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Biomedical subjects

T T Wang

Publications and source records attributed to T T Wang.

70 records · Page 4Linked to original sources
PubMed1986

Intracellular pteroylpolyglutamate hydrolase from human jejunal mucosa. Isolation and characterization.

Human jejunal intracellular pteroylpolyglutamate hydrolase was purified 30-fold from intestinal mucosa. The apparent molecular weight of the enzyme was 75,000 by Sephadex G-200 gel filtration, and the isoelectric point was at pH 8.0. The enzyme was maximally active at pH 4.5 and was unstable at increasing temperatures. Intracellular pteroylpolyglutamate hydrolase cleaved both terminal and internal gamma-glutamate linkages. In contrast, brush-border pteroylpolyglutamate hydrolase catalyzed the hydrolysis of only terminal gamma-glutamate linkages. The intracellular enzyme showed greatest affinity for the complete folic acid molecule with longer glutamate chains. Subcellular fractionation studies showed the intracellular enzyme was localized in lysosomes. These data show that the properties of human jejunal intracellular pteroylpolyglutamate hydrolase are distinct from those of the brush-border enzyme but are similar to the properties of intracellular pteroylpolyglutamate hydrolase described in other tissues.

Carboxypeptidases↗
PubMed1986

Pteroylpolyglutamate hydrolase from human jejunal brush borders. Purification and characterization.

Pteroylpolyglutamate hydrolase was solubilized with Triton X-100 from human jejunal mucosal brush borders and purified approximately 5,000-fold using organomercurial affinity chromatography, DEAE-cellulose chromatography, and gel filtration. The apparent molecular weight of the purified enzyme in the Triton micelle was estimated as 700,000 using Bio-Gel A-1.5m gel filtration. Sodium dodecyl sulfate/urea-polyacrylamide gel electrophoresis followed by Coomassie stain demonstrated two polypeptide bands at 145,000 and 115,000 daltons. The purified enzyme had an isoelectric point of 7.2, was maximally active at pH 5.5, and was stable above pH 6.5 and at temperatures up to 65 degrees C for at least 90 min. Human jejunal brush-border pteroylpolyglutamate hydrolase is an exopeptidase which liberated [14C]Glu as the sole labeled product of PteGlu2[14C]Glue (where PteGlun represents pteroylpolyglutamate), failed to liberate a radioactive product from PteGlu2[14C]GluLeu2, and released all possible labeled PteGlun products during incubation with Pte[14C]GluGlu6 with the accumulation of Pte[14C]Glu. PteGlu2, PteGlu3, and PteGlu7 were substrates, each with Km = 0.6 microM, whereas PteGlu was a weak inhibitor of the hydrolysis of PteGlu3 with Ki = 20 microM. Components of the pteroyl moiety, Glu, and short chain Glun in alpha or gamma linkages were not inhibitory. The enzyme was activated by Zn2+ or Co2+. The properties of brush-border pteroylpolyglutamate hydrolase are different from those described for the soluble intracellular pteroylpolyglutamate hydrolase in other species and in human mucosa, yet are consistent with previous data on the process of hydrolysis of PteGlun in the intact human intestine.

Carboxypeptidases↗
PubMed1986

Pattern recognition analysis of in vivo enzyme-substrate fluorescence velocities in microorganism detection and identification.

A spectrometric technique is presented that combines most of the important criteria necessary for efficient detection and identification of microorganisms. These criteria include simplicity of experimental design, various degrees of sensitivity and selectivity, convenience, and total reaction times of less than 15 min. The study takes advantage of the inherent extracellular enzymes present in living as opposed to dead, non-enzyme-producing organisms. Sequentially these are harnessed in in vivo reactions with a substrate containing a select organic functional group that is known to be cleaved or hydrolyzed by a certain enzyme. The substrate is tailored so that one of the products can be induced to fluoresce, and by using a conventional spectrofluorimeter the rate at which the fluorescence appears can be recorded. By subjecting the same bacterial sample to a number of different enzyme substrates, a pattern of fluorescence response rates emerges from a 7 by 7 microorganism-substrate matrix. Detection limits ranged from 3.6 X 10(2) to 3.5 X 10(8) cells per ml for the Bacillus globigii-indoxyl acetate and Escherichia coli-diacetylfluorescein pairs, respectively. The specificity and versatility of the method for bacterial determination is demonstrated in probing different bacterial enzymes through their spectrally active metabolic products.

