid protease for 24 hours at 37 degrees and pH 4.0. Kinin formation by crystalline trypsin and human pancreatic kallikrein also was compared. Citrated rat plasma first was precipitated with 43% ammonium sulfate. The kininogen fractions then were subjected to a series of gel filtration ion exchange chromatographic columns that included G-200 Sephadex, G-200: G-100 Sephadex interconnected columns, DEAE-A50 Sephadex, and hydroxylapatite. The kininogen fractions finally were subjected to preparative polyacrylamide gel electrophoresis, resulting in a final purification of 92.9-fold compared to the initial rat plasma. A single major kininogen protein band and a minor band of protein impurity were obtained on disc gel electrophoresis. Only the pancreatic kallikrein did not form kinin from this purified kininogen. The apparent molecular weight was estimated by SDS polyacrylamide gel technique to be 110,000.

Ammonium Sulfate↗
PubMed1979

Purification and properties of a phosphorylase (phosphoprotein) phosphatase associated with an alkaline phosphatase of Mr 35000 from bovine adrenal cortex.

A metal-ion-independent, nonspecific phosphoprotein phosphatase (Mr = 35000) which represents the major phosphorylase phosphatase activity in bovine adrenal cortex has been purified to apparent homogeneity. An alkaline phosphatase activity (p-nitrophenyl phosphate as a substrate) of the same molecular weight, which requires both a metal ion (Mg2+ greater than Mn2+ greater than Co2+) and a sulfhydryl compound for activity, has been found to co-purify with the phosphoprotein phosphatase throughout the purification procedures. Characterization of the phosphoprotein and the alkaline phosphatase activities with respect to their catalytic properties, substrate and metal ion specificities, relationship with large molecular forms of the enzymes and responses to various effectors has been carried out. The results indicate that the phosphoprotein phosphatase can be converted by pyrophosphoryl compounds (e.g. PPi and ATP) to a metal-ion-dependent form which, subsequently, can be reactivated by Co2+ greater than Mn2+ but not by Mg2+ or Zn2+. The results also indicate that, although the phosphoprotein and the alkaline phosphatase activities are closely associated, they exhibit distinct physical and catalytic properties. Discussions concerning whether these two activities represent two different forms of the same protein or two different yet very similar polypeptide chains have been presented.

Adrenal Cortex↗
PubMed1979

Isolation and partial characterization of kinin-like peptides formed by acid protease from murine fibroblast L-929.

A kinin-forming acid protease was isolated and purified from a stationary cell culture of murine fibroblasts L-929 (Biochem. Pharmacol. 26: 1187, 1977). The enzyme formed kinins at an optimal pH of 4.0 from Murphy-Sturm lymphosarcoma tissue and rat plasma kininogen substrate. This study reports on the isolation and partial chemical characterization of two fibroblast kinins. The 12,000 g fraction of L-929 fibroblast homogenates was incubated with rat plasma at 37 degrees, pH 4.0, for 92 hours, and the kinins extracted with ethyl alcohol-p-toluene sulfonic acid. Two fibroblast kinins, FKI and FKII, were separated and purified on the following chromatographic columns: G-25 Sephadex (1 X 115 cm), CM-C50 Sephadex (2.5 X 15 cm), and Biogel P-4 (1 X 115 cm). Kinin activity was bioassayed on the perfused isolated rat uterus preparation. The purity of the kinins was ascertained by thin layer chromatography coupled with dansylation. The estimated molecular weight of FKI was 1450 with a 14-amino acid composition (determined by automatic AA analysis) that included AspThrSerProGluGlyAlaValLeuTyrPheLysHisArg. FKII had an estimated molecular weight of 1000 with a 12-amino acid composition including AspSerProGluGlyAlaLeuTyrLysHisArg. The kinins were relatively rich in arginine and did not contain the bradykinin sequence.

Amino Acids↗
PubMed1979

The kinin-forming acid protease system in murine fibroblasts L-929.

