Associations of polymorphisms in the promoter I of bovine acetyl-CoA carboxylase-alpha gene with beef fatty acid composition.

The objectives of this study were to identify single nucleotide polymorphisms (SNPs) in the promoter I (PI) region of the bovine acetyl-CoA carboxylase-alpha (ACACA) gene and to evaluate the extent to which they were associated with lipid-related traits. Eight novel SNPs were identified, which were AJ276223:g.2064T>A (SNP1), g.2155C>T (SNP2), g.2203G>T (SNP3), g.2268T>C (SNP4), g.2274G>A (SNP5), g.2340A>G (SNP6), g.2350T>C (SNP7) and g.2370A>G (SNP8). Complete linkage disequilibrium was observed among SNP1, 2, 4, 5, 6 and 8. Phenotypic data were collected from 573 cross-bred steers with six sire breeds, including Hereford, Angus, Brangus, Beefmaster, Bonsmara and Romosinuano. The genotypes of SNP1/2/4/5/6/8 were significantly associated with adjusted backfat thickness. The genotypes of SNP3 were significantly associated with triacylglycerol (TAG) content and fatty acid composition of longissimus dorsi muscle (LM) in Brangus-, Romosinuano- and Bonsmara-sired cattle. Cattle with g.2203GG genotype had greater concentrations of TAG, total lipid, total saturated fatty acid and total monounsaturated fatty acid than did cattle with g.2203GT genotype. The genotypes of SNP7 were significantly associated with fatty acid composition of LM. Cattle with genotype g.2350TC had greater amounts of several fatty acids in LM than did cattle with genotype g.2350CC. Our results suggested that the SNPs in the PI region of ACACA gene are associated with variations in the fatty acid contents in LM.

Effects of breed, sex and halothane genotype on fatty acid composition of triacylglycerols and phospholipids in pork longissimus muscle.

The objective of this study was to estimate the effects of breed, sex, and halothane (HAL-1843) genotype on fatty acid composition of triacylglycerols (TAG) and phospholipids (PL) extracted from porcine longissimus muscle (LM). Purebred Yorkshire (n = 131), Duroc (n = 136), Hampshire (n = 49), Spotted (n = 35), Chester White (n = 74), Poland China (n = 51), Berkshire (n = 169) and Landrace (n = 82) pigs (n = 727; 427 barrows and 300 gilts) from the 1994 and 2001 National Barrow Show Sire Progeny Tests were used. For statistical analyses, a mixed model was used that included fixed effects of breed, sex, HAL-1843(TM) genotype, year, slaughter date within each year, interaction of breed x sex and random effects of sire and dam within breed. Breeds and sex were significantly associated with the percentages of the majority fatty acids in TAG. Duroc pigs had greater total saturated fatty acids (SFA) and lower total monounsaturated fatty acids (MUFA) (p < 0.05) contents than did pigs of all other breeds except Berkshire (p > 0.05). The concentration of total polyunsaturated fatty acids (PUFA) was the greatest in Hampshire pigs (p < 0.05). The content of total SFA was greater (p < 0.01), whereas the concentrations of total MUFA and PUFA were lower (p < 0.01) in barrows than those in gilts. The contents of major SFA in PL did not differ significantly among pigs from different breeds and sex groups. However, breed and sex significantly affected the concentrations of major MUFA and PUFA in PL and strong negative correlation between the total contents of MUFA and PUFA in PL was observed in the current study. Chester White pigs had greater total MUFA and lower total PUFA contents (p < 0.05) in PL than did pigs of all other breeds except Spotted (p > 0.05). In contrast to breed and sex effects, the concentrations of fatty acids in PL were more affected by HAL-1843 genotype than those in TAG. The content of C16:0, a major SFA in PL, differed significantly in pigs with different HAL-1843 genotypes. In conclusion, these results suggest that breed and sex are important sources of the variations for fatty acid composition of TAG and PL in LM.

DNA polymorphisms in bovine fatty acid synthase are associated with beef fatty acid composition.