Bacillus↗
PubMed1985

Comparison of folate conjugase activities in human, pig, rat and monkey intestine.

Folate conjugase, an intestinal enzyme that hydrolyzes dietary polyglutamyl folate to the absorbable monoglutamyl derivative, is present in two locations in human jejunal mucosa: one on the brush border membrane, the other soluble and intracellar. Although the brush border enzyme has not been found in the rodent, a recent study demonstrated both brush border and intracellular folate conjugases in the pig with properties similar to the human enzymes. To confirm and expand these data, we compared folate conjugase activities in intestinal brush border and 30,000 X g supernatant fractions in human, pig, rat and monkey mucosa. In both the human and pig, brush border folate conjugase was active from pH 4.5 to 8.5, and activity was significantly increased by zinc acetate. In contrast, folate conjugase activity was negligible in rat and monkey mucosal brush borders. Intracellular folate conjugase was maximally active at pH 4.5 in the human, pig and monkey, whereas this enzyme was equally active from pH 4.0 to 7.5 in the rat. In each species, supernatant activity at pH 4.5 was completely inhibited by p-hydroxymercuribenzoate. Although folate conjugase was quantitatively more active in the human than in the pig intestine, the brush border and intracellular enzymes exhibited similar properties. Sucrase, lactase and aminopeptidase activities were also similar in the two species. Our data show marked species differences in mucosal folate conjugase activities and indicate that the pig may be a suitable experimental animal for further studies of folate hydrolysis and absorption in humans.

Acetates↗
PubMed1984

Formation of cyanide from carbon 1 of 1-aminocyclopropane-1-carboxylic acid during its conversion to ethylene.

It has been shown that 1-aminocyclopropane-1-carboxylic acid (ACC) is the immediate precursor of ethylene, which is derived from C-2 and C-3 of ACC. When [1-(14)C]ACC was administered to etiolated mungbean (Vigna radiata) hypocotyls, approximately 16% of the ACC was converted to ethylene and about 10% of the radioactivity was converted to [(14)C]asparagine in 7 hr. In etiolated epicotyls of common vetch (Vicia sativa), after 7 hr about 14% of the ACC was converted to ethylene and 16% of the radioactivity was converted to beta-cyanoalanine plus gamma-glutamyl-beta-cyanoalanine. Itis known that in most plants cyanide is metabolized to asparagine via the intermediate beta-cyanoalanine, whereas in a fewplants such as V. sativa, beta-cyanoalanine is converted to the conjugate gamma-glutamyl-beta-cyanoalanine. We confirmed that [(14)C]cyanide was metabolized into [(14)C]asparagine in mungbean and into [(14)C]cyanoalanine plus its conjugate in V. sativa. Moreover, after feeding plant tissue with [1-(14)C]ACC, [(14)C]asparagine isolated from mungbean and beta-[(14)C]cyanoalanine from V. sativa were labeled in the C-4 position, as would be expected if these two compounds were derived from [(14)C]cyanide. When the conversion of ACC to ethylene in V. sativa tissue was inhibited by high temperature (41 degrees C), the conversion of [1-(14)C]ACC to beta-[(14)C]cyanoalanine and gamma-glutamyl-beta-[(14)C]cyanoalanine was similarly inhibited. When [carboxyl-(14)C]ACC was administered to mungbean and V. sativa, (14)CO(2) was recovered in an amount equivalent to the amount of ethylene produced. These data indicate that in the conversion of ACC to ethylene the carboxyl group yields CO(2), and C-1 is released as HCN.

Journal Article↗
PubMed1979

Volatile N-nitrosamines in the urine of normal donors and of bladder cancer patients.

Volatile N-nitrosamines were detected in the urine of male volunteers with gas-liquid and high-pressure liquid chromatography interfaced to the thermal energy analyzer. Of 50 samples from normal males, 10 contained nitrosodimethylamine (0.02 to 0.10 micrograms/liter), 6 contained nitrosodiethylamine (0.02 to 3.10), 9 contained nitrosomorpholine (0.006 to 0.67), and none contained nitrosodibutylamine. Of 4 samples from bladder cancer patients, 2 contained nitrosodibutylamine (0.35 and 0.66). Cigarette smoking did not appear to be related to the pattern or amount of urinary volatile N-nitrosamines. The possibility that the N-nitrosamines arise from the diet or from endogenous production is considered.

