Glutamine Synthetase of Rat Cerebral Cortex: Intracellular Distribution and Structural Latency

Oz, Sellinger; Fd, Verster

doi:10.1016/s0021-9258(18)60237-6

Cited by 119 publications

(6 citation statements)

References 42 publications

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“…The RNA content of the microsomal pellets isolated by centrifugation in water (Table III) was intermediate between the values reported by Toschi (7) and those reported more recently by Wherrett and McIlwain (9). The calculated ratio (RNA)/(protein + RNA) of 0.11 was considerably higher than the ratio of 0.03 obtained for pellets isolated in sucrose (14). A value of 0.12 was calculated for the ratio RNA/protein, in agreement with values reported by Ernster et al (40) and Dallner et al (41) for this ratio in subfractions isolated by treatment of rat liver microsomes with deoxycholate (40) or by layering techniques (41), but it is higher by about 350 per cent than the corresponding ratio for guinea pig cerebral microsomes isolated in sucrose (9).…”

Section: Composition Of Cerebral Microsomal Pellet Isolated In Watercontrasting

confidence: 71%

“…The soluble marker enzyme, lactic dehydrogenase, was found to be distributed similarly in both centrifugation media. As noted previously (14), glutamine synthetase retained its association with the microsomal membranes to a much greater degree when pellets were isolated in water than when they were isolated in sucrose. The relative specific activity of this enzyme in the pellet isolated in water was 5.4 as compared with 1.4 in the pellet isolated in sucrose.…”

Section: Morphological Findingssupporting

confidence: 66%

“…The differential behavior of two cerebral enzymes, glutamine synthetase and esterase (substrate, 0-nitrophenyl acetate), toward homogenization and centrifugation in increasing concentrations of sucrose has been described previously (14). These studies showed that the maximal degree of attachment of glutamine synthetase to the membranes of the microsomal fraction was obtained when sucrose-free media were used for the isolated of the fraction.…”

mentioning

confidence: 80%

“…Glutamine synthetase was determined as described previously (14). One unit of activity refers to the amount of enzyme required to produce 1 #mole of L-glutamylhydroxamate per hour.…”

Section: Methodsmentioning

confidence: 99%

“…The entire procedure was accomplished in less than 30 seconds. The tissue was homogenized in approximately 4 volumes of medium as previously described (14). The homogenate was centrifuged for 10 minutes at 25,000 g. The resulting sediments (fraction NML) were resuspended in the respective media for isolation and were centrifuged for an additional I0 minutes at 25,000 g. The supernatants (fraction PS) were combined, diluted to give a 1 to 10 suspension, and centrifuged at 145,000 g for 60 minutes to yield a microsomal pellet (fraction P) and a supernatant (fraction S).…”

Section: Methodsmentioning

confidence: 99%

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Morphological and Biochemical Correlates of Cerebral Microsomes

Verster

Sellinger

Harkin

1965

The Journal of Cell Biology

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Microsomal fractions, both homogeneous in appearance and functionally operative, were isolated from a homogenate of rat cerebral cortex by fractionation in water. The preparations thus obtained contain the membranous elements of the endoplasmic reticulum, synaptic vesicles, and ribosomes. Esterase, ATPase, and glutamine synthetase were found to be present and fully functional in the microsomal fractions isolated in water. The contamination of the water-isolated microsomal fractions by mitochondria and lysosomes was found to be considerably lower than in microsomal pellets isolated in sucrose. The contamination by nerve ending particles, as judged by electron microscopy and by the levels of soluble lactic dehydrogenase entrapped in the cytoplasm of the particles, was also low. Most of the contamination by mitochondria and nerve ending particles could be removed by treatment of the microsomal pellet with 150 mM NaC1. Resistant to elution by this treatment is the lysosomal contamination as well as microsomal esterase and ATPase. Glutamine synthetase, on the other hand, was almost totally solubilized. Microsomal preparations isolated in water are also shown to contain amounts of protein, RNA, phospholipid, and ganglioside comparable to those found in microsomal preparations isolated in sucrose.The microsomal fraction isolated from mammalian tissues by high speed centrifugation has not been unequivocally characterized, for it has often been found to consist of disrupted fragments of intracellular materials, including the cellular envelope, that connot readily be compared with, or identified with, intact structures (1, 2). This is particularly true for fractions isolated from brain, owing to the complex and heterogeneous organization of this organ. In contrast to brain is the liver, for instance, from which fractions can be isolated which are much more homogeneous as a eonsequence of the less complex organization of this organ. Palade and Siekevitz (3) have demonstrated that in rat liver the microsomal fraction consists preponderantly of vesicular and tubular fragments of the intracellular endoplasmic reticulum and of ribosomal granules. Hanzon and Toschi (4) described a similar composition for the microsomal fraction of brain tissue. However, more recent studies by de Robertis et al. (5) and by Whittaker (6) have revealed that the microsomal fraction of brain, as usually isolated in sucrose media, consists of a rather heterogeneous popula-69