The objective of this study was to identify single nucleotide polymorphisms (SNPs) in the thioesterase (TE) domain of the bovine fatty acid synthase (FASN) gene and to evaluate the extent to which they were associated with beef fatty acid composition. The four exons in FASN that encode for the TE domain were sequenced, and three SNPs, AF285607:g.17924A>G, g.18663T>C and g.18727C>T, were identified. Purebred Angus bulls (n = 331) were classified into three genotype groups, g.17924AA (n = 121), g.17924AG (n = 168) and g.17924GG (n = 42). The g.17924A>G genotype was significantly associated with fatty acid composition of longissimus dorsi muscle of Angus bulls. Cattle with the g.17924GG genotype had lower myristic acid (C14:0; P < 0.0001), palmitic acid (C16:0, P < 0.05) and total saturated fatty acid contents (P < 0.01), greater health index (P < 0.001), oleic acid content (C18:1; P < 0.001) and total monounsaturated fatty acid concentration (P < 0.01) in the total lipids and triacylglycerols fraction than did those with the g.17924AA genotype. Because of the linkage disequilibrium between SNPs g.17924A>G and g.18663T>C, similar significant associations of fatty acid contents with the g.18663T>C genotypes were observed. In conclusion, the SNPs g.17924A>G and g.18663T>C may be used as DNA markers to select breeding stock that have a healthier fatty acid composition.

Effects of breed, sex, and halothane genotype on fatty acid composition of pork longissimus muscle.

The objective of this study was to estimate the effects of breed, sex, and halothane genotype on fatty acid composition and several fatty acid indices of lipid extracted from porcine LM. Purebred Yorkshire (n = 436), Duroc (n = 353), Hampshire (n = 218), Spotted (n = 187), Chester White (n = 173), Poland China (n = 124), Berkshire (n = 256), and Landrace (n = 187) pigs (n = 1,934; 1,128 barrows and 806 gilts) from 1991, 1992, 1994, and 2001 National Barrow Show Sire Progeny Tests were used. Pigs were classified as the HAL-1843 normal (NN) genotype (n = 1,718) or the HAL-1843 carrier (Nn) genotype (n = 216). For statistical analysis, a mixed model was used that included fixed effects of breed, sex, halothane genotype, test, slaughter date, interaction of breed x sex, and random effects of sire and dam within breed. Breed significantly affected the concentration of individual fatty acids, total lipid content, and the values of several fatty acid indices of LM. Duroc pigs had the greatest (P < 0.01) content of total SFA. Total MUFA concentration in Poland China pigs was greater (P < 0.05) than in all other breeds except the Spotted (P > 0.05). The concentrations of total PUFA were greater (P < 0.01) in Hampshire, Landrace, and Yorkshire pigs compared with those of other breeds. Significant sex differences for individual fatty acids were detected. Compared with gilts, barrows had greater (P < 0.01) concentrations of SFA and MUFA but lower (P < 0.01) total PUFA. Halothane genotype was a significant source of variation for the percentages of some fatty acids. Pigs with the carrier (Nn) genotype had lower concentrations of SFA (P < 0.05) and MUFA (P < 0.01) but a greater concentration of PUFA (P < 0.01) compared with NN pigs. There were significant negative correlations between total lipid content and individual PUFA and significant positive correlations between lipid concentration and most individual SFA and MUFA. In conclusion, the results suggest that breed and sex are important sources of variation for fatty acid composition of LM.

Influence of lipid content on pork sensory quality within pH classification.

The objective of this project was to determine the contribution of lipid content to textural and sensory properties of fresh pork within defined pH classifications. Pigs (n = 1,535; from 248 sires and 836 dams) from the 1991, 1992, and 1994 National Barrow Show Sire Progeny Test were used in this study. The test included purebred Berkshire (107), Chester White (113), Duroc (249), Hampshire (220), Landrace (165), Poland China (101), Spotted (181), and Yorkshire (399) barrows (901) and gilts (634). Diets were uniform across breeds within test. The halothane (Hal 1843) genotype (1346 NN and 189 Nn) was determined. Pigs were slaughtered at 105 kg of BW, and samples of the LM were obtained from each carcass at the 10th rib. Star probe, sensory traits, and lipid content were determined on the LM from each pig. A pH classification of LM was assigned as follows: class A, > 5.95, n = 186; class B, > or = 5.80 to 5.95, n = 236; class C, > or = 5.65 to 5.80, n = 467; class D, > or = 5.50 to 5.65, n = 441; class E, < 5.50, n = 205. Data were analyzed using a mixed linear model including pH classification, test, sex, halothane genotype, breed, and breed x sex interaction as fixed effects, with sire and dam within breed included as random effects. Correlations were determined within pH class. Lipid content was a significant source of variation for models predicting star probe values in class C and D and for chewiness in class B, C, and D. Increasing lipid content tended to increase sensory tenderness in pH class D. Sensory tenderness was not affected by lipid content in pH class A, B, or E. Lipid content was not a significant source of variation for juiciness scores within any pH class. Intramuscular lipid is correlated with sensory texture traits primarily in classes C and D. Within class C and D, correlations indicate that increasing lipid content is associated with high sensory tenderness, low sensory chewiness, and low star probe values. It is concluded that lipid content is a small source of variation in texture and tenderness of pork loin with pH between 5.80 and 5.50, but not at a greater or lesser pH.

