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At least 19 recordsLinked to original sources
PubMed1977

Purification and some characteristics of liver cytosol cornin, an antimitotic substance from rat liver cytosol.

Further purification and characterization are reported on rat cytosol cornin (RLCC), an antimitotic substance. Fraction I (purified RLCC) was purified more than 10-fold from crude RLCC with Sephadex G-50 column chromatography and showed a remarkable inhibitory effect on division of inseminated sea urchin eggs and mouse fibroblast cells. Fraction I was observed as one spot, and the molecular weight was estimated to be about 25,000 by thin layer gel filtration. Fraction I contained protein (92%) and RNA (8%), but the antimitotic activity was scarcely affected by treatment by pancreatic RNase. The protein of Fraction I was separated into two bands by SDS-polyacrylamide gel electrophoresis, and the molecular weight was estimated as 10,000 and 15,000, respectively. The 50% inhibition dose of Fraction I on the first division of inseminated sea urchin eggs and on proliferation of mouse L cells was about 2.5 X 10(-5) g/ml and 5 X 10(-4) g/ml, respectively. The yield of fraction I was about 35 mg from 100 g rat liver.

Amino Acids↗
PubMed1978

Activation of epinephrine and glucagon-sensitive adenylate cyclases of rat liver by cytosol protein factors. Role in loss of enzyme activities during preparation of particulate fractions, quantitation and partial characterization.

The role of cytosol components in the loss of rat liver adenylate cyclase activity which occurs during the preparation of particulate fractions from crude homogenates was studied. Epinephrine (5 micron)-, glucagon (10 micron)-, and fluoride (5 mM)- stimulated activities of twice-washed particulates were 31%, 58% and 67% of the homogenate activities, respectively. Addition of cytosol (100,000 X g supernatant devoid of adenylate cyclase activity) restored these activities to 82%, 88% and 80%. Cytosol also increased particulate basal activity from 60% of homogenate activity to 98%. The cytosol components capable of increasing adenylate cyclase activity were heat labile, nondialyzable, stable to freezing at -20 degrees, resistant to change of pH between 2 and 12, and unaffected by EGTA and NAD. Pretreatment with pepsin destroyed the effects of cytosol on both epinephrine- and glucagon-sensitive activities, whereas trypsin destroyed the effect of cytosol only on epinephrine-sensitive activity. The cytosol effect on adenylate cyclase was specific, since several purified proteins and ubiquitin, did not stimulate enzyme activity. Only part of the cytosol effect could be attributed to its GTP content. GTP at the concentration present in cytosol stimulated epinephrine-sensitive activity but significantly less than did cytosol, while GTP had no effect on glucagon-sensitive activity. Dialyzed cytosol retained its effectiveness even after removal of most (97%) of its GTP to a concentration where GTP had only a minimal effect on epinephrine-sensitive activity. Cytosol, unlike GTP, stimulated rather than inhibited activation by fluoride. Cytosol thus appears to contain at least two different protein components, which increase the activity of the two hormone-sensitive adenylate cyclases and presumably account in part for losses of adenylate cyclase activities seen during the preparation of particulates from homogenates.

Adenylyl Cyclases↗
PubMed1978

Subcellular metabolite concentrations. Dependence of mitochondrial and cytosolic ATP systems on the metabolic state of perfused rat liver.

Mitochondrial and cytosolic contents of adenine nucleotides and phosphate were measured in perfused rat livers employing a technique of fractionation of freeze-fixated tissue in non-aqueous solvents. From the subcellular contents the mitochondrial and cytosolic concentrations of ATP, ADP, AMP and phosphate and the phosphorylation potentials of the subcellular ATP systems were calculated. An attempt was made to elucidate the relationship between mitochondrial and cytosolic adenine nucleotide systems and the dependency on the metabolic state of the liver. The following results were obtained: 1. Under all metabolic conditions studied the mitochondrial ATP/ADP ratios were considerably lower than the cytosolic ratios (mitochondria: 0.1-0.7; cytosol: 2-11). 2. The ATP/ADP ratios calculated from overall tissue contents reflect mainly the cytosolic ratios. 3. An inverse relationship was found between mitochondrial and cytosolic ATP/ADP ratios, i.e. when the mitochondrial ratios tended to increase, the cytosolic ratios decreased and vice versa. 4. The phosphorylation potentials calculated from the subcellular concentrations were higher in the cytosol than in the mitochondria. The potential difference varied between 11 and 3 kj/mol in livers from fed and starved rats, respectively. 5. In the presence of mitochondrial inhibitors, i.e. amytal, dinitrophenol and carboxyatractyloside, the potential difference between the subcellular ATP systems decreased predominantly due to an increase in the mitochondrial ATP/ADP ratios. 6. A correlation between mitochondrial ATP/ADP ratios and the respiratory rates was not observed, but the subcellular ratios appeared to correlate with the rate of glycolysis. When the rate of lactate + pyruvate production was increased, the cytosolic ATP/ADP ratios were increased, too, whereas the mitochondrial ratios tended to decrease. 7. The adenine nucleotides in the cytosol appear to be in near equilibrium catalysed by the adenylate kinase. In the mitochondria, the AMP concentration is much lower than to be expected under equilibrium conditions. These results were discussed with respect to rate control of processes involved in ATP generation, i.e. oxidative phosphorylation, adenine nucleotide translocation and glycolysis.

