28 results on '"Su TZ"'
Search Results
2. Circular Permutation Prediction Reveals a Viable Backbone Disconnection for Split Proteins: An Approach in Identifying a New Functional Split Intein
- Author
-
Lee, Yun-Tzai, primary, Su, Tz-Hsiang, additional, Lo, Wei-Cheng, additional, Lyu, Ping-Chiang, additional, and Sue, Shih-Che, additional
- Published
- 2012
- Full Text
- View/download PDF
3. Correlations between cytoplasmic CSE1L in neoplastic colorectal glands and depth of tumor penetration and cancer stage
- Author
-
Tai Cheng-Jeng, Su Tzu-Cheng, Jiang Ming-Chung, Chen Hung-Chang, Shen Shing-Chuan, Lee Woan-Ruoh, Liao Ching-Fong, Chen Ying-Chun, Lin Shu-Hui, Li Li-Tzu, Shen Ko-Hung, Yeh Chung-Min, Yeh Kun-Tu, Lee Ching-Hsiao, Shih Hsin-Yi, and Chang Chun-Chao
- Subjects
Colorectal cancer ,CSE1L ,Cytoplasm ,Invasion ,Metastasis ,Nucleus ,Medicine - Abstract
Abstract Background Colorectal carcinomas spread easily to nearby tissues around the colon or rectum, and display strong potential for invasion and metastasis. CSE1L, the chromosome segregation 1-like protein, is implicated in cancer progression and is located in both the cytoplasm and nuclei of tumor cells. We investigated the prognostic significance of cytoplasmic vs. nuclear CSE1L expression in colorectal cancer. Methods The invasion- and metastasis-stimulating activities of CSE1L were studied by in vitro invasion and animal experiments. CSE1L expression in colorectal cancer was assayed by immunohistochemistry, with tissue microarray consisting of 128 surgically resected specimens; and scored using a semiquantitative method. The correlations between CSE1L expression and clinicopathological parameters were analyzed. Results CSE1L overexpression was associated with increased invasiveness and metastasis of cancer cells. Non-neoplastic colorectal glands showed minimal CSE1L staining, whereas most colorectal carcinomas (99.2%, 127/128) were significantly positive for CSE1L staining. Cytoplasmic CSE1L was associated with cancer stage (P=0.003) and depth of tumor penetration (P=0.007). Cytoplasmic CSE1L expression also correlated with lymph node metastasis of the disease in Cox regression analysis Conclusions CSE1L regulates the invasiveness and metastasis of cancer cells, and immunohistochemical analysis of cytoplasmic CSE1L in colorectal tumors may provide a useful aid to prognosis.
- Published
- 2013
- Full Text
- View/download PDF
4. Robust Background Subtraction with Shadow and Highlight Removal for Indoor Surveillance
- Author
-
Hu Jwu-Sheng and Su Tzung-Min
- Subjects
Telecommunication ,TK5101-6720 ,Electronics ,TK7800-8360 - Abstract
This work describes a robust background subtraction scheme involving shadow and highlight removal for indoor environmental surveillance. Foreground regions can be precisely extracted by the proposed scheme despite illumination variations and dynamic background. The Gaussian mixture model (GMM) is applied to construct a color-based probabilistic background model (CBM). Based on CBM, the short-term color-based background model (STCBM) and the long-term color-based background model (LTCBM) can be extracted and applied to build the gradient-based version of the probabilistic background model (GBM). Furthermore, a new dynamic cone-shape boundary in the RGB color space, called a cone-shape illumination model (CSIM), is proposed to distinguish pixels among shadow, highlight, and foreground. A novel scheme combining the CBM, GBM, and CSIM is proposed to determine the background which can be used to detect abnormal conditions. The effectiveness of the proposed method is demonstrated via experiments with several video clips collected in a complex indoor environment.
- Published
- 2007
5. Synthesis and SAR of tolylamine 5-HT6 antagonists.
- Author
-
Singer JM, Wilson MW, Johnson PD, Graham SR, Cooke LW, Roof RL, Boxer PA, Gold LH, Meltzer LT, Janssen A, Roush N, Campbell JE, Su TZ, Hurst SI, Stoner CL, and Schwarz JB
- Subjects
- Animals, Chemistry, Organic methods, Chemistry, Pharmaceutical methods, Drug Design, Ethers chemistry, Inhibitory Concentration 50, Kinetics, Models, Chemical, Rats, Serotonin Receptor Agonists chemistry, Serotonin Receptor Agonists pharmacology, Structure-Activity Relationship, Amines chemistry, Receptors, Serotonin chemistry, Serotonin Receptor Agonists chemical synthesis
- Abstract
The synthesis and SAR of tolylamines with 5-HT(6) receptor antagonist activity is presented. The amine, core aromatic, peripheral aromatic, and ether linker moieties of HTS hit 1 were modulated and the effect on potency at 5-HT(6) examined. Tolylpiperidine ether 9h was found to possess desirable pharmacokinetic (PK) properties, and was also shown to enhance cognition in the rat novel object recognition paradigm.
- Published
- 2009
- Full Text
- View/download PDF
6. Identification of the alpha2-delta-1 subunit of voltage-dependent calcium channels as a molecular target for pain mediating the analgesic actions of pregabalin.
- Author
-
Field MJ, Cox PJ, Stott E, Melrose H, Offord J, Su TZ, Bramwell S, Corradini L, England S, Winks J, Kinloch RA, Hendrich J, Dolphin AC, Webb T, and Williams D
- Subjects
- Amino Acid Sequence, Animals, Arginine genetics, Arginine metabolism, Autoradiography, Base Sequence, Calcium Channels chemistry, Calcium Channels genetics, Calcium Channels, N-Type metabolism, Cell Line, Chlorocebus aethiops, Constriction, Pathologic, Female, Formaldehyde, Ion Channel Gating drug effects, Male, Mice, Mice, Transgenic, Mutation genetics, Pain genetics, Pregabalin, Protein Binding, Protein Subunits chemistry, Protein Subunits genetics, Protein Subunits metabolism, Swine, gamma-Aminobutyric Acid metabolism, gamma-Aminobutyric Acid therapeutic use, Analgesics therapeutic use, Calcium Channels metabolism, Pain drug therapy, Pain metabolism, gamma-Aminobutyric Acid analogs & derivatives
- Abstract
Neuropathic pain is a debilitating condition affecting millions of people around the world and is defined as pain that follows a lesion or dysfunction of the nervous system. This type of pain is difficult to treat, but the novel compounds pregabalin (Lyrica) and gabapentin (Neurontin) have proven clinical efficacy. Unlike traditional analgesics such as nonsteroidal antiinflammatory drugs or narcotics, these agents have no frank antiinflammatory actions and no effect on physiological pain. Although extensive preclinical studies have led to a number of suggestions, until recently their mechanism of action has not been clearly defined. Here, we describe studies on the analgesic effects of pregabalin in a mutant mouse containing a single-point mutation within the gene encoding a specific auxiliary subunit protein (alpha2-delta-1) of voltage-dependent calcium channels. The mice demonstrate normal pain phenotypes and typical responses to other analgesic drugs. We show that the mutation leads to a significant reduction in the binding affinity of pregabalin in the brain and spinal cord and the loss of its analgesic efficacy. These studies show conclusively that the analgesic actions of pregabalin are mediated through the alpha2-delta-1 subunit of voltage-gated calcium channels and establish this subunit as a therapeutic target for pain control.
