GS Levels Research Articles

Inorganic nitrogen assimilation in the non‐N2‐fixing cyanobacterium Phormidium laminosum. I. Cellular levels of glutamine synthetase and NADPH‐dependent glutamate dehydrogenase

Cell‐free extracts of nitrate‐grown as well as of ammonium‐grown cells of the filamentous non‐nitrogen‐fixing cyanobacterium Phormidium laminosum (strain OH‐1‐p.Cl1) showed detectable levels of both glutamine synthetase (GS, EC 6.3.1.2) and NADPH‐dependent glutamate dehydrogenase (GDH, EC 1.4.1.4) activities. The GS level of nitrate‐grown cells was higher than that of ammonium‐grown cells, whereas the GDH level was higher in ammonium‐grown cells and depended on the external ammonium concentration. When nitrate‐grown cells were transferred to an ammonium‐containing medium, a decrease of GS and an increase of GDH specific activities occurred, even in the presence of nitrate. Conversely, when ammonia‐grown cells were transferred to a nitrate‐containing medium, an increase of GS and a decrease of GDH‐specific activities took place. Both these effects were inhibited by chloramphenicol and were probably mediated by de novo protein synthesis. When either cell type was transferred to a medium without nitrogen source, the specific activities of both enzymes increased. When nitrate‐grown cells were transferred to nitrate medium with L‐methionine‐DL‐sulphoximine (MSX) added, the specific activity of GDH also increased. Here we present some evidence that, under certain conditions of nitrogen availability, GDH would play a minor role in ammonium assimilation.

Glutamine Synthetase of Streptomyces cattleya: Purification and Regulation of Synthesis

Glutamine synthetase (GS; EC 6.3.1.2) from Streptomyces cattleya was purified using a single affinity-gel chromatography step, and some of its properties were determined. Levels of GS in S. cattleya cells varied by a factor of 8 depending upon the source of nitrogen in the growth medium. Of 24 nitrogen sources examined only glutamine or NH4Cl utilization resulted in very low GS activity. Addition of NH4Cl to a culture with high GS levels appeared to stop further synthesis and resulted in a progressive decrease in the specific activity of the enzyme. The GS inhibitor methionine sulphoximine (MSX) inhibited GS activity but had no effect on exponentially growing cells. The presence of MSX either lengthened or shortened the period between spore inoculation and initiation of exponential growth, depending on the source of nitrogen. In glutamine minimal medium MSX produced earlier and more efficient spore germination while in glutamate or nitrate minimal medium germination was delayed by its presence.

Catabolism and Nitrogen Control in Escherichia coli

It would appear from these studies that nitrogen control reflects the catabolic capacity of the cell and that utilizable nitrogen sources and some carbon sources are, to some extent, in competition for this capacity. The series of catabolic events initiated by addition of D-amino acids or by growth on aldol sugars, in the presence of ammonia nitrogen in the growth medium, provide an opportunity for study of the positive aspect of nitrogen control under conditions where negative control predominates. This approach may eventually clarify the apparent interactions between the modification cascade components, PII and UT/UR, with the nitrogen regulatory gene, glnG. The utilization of nutrients by E. coli seems less a matter of energy than of expeditious use of whatever is offered in the diet. A comparison of the rate of increase of GS on cultural downshift with the rate of increase following D-glutamate addition would suggest that control by nitrogen limitation is about eight times more effective than positive activation by D-glutamate in the presence of ammonia nitrogen. This observation is consistent with the finding of an additive effect for the D-amino acids which can function as positive activators in GS regulation. It has been demonstrated for the wild-type organism that the increase in GS level generated by a mixture of D-glutamate, D-lysine, D-threonine, and glycine approximates the increase in GS level observed during step-down of the culture from an ammonia-sufficient to an ammonia-limited condition. This observation further supports the physiologic relevance of the effect of D-amino acids in nitrogen control and suggests that the apparent derepression of GS observed upon exhaustion of the ammonia nitrogen supply represents a composite of positive activation generated as alternative catabolic functions assume a greater importance. As might be expected, addition of D-glutamate to cells at the point of ammonia exhaustion had no additional positive effect. Following a downshift from glucose-ammonia-glutamine to glucose-glutamine cultural conditions, only the level of GS increases during the initial 60-minute observation period. This finding suggests that glutamine catabolism may, like D-threonine, D-lysine, and glycine, bypass the positive activation of GDH and GAT controls. The likely possibility in that the increases observed for GAT and GDH depend on D-glutamate as specific inducer. There are several instances where D-amino acids function as inducers of L-amino acid dehydrogenases and where amino acid racemase activity is directly coupled to flavoprotein dehydrogenases.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenylylation of Bacterial Glutamine Synthetase: Physiological Significance