Components of an acid protease kinin-forming enzyme system were isolated and purified from the murine fibroblast L-929 cell line grown in stationary cell culture. The enzyme, was purified from the 10,000 g supernatant cell fraction by sequential passage through G-200 Sephadex, hydroxylapatite, DEAE-A50 Sephadex and affinity chromatography columns. The specific activity was determined on a rat plasma kininogen purified on DEAE and G-100 Sephadex columns as assayed for kinin-forming activity on the isolated perfused rat uterus. Both enzyme and substrate showed a single band pattern by disc gel electrophoresis technique. Optimum protease activity was obtained at pH 3.8, and kinin release was both time- and enzyme-dependent at 37 degrees. The molecular weight of the protease and kininogen, estimated on a G-200 Sephadex column, was 39,000 and 115,000 respectively. Two fibroblast kinins were isolated from a mixture of 12,000 g fraction of fibroblast homogenate and rat plasma incubated at 37 degrees, pH 4.0 for 92 hours. The two fibroblast kinins (I, II) were separated and purified on G-25 Sephadex, CM-C50 Sephadex, and Biogel P-4 columns. The purity of the kinins was ascertained by thin layer chromatography. The molecular weight of fibroblast kinin I was estimated to be 1450 with a 14-amino acid composition consisting of AspThrSerProGluGlyAlaVal-LeuTyrPheLysHisArg. Fibroblast kinin II had an estimated molecular weight of 1000 with a 12-amino acid composition that included AspSerProGluGlyAlaLeuTyrLysHisArg. The kinins were relatively rich in arginine and did not contain the bradykinin sequence.

Amino Acids↗
PubMed1978

The role of ATP and divalent cations in the regulation of a cardiac phosphorylase phosphatase (phosphoprotein phosphatase) of Mr = 35,000.

The effects of ATP and divalent cations on a divalent cation-independent phosphorylase phosphatase of Mr = 35,000 (phosphatase S) purified from canine cardiac muscle have been studied. The enzyme can be rapidly inactivated by ATP or other nucleoside di- and triphosphates and PPi, but not by AMP, adenosine, adenine, Pi, EDTA, ethylene glycol bis(beta-aminoethyl ether)N,N' -tetraacetic acid, 1,10-phenanthroline, or 8-hydroxyquinoline. After removing the inactivating agent, such as ATP or PPi, by gel filtraiton followed by exhaustive dialysis, the inactivated enzyme (apophosphatase S) can be reactivated by preincubating with Mn2+ or Co2+, but not with Mg2+, Ca2+, Ni2+, Zn2+, Fe2+, Cu2+, Ba2+, Hg2+, Pb2+, or Cd2+. The Mn2+ -reactivated enzyme, which is less active than the Co2+ -reactivated enzyme, can be again inactivated by preincubating with ATP. The present findings indicate that phosphatase S contains a tightly bound divalent cation, probably Mn2+, in the active site. ATP and PPi, due to their structural similarity to the phosphoprotein substrate and their ability to chelate metal ions, can readily enter the active site to remove the divalent cation(s) essential for the catalytic function. The present findings also indicate that phosphatase S, a common catalytic subunit of several larger molecular forms of nospecific phosphoprotein phosphatase in cardiac muscle, can exist in two interconvertible forms, a metallized form (active) and a demetallized form (inactive). ATP and metal ions may regulate this class of isozymes by mediating the interconversions.

Adenosine Triphosphate↗
PubMed1978

Specificity of salivary-bacterial interactions: role of terminal sialic acid residues in the interaction of salivary glycoproteins with Streptococcus sanguis and Streptococcus mutans.

Four highly purified salivary glycoproteins were used to study salivary-bacterial interactions. One pair of glycoproteins was mucin-like in composition, whereas the second pair was not. By an agglutination assay, it was found that only the mucin-glycoproteins agglutinated Streptococcus sanguis and S. mutans. Removal of sialic acid from these molecules resulted in a loss of agglutination of S. sanguis but not of S. mutans. The agglutination phenomenon was shown to require a salivary macromolecule of at least 150,000 daltons.

Agglutination↗
PubMed1977

Dissociation of phosphohistone phosphatases from canine heart.

Phosphohistone phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) of canine heart extract has been separated by DEAE-cellulose chromatography into 4 molecular forms, namely phosphatases A (Mr = 156 000), B (Mr = 161 000), C (Mr = 95 600) and U (Mr = 61 000). ATP inhibited phosphatase A, stimulated phosphatase B and did not significantly affect phosphatase C activity. Phosphatase U requires Mn2+ for activity, under which condition ATP is inhibitory. Phosphatases A, B and C, but not phosphatase U, were dissociated by ethanol into catalytic subunits that were inhibited by ATP, insensitive to Mn2+, and had a common molecular weight of 34 800 (phosphatase S). The dissociation was accompanied by an increase of enzymic activity. Chromatography of the ethanol-treated 55% (NH4)2SO4 fraction of canine heart extract on DEAE-cellulose demonstrated that the multiple forms of phosphohistone phosphatase could be reduced to two forms: phosphatase U and phosphatase S, which may represent two basic constituents of the multiple forms of phosphohistone phosphatase in canine heart.

Adenosine Triphosphate↗