- Published
- 2006
- Full Text
- View/download PDF
7. Heteroaromatic side-chain analogs of pregabalin.
- Author
-
Schelkun RM, Yuen PW, Wustrow DJ, Kinsora J, Su TZ, and Vartanian MG
- Subjects
- Animals, Anticonvulsants chemical synthesis, Disease Models, Animal, Drug Design, Drug Evaluation, Preclinical, In Vitro Techniques, Mice, Mice, Inbred DBA, Molecular Structure, Pregabalin, Stereoisomerism, gamma-Aminobutyric Acid chemical synthesis, gamma-Aminobutyric Acid chemistry, gamma-Aminobutyric Acid pharmacology, Anticonvulsants chemistry, Anticonvulsants pharmacology, Seizures prevention & control, gamma-Aminobutyric Acid analogs & derivatives
- Abstract
A series of heteroaromatic analogs of pregabalin has been identified that possess anticonvulsant activity in the DBA/2 mouse model. The methods of synthesis and preliminary pharmacology are discussed herein.
- Published
- 2006
- Full Text
- View/download PDF
8. The design and synthesis of human branched-chain amino acid aminotransferase inhibitors for treatment of neurodegenerative diseases.
- Author
-
Hu LY, Boxer PA, Kesten SR, Lei HJ, Wustrow DJ, Moreland DW, Zhang L, Ahn K, Ryder TR, Liu X, Rubin JR, Fahnoe K, Carroll RT, Dutta S, Fahnoe DC, Probert AW, Roof RL, Rafferty MF, Kostlan CR, Scholten JD, Hood M, Ren XD, Schielke GP, Su TZ, Taylor CP, Mistry A, McConnell P, Hasemann C, and Ohren J
- Subjects
- Animals, Benzofurans chemistry, Calcium antagonists & inhibitors, Calcium metabolism, Cells, Cultured, Crystallography, X-Ray, Drug Design, Drug Evaluation, Preclinical, Enzyme Inhibitors chemistry, Glutamic Acid drug effects, Glutamic Acid metabolism, Humans, In Vitro Techniques, Models, Molecular, Molecular Structure, Neurons cytology, Neurons drug effects, Rats, Rats, Inbred Lew, Stereoisomerism, Structure-Activity Relationship, Sulfonamides chemistry, Benzofurans chemical synthesis, Benzofurans therapeutic use, Enzyme Inhibitors chemical synthesis, Enzyme Inhibitors therapeutic use, Neurodegenerative Diseases drug therapy, Sulfonamides chemical synthesis, Sulfonamides therapeutic use, Transaminases antagonists & inhibitors
- Abstract
The inhibition of the cytosolic isoenzyme BCAT that is expressed specifically in neuronal tissue is likely to be useful for the treatment of neurodegenerative and other neurological disorders where glutamatergic mechanisms are implicated. Compound 2 exhibited an IC50 of 0.8 microM in the hBCATc assays; it is an active and selective inhibitor. Inhibitor 2 also blocked calcium influx into neuronal cells following inhibition of glutamate uptake, and demonstrated neuroprotective efficacy in vivo. SAR, pharmacology, and the crystal structure of hBCATc with inhibitor 2 are described.
- Published
- 2006
- Full Text
- View/download PDF
9. Mediation of highly concentrative uptake of pregabalin by L-type amino acid transport in Chinese hamster ovary and Caco-2 cells.
- Author
-
Su TZ, Feng MR, and Weber ML
- Subjects
- Amino Acid Transport System y+ physiology, Animals, CHO Cells, Caco-2 Cells, Cricetinae, Fusion Regulatory Protein 1, Light Chains physiology, GABA Plasma Membrane Transport Proteins, Humans, Large Neutral Amino Acid-Transporter 1 physiology, Male, Membrane Transport Proteins physiology, Pregabalin, Rats, Rats, Wistar, gamma-Aminobutyric Acid pharmacology, Amino Acid Transport Systems physiology, gamma-Aminobutyric Acid analogs & derivatives, gamma-Aminobutyric Acid pharmacokinetics
- Abstract
Pregabalin (PGB) is a novel drug under development for the treatment of epilepsy, neuropathic pain, fibromyalgia, and generalized anxiety disorder. In this study, we investigated PGB transport in rats, mammalian cell lines, and Xenopus laevis oocytes. In contrast to gabapentin (GBP), PGB absorption in rats showed unique linear pharmacokinetics. PGB entered CHO and Caco-2 cells predominately via Na(+)-independent processes. Uptake of PGB was mutually exclusive with leucine, GBP and 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid, the substrates preferential for system L. The preloaded PGB in CHO cells was exchangeable with leucine, but at a lower exchange rate than that of leucine and GBP. Dixon plots showed competitive inhibition of leucine uptake by PGB, with a K(i) value very close to the K(m) value for PGB uptake (377 versus 363 microM). At an extracellular concentration of 300 microM, the intracellular PGB concentration in CHO cells reached 1.5- and 23-fold higher than that of GBP and leucine, respectively. In contrast, at clinically relevant concentrations, PGB seemed not to interact with GABA transport in GAT1, GAT2, and GAT3 cell lines, system y(+), b(0,+), B(0,+), and B(0) transport activities in Caco-2 and NBL-1 cells, and the b(0,+)-like transport activity in rBAT cRNA-injected X. laevis oocytes. Taken together, these results suggest that L-type transport is the major transport route for PGB and GBP uptake in mammalian cells. The differential affinity of PGB and GBP at L-type system leads to more concentrative accumulation of PGB than GBP, which may facilitate PGB transmembrane absorption in vivo.
