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  Partner: UNT Libraries
 Department: Department of Biological Sciences
 Degree Discipline: Biochemistry
N-Acylethanolamines and Plant Phospholipase D

N-Acylethanolamines and Plant Phospholipase D

Date: December 1998
Creator: Brown, Shea Austin
Description: Recently, three distinct isoforms of phospholipase D (PLD) were identified in Arabidopsis thaliana. PLD α represents the well-known form found in plants, while PLD β and γ have been only recently discovered (Pappan et al., 1997b; Qin et al., 1997). These isoforms differ in substrate selectivity and cofactors required for activity. Here, I report that PLD β and γ isoforms were active toward N-acylphosphatidylethanolamine (NAPE), but PLD α was not. The ability of PLD β and γ to hydrolyze NAPE marks a key difference from PLD α. N-acylethanolamines (NAE), the hydrolytic products of NAPE by PLD β and γ, inhibited PLD α from castor bean and cabbage. Inhibition of PLD α by NAE was dose-dependent and inversely proportional to acyl chain length and degree of unsaturation. Enzyme kinetic analysis suggested non-competitive inhibition of PLD α by NAE 14:0. In addition, a 1.2-kb tobacco (Nicotiana tabacum L.) cDNA fragment was isolated that possessed a 74% amino acid identity to Arabidopsis PLD β indicating that this isoform is expressed in tobacco cells. Collectively, these results provide evidence for NAE producing PLD activities and suggest a possible regulatory role for NAE with respect to PLD α.
Contributing Partner: UNT Libraries
Cottonseed Microsomal N-Acylphosphatidylethanolamine Synthase: Identification, Purification and Biochemical Characterization of a Unique Acyltransferase

Cottonseed Microsomal N-Acylphosphatidylethanolamine Synthase: Identification, Purification and Biochemical Characterization of a Unique Acyltransferase

Date: December 1998
Creator: McAndrew, Rosemary S. (Rosemary Smith)
Description: N-Acylphosphatidylethanoiamine (NAPE) is synthesized in the microsomes of cotton seedlings by a mechanism that is possibly unique to plants, the ATP-, Ca2+-, and CoA-independent acylation ofphosphatidylethanolamine (PE) with unesterified free fatty acids (FFAs), catalyzed by NAPE synthase. A photoreactive free fatty acid analogue, 12-[(4- azidosalicyl)amino]dodecanoic acid (ASD), and its 125I-labeled derivative acted as substrates for the NAPE synthase enzyme.
Contributing Partner: UNT Libraries
Nucleotide Inhibition of Glyoxalase II

Nucleotide Inhibition of Glyoxalase II

Date: May 1999
Creator: Gillis, Glen S
Description: The glyoxalase system mediates the conversion of methylglyoxal, a toxic ketoaldehyde, to D-lactic acid. The system is composed of two enzymes, glyoxalase I (Glo-I) and glyoxalase II (Glo-II), and exhibits an absolute requirement for a catalytic quantity of glutathione (GSH). Glo-I catalyzes the isomerization of a hemithioacetal, formed non-enzymatically from methylglyoxal and GSH, to the corresponding a -D-hydroxyacid thioester, s-D-lactoylglutathione (SLG). Glo-II catalyzes the irreversible breakdown of SLG to D-lactate and GSH. We have observed that ATP or GTP significantly inhibits the Glo-II activity of tissue homogenates from various sources. We have developed a rapid, one step chromatography procedure to purify Glo-II such that the purified enzyme remains "sensitive" to inhibition by ATP or GTP (Glo-II-s). Studies indicate that inhibition of Glo-II-s by nucleotides is restricted to ATP, GTP, ADP, and GDP, with ATP appearing most effective. Kinetics studies have shown that ATP acts as a partial non-competitive inhibitor of Glo-II-s activity, and further suggest that two kinetically distinguishable forms of the enzyme exist. The sensitivity of pure Glo-II-s to nucleotide inhibition is slowly lost on storage even at -80° C. This loss is accelerated at higher temperatures or in the presence of ATP. Kinetics studies on the resultant "insensitive" ...
Contributing Partner: UNT Libraries
Conformational Studies of Myosin and Actin with Calibrated Resonance Energy Transfer

Conformational Studies of Myosin and Actin with Calibrated Resonance Energy Transfer