Circulating ghrelin concentrations fluctuate relative to nutritional status and influence feeding behavior in cattle.

The objective of these experiments was to establish the relationship of plasma ghrelin concentrations with feed intake and hormones indicative of nutritional state of cattle. In Exp.1, 4 steers (BW 450 +/- 14.3 kg) were used in a crossover design to compare plasma ghrelin concentrations of feed-deprived steers with those of steers allowed to consume feed and to establish the relationship of plasma ghrelin concentrations with those of GH, insulin (INS), glucose (GLU), and NEFA. After adaptation to a once-daily feed offering (0800), 2 steers continued the once-daily feeding schedule (FED), whereas feed was withheld from the other 2 steers (FAST). Serial blood samples were collected via indwelling jugular catheter from times equivalent to 22 h through 48 h of feed deprivation. Average plasma ghrelin concentrations were greater (P < 0.001) in FAST compared with FED (690 and 123 +/- 6.5 pg/mL) steers. Average plasma ghrelin concentrations for FED steers prefeeding were elevated (P < 0.001) when compared with those postfeeding (174 and 102 +/- 4.2 pg/mL, respectively). Average plasma GH concentration was elevated (P < 0.05) for FAST steers compared with FED steers. Plasma GLU concentrations were not different; however, for FAST steers, NEFA concentrations were elevated (P < 0.001) and INS concentrations were decreased (P < 0.001). In Exp. 2, 4 steers (BW 416 +/- 17.2 kg) were used in a crossover design to determine the effects of i.v. injection of bovine ghrelin (bGR) on plasma GH, INS, GLU, and NEFA concentrations; length of time spent eating; and DMI. Steers were offered feed once daily (0800). Serial blood samples were collected from steers via indwelling jugular catheter. Saline or bGR was injected via jugular catheter at 1200 and 1400. A dosage of 0.08 microg/kg of BW bGR was used to achieve a plasma ghrelin concentration similar to the physiological concentration measured in a FAST steer in Exp. 1 (1,000 pg/mL). Injection of bGR resulted in elevated (P < 0.005) plasma GH concentrations after the 1200 but not the 1400 injection. Plasma INS, GLU, and NEFA concentrations were not affected by bGR injection. For the combined 1-h periods postinjection, length of time spent eating was greater (P = 0.02) and DMI tended to be increased (P = 0.06) for bGR steers. These data are consistent with the hypothesis that ghrelin serves as a metabolic signal for feed intake or energy balance in ruminants.

Feeding 25-hydroxyvitamin D3 to improve beef tenderness.