- Published
- 2005
- Full Text
- View/download PDF
10. Novel cyclopropyl beta-amino acid analogues of pregabalin and gabapentin that target the alpha2-delta protein.
- Author
-
Schwarz JB, Gibbons SE, Graham SR, Colbry NL, Guzzo PR, Le VD, Vartanian MG, Kinsora JJ, Lotarski SM, Li Z, Dickerson MR, Su TZ, Weber ML, El-Kattan A, Thorpe AJ, Donevan SD, Taylor CP, and Wustrow DJ
- Subjects
- Administration, Oral, Amines chemistry, Amines pharmacology, Amino Acid Transport System L metabolism, Animals, Anticonvulsants chemical synthesis, Anticonvulsants chemistry, Anticonvulsants pharmacology, Biological Transport, Active, Blood-Brain Barrier metabolism, CHO Cells, Calcium Channels metabolism, Cricetinae, Cricetulus, Cyclization, Cyclohexanecarboxylic Acids chemistry, Cyclohexanecarboxylic Acids pharmacology, Cyclopropanes chemistry, Cyclopropanes pharmacology, Gabapentin, In Vitro Techniques, Injections, Intraventricular, Ion Channel Gating, Male, Mice, Mice, Inbred DBA, Nitriles chemistry, Pregabalin, Protein Subunits metabolism, Rats, Rats, Sprague-Dawley, Swine, gamma-Aminobutyric Acid chemistry, gamma-Aminobutyric Acid pharmacology, Amines chemical synthesis, Amino Acids chemistry, Calcium Channels drug effects, Cyclohexanecarboxylic Acids chemical synthesis, Cyclopropanes chemical synthesis, gamma-Aminobutyric Acid analogs & derivatives, gamma-Aminobutyric Acid chemical synthesis
- Abstract
As part of a program aimed at generating compounds with affinity for the alpha(2)-delta subunit of voltage-gated calcium channels, several novel beta-amino acids were prepared using an efficient nitroalkane-mediated cyclopropanation as a key step. Depending on the ester that was chosen, the target amino acids could be prepared in as few as three steps. The cyclopropyl amino acids derived from ketones proved to be potent binders of the alpha(2)-delta subunit of voltage-gated calcium channels, but did not interact with the large neutral amino acid system L (leucine) transporter. Anticonvulsant effects were observed in vivo with compound 34 but only after intracerebroventricular (icv) administration, presumably due to inadequate brain concentrations of the drug being achieved following oral dosing. However, pregabalin 1 was active in the DBA/2 model after oral (and icv) dosing, supporting a hypothesis that active transport is a prerequisite for such zwitterionic species to cross the blood-brain barrier.
- Published
- 2005
- Full Text
- View/download PDF
11. Cloning and tissue-specific expression of spliced variants of the rat organic anion transporter (rOAT-K).
- Author
-
Wolff MW and Su TZ
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Molecular Sequence Data, Organ Specificity, Protein Isoforms, Rats, Sequence Alignment, Alternative Splicing, Organic Anion Transport Protein 1 genetics
- Abstract
During the last decade, molecular cloning has identified several families of multispecific organic ion transporters mediating the renal and hepatic elimination of organic ions. Clinically, these transporters play important roles in the renal tubular secretion and reabsorption of various drugs. They are also in part responsible for the drug pharmacologic responses, drug-drug interactions, and drug nephrotoxicity. This study describes 12 novel isoforms of the rat kidney organic anion transporter rOAT-K. These isoforms are spliced variants of the same gene arising from alternative splicing of six regions defined as A-F. The two previously reported isoforms rOAT-K1 and rOAT-K2 were also found to be spliced variants of this gene. The open reading frames of the 12 isoforms encode a range of 352-670 amino acid proteins. Tissue distribution studies showed that the majority of the isoforms are kidney- and liver-specific.
- Published
- 2002
- Full Text
- View/download PDF
12. Tissue-specific expression and gabapentin-binding properties of calcium channel alpha2delta subunit subtypes.
- Author
-
Gong HC, Hang J, Kohler W, Li L, and Su TZ
- Subjects
- Amino Acids metabolism, Animals, Anticonvulsants metabolism, Binding Sites, Calcium Channels chemistry, Calcium Channels genetics, Cell Line, Gabapentin, Humans, Mice, Protein Binding, Protein Isoforms, Protein Structure, Secondary, Protein Subunits, Radioligand Assay, Tissue Distribution, Acetates metabolism, Amines, Calcium Channels metabolism, Cyclohexanecarboxylic Acids, gamma-Aminobutyric Acid
- Abstract
We report here the tissue-specific expression and gabapentin-binding properties of calcium channel alpha2delta subunits. Northern blot analysis demonstrated that human alpha2delta-1, -2, and -3 mRNA all had high levels of expression in brain, heart and skeletal muscle. However, the highest expression of human alpha2delta-2 mRNA was found in lung. Human alpha2delta-1, -2, and -3 mRNAs were detected in all portions of brain tested. Western blotting revealed that alpha2delta-2 protein was predominantly expressed in cerebellar cortex (brain) and undetectable in lung. The dissociation between mRNA and protein levels of human alpha2delta-2 in lung suggests possible post-transcriptional regulation. Although mouse alpha2delta-1 proteins exhibited a similar tissue distribution profile as that of human, tissue distribution of mouse alpha2delta-2 and -3 mRNA revealed a different profile. Mouse alpha2delta-3 mRNA was restricted to brain and mouse alpha2delta-2 mRNA was not detectable in lung. Gel electrophoresis under a reduced condition resulted in a mobility shift of both alpha2delta-1 and alpha2delta-2 proteins, suggesting that alpha2 and delta of alpha2delta-2 protein are linked by disulfide bond as are alpha2 and delta of alpha2delta-1. Scatchard plots revealed a single population of gabapentin binding sites for human alpha2delta-2 with the KD value twofold higher than that of porcine alpha2delta-1 (156 +/- 25 nm vs. 72 +/- 9 nm). Inhibition of gabapentin binding to alpha2delta-2 by selected amino acids and gabapentin analogs produced a binding profile similar, but not identical to that of alpha2delta-1.