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Date: May 2000
Creator: Xu, Jin
Description: Resonance energy transfer was employed to study the conformational changes of actomyosin during ATP hydrolysis. To calibrate the technique, the parameters for resonance energy transfer were defined. With conformational searching algorithms to predict probe orientation, the distances measured by resonance energy transfer are highly consistent with the atomic models, which verified the accuracy and feasibility of resonance energy transfer for structural studies of proteins and oligonucleotides. To study intramyosin distances, resonance energy transfer probes were attached to skeletal myosin's nucleotide site, subfragment-2, and regulatory light chain to examine nucleotide analog-induced structural transitions. The distances between the three positions were measured in the presence of different nucleotide analogs. No distance change was considered to be statistically significant. The measured distance between the regulatory light chain and nucleotide site was consistent with either the atomic model of skeletal myosin subfragment-1 or an average of the three models claimed for different ATP hydrolysis states, which suggested that the neck region was flexible in solution. To examine the participation of actin in the powerstroke process, resonance energy transfer between different sites on actin and myosin was measured in the presence of nucleotide analogs. The efficiencies of energy transfer between myosin catalytic domain and actin ...
Contributing Partner: UNT Libraries
Noncovalent crosslinking of SH1 and SH2 to detect dynamic flexibility of the SH1 helix

Noncovalent crosslinking of SH1 and SH2 to detect dynamic flexibility of the SH1 helix

Date: August 2000
Creator: Park, Hyunguk
Description: In this experiment, fluorescent N- (1-pyrenyl) iodoacetamide modified the two reactive thiols, SH1 (Cys 707) and SH2 (Cys 697) on myosin to detect SH1-SH2 a -helix melting. The excimer forming property of pyrene is well suited to monitor the dynamics of the SH1 and SH2 helix melting, since the excimer should only form during the melted state. Decreased anisotropy of the excimer relative to the monomeric pyrene fluorescence is consistent with the disordering of the melted SH1-SH2 region in the atomic model. Furthermore, nucleotide analogs induced changes in the anisotropy of the excimer, suggesting that the nucleotide site modulates the flexibility of SH1-SH2 region.
Contributing Partner: UNT Libraries
Palmitoyl-acyl carrier protein thioesterase in cotton (Gossypium hirsutum L.): biochemical and molecular characterization of a major mechanism for the regulation of palmitic acid content.

Palmitoyl-acyl carrier protein thioesterase in cotton (Gossypium hirsutum L.): biochemical and molecular characterization of a major mechanism for the regulation of palmitic acid content.

Date: August 2001
Creator: Huynh, Tu T
Description: The relatively high level of palmitic acid (22 mol%) in cottonseeds may be due in part to the activity of a palmitoyl-acyl carrier protein (ACP) thioesterase (PATE). In embryo extracts, PATE activity was highest at the maximum rate of reserve accumulation (oil and protein). The cotton FatB mRNA transcript abundance also peaked during this developmental stage, paralleling the profiles of PATE enzyme activity and seed oil accumulation. A cotton FatB cDNA clone was isolated by screening a cDNA library with a heterologous Arabidopsis FatB probe (Pirtle et al., 1999, Plant and Cell Physiology 40: 155-163). The predicted amino acid sequence of the cotton PATE preprotein had 63% identity to the Arabidopsis FatB thioesterase sequence, suggesting that the cotton cDNA clone probably encoded a FatB-type thioesterase. When acyl-CoA synthetase-minus E. coli mutants expressed the cotton cDNA, an increase in 16:0 free fatty acid content was measured in the culture medium. In addition, acyl-ACP thioesterase activity assays in E. coli lysates revealed that there was a preference for palmitoyl-ACP over oleoyl-ACP in vitro, indicating that the cotton putative FatB cDNA encoded a functional thioesterase with a preference for saturated acyl-ACPs over unsaturated acyl-ACPs (FatA). Overexpression of the FatB cDNA in transgenic cotton ...
Contributing Partner: UNT Libraries
Plastidial carbonic anhydrase in cotton (Gossypium hirsutum L.): characterization, expression, and role in lipid biosynthesis

Plastidial carbonic anhydrase in cotton (Gossypium hirsutum L.): characterization, expression, and role in lipid biosynthesis