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Section: Composition Of Cerebral Microsomal Pellet Isolated In Watercontrasting

confidence: 71%

Section: Morphological Findingssupporting

confidence: 66%

mentioning

confidence: 80%

“…Glutamine synthetase was determined as described previously (14). One unit of activity refers to the amount of enzyme required to produce 1 #mole of L-glutamylhydroxamate per hour.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Morphological and Biochemical Correlates of Cerebral Microsomes

Verster

Sellinger

Harkin

1965

The Journal of Cell Biology

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Inhibition of glutamine transport into mitochondria protects astrocytes from ammonia toxicity

Pichili

Rao

Jayakumar

et al. 2007

Glia

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Hepatic encephalopathy (HE) is a major neurological complication that occurs in the setting of severe liver failure. Ammonia is a key neurotoxin implicated in this condition, and astrocytes are the principal neural cells histopathologically and functionally affected. Although the mechanism by which ammonia causes astrocyte dysfunction is incompletely understood, glutamine, a by-product of ammonia metabolism, has been strongly implicated in many of the deleterious effects of ammonia on astrocytes. Inhibiting mitochondrial glutamine hydrolysis in astrocytes mitigates many of the toxic effects of ammonia, suggesting the involvement of mitochondrial glutamine metabolism in the mechanism of ammonia neurotoxicity. To determine whether mitochondriaare indeed the organelle where glutamine exerts its toxic effects, we examined the effect of L-histidine, an inhibitor of mitochondrial glutamine transport, on ammonia-mediated astrocyte defects. Treatment of cultured astrocytes with L-histidine completely blocked or significantly attenuated ammonia-induced reactive oxygen species production, cell swelling, mitochondrial permeability transition, and loss of ATP. These findings implicate mitochondrial glutamine transport in the mechanism of ammonia neurotoxicity.

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The intracellular distribution of glutamine synthetase in rat liver and the effect of metals on its activity

Hsu

Tappel

1964

J. Cell. Comp. Physiol.

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A N D A. L. TAPPEL Differential centrifugation in 0.25 M sucrose of rat brain (Waelsch, '59 and Sellinger and de Balbian Verster, '62) and rat liver (Wu, '63b) indicate that in both tissues almost half of the total glutamine synthetase is associated with the microsomal fraction, with the remaining being distributed among the soluble, nuclear and mitochondrial fractions. In the microsomal fraction, the enzyme is loosely bonded onto the outer surface of the vesicular membranes from which place it can be solubilized with NaCl in concentration lower than that of physiological saline (Wu, '63b) and with EDTA which also has an activating effect (Sellinger and de Balbian Verster, '62 and Wu, '63b). This effect appears to be an indirect one as a two-fold increase of total glutamine synthetase activity occurred only when EDTA was incorporated into the homogenizing medium but not when added to an isolated fraction (Sellinger and de Balbian Verster, '62).In the present study, rat livers were fractionated in 0.25 M sucrose and the intracellular distribution of glutamine synthetase was determined and compared with that of enzymes selected as markers for the various subcellular organelles including lysosomes and uricase particles. The possibility that the activating effect of EDTA was due to removal of inhibitory metals was tested. tase, eight livers were fractionated, yielding six major fractions: a nuclear and cell debris fraction sedimented at 480 X g for 10 min, a mitochondrial fraction at 5000 X g for 10 min, a 'light mitochondrial" fraction at 16,300 X g for 20 min, a "heavy microsomal fraction" at 30,000 X g for 30 min, and a "light microsomal fraction" at 100,000 X g for one hour. The supernatant obtained after the last centrifugation was termed the soluble fraction. To obtain data on the effects of metals on synthetase activity, the cation under study was added to the 0.25 M sucrose suspending medium in which the livers were homogenized an3 fractionated.The activity of glutamine synthetase was determined according to the colorimetric assay outlined by Wu ('63a). The oxidation of alcohol by alcohol dehydrogenase at pH 8.5 was measured by the increase in DPNH at 340 mH. Glutamic dehydrogenase was assayed in buffer of pH 6.8 and 0.01 M in KCN and the amount of DPNH oxidized when glutamic acid is formed from a-keto glutaric acid and ammonia was taken as a measure of enzyme activity. The activity of glucose-6-phosphatase was measured at pH 6.5 with glucose-6-phosphate as substrate. Uricase was assayed at pH 9.0 by measuring the decrease in absorption at 290 mv as uric acid is converted to allantoin. Acid phosphatase, @-glucuronidase and aryl sulfatase were assayed simultaneously at pH 5.0 with an autoanalyzer program using the substrates, phenolphthalein phosphate, phenolphthalein glucuronide and nitrocatechol sulfate, respectively. Protein values were obtained by Miller's method ('59).

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Glutamine Synthetase of Rat Cerebral Cortex: Intracellular Distribution and Structural Latency

Cited by 119 publications

References 42 publications

Morphological and Biochemical Correlates of Cerebral Microsomes

Morphological and Biochemical Correlates of Cerebral Microsomes

Inhibition of glutamine transport into mitochondria protects astrocytes from ammonia toxicity

The intracellular distribution of glutamine synthetase in rat liver and the effect of metals on its activity

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