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S X Lin

Publications and source records attributed to S X Lin.

At least 73 records · Page 4Linked to original sources
PubMed1992

Subunit identity of the dimeric 17 beta-hydroxysteroid dehydrogenase from human placenta.

Human placental 17 beta-hydroxysteroid dehydrogenase has been purified with a new rapid procedure based on fast protein liquid chromatography, yielding quantitatively a homogeneous preparation with high specific activity catalyzing the oxidation of 7.2 mumol of estradiol/min/mg of enzyme protein at 23 degrees C, pH 9.2. This preparation was shown to have a subunit mass of 34.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis while having a molecular mass of 68 kDa by both Superose-12 gel-filtration and native pore gradient gel electrophoresis. When 17 beta-hydroxysteroid dehydrogenase was expressed in HeLa cells or overproduced in insect cells using the baculovirus expression system, both from its cDNA encoding a protein of 34 kDa, the enzyme had the same migration in native and sodium dodecyl sulfate-gel electrophoresis as the purified one from human placenta and eluted from the Superose-12 column at the same elution volume. Moreover, all the above forms of this enzyme have similar specific activity. These results clearly demonstrate the identity of the three enzyme forms. The enzyme produced from the cDNA is expressed as a dimer, and its two subunits are identical. 17 beta-Hydroxysteroid dehydrogenase subunit identity is thus proved. The NH2-terminal analysis revealed a unique sequence of Ala-Arg-Thr-Val-Val-Leu-Ile for the purified enzyme from placenta, further confirming the above conclusion.

17-Hydroxysteroid Dehydrogenases↗
PubMed1992

Higher specific activity of the Escherichia coli glutamyl-tRNA synthetase purified to homogeneity by a six-hour procedure.

The glutamyl-tRNA synthetase (EC 6.1.1.17) of Escherichia coli was purified to homogeneity from the overproducing strain DH5 alpha(pLQ7612) by a two-step procedure that takes only about 6 h and yields 10 mg of enzyme per gram of wet cells. The process consists of a two-phase polyethylene glycol-dextran partition, the top phase of which is diluted and directly applied to an anion-exchange FPLC MonoQ column. The purified enzyme has a specific activity about twice that of the same enzyme purified to homogeneity by the lengthy conventional procedure from either a normal strain or this overproducing strain. This difference is discussed in relation to the generation of microheterogeneity in proteins during their purification.

Bacterial Proteins↗
PubMed1992

Immunolocalization of cytoplasmic dynein to lysosomes in cultured cells.

Polyclonal antisera have been raised against cytoplasmic dynein purified from calf brain and rat testis. These antibodies reacted most strongly with the 74 kDa dynein intermediate chain, but also recognized the 410 kDa heavy chain, and the 150 and 45 kDa polypeptides previously observed to copurify with cytoplasmic dynein from rat tissues. Localization studies were performed by indirect immunofluorescence microscopy using a fibroblast cell line. Dynein-specific staining appeared vesicular, distributed throughout the cell, but more concentrated near the nucleus. Double-labeling studies using fluorescent markers for membranous organelles indicated a co-localization of dynein with lysosomes. The distribution of the dynein-positive lysosomes was disrupted by treatment of the cells with microtubule-active drugs, and by acidification of the cytoplasm. Comparison of the distribution of lysosomes with peripheral microtubules indicated a high degree of coincidence. These results are consistent with the hypothesis that cytoplasmic dynein is involved in retrograde-directed movement of membranous organelles. In mitotic cells, dynein staining was also apparent along the microtubules of the mitotic apparatus, though vesicular staining was still conspicuous. The presence of dynein on vesicles as well as on spindle microtubules indicates that dynein distribution between these compartments may be regulated by distinct binding proteins.

Adenosine Triphosphatases↗
PubMed1991

Arginyl-tRNA synthetase from Escherichia coli affinity labeling with 3'-oxidized tRNA(Arg).