- Published
- 2001
- Full Text
- View/download PDF
13. Structural requirement of the calcium-channel subunit alpha2delta for gabapentin binding.
- Author
-
Wang M, Offord J, Oxender DL, and Su TZ
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Binding Sites genetics, COS Cells, Calcium Channels genetics, DNA Primers genetics, Disulfides chemistry, Gabapentin, In Vitro Techniques, Molecular Sequence Data, Mutagenesis, Site-Directed, Peptide Mapping, Point Mutation, Protein Structure, Quaternary, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Sequence Deletion, Swine, Transfection, Acetates metabolism, Amines, Anticonvulsants metabolism, Calcium Channels chemistry, Calcium Channels metabolism, Cyclohexanecarboxylic Acids, gamma-Aminobutyric Acid
- Abstract
Gabapentin [Neurontin, 1-(aminomethyl)cyclohexaneacetic acid] is a novel anticonvulsant drug with a high binding affinity for the Ca(2+)-channel subunit alpha(2)delta. In this study, the gabapentin-binding properties of wild-type and mutated porcine brain alpha(2)delta proteins were investigated. Removal of the disulphide bonds between the alpha(2) and the delta subunits did not result in a significant loss of gabapentin binding, suggesting that the disulphide linkage between the two subunits is not required for binding. Singly expressed alpha(2) protein remained membrane associated. However, alpha(2) alone was unable to bind gabapentin, unless the cells were concurrently transfected with the expression vector for delta, suggesting that both alpha(2) and delta are required for gabapentin binding. Using internal deletion mutagenesis, we mapped two regions [amino acid residues 339-365 (DeltaF) and 875-905 (DeltaJ)] within the alpha(2) subunit that are not required for gabapentin binding. Further, deletion of three other individual regions [amino acid residues 206-222 (DeltaD), 516-537 (DeltaH) and 583-603 (DeltaI)] within the alpha(2) subunit disrupted gabapentin binding, suggesting the structural importance of these regions. Using alanine to replace four to six amino acid residues in each of these regions abolished gabapentin binding. These results demonstrate that region D, between the N-terminal end and the first putative transmembrane domain of alpha(2), and regions H and I, between the putative splicing acceptor sites (Gln(511) and Ser(601)), may play important roles in maintaining the structural integrity for gabapentin binding. Further single amino acid replacement mutagenesis within these regions identified Arg(217) as critical for gabapentin binding.
- Published
- 1999
14. Troglitazone, an antidiabetic agent, inhibits cholesterol biosynthesis through a mechanism independent of peroxisome proliferator-activated receptor-gamma.
- Author
-
Wang M, Wise SC, Leff T, and Su TZ
- Subjects
- 3T3 Cells, Animals, CHO Cells, Cell Line, Cholesterol genetics, Cricetinae, Cycloheximide pharmacology, Dactinomycin pharmacology, Mice, Nucleic Acid Synthesis Inhibitors pharmacology, Protein Synthesis Inhibitors pharmacology, RNA antagonists & inhibitors, Troglitazone, Anticholesteremic Agents pharmacology, Cholesterol biosynthesis, Chromans pharmacology, Hypoglycemic Agents pharmacology, Receptors, Cytoplasmic and Nuclear physiology, Thiazoles pharmacology, Thiazolidinediones, Transcription Factors physiology
- Abstract
Troglitazone is an antidiabetic agent of the thiazolidinedione family. It is generally believed that thiazolidinediones exert their insulin-sensitizing activity through activation of peroxisome proliferator-activated receptor-gamma (PPAR-gamma), a member of the steroid nuclear receptor superfamily. In the present study, we examined the effect of troglitazone on cholesterol biosynthesis in cultured Chinese hamster ovary (CHO) cells. Troglitazone inhibited biosynthesis of cholesterol, but not that of total sterols, in a dose-dependent manner, with a half-maximal concentration (IC50) value of 8 micromol/l. At 20 micromol/l, troglitazone inhibited cholesterol biosynthesis by more than 80%, resulting in the accumulation of lanosterol and several other sterol products. This inhibitory effect observed in CHO cells was also reproduced in HepG2, L6, and 3T3-L1 cells, suggesting that there is a common pathway for this troglitazone action. One hour after removal of troglitazone from the culture medium, disappearance of the accumulated sterols was accompanied by restored cholesterol synthesis, indicating that those accumulated sterols are precursors of cholesterol. PPAR-gamma reporter assays showed that PPAR-gamma activation by troglitazone was completely blocked by actinomycin D and cycloheximide. In contrast, the inhibition of cholesterol synthesis by troglitazone remained unchanged in the presence of the above compounds, suggesting that this inhibition is mechanistically distinct from the transcriptional regulation by PPAR-gamma. Like troglitazone, two other thiazolidinediones, ciglitazone and englitazone, exhibited similar inhibitory effect on cholesterol synthesis; however, other known PPAR-gamma ligands such as BRL49653, pioglitazone, and 15-deoxy-delta(12,14)-prostaglandin J2 showed only weak or no inhibition. The dissociation of PPAR-gamma binding ability from the potency for inhibition of cholesterol synthesis further supports the conclusion that inhibition of cholesterol biosynthesis by troglitazone is unlikely to be mediated by PPAR-gamma.
- Published
- 1999
- Full Text
- View/download PDF
15. Troglitazone increases system A amino acid transport in 3T3-L1 cells.
- Author
-
Su TZ, Wang M, Oxender DL, and Saltiel AR
- Subjects
- 3T3 Cells, Adipocytes drug effects, Adipocytes metabolism, Amino Acid Transport Systems, Aminoisobutyric Acids metabolism, Animals, Cell Differentiation, Insulin pharmacology, Mice, Troglitazone, Amino Acids metabolism, Carrier Proteins drug effects, Chromans pharmacology, Hypoglycemic Agents pharmacology, Thiazoles pharmacology, Thiazolidinediones
- Abstract
System A is one of the most highly regulated transport systems for transport of neutral amino acids into mammalian cells. Stimulation of uptake of alpha-[3H]methylaminoisobutyric acid (MeAIB), a nonmetabolizable system A substrate, by a novel insulin-sensitizing agent, troglitazone, in 3T3-L1 adipocytes was investigated. Treating adipocytes with troglitazone alone resulted in a time- and dose-dependent increase in the uptake of MeAIB. The peak stimulation appeared about 24 h after troglitazone addition. Both troglitazone- and insulin-stimulated transport activities increased markedly after the induction of differentiation of preadipocytes into adipocytes, and declined to a steady state level in adipocytes. The stimulated MeAIB uptake exhibited substrate specificity typical of system A and was mediated by a single component as determined by Eadie-Hofstee plots. The stimulation by troglitazone and that by insulin were similarly sensitive to actinomycin D and cycloheximide, suggesting that both agents may induce de novo synthesis of the same type of system A transport. Apart from the insulin-independent effect, troglitazone also showed an insulin-dependent action characterized by enhanced sensitivity to insulin. The synergistic stimulation of MeAIB uptake by coadministration of insulin and troglitazone was most prominent at the early stages of adipocyte differentiation. Pretreating cells with troglitazone during the differentiation attenuated the sensitivity of insulin to inhibition by actinomycin D, suggesting that troglitazone may enhance the insulin action by stabilizing messenger RNA involved in system A function.