Adenosine Diphosphate↗
PubMed1979

Lipid requirements for the aggregation of CTP:phosphocholine cytidylyltransferase in rat liver cytosol.

Two forms of CTP:phosphocholine cytidylyltransferase were identified in rat liver cytosol by gel filtration chromatography. The low molecular weight form (L form) is the major form in fresh cytosol. The enzyme associates into a high molecular weight form (H form) upon storage of the cytosol at 4 degrees C. Aggregation of the purified L form of cytidylyltransferase is caused by total rat liver lipids, neutral lipids, diacylglycerol, or phosphatidylglycerol. Diacylglycerol was the only lipid isolated from the rat liver that caused aggregation of the purified enzyme. Although the addition of diacylglycerol to the cytosol did not change the amount of aggregation of the enzyme, a 2.5-fold increase in H form was observed in cytosol pretreated with phospholipase C, or in cytosol from rats fed a high cholesterol diet. In both of these cytosolic preparations, the concentration of diacylglycerol was elevated twofold. Phosphatidylglycerol did not seem to affect the association of the enzyme in cytosol since it is present in very low concentrations in the rat liver cytosol, and its degradation in cytosol by a specific phospholipase did not affect the rate of aggregation. The results suggest that diacylglycerol in an appropriate form is required for association of cytidylyltransferase in rat liver cytosol.

Animals↗
PubMed1979

A comparison of the glucocorticoid receptor in cytosol from rat liver and hippocampus.

The [3H]corticosterone- and [3H]dexamethasone-binding proteins in cytosol from liver and hippocampus of the rat were compared by isoelectric focusing analysis in slabs of polyacrylamide gel. A single peak of radioactivity with a pI of 6.1--6.2 was obtained during analysis of cytosol from both liver and hippocampus using either corticosterone or dexamethasone as radiolabelled ligand, provided the tissue was carefully perfused with buffer prior to preparation of cytosol. Rat serum or insufficiently perfused tissue contained a corticosterone-binding component with pI of 5.2--5.5 representing corticosteroid-binding globulin. Limited trypsin digestion resulted in fragmentation of the dexamethasone- and corticosterone-binding protein in cytosol from liver and hippocampus. Incubation of radiolabelled cytosol with 0.5 microgram of trypsin/A280--310nm of cytosol gave a sharp radioactive peak with a pI of 5.9--6.1 when analyzed by isoelectric focusing; when 5.0 microgram of trypsin/A280--310nm of cytosol was used, a double peak with pI values of 5.9--6.1 and 6.3--6.5, respectively, was seen. The same trypsin-induced peaks were seen with both [3H]dexamethasone and [3H]corticosterone as ligands. The substrate specificity and the sensitivity of this glucocorticoid binder for limited trypsin digestion is in good agreement with what was previously found for the glucocorticoid receptor in rat liver cytosol. It is concluded that cytosol from liver and hippocampus contains an identical or very similar receptor for glucocorticoid hormones.

Animals↗
PubMed1975

[Interaction of norethindrone on estrogen and progesterone receptors in the rabbit uterine cytosol (author's transl)].