The covalent modification of E. coli arginyl-tRNA synthetase by the 2',3'-dialdehyde derivative of tRNA(Arg) (tRNA(oxArg)) resulted in the complete inactivation of the ATP-PPi exchange and aminoacylation activities of the enzyme. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the ArgRS-tRNA(oxArg) covalent complexes indicated that two bands simultaneously appeared on the gel parallel with inactivation corresponding to different higher molecular weights. This result was different from that of the other aminoacyl-tRNA synthetase labeling systems as previously reported. Upon the ribonuclease treatment of the modified ArgRS, less than 15% of both the initial ATP-PPi exchange and aminocylation activities were recovered. During the whole process of labeling and RNase treatment, the two activities of the enzyme were closely associated.

Affinity Labels↗
PubMed1990

Demonstration of a slow conformational change in liver glucokinase by fluorescence spectroscopy.

A slow interconversion between two enzyme forms of rat liver glucokinase has been previously inferred from kinetic assay studies. Two different conformations of the enzyme-substrate complex have now been directly demonstrated by an intrinsic fluorescence enhancement in glucokinase upon addition of glucose. The transition between these two conformations upon glucose addition is measurably slow in the presence of glycerol, with a glucose-dependent half-life of 0.5 to more than 10 min. In the presence of 5% glycerol, the forward and reverse isomerization rate constants are 2.6 x 10(-2) s-1 and 8.5 x 10(-4) s-1, respectively. Correspondingly, the overall equilibrium dissociation constant (0.13 mM) is more than 30-fold lower than the first binding step, i.e. the affinity for glucose is greatly increased. This result was also verified by equilibrium titration of the enzyme with glucose. A similar slow transition was analyzed in the presence of 30% glycerol and observed without glycerol. The dilution of stock glucokinase to promote glucose dissociation from the enzyme showed an exponential fluorescence decay, exactly the reverse phenomenon of glucose addition. The deduced rate constant for the reverse reaction coincided with that calculated from the association results. The conformational change is specific for glucose and responsible for the generation of the kinetic cooperativity of this monomeric enzyme, thus playing a regulatory role in the uptake of glucose in liver.

Animals↗
PubMed1989

Stoichiometry of slow binding of palmitoyl-CoA to liver glucokinase.

The interaction of palmitoyl-CoA with porcine glucokinase was studied by the gel permeation technique. The finding that glucokinase "bound" up to 60 molecules was unexpected from the specific inhibition of rat glucokinase by long chain acyl-CoA (Tippett & Neet, J. Biol. Chem. (1982) 287, 12839-12845). Sephacryl S-200 gel filtration in the presence of palmitoyl-CoA demonstrated a protein peak without enzyme activity that was eluted earlier than the active enzyme peak, indicating a large molecular weight shift for the inactivated enzyme form and confirming a large number (greater than or equal to 30) of associated palmitoyl-CoA molecules. The binding was also verified by analyzing the absorption characteristics of the inactivated enzyme peak. In the presence of glycerol, the size of the inactivated peak greatly decreased, but the separation between the two peaks remained unchanged. Therefore, the amphiphile bound predominantly to the inactive enzyme and not to the active form, suggesting that the rapid inhibitory interactions between palmitoyl-CoA and glucokinase previously observed are specific. Parallel enzyme activity studies showed that in the time range of the column experiments (4-20 h), both the rat and pig enzyme were greatly inactivated (greater than 90%) in the presence of palmitoyl-CoA (15 microM) in the absence of glycerol. This slow inactivation is different from the immediate specific inhibition previously reported and depends on both enzyme and palmitoyl-CoA concentrations. The presence of up to 20% glycerol slowed this inactivation process. These results demonstrated that even below the critical micelle concentration, partial inactivation of glucokinase occurs in the presence of palmitoyl-CoA over a long period of time.

Acyl Coenzyme A↗
PubMed1989

[Content, location and action site of enkephalins in arterial walls].