- Published
- 1998
- Full Text
- View/download PDF
16. Regulation of system A amino acid transport in 3T3-L1 adipocytes by insulin.
- Author
-
Su TZ, Wang M, Syu LJ, Saltiel AR, and Oxender DL
- Subjects
- 3T3 Cells, Adipocytes metabolism, Androstadienes pharmacology, Animals, Biological Transport, Chromones pharmacology, Enzyme Inhibitors pharmacology, Kinetics, Mice, Morpholines pharmacology, Phosphoinositide-3 Kinase Inhibitors, Signal Transduction, Wortmannin, beta-Alanine analogs & derivatives, beta-Alanine metabolism, Adipocytes drug effects, Amino Acids metabolism, Carrier Proteins metabolism, Insulin pharmacology
- Abstract
The insulin-stimulated uptake of 2-(methylamino)isobutyric acid (MeAIB), a nonmetabolizable substrate for system A, in 3T3-L1 adipocytes was investigated. As cells took on a more adipogenic phenotype, the insulin-stimulated versus the saturable basal MeAIB uptake increased by 5-fold. The induced transport activity showed properties characteristic of system A, with a Km value of 190 microM. The half-life of the induced system A activity was independent of de novo mRNA and protein synthesis and was not accelerated by ambient amino acids, therefore, it was mechanistically distinct from the previously described adaptive and hormonal regulation of system A. Inhibition of mitogen-activated protein kinase kinase by PD98059, Ras farnesylation by PD152440 and B581, p70(S6K) by rapamycin, and phosphatidylinositol 3-kinase (PI 3'-K) by wortmannin and LY294002 revealed that only wortmannin and LY294002 inhibited the insulin-induced MeAIB uptake with IC50 values close to that previously reported for inhibition of PI 3'-K. These results suggest that the Ras/mitogen-activated protein kinase and pp70(S6K) insulin signaling pathways are neither required nor sufficient for insulin stimulation of MeAIB uptake, and activation of PI 3'-K or a wortmannin/LY294002-sensitive pathway may play an important role in regulation of system A transport by insulin in 3T3-L1 cells.
- Published
- 1998
- Full Text
- View/download PDF
17. A summary of mechanistic hypotheses of gabapentin pharmacology.
- Author
-
Taylor CP, Gee NS, Su TZ, Kocsis JD, Welty DF, Brown JP, Dooley DJ, Boden P, and Singh L
- Subjects
- Acetates pharmacokinetics, Analgesics pharmacology, Analgesics therapeutic use, Animals, Anti-Anxiety Agents administration & dosage, Anti-Anxiety Agents therapeutic use, Anticonvulsants pharmacology, Anticonvulsants therapeutic use, Brain drug effects, Brain physiology, Calcium Channels chemistry, Calcium Channels drug effects, Calcium Channels physiology, Gabapentin, Humans, Models, Neurological, Neuroprotective Agents pharmacology, Neuroprotective Agents therapeutic use, Neurotransmitter Agents physiology, Pain, Sodium Channels physiology, Synapses drug effects, Synapses physiology, Tissue Distribution, Acetates pharmacology, Acetates therapeutic use, Amines, Cyclohexanecarboxylic Acids, gamma-Aminobutyric Acid metabolism
- Abstract
Although the cellular mechanisms of pharmacological actions of gabapentin (Neurontin) remain incompletely described, several hypotheses have been proposed. It is possible that different mechanisms account for anticonvulsant, antinociceptive, anxiolytic and neuroprotective activity in animal models. Gabapentin is an amino acid, with a mechanism that differs from those of other anticonvulsant drugs such as phenytoin, carbamazepine or valproate. Radiotracer studies with [14C]gabapentin suggest that gabapentin is rapidly accessible to brain cell cytosol. Several hypotheses of cellular mechanisms have been proposed to explain the pharmacology of gabapentin: 1. Gabapentin crosses several membrane barriers in the body via a specific amino acid transporter (system L) and competes with leucine, isoleucine, valine and phenylalanine for transport. 2. Gabapentin increases the concentration and probably the rate of synthesis of GABA in brain, which may enhance non-vesicular GABA release during seizures. 3. Gabapentin binds with high affinity to a novel binding site in brain tissues that is associated with an auxiliary subunit of voltage-sensitive Ca2+ channels. Recent electrophysiology results suggest that gabapentin may modulate certain types of Ca2+ current. 4. Gabapentin reduces the release of several monoamine neurotransmitters. 5. Electrophysiology suggests that gabapentin inhibits voltage-activated Na+ channels, but other results contradict these findings. 6. Gabapentin increases serotonin concentrations in human whole blood, which may be relevant to neurobehavioral actions. 7. Gabapentin prevents neuronal death in several models including those designed to mimic amyotrophic lateral sclerosis (ALS). This may occur by inhibition of glutamate synthesis by branched-chain amino acid aminotransferase (BCAA-t).
- Published
- 1998
- Full Text
- View/download PDF
18. Glutamine transport in cerebellar granule cells in culture.
- Author
-
Su TZ, Campbell GW, and Oxender DL
- Subjects
- Animals, Animals, Newborn, Arginine metabolism, Astrocytes cytology, Astrocytes metabolism, Biological Transport, Cells, Cultured, Cerebellum cytology, Cerebral Cortex metabolism, Fetus, Hydrogen-Ion Concentration, Kinetics, Neurons cytology, Rats, Cerebellum metabolism, Glutamine metabolism, Neurons metabolism
- Abstract
In the present study, uptake of glutamine by rat cerebellar granule cells, a predominantly glutamatergic nerve cell population, has been investigated. Glutamine is taken up by granule cells via at least three transport systems, A, ASC and L. The L-type low affinity system (K(m) = 2.6 mM) is the major transport system in the absence of Na+. The systems A and ASC represent the Na(+)-dependent transport routes, both with almost identical high affinity for glutamine (K(m) = 0.26 mM). Similar transport systems for glutamine are also found in cerebral cortical neurons, a predominantly GABAergic nerve cell population, and cerebral cortical astrocytes. The glutamine transport properties in granule cells, however, show a series of differences from that of cortical neurons and astrocytes: (1) uptake of glutamine by granule cells is primarily mediated by system A (54%), while contributions by system A in cortical neurons and astrocytes are less than 30%; (2) granule cells exhibit strikingly higher transport efficiency for glutamine (V(max)/K(m) = 20 min(-1) for system A as compared to the V(max)/K(m) ratio of 5 min(-1) in cortical neurons and astrocytes), and (3) the initial uptake rates and the steady-state accumulation levels of glutamine are two- to threefold higher in granule cells than that of cortical neurons and astrocytes. These results taken together suggest that in accordance with the important need to replenish the neurotransmitter pool of glutamate, glutamatergic neurons exhibit highly efficient transport systems to accumulate glutamine, one of the major precursors of glutamate.
