Guanosine triphosphate cyclohydrolase activity in rat tissues.

The GTP cyclohydrolase activity of rat tissues has been studied by means of the measurement of formic acid release and neopterin synthesis from GTP. After gel filtration of a 45%-satd.-(NH4)2SO4 fraction of liver homogenates, three enzyme fractions were separated and named A1, A2 and A3 according to the order of their elution. Fractions A1 and A3 displayed an 8-formyl-GTP deformylase activity; no proof of cyclized product has yet been established. This activity was heat-labile and required Mg2+ for maximal activity. Fraction A2 displayed a 'neopterin-synthetase' activity, with dihydroneopterin triphosphate and formic acid formed in stochiometric amounts. Fraction A1 isolated from heat-treated homogenates also produced dihydroneopterin triphosphate. Neopterin synthetase activity in fractions A1 and A2 was heat-resistant and inhibited by Mg2+. In liver the A2 fraction represented 70-75% of the neopterin synthetase capacity and was inhibited by reduced pterines (sepiapterin, dihydrobiopterin and tetrahydrobiopterin) and to a lesser extent by reduced forms of folic acid. In kidney and brain, fraction A1 and A3 GTP 8-formylhydrolase activities were found in significant amounts, in contrast with the neopterin synthetase activity, which was low and appeared to be confined to the A1 fraction.     

Adenosine triphosphate:creatine phosphotransferase of rat cerebral cortex: evidence for a bimodal subcellular localization

—(1) ATP: creatine phosphotransferase of rat cerebral cortex is soluble to the extent of 57 per cent when the tissue is homogenized in 0.25 M-sucrose and 80 per cent when distilled water is used for tissue dispersion. Among particulate fractions, the crude mitochondria] fraction contains the highest percentage of enzyme activity. (2) Discontinuous sucrose gradient fractionation of the crude mitochondrial fraction yields about 55 per cent of the particulate activity in the nerve ending fractions and 24 per cent in the mitochondrial pellet. (3) Rupturing of the nerve-ending particles by a moderate osmotic shock designed to spare the mitochondria results in about 60 per cent of the ATP:creatine phosphotransferase becoming soluble, the remainder preserving the association with heavy particles, presumably mitochondria. (4) Subfractionation of the microsomal fraction on a discontinuous sucrose gradient reveals that this particulate component of the enzyme is an adsorption artifact. (5) The overall evidence points to at least two distinct subcellular localizations of the enzyme in rat brain cortex, a major soluble component and a particulate component. It has not been unequivocally shown whether the latter, in turn, reflects the presence of a single, mitochondrial component or whether the soluble matrix of the nerve ending particles represents a third locale for the enzyme.     

Guanosine 5'-(gamma-thio)triphosphate stimulates arachidonic acid liberation in permeabilized rat peritoneal mast cells

The exocytotic histamine secretion from ATP-permeabilized and Mg-resealed rat peritoneal mast cells is markedly enhanced by the addition of guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) at a concentration of 100 uM. GTP gamma S also caused a great enhancement of arachidonic acid liberation from these cells. The level of released arachidonic acid in permeabilized cells enhanced by GTP gamma S in the absence of Ca2+ was nearly equal to the level of permeabilized cells incubated in the presence of Ca2+ but without GTP gamma S, suggesting the Ca2+ sparing effect of GTP gamma S. From the time sequential changes in the [3H]arachidonate radioactivities in various phospholipids, it is conceivable that nucleotide-dependent arachidonic acid release was mediated via phospholipase A2 pathway. The entrapment of a diacylglycerol (DG) lipase inhibitor, RHC 80267, caused suppression of both Ca2+- and guanine nucleotide-dependent arachidonic acid liberation in mast cells, indicating contribution of DG lipase pathway for arachidonic acid generation.     

Metabolism of 5-hydroxylysine in Pseudomonas fluorescens.

Hydroxylysine is metabolized via two routes by a Pseudomonas fluorescens strain as shown by the oxidation of selected intermediates. Hydroxy-L-lysine is oxidized via a pathway analogous to the monooxygenase pathway for L-lysine, and data suggest that at least some of tthe enzymes are those involved in the metabolism of L-lysine. Hydroxy-L-lysine is also converted by a racemase to allohydroxy-D-lysine, which is then degraded via a pathway analogous to, but different from, that described for D-lysine, involving hydroxy-L-pipecolate, 2-amino-5-hydroxyadipate, and 2-hydroxyglutarate. Data obtained with mutants unable to oxidize L-pipecolate suggest that the enzymes for the metabolism of hydroxy-L-pipecolate are distinct from those for L-pipecolate. Studies on D- and L-lysine degradation have shown that the previously described pathways for these compounds are present in this soil pseudomonad.     