The objective of this trial was to determine if a single oral bolus of 25-hydroxyvitamin D3 (25-OH D3) given at various times before slaughter would enhance the tenderness of beef loin steaks. One hundred eight crossbred steers were allotted to 18 pens so that the mean weight of the cattle in each pen was similar. Treatments (25-OH D3 dose [62.5 or 125 mg]) and time of administration of the single oral bolus (4, 7, 21, or 35 d before slaughter) were assigned randomly to each pen of steers. Serial plasma samples were collected at each bolus administration time for control animals. For steers assigned to a treatment group, a baseline blood sample was collected before bolus administration and at each subsequent administration when other treatment groups received their bolus. Plasma samples were assayed for 25-OH D3 and calcium concentrations. Troponin-T degradation and Warner-Bratzler shear force were measured as indicators of tenderness for loin steaks collected at slaughter and aged for 6 or 14 d postmortem. Muscle samples, collected concurrently, were assayed for 25-OH D3 and calcium concentrations. A single oral bolus of 25-OH D3 was sufficient to increase plasma 25-OH D3 concentrations (P < 0.001) through slaughter, regardless of dose or time of bolus administration. The single oral bolus of 25-OH D3, however, did not increase plasma calcium concentrations (P > 0.05). As a result, neither troponin-T degradation nor Warner-Bratzler shear force was improved (P > 0.05) by treatment. Muscle 25-OH D3 concentrations were increased (P > 0.001) by treatment with 25-OH D3. Although sustained plasma 25-OH D3 concentrations did not increase plasma or muscle calcium at slaughter nor influence tenderness, the use of 25-OH D3 as a nutritional means of improving beef tenderness is in its infancy, and more research to delineate an effective dose and the potential interaction of seasonal exposure to ultraviolet light is warranted.

Developmental changes in cholesterol 7alpha- and 27-hydroxylases in the piglet.

Hepatic cholesterol 7alpha-hydroxylase (CYP7A) and sterol 27 hydroxylase activities were measured in fetal, newborn, suckling, and weaned piglets from 76 d into gestation to 49 d of age. Hepatic CYP7A activity was not detected in fetal microsomes, but it increased to 6.8 +/- 2.6 pmol/min x mg(-1) protein in suckling piglets at 21 d of age and to 18.2 +/- 2.5 in weaned piglets at 49 d of age. Hepatic CYP7A activity was not different between 49-d-old piglets weaned at 21 d and piglets suckled for 49 d (18.9 +/- 2.6 and 18.2 +/- 2.5 pmol/min x mg protein, respectively). Fasting for 14 h decreased CYP7A activity by 86% in both suckled and weaned piglets. Cholesterol 7alpha-hydroxylase activity remained decreased for at least 5 h after refeeding. Sterol 27-hydroxylase activity was also undetectable near birth, but was detectable by 21 d of age. Postnatally, sterol 27-hydroxylase activity was not influenced by age or suckling and weaning, as was CYP7A. Sterol 27-hydroxylase was decreased by 80% in piglets deprived of feed compared with piglets given free access. In contrast to CYP7A activity, 27-hydroxylase activity returned within 5 h after refeeding to levels observed in piglets given ad libitum access to feed. Similar to CYP7A enzyme activity, hepatic CYP7A mRNA was not detected in newborn piglets, but increased from 2.7 +/- 1.7 pg mRNA/microg RNA in suckling piglets at 21 d to 13.7 +/- 1.2 in 49-d-old piglets weaned at 21 d. As with enzyme activity, feed deprivation decreased CYP7A mRNA to barely detectable levels (< .5 pg/microg RNA), and which remained decreased for at least 5 h following refeeding (.6 +/- .3 and 2.67 +/- .4 pg mRNA/microg RNA for suckled and weaned piglets, respectively). In piglets allowed free access to feed, CYP7A mRNA concentrations were associated positively (P = .001) with enzyme activity. These results suggest that developmental regulation of CYP7A activity is the result of a pretranslational mechanism.

Resveratrol promotes atherosclerosis in hypercholesterolemic rabbits.

The hypothesis was tested that resveratrol, a compound in red wine, would inhibit atherosclerotic development in rabbits fed 0.5% cholesterol for 60 days. Rabbits were supplemented with or without oral resveratrol. During the study, body weights and food consumption were similar for the two groups. The lack of differences between liver weights and a series of serum parameters indicative of liver disease suggest that liver function was similar in the two groups. The diet produced hypercholesterolemia in both groups, but no differences in lipoprotein-cholesterol concentrations. The electrophoretic mobility of plasma low-density lipoprotein (LDL) and plasma LDL after induced oxidation also was not different between the groups. Staining of atherosclerotic lesions in the control and resveratrol-treated groups revealed that the resveratrol-treated rabbits had significantly more aortic surface area covered by atherosclerotic lesions (P < 0.02). Therefore, resveratrol promoted atherosclerotic development, rather than protect against it, by a mechanism that is independent of observed differences in gross animal health, liver function, plasma cholesterol concentrations, or LDL oxidative status.