Adult↗
PubMed1978

Modification of aspartic acid residues to induce trypsin cleavage.

1,2-Diaminoethane and diaminomethane were coupled to aspartic acid residues in small peptides by means of a water-soluble carbodiimide. The resulting modified side chains sufficiently resembled lysine for trypsin to cleave the peptides. Similar modification of glutamic acid residues in peptides gave little or no susceptibility to trypsin.

Amino Acid Sequence↗
PubMed1976

Acyl and amino intermediates in reactions catalysed by pig pepsin. Analysis of transpeptidation products.

The action of pig pepsin on a variety of small peptides including Leu-Trp-Met-Arg, Leu-Trp-Met, Leu-Leu-NH2, benzyloxycarbonyl-Phe-Leu and Gly-Leu-Tyr was studied. Leu-Leu-Leu was found to be the major product from the substrates Leu-Trp-Met-Arg and Leu-Trp-Met, indicating that the predominant reaction at pH 3.4 was a transpeptidation of the acyl-transfer type. Leu-Leu-Leu was also formed in high yield by amino transfer from benzyloxycarbonyl-Phe-Leu. Like the amino-transfer reactions the acyl transfer proceeded via a covalent intermediate, since [14C]leucine was not incorporated into transpeptidation products and did not exchange with enzyme-bound leucine in the presence of acceptors. With Leu-Trp-Met both acyl and amino transpeptidation products, namely Leu-Leu, Leu-Leu-Leu, Met-Met and Met-Met-Met, were formed in addition to methionine and leucine. With Leu-Trp-Met-Arg (1 mM) the pH optimum for the rates of hydrolysis and acyl transfer is about pH 3.4. At this pH the rate of acyl transfer exceeds that of hydrolysis; at pH 2, however, hydrolysis was faster than transfer. A comparison of the effect of the length of substrates and products on the reaction rates allows the conclusion that the binding site can extend over eight to nine amino acid residues. Although the experiments provide no conclusive evidence for or against the involvement of amino and/or acyl intermediates in the hydrolysis of long peptides and proteins, the high yield of transpeptidation reactions of both types observed with some substrates suggests a major role for the intermediates in pepsin-catalysed reactions. The results also show that when pig pepsin is used for the digestion of proteins for sequence work, the likelihood of the formation of transpeptidation products is considerable. In this way peptides not present in the original sequence could easily form in a reasonably good yield.

Amino Acid Sequence↗
PubMed1976

Effects of secondary binding by activator and inhibitor peptides on covalent intermediates of pig pepsin.

A number of peptides were found to increase the activity of pig pepsin towards small synthetic substrates. The activators increase transpeptidation of both the acyl-transfer and the amino-transfer types by as much as 45-fold. The effect on hydrolysis varies from inhibition to modest activation, but is always less than the effect on transpeptidation. The kinetics of substrate cleavage are the converse of non-competitive inhibition and show an increase in kcat. and no effect on Km values. Lineweaver-Burk plots of results obtained in the presence of the activators indicate a substrate activation at high substrate concentration. This appears to be a co-operative effect, since it is not observed in the absence of the activators. The activation is greatest at pH 4.7, less at pH 3.4, and at pH 2.0 is observable only with some of the activator peptides. The results show directly the effect of secondary binding on the catalytic efficiency of pepsin. The most effective activators are those that are most hydrophobic. The results suggest that binding in the secondary binding sites causes an increase in hydrophobicity in the catalytic site which results in increased stability of the acyl and amino intermediates, and preferential reaction with acceptors other than water. The implication that the present results strengthen the case for a role of covalent intermediates in the hydrolysis of good substrates (high kcat. values) is discussed.

Animals↗
PubMedDate not supplied

Differential effect of genistein on transforming growth factor beta 1 expression in normal and malignant mammary epithelial cells.

Our present study demonstrates that the isoflavone genistein exerts a differential response in normal and malignant breast epithelial cells. The addition of genistein results in a dose-dependent increase in TGF-beta 1 mRNA expression in normal human mammary epithelial cells (HMEC) but not in tumor-derived MCF-7 cells. Genistein inhibits the growth and also causes an increase in apoptosis of HMEC. The increased expression of TGF-beta 1 may contribute to the growth inhibitory as well as apoptotic effects of genistein on HMEC.

Apoptosis↗