Norethindrone (ENT), which is a representative in estrane series of progestogen, is not only strongly progestational but also estrogenic and in some cases, antiestrogenic. To understand progestational effect and antiestrogenic effect, the interactions of ENT on estrogen and progestogen receptors were studied in the uterine cytosol of white female rabbit. The 274,200 X G supernatant of uterine homogenate was used as cytosol. 3H-Estradiol, 3H-Progesterone, 3H-ENT or cold ENT were incubated with uterine cytosol at 4 degrees C for 2 hours. Results are as follows: 1. Sucrose gradient centrifugation [5 approximately 20% linear and 40,000 rpm (159,200 X G) for 16 hours at 4 degrees C]: ENT was bound to extrogen 8S receptor in immature rabbit uterus (Fig. 2 & 3), and to progestogen 8S receptor in estrogen primed rabbit uterus (Fig. 5). 2. Kinetic study, determined by dextran coated charcoal (0.001% dextran and 0.1% charcoal): (1) In the uterine cytosol of immature rabbit, 3H-estradiol-receptor binding was observed with Kd divide by 3.6 X 10-9 M and it was revealed that ENT was a competitive inhibitor to this binding with Ki divide by 2.6 X 10-6 M, as in Fig. 6. (2) 8S component, obtained by centrifugation of uterine cytosol (Fig. 1) in estrogen primed rabbit, binds 3H-progesterone with Kd divide by 8.1 X 10-10 M and Bm (maximal binding sites) divide by 5.0 X 10-8 M/mg of protein, and ENT was a competitive inhibitor in this binding with Ki divide by 2.3 X 10-9 M (FIG. 7 & 8). 3H-ENT-8S binding was demonstrated with Kd divide by 1.1 X 10-9 M and Bm divide by 8.7 X 10-8 M/mg of cytosol protein (Fig. 8). These results indicate: (a) ENT is bound to both estrogen and progestogen receptors in 8S macromolecules of uterine cytosol, (b) competitive inhibition of ENT to these bindings indicated that ENT is bound to these receptors at the steroid binding sites where estradiol and progesterone bind to, (c) ENT has much more affinity to progestogen receptor (Ki divide by 2.3 X 10-9 M) than to estrogen receptor (Ki divide 2.6 X 10-6 M), (d) while ENT is bound to progestogen and estrogen receptors at the same time, Bm of ENT (8.7 X 10-8 M/mg of cytosol protein) is more than Bm of progesterone (5.0 X 10-9 M/mg of cytosol protein), and Kd of ENT (1.1 X 10-9 M) was less than Ki of ENT (2.3 X 10-9 M) in the binding to progesterone-receptor. Biologically, while ENT is bound to progestogen -receptor with high affinity and to estrogen receptor with low affinity, ENT is actually progestational in low dose and antiestrogenic in high dose but the anti-estrogenicity seems to be incomplete in vivo as ENT may be metabolized to a potent estrogenic compound, ethinyl estradiol

Animals↗
PubMed1975

Enzymic N-acetylation of 2,4-toluenediamine by liver cytosols from various species.

1. 2,4-Toluenediamine was incubated with liver cytosol from various species, or cytosol from various tissues of the hamster or rabbit, in the presence of [1-14C]acetyl-CoA. N-Acetylation occurred selectively at the p-amino group of 2,4-toluenediamine and to a much lesser extent on the o-amino group. 2. In hamsters and rabbits the highest N-acetyltransferase activity was present in the liver cytosol, followed by kidney intestinal mucosa and lung cytosols. 3. Hamster liver cytosol had the greatest activity followed by liver cytosols from guinea-pig, rabbit, mouse and rat. With human liver cytosol only a trace of an N-acetyl derivative of 2,4-toluenediamine was found while dog liver cytosol showed no activity. 4. N-Acetyltransferase activity was maximal at pH 7-5 in mouse, pH 6-0 in rat and man, and pH 7-0 in rabbit liver cytosols. 5. There was a slight difference in the levels of N-acetyltransferases in males and females; the female mouse had more enzyme activity than the male, but the male rat had more enzyme activity than the female.

Acetylation↗
PubMed1978

Studies on brain cytosol neuraminidase. II. Extractability, solubility and intraneuronal distribution of the enzyme in pig brain.

The origin and properties of cytosolic neuraminidase (acylneuraminyl hydrolase, EC 3.2.1.18) from pig brain were studied. 1. The brain extracts containing the cytosol derived from neuronal bodies and glial cells carry 0.69 munits neuraminidase/g fresh tissue. The behaviour of neuraminidase during extraction closely paralleled that of authentic cytosolic enzyme, lactate dehydrogenase; whereas, it differed from that of the lysosomal enzymes, beta-hexosaminidase and beta-galactosidase, also found in the extracts. 2. Nerve endings from either crude or purified preparations, when treated by hypoosmotic shock, released neuraminidase activity up to a maximum of 1.25 munits/g fresh tissue. The behaviour of releasable neuraminidase was always identical to that of lactate dehydrogenase and very similar to that of ATPase and acetylcholinesterase. Typical lysosomal enzymes, however, such as beta-galactosidase and beta-hexosaminidase, behaved differently under the same conditions. This neuraminidase activity is thought to be derived from the cytosol of nerve endings. 3. The specific activity of neuraminidase in nerve-ending cytosol is 15--20 times that in neuronal body and glial cell cytosol. Some properties (pH, Km value, V/t relationship) of the cytosolic enzymes of different origin are similar; others (stability on standing at 4 degrees C; resistance to freezing and thawing) are different. Hypoionic solutions caused both cytosolic neuraminidases to slowly precipitate and to assume a stable insoluble form which was still active.