Contents of L-enkephalin (L-ENK) and M-enkephalin (M-ENK) in arteries of rabbit were measured by radioimmunoassay. Mean values of L-ENK and M-ENK (pg/mg of wet tissue) were 38.99 +/- 17.29 and 134.67 +/- 8.11 in the rabbit ear artery; 31.10 +/- 7.76 and 93.60 +/- 18.22 in the renal artery; 25.70 +/- 13.60 and 88.43 +/- 18.16 in the mesenteric artery, respectively. L-ENK in these arteries decreased significantly (P less than 0.001) after chemicosympathectomy by injection of 6-OHDA; ENK in the ear artery were reduced significantly (P less than 0.001) following the excision of the superior cervical ganglion or electrical-field stimulation, but not changed after injection of reserpine. Content of NE in the artery strip bath medium was measured by fluorimetric assay. NE in the medium was decreased significantly after administration of L-ENK or M-ENK. The results suggest that enkephalins may exist in the sympathetic nerve terminals innervating arterial walls and may be released by electrical-field stimulation. Enkephalins inhibit the contracting activity of artery strips probably by decreasing the release of NE from sympathetic nerve endings.

Animals↗
PubMed1988

Arginyl-tRNA synthetase from Escherichia coli, purification by affinity chromatography, properties, and steady-state kinetics.

A Blue Sephadex G-150 affinity column adsorbs the arginyl-tRNA synthetase of Escherichia coli K12 and purifies it with high efficiency. The relatively low enzyme content was conveniently purified by DEAE-cellulose chromatography, affinity chromatography, and fast protein liquid chromatography to a preparation with high activity capable of catalyzing the esterification of about 23,000 nmol of arginine to the cognate tRNA per milligram of enzyme within 1 min, at 37 degrees C, pH 7.4. The turnover number is about 27 s-1. The purification was about 1200-fold, and the overall yield was more than 30%. The enzyme has a single polypeptide chain of about Mr 70,000 and binds arginine and tRNA with 1:1 stoichiometry. For the aminoacylation reaction, the Km values at pH 7.4, 37 degrees C, for various substrates were determined: 12 microM, 0.9 mM, and 2.5 microM for arginine, ATP, and tRNA, respectively. The Km value for cognate tRNA is higher than those of most of the aminoacyl-tRNA synthetase systems so far reported. The ATP-PPi exchange reaction proceeds only in the presence of arginine-specific tRNA. The Km values of the exchange at pH 7.2, 37 degrees C, are 0.11 mM, 2.9 mM, and 0.5 mM for arginine, ATP, and PPi, respectively, with a turnover number of 40 s-1. The pH dependence shows that the reaction is favored toward slightly acidic conditions where the aminoacylation is relatively depressed.

Amino Acyl-tRNA Synthetases↗
PubMed1988

Interactions between Escherichia coli arginyl-tRNA synthetase and its substrates.

Interactions between Escherichia coli arginyl-tRNA synthetase and its substrates were extensively studied and distinctly demonstrated. Various approaches such as equilibrium dialysis, fluorescence titration, and substrate protection against heat inactivation of the enzyme were used for these studies. In the absence of other substrates, the equilibrium dissociation constants for arginine, ATP, and the cognate tRNA were about 70 microM, 0.85 mM, and 0.45 microM, respectively, at pH 7.5, in Tris buffer. The binding of arginine to the enzyme was affected neither by the presence of tRNA nor by the presence of ATP but was considerably enhanced when ATP and tRNA were both present at saturating concentrations. The dissociation constant in this case (about 16 microM) was very close to the Km (12 microM) for arginine during aminoacylation. The binding of ATP (the equilibrium dissociation constant KD approximately 0.85 mM) was not affected by the presence of arginine but was depressed in the presence of tRNA (KD became 3 mM). Arginyl-tRNA showed a dissociation constant of (4-5) X 10(-7) M which was not affected by the presence of a single other substrate. Possible explanations for the high Km for tRNA in the aminoacylation are discussed. Our results indicated pronounced interactions between substrates mediated by the enzyme under catalytic conditions. Periodate oxidation did not alter the tRNA binding to the enzyme. The oxidized tRNA still afforded protection against heat inactivation of the enzyme.