Date: August 2001
Creator: Hoang, Chau V.
Description: Recently, plastidial carbonic anhydrase (CA, EC 4.2.1.1) cDNA clones encoding functional CA enzymes were isolated from a nonphotosynthetic cotton tissue. The role of CA in photosynthetic tissues have been well characterized, however there is almost no information for the role of CA in nonphotosynthetic tissues. A survey of relative CA transcript abundance and enzyme activity in different cotton organs revealed that there was substantial CA expression in cotyledons of seedlings and embryos, both nonphotosynthetic tissues. To gain insight into the role(s) of CA, I examined CA expression in cotyledons of seedlings during post-germinative growth at different environmental conditions. CA expression in cotyledons of seedlings increased from 18 h to 72 h after germination in the dark. Seedlings exposed to light had about a 2-fold increase in CA activities when compared with seedlings kept in the dark, whereas relative CA transcript levels were essentially the same. Manipulation of external CO2 environments [zero, ambient (350 ppm), or high (1000 ppm)] modulated coordinately the relative transcript abundance of CA (and rbcS) in cotyledons, but did not affect enzyme activities. On the other hand, regardless of the external CO2 conditions seedlings exposed to light exhibited increase CA activity, concomitant with Rubisco activity and increased ...
Contributing Partner: UNT Libraries
Tobacco Phospholipase D β1: Molecular Cloning and Biochemical Characterization

Tobacco Phospholipase D β1: Molecular Cloning and Biochemical Characterization

Date: December 2002
Creator: Hodson, Jane E.
Description: Transgenic tobacco plants were developed containing a partial PLD clone in antisense orientation. The PLD isoform targeted by the insertion was identified. A PLD clone was isolated from a cDNA library using the partial PLD as a probe: Nt10B1 shares 92% identity with PLDβ1 from tomato but lacks the C2 domain. PCR analysis confirmed insertion of the antisense fragment into the plants: three introns distinguished the endogenous gene from the transgene. PLD activity was assayed in leaf homogenates in PLDβ/g conditions. When phosphatidylcholine was utilized as a substrate, no significant difference in transphosphatidylation activity was observed. However, there was a reduction in NAPE hydrolysis in extracts of two transgenic plants. In one of these, a reduction in elicitor- induced PAL expression was also observed.
Contributing Partner: UNT Libraries
Identification and quantification of lipid metabolites in cotton fibers: Reconciliation with metabolic pathway predictions from DNA databases.

Identification and quantification of lipid metabolites in cotton fibers: Reconciliation with metabolic pathway predictions from DNA databases.

Date: May 2004
Creator: Wanjie, Sylvia W.
Description: The lipid composition of cotton (Gossypium hirsutum, L) fibers was determined. Fatty acid profiles revealed that linolenate and palmitate were the most abundant fatty acids present in fiber cells. Phosphatidylcholine was the predominant lipid class in fiber cells, while phosphatidylethanolamine, phosphatidylinositol and digalactosyldiacylglycerol were also prevalent. An unusually high amount of phosphatidic acid was observed in frozen cotton fibers. Phospholipase D activity assays revealed that this enzyme readily hydrolyzed radioactive phosphatidylcholine into phosphatidic acid. A profile of expressed sequence tags (ESTs) for genes involved in lipid metabolism in cotton fibers was also obtained. This EST profile along with our lipid metabolite data was used to predict lipid metabolic pathways in cotton fiber cells.
Contributing Partner: UNT Libraries
Studies on actomyosin crossbridge flexibility using a new single molecule assay.

Studies on actomyosin crossbridge flexibility using a new single molecule assay.

Date: May 2004
Creator: Gundapaneni, Deepika
Description: Several key flexure sites exist in the muscle crossbridge including the actomyosin binding site which play important roles in the actomyosin crossbridge cycle. To distinguish between these sources of flexibility, a new single molecule assay was developed to observe the swiveling of rod about a single myosin. Myosins attached through a single crossbridge displayed mostly similar torsional characteristics compared to myosins attached through two crossbridges, which indicates that most of the torsional flexibility resides in the myosin subfragment-2, and thus the hinge between subfragment-2 and light meromyosin should contribute the most to this flexibility. The comparison of torsional characteristics in the absence and presence of ADP demonstrated a small but significant increase in twist rates for the double-headed myosins but no increase for single-headed myosins, which indicates that the ADP-induced increase in flexibility arises due to changes in the myosin head and verifies that most flexibility resides in myosin subfragment-2.
Contributing Partner: UNT Libraries
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