Ectopic Overexpression of Asparagine Synthetase in Transgenic Tobacco.

Here, we monitor the effects of ectopic overexpression of genes for pea asparagine synthetase (AS1) in transgenic tobacco (Nicotiana tabacum). The AS genes of pea and tobacco are normally expressed only during the dark phase of the diurnal growth cycle and specifically in phloem cells. A hybrid gene was constructed in which a pea AS1 cDNA was fused to the cauliflower mosaic virus 35S promoter. The 35S-AS1 gene was therefore ectopically expressed in all cell types in transgenic tobacco and constitutively expressed at high levels in both the light and the dark. Northern analysis demonstrated that the 35S-AS1 transgene was constitutively expressed at high levels in leaves of several independent transformants. Furthermore, amino acid analysis revealed a 10- to 100-fold increase in free asparagine in leaves of transgenic 35S-AS1 plants (construct z127) compared with controls. Plant growth analyses showed increases (although statistically insignificant) in growth phenotype during the vegetative stage of growth in 35S-AS1 transgenic lines. The 35S-AS1 construct was further modified by deletion of the glutamine-binding domain of the enzyme (gln[delta]AS1; construct z167). By analogy to animal AS, we reasoned that inhibition of glutamine-dependent AS activity might enhance the ammonia-dependent AS activity. The 3- to 19-fold increase in asparagine levels in the transgenic plants expressing gln[delta]AS1 compared with wild type suggests that the novel AS holoenzyme present in the transgenic plants (gln[delta]AS1 homodimer) has enhanced ammonia-dependent activity. These data indicate that manipulation of AS expression in transgenic plants causes an increase in nitrogen assimilation into asparagine, which in turn produces effects on plant growth and asparagine biosynthesis.

Modulation of glutamine synthetase gene expression in tobacco by the introduction of an alfalfa glutamine synthetase gene in sense and antisense orientation: molecular and biochemical analysis.

A glutamine synthetase (GS) cDNA isolated from an alfalfa cell culture cDNA library was found to represent a cytoplasmic GS. The full-length alfalfa GS1 coding sequence, in both sense and antisense orientation and under the transcriptional control of the cauliflower mosaic virus 35S promoter, was introduced into tobacco. Leaves of tobacco plants transformed with the sense construct contained greatly elevated levels of GS transcript and GS polypeptide which assembled into active enzyme. Leaves of the plants transformed with the antisense GS1 construct showed a significant decrease in the level of both GS1 and GS2 polypeptides and GS activity, but did not show any significant decrease in the level of endogenous GS mRNA. We have proposed that antisense inhibition using a heterologous antisense GS RNA occurs at the level of translation. Our results also suggest that the post-translational assembly of GS subunits into a holoenzyme requires an additional factor(s) and is under regulatory control.

Oats Tolerant of Pseudomonas syringae pv. tabaci Contain Tabtoxinine-beta-Lactam-Insensitive Leaf Glutamine Synthetases.

Pseudomonas syringae pv. tabaci, a commonly recognized leaf pathogen of tobacco, can infest the rhizosphere of many plants, including oats. Normal oat plants do not survive this infestation as a consequence of the complete and irreversible inactivation of all of their glutamine synthetases by tabtoxinine-beta-lactam (TbetaL), a toxin released by pv. tabaci. We have identified a population of oat (Avena sativa L. var Lodi) plants that are tolerant of pv. tabaci. The tolerant plants had no detectable TbetaL-detoxification mechanisms. Pathogen growth on these plant roots was not inhibited. These plants contain leaf glutamine synthetases (GS(1) and GS(2)) that were less sensitive to inactivation by TbetaL in vitro; these GSs have normal K(m) values for glutamate and ATP when compared with those of GS in control plants. Root glutamine synthetase of the tolerant plants was inactivated in vivo during infestation by the pathogen or by TbetaL in vitro. When growing without pv. tabaci, the tolerant plants contained normal levels of glutamine synthetase in their roots and leaves and normal levels of protein, ammonia, glutamate, and glutamine in their leaves. However, when the tolerant plants' rhizosphere was infested with pv. tabaci, the plant leaves contained elevated levels of glutamine synthetase activity, protein, ammonia, glutamate, and glutamine. No changes in glutamate dehydrogenase activity were detected in leaves and roots of pathogen-infested tolerant plants.