- Published
- 1997
- Full Text
- View/download PDF
19. Structure-activity relationships of cysteine-lacking pentapeptide derivatives that inhibit ras farnesyltransferase.
- Author
-
Leonard DM, Shuler KR, Poulter CJ, Eaton SR, Sawyer TK, Hodges JC, Su TZ, Scholten JD, Gowan RC, Sebolt-Leopold JS, and Doherty AM
- Subjects
- Amino Acids chemistry, Animals, Binding Sites, Insulin Antagonists pharmacology, Oocytes cytology, Oocytes drug effects, Phosphates chemistry, Rats, Structure-Activity Relationship, Xenopus, Alkyl and Aryl Transferases, Cysteine chemistry, Cysteine pharmacology, Enzyme Inhibitors chemical synthesis, Enzyme Inhibitors pharmacology, Oligopeptides chemical synthesis, Oligopeptides pharmacology, Transferases antagonists & inhibitors
- Abstract
Mutational activation of ras has been found in many types of human cancers, including a greater than 50% incidence in colon and about 90% in pancreatic carcinomas. The activity of both native and oncogenic ras proteins requires a series of post-translational processing steps. The first event in this process is the farnesylation of a cysteine residue located in the fourth position from the carboxyl terminus of the ras protein, catalyzed by the enzyme farnesyltransferase (FTase). Inhibitors of FTase are potential candidates for development as antitumor agents. Through a high-volume screening program, the pentapeptide derivative PD083176 (1), Cbz-His-Tyr(OBn)-Ser(OBn)-Trp-DAla-NH2, was identified as an inhibitor of rat brain FTase, with an IC50 of 20 nM. Structure-activity relationships were carried out to determine the importance of the side chain and chirality of each residue. This investigation led to a series of potent FTase inhibitors which lack a cysteine residue as found in the ras peptide substrate. The parent compound (1) inhibited the insulin-induced maturation of Xenopus oocytes (concentration: 5 pmol/oocyte), a process which is dependent on the activation of the ras pathway.
- Published
- 1997
- Full Text
- View/download PDF
20. Effects of anticonvulsant drug gabapentin on the enzymes in metabolic pathways of glutamate and GABA.
- Author
-
Goldlust A, Su TZ, Welty DF, Taylor CP, and Oxender DL
- Subjects
- Animals, Cytosol drug effects, Cytosol enzymology, Gabapentin, Glutamate Dehydrogenase antagonists & inhibitors, Glutamate Dehydrogenase metabolism, Glutamate-Ammonia Ligase antagonists & inhibitors, Glutamate-Ammonia Ligase metabolism, Glutaminase antagonists & inhibitors, Glutaminase metabolism, In Vitro Techniques, Kinetics, Male, Mitochondria drug effects, Mitochondria enzymology, Rats, Rats, Sprague-Dawley, Synaptosomes drug effects, Synaptosomes enzymology, Transaminases antagonists & inhibitors, Transaminases metabolism, Acetates pharmacology, Amines, Anticonvulsants pharmacology, Cyclohexanecarboxylic Acids, Glutamic Acid metabolism, gamma-Aminobutyric Acid metabolism
- Abstract
Gabapentin is a novel anticonvulsant drug. The anticonvulsant mechanism of gabapentin is not known. Based on the amino acid structure of gabapentin we explored its possible effects on glutamate and gamma-aminobutyric acid (GABA) metabolism in brain as they may relate to its anticonvulsant mechanisms of action. Gabapentin was tested for its effects on seven enzymes in the metabolic pathways of these two neurotransmitters: alanine aminotransferase (AL-T), aspartate aminotransferase (AS-T), GABA aminotransferase (GABA-T), branched-chain amino acid aminotransferase (BCAA-T), glutamine synthetase (Gln-S), glutaminase (GLNase), and glutamate dehydrogenase (GDH). In the presence of 10 mM gabapentin, only GABA-T, BCAA-T, and GDH activities were affected by this drug. Inhibition of GABA-T by gabapentin was weak (33%). The Ki values for inhibition of cytosolic and mitochondrial forms of GABA-T (17-20 mM) were much higher than the Km values for GABA (1.5-1.9 mM). It is, therefore, unlikely that inhibition of GABA-T by gabapentin is clinically relevant. As with leucine, gabapentin stimulated GDH activity. The GDH activity in rat brain synaptosomes was activated 6-fold and 3.4-fold, respectively, at saturating concentrations (10 mM) of leucine and gabapentin. The half-maximal stimulation by gabapentin was observed at approximately 1.5 mM. Gabapentin is not a substrate of BCAA-T, but it exhibited a potent competitive inhibition of both cytosolic and mitochondrial forms of brain BCAA-T. Inhibition of BCAA-T by this drug was reversible. The Ki values (0.8-1.4 mM) for inhibition of transamination by gabapentin were close to the apparent Km values for the branched-chain amino acids (BCAA) L-leucine, L-isoleucine, and L-valine (0.6-1.2 mM), suggesting that gabapentin may significantly reduce synthesis of glutamate from BCAA in brain by acting on BCAA-T.
- Published
- 1995
- Full Text
- View/download PDF
21. Transport of gabapentin, a gamma-amino acid drug, by system l alpha-amino acid transporters: a comparative study in astrocytes, synaptosomes, and CHO cells.