This chapter focuses on the adenylylation of bacterial glutamine synthetase. The enzyme glutamine synthetase catalyzes the synthesis of glutamine from ammonia and glutamate in an ATP-dependent reaction. In bacteria and higher plants, it also participates in net synthesis of glutamate from ammonia and 2-oxoglutarate by functioning in a cycle with glutamate synthase. Physiological studies of Hölzer and colleagues indicated that the GS of Escherichia coli was very rapidly adenylylated when the organism was shifted from N-limited medium to a medium containing excess ammonium. Strains of S. typhimurium (glnE) that lack the ability to adenlylate GS show a large growth defect upon shift from N-limited media, media in which they have accumulated high levels of GS, to media containing a high concentration of NH 4 +. This growth defect appears to be because of excessive GS activity after the shift because it is eliminated in glnE strains that also synthesize abnormally low amounts of GS in both N-limited and NH4+, it is prolonged in glnE strains that also synthesize abnormally high amounts of GS in NH4+ excess media and the latter strains continue to show a growth defect even after adaptation to NH 4 +, and glnE strains have normal amounts of both of the glutamatebiosynthetic enzymes under N-limited conditions and after NH 4 + upshift. At present, there appears to be no simple explanation for the distribution of this covalent modification. Although adenylylation of GS occurs widely among gram-negative bacteria, a more complete understanding of the physiological requirements for adenylylation of GS will contribute to understanding the basis for distribution of this covalent modification among the prokaryotes.

Randomized response estimates of problem use of alcohol among employed females.

As part of the evaluation of an alcoholism orientation program conducted in 12 federal agencies, 378 female employees were asked to provide personal information about problems associated with their use of alcohol. The optimized form of the unrelated question randomized response technique (RRT) was used to provide for a comparison of estimates of frequency of problem drinking obtained with guaranteed confidentiality of response versus estimates obtained using a conventional anonymous questionnaire. The estimated proportion of alcoholics or possible alcoholics among participants in the orientation program was 34.3% (+/- 4.9) by the RRT and 21.9% (+/- 3.6) by direct response. Significant underreporting of alcoholism was found among older respondents (greater than or equal to 36 years), lower GS level respondents (less than grade 6), those with low seniority (less than 8 years), and those never married. Further, significant underreporting was found among those who claimed they were told they had to attend the program versus others, and among those who claimed they were not curious about the topic of alcoholism versus others. The RRT is recommended for use in experimental situations where answers to sensitive questions are needed to evaluate program success. The necessity for caution in interpretation of apparent differences in respondent characteristics with respect to problems with alcohol use is discussed. Finally, the findings indicate the importance of providing avenues by which confidential treatment for alcoholism can be obtained.

Inter-Relationships Between Organizational Commitment and Job Characteristics, Job Satisfaction, Professional Behavior, and Organizational Climate

Commitment to the organization is an important behavioral dimension which can be utilized to evaluate employees' strength of attachment. Keeping employees highly committed is important, especially in not-forprofit firms whose salary scales may not be as competitive as industrial firms. Management is concerned with identifying those variables that are related to organizational commitment in order that they may design organizational strategies to maximize commitment levels. Results in a healthcare institution indicate that role conflict and role ambiguity are detrimental to commitment, while a participative climate, power, teamwork, reading professional journals, satisfaction with work and promotion opportunities, age, GS level, tenure, and length of professional employment are positively related to organizational commitment.