Enhancement of symbiotic dinitrogen fixation by a toxin-releasing plant pathogen.

An approximate doubling in plant growth, total plant nitrogen, nodulation, and overall dinitrogen fixation of alfalfa are the consequences of the action of a toxin delivered by a Pseudomonas infesting the alfalfa rhizosphere. The toxin, tabtoxinine-beta-lactam, inactivates selectively one form of glutamine synthetase in the nodules. Thus, normal glutamine synthetase-catalyzed ammonia assimilation is significantly impaired; yet these plants assimilated about twice the normal amount of nitrogen. How plants regulate dinitrogen fixing symbiotic associations is an important and unresolved question; the current results imply that the glutamine synthetase-catalyzed step in ammonia assimilation, a plant function, strongly influences overall dinitrogen fixation in legumes.

Adenine nucleotides as allosteric effectors of pea seed glutamine synthetase.

The effects of adenine nucleotides on pea seed glutamine synthetase (EC 6.3.1.2) activity were examined as a part of our investigation of the regulation of this octameric plant enzyme. Saturation curves for glutamine synthetase activity versus ATP with ADP as the changing fixed inhibitor were not hyperbolic; greater apparent Vmax values were observed in the presence of added ADP than the Vmax observed in the absence of ADP. Hill plots of data with ADP present curved upward and crossed the plot with no added ADP. The stoichiometry of adenine nucleotide binding to glutamine synthetase was examined. Two molecules of [gamma-32P]ATP were bound per subunit in the presence of methionine sulfoximine. These ATP molecules were bound at an allosteric site and at the active site. One molecule of either [gamma-32P]ATP or [14C]ADP bound per subunit in the absence of methionine sulfoximine; this nucleotide was bound at an allosteric site. ADP and ATP compete for binding at the allosteric site, although ADP was preferred. ADP binding to the allosteric site proceeded in two kinetic phases. A Vmax value of 1.55 units/mg was measured for glutamine synthetase with one ADP tightly bound per enzyme subunit; a Vmax value of 0.8 unit/mg was measured for enzyme with no adenine nucleotide bound at the allosteric site. The enzyme activation caused by the binding of ADP to the allosteric sites was preceded by a lag phase, the length of which was dependent on the ADP concentration. Enzyme incubated in 10 mM ADP bound approximately 4 mol of ADP/mol of native enzyme before activation was observed; the activation was complete when 7-8 mol of ADP were bound per mol of the octameric, native enzyme. The Km for ATP (2 mM) was not changed by ADP binding to the allosteric sites. ADP was a simple competitive inhibitor (Ki = 0.05 mM) of ATP for glutamine synthetase with eight molecules of ADP tightly bound to the allosteric sites of the octamer. Binding of ATP to the allosteric sites led to marked inhibition.

Self-protection of Pseudomonas syringae pv. "tabaci" from its toxin, tabtoxinine-beta-lactam.

An extracellular toxin, tabtoxinine-beta-lactam (T beta L), is produced by Pseudomonas syringae pv. "tabaci." This toxin irreversibly inhibits its target, glutamine synthetase; yet P. syringae pv. "tabaci" retains significant amounts of glutamine synthetase activity during toxin production in culture. As part of our investigation of the self-protection of P. syringae pv. "tabaci," we compared the effects of T beta L on Tox+ (T beta L-producing, insensitive to T beta L) and Tox- (T beta L nonproducing, sensitive to T beta L) strains. The extent of protection afforded to the Tox- strain when induced to adenylylate glutamine synthetase was tested. We concluded that an additional protection mechanism was required. A detoxification activity was found in the Tox+ strain which opens the beta-lactam ring of T beta L to produce the inactive, open-chain form, tabtoxinine. Whole cells of the Tox+ strain incubated for 24 h with [14C]T beta L (0.276 mumol/3 X 10(10) cells) contained [14C]tabtoxinine (0.056 mumol), and the medium contained T beta L (0.226 mumol). Extracts of spheroplasts of the Tox+ stain also converted T beta L to tabtoxinine, whereas extracts of the Tox- strain did not alter T beta L. The conversion was time dependent and stoichiometric and was destroyed by boiling for 30 min or by the addition of 5 mM EDTA. Penicillin, a possible substrate and competitive inhibitor of this lactamase activity, inhibited the conversion of T beta L to tabtoxinine. Periplasmic fluid did not catalyze the conversion of T beta L.