Amino Acyl-tRNA Synthetases↗
PubMed1987

Affinity labelling of E. coli leucyl-tRNA synthetase with 3'-oxidized tRNA(Leu).

The E. coli leucyl-tRNA synthetase (E.C. 6.1.1.4) was specifically labelled with 3'-oxidized tRNA(Leu) (tRNA(oxLeu)). The procedure involves a Schiff's base formation and its subsequent reduction by sodium cyanoborohydride. Stoichiometric inactivation of aminoacylation was achieved with the incorporation of 1 mol of tRNA(oxLeu) per mol LeuRS. On the other hand, the amino acid activation activity of LeuRS-tRNA(ox) complex was partially inhibited. After extensive digestion of the complex by pancreatic ribonuclease, the amino acid activation activity was fully recovered, while the aminoacylation activity was not restored at all.

Affinity Labels↗
PubMed1984

Fast kinetic study of yeast phenylalanyl-tRNA synthetase: role of tRNAPhe in the discrimination between tyrosine and phenylalanine.

An extensive study of the discrimination between phenylalanine and tyrosine by yeast phenylalanyl-tRNA synthetase was carried out in the presence of native tRNAPhe. Our results clearly show that, besides the previously reported dissociation of tyrosyl adenylate from the enzyme template [Lin, S. X., Baltzinger, M., & Remy, P. (1983) Biochemistry 22, 681-689], two more correction processes are involved in the rejection of tyrosine in the presence of tRNAPhe. A minor part of the misactivated tyrosine is indeed transferred to tRNAPhe, but the resulting misaminoacylated tRNA is very rapidly hydrolyzed (kh approximately equal to 60 s-1), as it has already been shown for other systems. However, the major part of the misactivated tyrosine is rejected as the result of a pretransfer correction consisting of the fast hydrolysis (k'h approximately equal to 20 s-1) of the enzyme-bound noncognate adenylate induced by the binding of native tRNAPhe. The transfer step itself is found to be non-specific, as the rate constant is almost the same for phenylalanine and tyrosine. This result is supported by the observation that tyrosine and phenylalanine are also transferred at the same rate to tRNAPheox-red. It is shown that the integrity of the 3'-terminal adenosine of the tRNA is critical for triggering the pretransfer hydrolysis of enzyme-bound noncognate aminoacyl adenylate. A detailed kinetic analysis is presented that shows that the observed rate constant of tRNAPhe tyrosylation and the rate of disappearance of enzyme-tyrosyl adenylate complex are in fact apparent rate constants.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acyl-tRNA Synthetases↗
PubMed1983

Yeast phenylalanyl-tRNA synthetase: symmetric behavior of the enzyme during activation of phenylalanine as shown by a rapid kinetic investigation.

The adenylation of phenylalanine catalyzed by phenylalanyl-tRNA synthetase was investigated in the absence of tRNA, by rapid kinetic measurements using 6-(p-toluidinyl)naphthalene-2-sulfonate (TNS) as a nonspecific fluorescent reporter group. It is shown that each protomer of the enzyme is able to catalyze independently the adenylation of phenylalanine by ATP, as well as the reversion by pyrophosphate, at least in the absence of tRNA. The kinetic rate constants of synthesis and pyrophosphorolysis are respectively found equal to 100 +/- 20 s-1 and 150 +/- 50 s-1. The symmetric behavior of the enzyme is consistent with a symmetric binding of 2 mol of phenylalanine to the enzyme as shown by equilibrium dialysis experiments. The affinity of phenylalanyladenylate for the enzyme could be characterized by an equilibrium constant of 0.2 x 10(9) M-1.

Adenosine Monophosphate↗