Glutamine synthetase activity in the developing secondary palate and induction by dexamethasone

Glutamine synthetase (EC 6.3.1.2) (GS) and glutamyltransferase (EC 2.3.2.1) (GT) specific activity were examined in developing A/Jax and C57BL/6J (C57) mouse fetal secondary palates. In addition, the induction of palatal GS was also examined after maternal injection of dexamethasone. Palatal GT activity was uniformly higher in A/J than C57 palates with both strains showing highest activity late on day 13 of gestation and a drop in activity by early day 14. In contrast, A/J palatal GS activity peaked transiently late on day 13, dropped by early day 14 and remained lower throughout the remaining period of palatal development. Palatal GS activity in C57 mouse fetuses, although failing to show a discrete transient peak of activity, remained at a constant elevated level from early day 13 to late day 14 and did not decrease until day 15 of gestation. These elevated levels of palatal GS and GT activity correspond to the gestation period of maximal palatal glycoconjugate biosynthesis. Thus, palatal GS activity may play an important regulatory role in the synthesis of these macromolecules. A/J and C57BL/6J mice exhibit different susceptibilities to glucocorticoid-induced cleft palate. However, maternal administration of a non-teratogenic dose of dexamethasone on either late day 12 or late day 13 resulted in a dramatic stimulation of both A/J and C57 fetal palatal GS but not GT activity when assay 18 h later. A/J palatal tissue responded to dexamethasone with greater induction of palatal GS activity than enzyme activity in C57 palates. Palatal GS, sensitive to glucocorticoid stimulation, may thus be an important link in expressing hormonal control of normal palatal differentiation.

Regulation of glutamine synthetase activity by adenylylation in the Gram-positive bacterium Streptomyces cattleya.

The enzymatic activity of glutamine synthetase [GS; L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] from the Gram-positive bacterium Streptomyces cattleya is regulated by covalent modification. In whole cells containing high levels of GS the addition of ammonium chloride leads to a rapid decline in GS activity. Crude extracts prepared from such ammonia-shocked cells had very low levels of GS activity as measured by biosynthetic and gamma-glutamyltransferase assays. Incubation of the crude extracts with snake venom phosphodiesterase restored GS activity. In cell extracts, GS was also inactivated by an ATP- and glutamine-dependent reaction. Radioactive labeling studies demonstrated the incorporation of an AmP moiety into GS protein upon modification. Our results suggest a covalent modification of GS in a Gram-positive bacterium. This modification appears to be adenylylation of the GS subunit similar to that found in the Gram-negative bacteria.

Regulation of Glutamine Synthetase Level in Isolated Pea Roots. II. The Effect of Exogenously Supplied Acids and Bases in Sugar-supplied and Sugar-starved Roots

Summary Exogenously supplied inorganic acids depress GS level in isolated pea roots cultivated in media containing sucrose but do not enhance the decrease in GS level caused by sugar starvation. The negative influence in sugar-supplied roots is not proportional to acid concentration. Bases incl. NH 4 OH counteract the negative effect of acids in sugar-supplied roots as well as the negative effect of NH 4 Cl. When applied alone, NH 4 OH does not influences GS level in sugar-supplied roots differently from KOH with the exception cf low concentrations, however in sugar-starved roots KOH does not affect GS level whereas NH 4 OH enhances the decrease caused by lack of sugar. There is no substantial difference between the effects of NH 4 Cl and (NH 4 ) 2 HPO 4 in sugar-starved roots; both salts significantly enhance the decrease in GS level with the depressing effect being accompanied by a decrease in the content of reducing sugars. These results confirm our earlier finding that the negative effect of ammonium salts of strong inorganic acids is in sugar-supplied roots mediated by increased H + concentration in the cells which counteracts the positive effect of sugars and simultaneously suggest that in sugar starved roots ammonium salts and ammonia enhance the decrease in GS level by enhaning sugar consumption. The results also indicate that some unknown factor(s) with positive effects on GS may be released in pea roots by increased H + concentration.

Glutamine synthetase in the avian diencephalon.

AbstractGlutamine synthetase (GS) activities are high in the nucleus rotundus of the avian diencephalon in late embryos and hatched chicks. The nucleus rotundus is known to be one component of a visual pathway in birds involving also the neural retina, the optic tectum and the cerebral ectostriatum. Since previous studies have shown that the retina, tectum and ectostriatum have high GS activities as well, the enzyme is considered to be clearly associated in some measure, although not exclusively, with processes subserving vision. Some of the possible normal functions for GS at specific neural sites are discussed.Hydrocortisone promotes GS levels in vitro in the embryonic diencephalon, just as GS activities are increased with this steroid in cultures of embryonic retina, tectum and cerebrum. The effects of steroid on GS in the diencephalon appear to be general and not restricted to the nucleus rotundus or the visual pathway.