Glycine metabolism in rat kidney cortex slices.

We have previously described a method for measuring the rotational diffusion of membrane proteins by using fluorescent triplet probes [Johnson & Garland (1981) FEBS Lett. 135, 252-256]. We now describe the criteria by which the suitability of such probes may be judged. In general, the greatest sensitivity is achievable with probes where the ratio of the quantum yields for prompt fluorescene (phi f) and triplet formation (phi t) are high, as with Rhodamine (phi f/phi t congruent to 10(3)). However, considerations of heat generation at the sample membrane, of time resolution of fast rotations and of irreversible bleaching of the fluorescent probe also apply. The immediate environment of a probe molecule at a membrane protein must also be important in determining the performance of a given probe. Nevertheless, we describe guidelines for evaluating the likely usefulness of fluorescent triplet probes in measurements of membrane protein rotation.     

Phosphodiesterase activator from rat kidney cortex.

Incubation of homogenates of rat renal cortex at 4 degrees resulted in increased cAMP phosphodiesterase activity; the increase was much more rapid in hypotonic medium than in one of physiological tonicity. cAMP phosphodiesterase activity did not increase with incubation of supernatant fractions (48,000 x g, 20 min) prepared from isotonic homogenates. Extraction of the isotonic particulate fraction with hypotonic buffer released an activator which increased cAMP phosphodiesterase activity of the supernatant fraction. The kidney phosphodiesterase activator differed from a heat-stable, calcium-dependent protein activator of phosphodiesterase in that it was destroyed by heating (90 degrees for 10 min) and was not inhibited by EGTA. The phosphodiesterases of rat renal cortex were partially resolved by chromatography on DEAE-Bio-Gel, and a cAMP phosphodiesterase that is sensitive to the kidney activator was identified. This phosphodiesterase was separable from that affected by a calcium-dependent phosphodiesterase activator from bovine brain and from cGMP-stimulated cAMP phosphodiesterase. As determined by sucrose density gradient centrifugation, after incubation with the kidney activator, the activated form of phosphodiesterase had a lower sedimentation velocity than did the unactivated form.     

Renin substrate in granules from rat kidney cortex.

1. Subcellular fractions of rat kidney cortex generated angiotensin I continuously over 2h when incubated at 37degreesC with rat renin, indicating the presence of renin substrate within cells in the renal cortex. 2. Renin substrate was located in highest specific concentration in particulate fractions. The particles containing renin substrate had a sedimentation velocity slightly lower than mitochondria and renin granules but greater than the microsomal fraction. 3. Isopycnic gradient centrifugation indicated a density of 1.190g/ml for the particles containing renin substrate, compared with 1.201 for renin granules, 1.177 for mitochondria, and 1.170 and 1.230 for lysosomes in the heavy-granule fraction. 4. In the liver, renin substrate was also found in particles, but these had a lower sedimentation rate than those from the kidney. 5. The molecular weights of renin substrate in kidney and liver granules and rat plasma were similar, namely 61000-62000. 6. On the basis of these biochemical findings, a mechanism for the intrarenal production of angiotensin, incorporating a subcellular reaction scheme, is proposed.     

Improved synthesis of 5-hydroxylysine (Hyl) derivatives.

The synthesis of 5-hydroxylysine (Hyl) derivatives for incorporation by solid-phase methodologies presents numerous challenges. Hyl readily undergoes intramolecular lactone formation, and protected intermediates often have poor solubilities. The goals of this work were twofold: first, develop a convenient method for the synthesis of O-protected Fmoc-Hyl; secondly, evaluate the efficiency of methods for the synthesis of O-glycosylated Fmoc-Hyl. The 5-O-tert-butyldimethylsilyl (TBDMS) fluoren-9-ylmethoxycarbonyl-Hyl (Fmoc-Hyl) derivative was conveniently prepared by the addition of tert-butyldimethylsilyl trifluoromethanesulfonate to copper-complexed Hyl[epsilon-tert-butyloxycarbonyl (Boc)]. The complex was decomposed with Na+ Chelex resin and the Fmoc group added to the alpha-amino group. Fmoc-Hyl(epsilon-Boc, O-TBDMS) was obtained in 67% overall yield and successfully used for the solid-phase syntheses of 3 Hyl-containing peptides. The preparation of Fmoc-Hyl[epsilon-Boc, O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)] was compared for the thioglycoside, trichloroacetimidate and Koenigs-Knorr methods. The most efficient approach was found to be Koenigs-Knorr under inverse conditions, where Fmoc-Hyl(epsilon-Boc)-OBzl and peracetylated galactosyl bromide were added to silver trifluoromethanesulfonate in 1,2-dichloroethane, resulting in a 45% isolated yield. Side-reactions that occurred during previously described preparations of glycosylated Hyl derivatives, such as lactone formation, loss of side-chain protecting groups, orthoester formation, or production of anomeric mixtures, were avoided here. Research on the enzymology of Lys hydroxylation and subsequent glycosylation, as well as the role of glycosylated Hyl in receptor recognition, will be greatly aided by the convenient and efficient synthetic methods developed here.     