Animals↗
PubMed1975

Evidence for a specific dihydrotestosterone-binding cytosol receptor in the human prostate.

A technique for the demonstration of a human steroid and organ-specific prostate cytosol receptor is described. Tissue from 17 patients with benign prostatic hypertrophy was incubated in Eagle's medium with 3H-testosterone (T) .06 nM following pre-incubation with an anti-androgen. The minces were homogenized and the cytosol fraction obtained. The cytosol was then fractionated on a duo-gel column of G-50 Sephadex over Bio-gel A 1.5m and fractions counted for 3H, assayed for protein and plotted graphically. Three main peaks were seen. Only the second peak (approximately 150,000 mol wt), which contained predominantly dihydrotestosterone (DHT), was significantly and reproducibly inhibited by 2.1 muM cyproterone acetate (Cyp A) pre-incubation. This inhibiton was considered a specific indicator for receptor since Cyp A at muM had little effect (less than 10%) on 3H-DHT binding to plasma. Similar results were noted for other anti-androgens tested, but cortisol and etiocholanolone had no effects on 3H-5alpha DHT binding to prostate cytosol. Steroid-protein peaks for fractionated human thyroid, muscle, and spleen cytosol were not inhibited by Cyp A. Fractionated kidney cytosol contained a 3H-T-binding protein peak which was significantly decreased by Cyp A. Pronase incubation and heating at 50 C for 30 min both resulted in either a significant decrease or complete loss of receptor. Gel filtration analysis of 3H-cytosol derived from human prostate minces pre-incubated with and without Cyp A, provides a relatively rapid technique for the demonstration of cytosol receptor in approximately 80% of prostates from patients with benign prostatic hypertrophy.

Aged↗
PubMed1975

Metal ion dependence of the binding of triiodothyronine by cytosol proteins of bullfrog tadpole tissues.

The binding of triiodothyronine by Rana catesbeiana tadpole tail fin, tail muscle, kidney, and liver cytosol was studied using dextran-coated charcoal to separate bound and free hormone. A metal ion dependency was suggested by the fact that EDTA decreased the binding of triiodothyronine 80 to 90% in tail fin and tail muscle cytosol. Inhibition of binding in kidney or liver was less, 40 to 50%. This inhibition could be restored by adding an excess of divalent cations with an order of potency of Mn2+ greater than Ca2+ congruent to Co2+ greater than Sr2+ greater than Ba2+ greater than Mg2+. Other chelators, e.g. o-phenanthroline, 8-hydroxyquinoline, and ethylene glycol bis(beta-aminoethylether)-N,N'-tetraacetate also decreased the binding of triiodothyronine, whereas citrate, oxalate, imidazole, and glycine had no effect. The triiodothyronine binding capacity of tail fin cytosol was reduced by EDTA treatment and dialysis against buffer. Ca2+ in the 1 to 10 mM range and Mn2+ at 1 mM could restore the binding to normal levels. Higher Mn2+ increased binding 70% above normal or to Ca2+-restored levels. The triiodothyronine cytosol binding activity was nondialyzable, heat-labile. pH-dependent, pronase-digestible, but unaffected by incubation with trypsin, RNase, and DNase, suggesting that the cytosol binding sites are acidic proteins. Scatchard analysis of triiodothyronine binding by the cytosol of different tissues, revealed Kassoc of 7.1 x 10(6) M(-1), 11.6 x 10(6) M(-1), 3.6 X 10(6) M(-1), and 68.0 x 10(6) M(-1) for tail fin, tail muscle, kidney, and liver cytosol, respectively. The corresponding maximal binding capacities in picomoles per mg of crude cytosol protein in these four tissues were 10.4, 0.86, 1.3, and 0.04, respectively.

Animals↗
PubMed1975

Involvement of cytosol proteins in oleate activation of rabbit liver fructose-1,6-diphosphatase.