Inactivation of pea seed glutamine synthetase by the toxin, tabtoxinine-beta-lactam.

Glutamine synthetase of plants is the physiological target of tabtoxinine-beta-lactam, a toxin produced by several disease-causing pathovars of Pseudomonas syringae. This toxin, a unique amino acid, is an active site-directed, irreversible inhibitor of glutamine synthetase from pea. ATP is required for inactivation. Neither ADP, AMP, nor adenosine 5'-(beta,gamma-methylene)triphosphate (AMP-PCP) supports inactivation. Adenyl-5'-yl imidophosphate (AMP-PNP) is slowly hydrolyzed by glutamine synthetase to produce adenyl-5'-yl phosphoramidate (AMP-PN) and inorganic phosphate as identified by 31P NMR spectroscopic analysis. AMP-PNP also supports a slow inactivation of glutamine synthetase by tabtoxinine-beta-lactam. These data are consistent with gamma-phosphate transfer being involved in the inactivation. Completely inactivated glutamine synthetase has 0.9 mumol of toxin bound/mumol of subunit. One mumol of ATP is bound per mumol of subunit of glutamine synthetase in the absence of either the toxin or another active site-directed inhibitor, methionine sulfoximine; whereas, a 2nd mumol of either [alpha- or gamma-32P]ATP is bound per mumol of subunit when glutamine synthetase is incubated in the presence of either toxin or methionine sulfoximine until all enzyme activity is lost. These data suggest that the gamma-phosphate hydrolyzed from ATP during inactivation remains with the enzyme-inhibitor complex, as well as the ADP. The open chain form, tabtoxinine, was neither a reversible nor an irreversible inhibitor of glutamine synthetase, suggesting that the beta-lactam ring is necessary for inhibition. The inactivation of glutamine synthetase with tabtoxinine-beta-lactam is pseudo-first-order when done in buffer containing 15% (v/v) ethylene glycol. The rate constant for this reaction is 3 X 10(-2) S-1, and the Ki for the toxin is 1 mM. Removal of the ethylene glycol from the buffer allows the reaction to proceed in a non-first-order manner with the apparent rate constant decreasing with time. As the enzyme is inactivated in these conditions, the binding affinity for the toxin appears to decrease, while the Km observed for glutamate does not change.

Effects of Tabtoxinine-beta-Lactam on Nitrogen Metabolism in Avena sativa L. Roots.

The effects of tabtoxinine-beta-lactam (T-beta-L) on nitrate uptake and glutamine synthetase (GS) and nitrate reductase (NR) activities in roots of Avena sativa seedlings were determined. Seven-day-old oat seedlings placed in a 10 mm KNO(3) and 0.5 mm T-beta-L solution for 24 hours took up T-beta-L and lost approximately 90% of their root GS activity. [(3)H]-T-beta-L taken up by roots of seven-day-old oat seedlings was associated with GS immunoprecipitated from the extract of these roots. Total nitrate uptake and in vivo NR activity were decreased approximately 50% in the T-beta-L treated roots. However, T-beta-L uptake did not affect the induction phases of nitrate uptake or reduction, nor did it inhibit in vitro NR activity. Thus, the decrease in nitrate uptake and reduction is a secondary effect of T-beta-L action. Roots of seven-day-old oat seedlings were inoculated with Pseudomonas syringae pv tabaci (Tox+) and the pathogen population in the rhizosphere was estimated by dilution plate count; 6 x 10(13) bacteria were recovered after 3 days, as compared to the original inoculation with 7 x 10(9) bacteria, indicating a significant growth of the pathogen in the rhizosphere. The bacteria recovered from the rhizosphere caused chlorosis in tobacco leaves and produced T-beta-L in culture; 1 x 10(14) bacteria were recovered from roots of seedlings inoculated with P. syringae pv tabaci (Tox-) using the same inoculation and assay procedure as for the pv tabaci (Tox+). Extracts of surface-sterilized roots previously inoculated with P. syringae pv tabaci (Tox+) did not produce viable bacterial cultures when plated out on a complete medium. Oat seedlings growing in sand culture and inoculated with P. syringae pv tabaci (Tox+) had developed chlorosis, and root GS activity had declined to less than 10% of controls after 3 days. Conversely, seedlings inoculated with P. syringae pv tabaci (Tox-) never developed chlorosis and maintained normal levels of GS activity. All oat plants inoculated with P. syringae pv tabaci (Tox+) died within 7 days after inoculation as compared to the plants inoculated with P. syringae pv tabaci (Tox-) which grew to maturity.