Guanosine triphosphate - dependent autocatalytic phosphorylation of peptide initiation factor 2 from rat liver

If incubated with [γ-³²P]guanosine triphosphate, homogenous peptide initiation factor 2 from rat liver liberates the terminal phosphate from GTP and utilizes 25–40% of it for its own phosphorylation. This phosphorylation is apparently autocatalytic since it does not require any additional proteins. Only the γ-subunit of the factor becomes phosphorylated by an acid-labile bond and the reaction is significantly inhibited by initiator Met-tRNA_F.     

Gluconeogenesis in developing rat kidney cortex

1. Gluconeogenesis in developing rat kidney cortex was studied by assaying the activities of two enzymes, glucose 6-phosphatase and phosphoenolpyruvate carboxykinase, and by measuring glucose formation in tissue slices. 2. Glucose 6-phosphatase and phosphoenolpyruvate carboxykinase are present in late foetal (21-22-day-old) tissue and increase rapidly postnatally. Maximum activity of phosphoenolpyruvate carboxykinase occurs at 7 days of age, followed by a decline to the adult level. Glucose 6-phosphatase activity rises during the first 2 postnatal weeks and then declines. 3. Late foetuses synthesize glucose from both pyruvate and l-glutamate. The rate increases during the first 2 weeks to above adult levels. Synthesis is always higher from pyruvate than from glutamate. 4. The effect of 24hr. starvation was studied in perinatal animals. The results indicate that the ability to increase the rate of glucose synthesis as a result of starvation is not present at birth, but develops some time after the second postnatal day.     

Guanosine triphosphate analysis: PEI-thin layer purification and luciferin-luciferase liquid scintillation quantitation

A novel method to purify and quantitate GTP is described. This method is simple to run and sensitive to detect GTP in picomole quantities. It is based on purfication and isolation of GTP on PEI-thin layer chromatograms and quantitation with the firefly luciferin-luciferase system.GTP characteristically gives a delayed (about 2 min) peak light emission similar to adenosine tetraphosphate (about 6 min) but different from ATP which gives a peak light emission value at zero time. The light emission from each of these three nucleotides is characteristically different from one another such that contamination between these three nucleotides can be detected by their peak light emission times. This method has been applied to analyze GTP in biological preparations which appeared to be free of detectable concentration(s) of interfering substances(s).     

Stimulation of gluconeogenesis by somatostatin in rat kidney cortex slices.

The effect of somatostatin on gluconeogenesis was studied in kidney cortex slices. Addition of somatostatin (2 μg) stimulated gluconeogenesis from lactate, pyruvate and glutamine by 42%, 50% and 68% respectively. Stimulation of glucose synthesis from lactate by somatostatin was found to be linear with time and dose dependent between 0.1 and 20 μg. Somatostatin-stimulated gluconeogenesis was inhibited by phentolamine (10 μM) but not by propranolol (10 μM) suggesting that somatostatin action is mediated by α-adrenergic stimuli.     

Guanosine triphosphate complex in rod outer segments of the frog retina

It is shown that nearly 70% water--soluble protein of the frog retina outer segments (ROS) consist of three polypeptides with molecular weights 39 000, 36 000 and less than 15 000 daltons. These proteins are present in equal proportions and are, apparently, the subunits of a tightly bound protein complex. The subunit of 39 000 daltons is responsible for guanyl nucleotides binding. Parameters of the investigated GTP-binding complex are similar to transducyn which transmits excitation from bleached rhodopsin to PDE molecules in the bovine retina ROS. The thermodynamic state of GTP-binding protein in frog retina ROS depends on the functional state of the photoreceptor membrane, as shown by microcalorimetric method.