Dialyzed rabbit liver cytosol was specifically freed of endogenous fructose-1,6-diphosphatase by immunoadsorption on a column of Sepharose-immobilized anti-fructose-1,6-diphosphatase. This material increased the specific activity of homogeneous enzyme to the maximal rate observed with EDTA and shifted the pH optimum from 8.4 to 7.4. With oleate or other fatty acids as activators, the hydrolysis of fructose-1,6-diphosphatase by enzyme, at neutral pH, showed nonlinear initial rates dropping to lower linear rates. Cytosol activator acted synergistically with oleate both to increase neutral enzyme activity and to maintain the high initial catalytic rates. After sucrose density centrifugation or gel filtration, the cytosol had no effect by itself, but still potentiated oleate activation. The factor was destroyed by treatment with subtilisin or trypsin, but all attempts to identify a unique protein component in cytosol were unsuccessful. The presence of Na dodecyl-SOJ, deoxycholate, or urea did not improve the resolution of the factor, but these compounds did lower the K50 for activation by cytosol. Since fatty acids are the only unique compounds which have been isolated from cytosol which activated fructose-1,6-diphosphatase, it appears that soluble proteins can act as natural carriers for the fatty acids. This was supported by the fact that both dialyzed rabbit alpha-globulins and muscle phosphofructokinase also acted synergistically with oleate in a manner similar to cytosol. Phosphatidic acid and phosphatidylserine activated fructose-1,6-diphosphatase, and their action was synergistic with oleate. Glutathione (1 mM) activated the enzyme 5-fold at pH 7.3 and its effects were additive with oleate and cytosol or alpha-globulins.

Albumins↗
PubMed1978

Characterization of protein kinases from adrenal medulla. A study of cytosol and nuclear enzymes.

Since phosphorylation of chromosomal proteins by cyclic AMP-dependent protein kinases (EC 2.7.1.37) enhances template activity of adrenal medulla chromatin (9), we have studied the properties and regulation of protein kinases isolated from chromaffin cell cytosol and nuclei. DEAE-cellulose chromatography revealed three peaks of kinase activity in the nucleus (nPKI, nPKII, nPKIII) and two in the cytosol (cPKI, cPKII). The three nuclear enzymes, as well as cPKII, did not require cyclic AMP to express their catalytic activity. nPKI and nPKIII preferred acidic substrates as PO3-4 acceptors, while nPKII and the cytosol enzymes preferred basic PO3-4 acceptors. Enzyme recombination experiments using protein kinase regulatory subunits from cytosol suggested that cPKII was the catalytic subunit of cPKI. In contrast, the nuclear enzymes were not catalytic subunits of the cyclic AMP-dependent protein kinase in the cytosol (cPKI). Only the cytosol protein kinases could be inhibited by endogenous heat-stable protein kinase inhibitors. The nuclear and cytosol cyclic AMP-independent protein kinases were distinguishable on the basis of their sedimentation constants as well as Mc2+ and Mn2+ requirements.

Adrenal Medulla↗
PubMed1976

Androgen and estrogen receptors in brain cytosol from male, female, and testicular feminized (tfm/y hermaphrodite) mice.

Specific binding of [3H] 5alpha-dihydrotestosterone (DHT) and [3H] estradiol by cytoplasmic extracts from whole brain of castrated male, female, and androgen-insensitive, testicular feminized (tfm/y male-female), mice has been investigated using glycerol gradient centrifugation and charcoal assay. Mouse brain cytosol contains macromolecules with the characteristics of steroid hormone receptors, binding preferentially with high-affinity androgens or estrogens. Both DHT- and estradiol-receptor complexes migrate at 8-9 S in gradients at low ionic strength and at 4-5 S in gradients containing 0.5M KCl. KD's (mean +/- SE) for DHT binding by brain cytosol from castrated males, females, and tfm/y male-female are 1.1 +/- 0.4, 0.9 +/- 0.4, and 0.8 +/- 0.1 X 10(-9)M, respectively. DHT binding activity in brain cytosol from tfm/y male-female mice is reduced to about 20-30% of that from their normal littermates, as is the case for tfm/y male-female kidney cytosol. The residual androgen receptor in tfm/y male-female brain cytosol has normal sedimentation properties. Unlike the situation for androgen binding, the number of estradiol binding sites is comparable in brain cytosol from male, female, and tfm/y male-female mice. KD's (mean +/- SE) for estradiol binding are 1.6 +/- 0.5 X 10(-10)M for castrated males, 2.4 +/- 0.4 X 10(-10)M for females, and 1.8 +/- 0.4 X 10(-10)M for tfm/y male-female. Cross-competition experiments with unlabeled estradiol, DHT, or testosterone, have shown a difference in the degree of specificity of the androgen and estrogen receptors, the estrogen receptor having considerably more specificity. For the interaction of estradiol with the androgen receptor, the Ki is 8-9 X 10(-9)M. The decrease in the number of DHT binding sites in the brain of tfm/y male-female mice without a concomitant decrease in estradiol binding sites, and the different specificities of the two sites, point to the existence of distinct androgen and estrogen receptor molecules in mouse brain cytosol.