- Author
-
Su TZ, Lunney E, Campbell G, and Oxender DL
- Subjects
- Amino Acid Transport Systems, Amino Acids pharmacology, Animals, Arginine metabolism, Binding, Competitive, Biological Transport drug effects, Cricetinae, Gabapentin, Glutamic Acid metabolism, Kinetics, Leucine metabolism, Male, Rats, Rats, Sprague-Dawley, gamma-Aminobutyric Acid metabolism, Acetates metabolism, Amines, Astrocytes metabolism, CHO Cells metabolism, Carrier Proteins metabolism, Cyclohexanecarboxylic Acids, Synaptosomes metabolism
- Abstract
The system L transporter is generally considered to be one of the major Na(+)-independent carriers for large neutral alpha-amino acids in mammalian cells. However, we found that cultured astrocytes from rat brain cortex accumulate gabapentin, a gamma-amino acid, predominately by this alpha-amino acid transport system. Uptake of gabapentin by system L transporter was also examined in synaptosomes and Chinese hamster ovary (CHO) cells. The inhibition pattern displayed by various amino acids on gabapentin uptake in astrocytes and synaptosomes corresponds closely to that observed for the system L transport activity in CHO cells. Gabapentin and leucine have Km values that equal their Ki values for inhibition of each other, suggesting that leucine and gabapentin compete for the same system L transporter. By contrast, gabapentin exhibited no effect on uptake of GABA, glutamate, and arginine, indicating that these latter three types of brain transporters do not serve for uptake of gabapentin. A comparison of computer modeling analysis of gabapentin and L-leucine structures shows that although the former is a gamma-amino acid, it can assume a conformation that can resemble the L-form of a large neutral alpha-amino acid such as L-leucine. The steady-state kinetic study in astrocytes and CHO cells indicates that the intracellular concentrations of gabapentin are about two to four times higher than that of leucine. The uptake levels of these two substrates are inversely related to their relative exodus rates. The concentrating ability by system L observed in astrocytes is consistent with the substantially high accumulation gradient of gabapentin in the brain tissue as determined by microdialysis.
- Published
- 1995
- Full Text
- View/download PDF
22. Chinese hamster ovary mRNA-dependent, Na(+)-independent L-leucine transport in Xenopus laevis oocytes.
- Author
-
Su TZ, Logsdon CD, and Oxender DL
- Subjects
- Animals, Binding, Competitive, Biological Transport, CHO Cells, Cricetinae, Female, Kinetics, Microinjections, Substrate Specificity, Xenopus laevis, Leucine metabolism, Oocytes metabolism, Poly A metabolism, RNA, Messenger metabolism, Sodium metabolism
- Abstract
In freshly prepared uninjected folliculated oocytes, Na(+)-independent leucine uptake is mediated predominantly by a system L-like transport system. Removal of follicular cells, however, results in an irreversible loss of this transport activity. When total poly(A)+ mRNA derived from Chinese hamster ovary (CHO) cells was injected into prophase-arrested stage V or VI Xenopus laevis oocytes, enhanced expression of Na(+)-independent leucine transport was observed. The injected mRNAs associated with increased levels of leucine uptake were between 2 and 3 kb in length. The newly expressed leucine transport activity exhibited important differences from the known characteristics of system L, which is the dominant Na(+)-independent leucine transporter in CHO cells as well as in freshly isolated folliculated oocytes. The CHO mRNA-dependent leucine uptake in oocytes was highly sensitive to the cationic amino acids lysine, arginine, and and ornithine (> 95% inhibition). As with the leucine uptake, an enhanced lysine uptake was also observed in size-fractionated CHO mRNA-injected oocytes. The uptakes of leucine and lysine were mutually inhibitable, suggesting that the newly expressed transporter was responsible for uptakes of both leucine and lysine. The inhibition of uptake of lysine by leucine was Na+ independent, thus clearly distinguishing it from the previously reported endogenous system y+ activity. Furthermore, the high sensitivity to tryptophan of the CHO mRNA-dependent leucine transport was in sharp contrast to the properties of the recently cloned leucine transport-associated gene from rat kidney tissue, although leucine transport from both sources was sensitive to cationic amino acids. Our results suggest that there may be a family of leucine transporters operative in different tissues and possibly under different conditions.
- Published
- 1992
- Full Text
- View/download PDF
23. Carbon-starvation induction of the ugp operon, encoding the binding protein-dependent sn-glycerol-3-phosphate transport system in Escherichia coli.
- Author
-
Su TZ, Schweizer HP, and Oxender DL
- Subjects
- Alkaline Phosphatase metabolism, Biological Transport, Carbon metabolism, Catechol 2,3-Dioxygenase, Cloning, Molecular, Escherichia coli genetics, Genes, Regulator, Oxygenases metabolism, Phosphates metabolism, Plasmids, beta-Galactosidase metabolism, Dioxygenases, Escherichia coli metabolism, Gene Expression Regulation, Bacterial, Glycerophosphates metabolism, Operon
- Abstract
The gene products of the ugp operon of Escherichia coli are responsible for the uptake of sn-glycerol-3-phosphate and certain glycerophosphodiesters. The regulation of ugp is mainly phoBR-dependent. Significant expression, however, can be observed even in the presence of high concentrations of phosphate, a condition which normally completely represses pho expression. Pho-independent ugp expression was found to be derepressed during the late logarithmic growth phase due to carbon starvation. Among different carbon sources tested, glucose caused the most complete repression. Addition of cAMP prevented glucose repression, indicating that a cAMP-CRP control mechanism may be directly or indirectly involved in the carbon-starvation response. This conclusion is supported by the fact that pho-independent ugp expression correlated with the presence of the cya and crp gene products.
- Published
- 1991
- Full Text
- View/download PDF
24. A novel phosphate-regulated expression vector in Escherichia coli.
- Author
-
Su TZ, Schweizer H, and Oxender DL
- Subjects
- Gene Expression Regulation, Bacterial drug effects, Glucose pharmacology, Glycerophosphates metabolism, Plasmids, Promoter Regions, Genetic, Genetic Vectors, Phosphates pharmacology
- Abstract
The ugp promoter (pugp) responsible for expression of the binding-protein-dependent sn-glycerol-3-phosphate transport system in Escherichia coli was cloned into a small multicopy plasmid pTER5, a derivative of pBR322, between the transcription terminators rpoCt and tL1. The resulting expression vector, pPH3, permits convenient insertion of structural genes containing their own translational-initiation regions, into the multiple-cloning site derived from the pUC19 plasmid. The efficiency and regulatory properties of pugp were measured using xylE and lacZ as reporter genes, which code for the corresponding enzymes catechol-2,3-dioxygenase (C23O) and beta-galactosidase (beta Gal), respectively. Enzyme activities were virtually completely repressed in the presence of excess inorganic phosphates (Pi) and high concentrations of glucose. Maximal induction was observed at limiting Pi (less than 0.1 mM) and normal levels of glucose (0.2-0.4%). The maximum expression of the pugp-directed beta Gal synthesis was approx. 80% of that directed by strong ptac. When the xylE gene was maximally expressed, the induced enzyme constituted approx. 50% of total cellular protein as judged by laser densitometry following sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. These results suggest the usefulness of the pugp in expression vectors for strong, but controlled, expression of cloned genes in E. coli. This Pi controlled vector can be adapted to large-scale fermentation by using Pi-limiting growth conditions.