Role of glutamine synthetase adenylylation in the self-protection of Pseudomonas syringae subsp. "tabaci" from its toxin, tabtoxinine-beta-lactam.

Selected pathovars of Pseudomonas syringae produce an extracellular phytotoxin, tabtoxinine-beta-lactam, that irreversibly inhibits its known physiological target, glutamine synthetase (GS). Pseudomonas syringae subsp. "tabaci" retains significant amounts of glutamine synthetase activity during toxin production in culture. As part of our investigation of the self-protection mechanism(s) used by these pathovars, we have determined that GS becomes adenylylated after toxin production is initiated and that the serine released from the zinc-activated hydrolysis of tabtoxin is a factor in the initiation of this adenylylation. The adenylylation state of this GS was estimated to range from E5.0-7.5. The irreversible inactivation by tabtoxinine-beta-lactam of unadenylylated and adenylylated glutamine synthetase purified from P. syringae subsp. "tabaci" was investigated. Adenylylated GS was inactivated by tabtoxinine-beta-lactam at a slower rate than was unadenylylated enzyme. Adenylylated GS (E7.5-10.5) was significantly protected from this inactivation in the presence of the enzyme effectors, AMP, Ala, Gly, His, and Ser. Thus, the combination of the adenylylation of GS after toxin production is initiated and the presence of the enzyme effectors in vivo could provide part of the self-protection mechanism used by subsp. "tabaci".

Metabolism of acridine by rat-liver enzymes.

Metabolism of acridine by S10 fractions from control adult male Sprague-Dawley rats produces mainly 9- acridone , apparently catalyzed by aldehyde oxidase ( ED1 .2.3.1). In contrast, the predominant metabolic product produced by the corresponding S10 fraction of PCB-induced liver enzymes is a dihydrodiol (either the 2,3- or 3,4-isomer) presumably derived from an epoxide. Several minor metabolites of unknown structure are also formed. During in vitro reactions aldehyde oxidase requires neither atmospheric oxygen nor NADPH. Acridine has been reported to be mutagenic to Salmonella typhimurium, but only in the absence of PCB-induced activating enzymes. It also has been reported to produce chromosomal aberrations in cultured Chinese hamster cells both with and without enzymatic activation. While a connection between aldehyde oxidase catalysis and mutagenic action of acridine has not been established, the extensive metabolic potential of this compound implies that complete description of mutagenicity will be difficult.

Rhythms in glutamine synthetase activity, energy charge, and glutamine in sunflower roots.

Roots of sunflower plants (Helianthus annuus L. var. Mammoth Russian) subjected to L12:D12, L18:D6, and L12:D12 followed by continuous light all display rhythms of about 12 hours for glutamine synthetase (GS) activity (transferase reaction) with one peak in the ;light phase' and one in the ;dark phase.' Root energy charge (EC = ATP+(1/2)ADP/ATP+ADP+AMP) is directly correlated with GS, but the GS rhythm is better explained as the result of a rhythmic adenine nucleotide ratio (ATP/ADP+AMP) that regulates enzyme activity through allosteric modification. When L12:D12 plants are subjected to free-running conditions in continuous darkness, only diurnal rhythms for GS and EC, with peaks in the dark phase, remain. The 12-hour root rhythms for GS and EC appear to be composed of two alternating rhythms, one a diurnal, light-dependent, incompletely circadian light phase rhythm and the other a light-independent, circadian dark phase rhythm.Only glutamine, of the root amino acids, displays cyclical changes in concentration, maintaining under all conditions a 12-hour rhythm that is consistently synchronized with, but nearly always inversely correlated with, GS and EC rhythms.