Androgen-Insensitivity Syndrome↗
PubMed1978

The influence of brain cytosol on RNA synthesis and RNA products of isolated mouse brain nuclei.

The incubation of isolated nuclei obtained from 10-day-old mouse brain in the presence of brain cell cytosol resulted in an increase in the synthesis of RNA. Under conditions of saturating concentrations of nucleoside triphosphates, the influence of cytosol could not be duplicated by the addition of cyclic nucleotides. The stimulatory activity of cytosol on brain nuclear RNA synthesis could not be attributed to either alterations in the permeability of the nuclear envelope or an increased uptake of radioactively-labeled precursors. Sucrose gradient analysis demonstrated that the RNA products synthesized by nuclei isolated from 10-day-old and adult mouse brain were of a relatively low molecular weight. However, the addition of cytosol resulted in a significant increase in the size of the RNA transcripts. In contrast to the observations with 10-day-old and adult brain nuclei, the RNA from 2-day-old mouse brain nuclei was larger in size and relatively unaffected by the presence of cytosol. Although cytosol caused an increase in the amounts of poly[A]-RNA in nuclei of 2-day-old and adult animals, no comparable effect could be measured in nuclei from 10-day-old brain tissue.

Animals↗
PubMed1979

Calcium-induced erythrocyte membrane changes. The role of adsorption of cytosol proteins and proteases.

Changes in the membranes of human red cells similar to those of certain hemolytic anemias were produced by calcium in three model systems and found to result from membrane adsorption of cytosol proteins and from proteolysis. Proteins of the cytosol adsorbed to human erythrocyte membranes in the presence of calcium and extractable by EDTA were compared to those of the total cytosol by polyacrylamide gel electrophoresis and by isoelectric focusing. Catalase (EC 1.11.1.6) and band 8 were adsorbed to the membranes from the supernatant cytosol with calcium. Band 8 was a normal constitutent of the cytosol, apparently a single chain of molecular weight 24,000 with a pI of 5.35. Other calcium-induced membrane changes could be demonstrated to be due to cytosol protease(s) adsorbed to the membrane in the presence of calcium and extractable with EDTA. When membranes were incubated with the proteases and calcium the decrease in bands 1,2,3 and 4.1 and the appearance of multiple low molecular weight peptides typical of calcium-induced membrane effects resulted.

Blood Proteins↗
PubMed1976

The purification and properties of the glutamine synthetase from the cytosol of Soya-bean root nodules.

The major portion of glutamine synthetase activity in root nodules of soya-bean plants is associated with the cytosol rather than with Rhizobium japonicum bacteroids. Glutamine synthetase accounts for about 2% of the total soluble protein in nodule cytosol. Glutamine synthetase from nodule cytosol has been purified by a procedure involving fractionation with protamine sulphate, ammonium sulphate and polypropylene glycol, chromatography on DEAE-Bio-Gel A and Bio-Gel A-5m and affinity chromatography on glutamate-agarose columns. The purified preparation appeared to be homogeneous in the analytical ultracentrifuge. From sedimentation-equilibrium experiments a mol. wt. of about 376000 was determined for the native enzyme and 47300 for the enzyme in guanidinium chloride. From these data and measurements of electron micrographs, we have concluded that glutamine synthetase from nodule cytosol consists of eight subunits arranged in two sets of planar tetramers which form a cubical configuration with dimensions of about 10 nm (100 A) across each side. Glutamine synthetase from nodule cytosol has a higher glycine and proline content and a lower content of phenylalanine than the glutamine synthetase that has been prepared from pea seed. The cytosol enzyme contains four half-cystine molecules per subunit, which is in contrast with two reported for the enzyme from pea seed. Enzyme activity is striking influenced by the relative proportion of Mg2+ and Mn2+ in the assay medium. Activity is inhibited by feedback inhibitors and is influenced by energy charge.

Amino Acids↗