- Published
- 1990
- Full Text
- View/download PDF
25. A multisite-directed mutagenesis using T7 DNA polymerase: application for reconstructing a mammalian gene.
- Author
-
Su TZ and el-Gewely MR
- Subjects
- Animals, Base Sequence, Chromosome Deletion, Molecular Sequence Data, Oligonucleotide Probes, Pituitary Gland metabolism, Plasmids, Restriction Mapping, Swine, DNA-Directed DNA Polymerase metabolism, Genes, Growth Hormone genetics, Mutation, T-Phages enzymology
- Abstract
A method to introduce multiple mutations and to reconstruct genes, using a single oligodeoxyribonucleotide and DNA polymerase with high processivity, such as modified T7 DNA polymerase [Tabor and Richardson, Proc. Natl. Acad. Sci. USA 84 (1987a) 4767-4771], is described. A eukaryotic cDNA, coding for porcine growth hormone (pGH), was reconstructed in this study to delete 75 bp and to introduce a G----A transition. The deletion removes 75 bp and brings an ATG just upstream from the codon for the first amino acid in the mature protein. Moreover, the G----A substitution creates a new PvuII restriction site to facilitate further manipulation of the gene. Maximum mutation frequency with this multisite-directed mutagenesis is reached within 15 min with an efficiency approaching 50%, when using the modified T7 DNA polymerase. No multisite-directed mutants were obtained when T4 DNA polymerase or Klenow (large) fragment of DNA polymerase I were used. The described method is also applicable to simple single site-directed mutations as well as to more complex gene reconstruction strategies.
- Published
- 1988
- Full Text
- View/download PDF
26. [Regulation in the expression of alpha-galactosidase gene in raf operon in Escherichia coli].
- Author
-
Su TZ, Qi S, Yun WH, and Xiu L
- Subjects
- Gene Expression Regulation, Escherichia coli genetics, Galactosidases genetics, Operon, alpha-Galactosidase genetics
- Abstract
The alpha-galactosidase, coded for by the first structural gene rafA in the plasmid determined raf operon was an inducible enzyme. In contrast to lac or mel operon, raf operon has more strict structural specificity for inducers. The enzyme can be induced by melibiose and raffinose, or weakly by D-galactose, but not by structurally related sugars such as lactose, PNPG etc.. The alpha-galactosidase forming capacity as function of growth curve reached a single peak at the end of the logarithmic phase of the growth. The structure and regulation of raf operon is similar to those of lac operon. The repressormor-mediated negative control plays a major role in the regulation of raf operon, and cAMP-CAP mediated positive control is also involved in the regulation. When 0.4% glucose was added into the medium with other carbon sources, the expression of the enzyme was repressed by 2-3 fold. Transient catabolite repression has been observed neither in inducible nor constitutive alpha-galactosidase expression. Based on alpha-galactosidase assay, in mutant strains CA8306(cya) and CA8445 (cya, crp) the expression level of raf operon was only 9% and 2.5% of that in wild type strain respectively. The glucose effect or the repression in cya mutant can be abolished by 1-5 mmol cAMP. The constitutive alpha-galactosidase expression in cya and cry double mutant (CA8445) remains repressible by glucose, but irreversible by cAMP, suggesting cAMP-CAP complex is not the exclusive mediator of the catablite repression.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1989
27. Identification of livG, a membrane-associated component of the branched-chain amino acid transport in Escherichia coli.
- Author
-
Nazos PM, Mayo MM, Su TZ, Anderson JJ, and Oxender DL
- Subjects
- Biological Transport, Active, Cell Membrane metabolism, DNA Restriction Enzymes, Escherichia coli drug effects, Escherichia coli metabolism, Ethyl Methanesulfonate pharmacology, Genetic Complementation Test, Genotype, Mutation, Plasmids, Species Specificity, Amino Acids metabolism, Bacterial Proteins, Carrier Proteins genetics, Escherichia coli genetics, Escherichia coli Proteins, Genes, Genes, Bacterial
- Abstract
Branched-chain amino acids are transported into Escherichia coli by two osmotic shock-sensitive systems (leucine-isoleucine-valine and leucine-specific transport systems). These high-affinity systems consist of separate periplasmic binding protein components and at least three common membrane-bound components. In this study, one of the membrane-bound components, livG, was identified. A toxic analog of leucine, azaleucine, was used to isolate a large number of azaleucine-resistant mutants which were defective in branched-chain amino acid transport. Genetic complementation studies established that two classes of transport mutants with similar phenotypes, livH and livG, were obtained which were defective in one of the membrane-associated transport components. Since the previously cloned plasmid, pOX1, genetically complemented both livH and livG mutants, we were able to verify the physical location of the livG gene on this plasmid. Recombinant plasmids which carried different portions of the pOX1 plasmid were constructed and subjected to complementation analysis. These results established that livG was located downstream from livH with about 1 kilobase of DNA in between. The expression of these plasmids was studied in minicells; these studies indicate that livG appears to be membrane bound and to have a molecular weight of 22,000. These results establish that livG is a membrane-associated component of the branched-chain amino acid transport system in E. coli.
- Published
- 1985
- Full Text
- View/download PDF
28. 4-methyleneglutamine synthetase: a new amide synthetase present in germinating peanuts.
- Author
-
Winter HC, Su TZ, and Dekker EE
- Subjects
- Adenosine Triphosphate metabolism, Arachis, Chromatography, DEAE-Cellulose, Glutamates biosynthesis, Kinetics, Ligases metabolism, Substrate Specificity, Amide Synthases, Ligases analysis, Plants enzymology
- Abstract
Enzymatic activity which catalyzes the synthesis of 4-methyleneglutamine from 4-methyleneglutamic acid + ammonia was detected in and partially purified from cotyledons of peanut seeds germinated 5 to 7 days. This activity was separated from glutamine and asparagine synthetases by ammonium sulfate precipitation and DEAE-cellulose chromatography. The enzyme is distinct from these other amide synthetases in its substrate specificity, lack of amide/hydroxylamine exchange, and use of ammonium ion as amide donor together with formation of AMP from ATP. The activity is quite labile in solution, but is retained as a precipitate in ammonium sulfate or when frozen in 12.5% glycerol at -77 degrees C. This activity might be responsible for catalyzing the rapid synthesis of 4-methyleneglutamine which occurs in germinating peanuts.
- Published
- 1983
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.