LOCALIZATION OF GLUCOSE, GLUCONATE, AND GLUCOSE-6-PHOSPHATE OXIDATION SYSTEMS IN EXTRACTS OF PSEUDOMONAS FLUORESCENS

1958 ◽  
Vol 4 (1) ◽  
pp. 1-7 ◽  
Author(s):  
R. G. Eagon
Keyword(s):  
Pseudomonas Fluorescens ◽  
Particulate Fraction ◽  
Supernatant Fraction ◽  
Oxidation Of Glucose

Cell-free extracts of Pseudomonas fluorescens, strain OSU 64, derived by means of sonic oscillation and alumina grinding, were assayed to determine the localization of enzymes necessary for the oxidation of glucose, gluconate, and glucose-6-phosphate. Glucose and gluconate were oxidized to 2-ketogluconate by both the supernatant and particulate fractions of the extracts. Approximately 40% of the activity for the oxidation of glucose was demonstrated to be in the supernatant fraction and 60% in the particulate fraction. Activity for the oxidation of gluconate was found to be located with approximately 50% in each fraction. Oxidation of glucose-6-phosphate was observed only in the soluble portion.

INCORPORATION OF IODIDE INTO ORGANIC COMPOUNDS IN THE GUINEA PIG THYROID

1963 ◽  
Vol 43 (1) ◽  
pp. 110-118 ◽  
Author(s):  
R. Ekholm ◽  
T. Zelander ◽  
P.-S. Agrell
Keyword(s):  
Guinea Pig ◽  
Protein Binding ◽  
Organic Compounds ◽  
Guinea Pigs ◽  
Microsomal Fraction ◽  
Thyroid Tissue ◽  
Particulate Fraction ◽  
Supernatant Fraction ◽  
Hydrolysis Of

ABSTRACT Guinea pigs, kept on a iodine-sufficient diet, were injected with Na131I and the thyroids excised from 45 seconds to 5 days later. The thyroid tissue was homogenized and separated into a combined nuclear-mitochondrial-microsomal fraction and a supernatant fraction by centrifugation at 140 000 g for one hour. Protein bound 131iodine (PB131I) and free 131iodide were determined in the fractions and the PB131I was analysed for monoiodotyrosine (MIT), diiodotyrosine (DIT) and thyroxine after hydrolysis of PB131I. As early as only 20 minutes after the Na131I-injection almost 100% of the particulate fraction 131I was protein bound. In the supernatant fraction the protein binding was somewhat less rapid and PB131I values above 90% of total supernatant 131I were not found until 3 hours after the injection. In all experiments the total amount of PB131I was higher in the supernatant than in the corresponding particulate fraction. The ratio between supernatant PB131I and pellet PB131I was lower in experiments up to 3 minutes and from 2 to 5 days than in experiments of 6 minutes to 20 hours. Hydrolysis of PB131I yielded, even in the shortest experiments, both MIT and DIT. The DIT/MIT ratio was lower in the experiments up to 2 hours than in those of 3 hours and over.

Cyclic AMP phosphodiesterase activity in mouse pancreatic islets Effects of calmodulin and phospholipids

1986 ◽  
Vol 111 (4) ◽  
pp. 533-538 ◽  
Author(s):  
Kirsten Capito ◽  
Carl Jørgen Hedeskov ◽  
Peter Thams
Keyword(s):  
Enzyme Activity ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Pancreatic Islets ◽  
Total Activity ◽  
Particulate Fraction ◽  
Supernatant Fraction ◽  
Phosphodiesterase Activity ◽  
Cyclic Amp Phosphodiesterase ◽  
Mouse Pancreatic Islets

Abstract. The activity of cyclic AMP phosphodiesterase in mouse pancreatic islets was investigated. 85% of the total activity was found in a 27 000 g supernatant fraction. The phosphodiesterase activity in the supernatant fraction, but not in the particulate fraction, was stimulated approximately 20% by Ca2+ (10−5m) and calmodulin (1 μm). The Km (cyclic AMP) of the unstimulated enzyme in the supernatant fraction was 20 μm, and the Vmax was 2 nmol/min × mg protein−1. The possible influence of a range of phospholipids was investigated. PI* and PS (150 μg/ml) inhibited the enzyme 20–30% both in the absence and presence of Ca2+/calmodulin, whereas PE, PC and PA did not affect the enzyme activity. ATP (1 mm) did not affect the particulate or supernatant fraction phosphodiesterase either in the absence or presence of Ca2+/calmodulin or Ca2+/phospholipid. It is concluded that, contrary to islet adenylate cyclase, islet cyclic AMP phosphodiesterase may be regulated by Ca2+/calmodulin.

Cyclic GMP phosphodiesterase and guanylate cyclase activities in rabbit ovaries and the effect of in-vivo stimulation with LH

1984 ◽  
Vol 101 (3) ◽  
pp. 305-310 ◽  
Author(s):  
V. V. Patwardhan ◽  
A. Lanthier
Keyword(s):  
Guanylate Cyclase ◽  
Cyclic Gmp ◽  
The Other ◽  
Particulate Fraction ◽  
Cyclase Activity ◽  
Supernatant Fraction ◽  
Phosphodiesterase Activity ◽  
Significant Drop ◽  
Guanylate Cyclase Activity

ABSTRACT The activities of guanylate cyclase and cyclic GMP (cGMP) phosphodiesterase, enzymes that are responsible for maintaining tissue levels of cGMP, were determined in the ovaries of rabbits killed without treatment or 4 h after administration of LH. Ovarian activities of the two enzymes were determined in the 100 000 g supernatant fraction (cytosol) and the resulting pellet (particulate fraction). Significant phosphodiesterase and cyclase activities were detected in both the cytosol and particulate fractions. Administration of LH had no significant effect on phosphodiesterase activity in either of the tissue fractions. On the other hand, LH caused a significant drop in guanylate cyclase activity in the cytosol and particulate fractions. This drop in the cyclase activity may be the cause of the decreased rabbit ovarian concentrations of cGMP that we have previously observed after LH stimulation. J. Endocr. (1984) 101, 305–310

scholarly journals The role of multivalent cations in the uptake and oxidation of glucose by Pseudomonas fluorescens (Short Communication)

1973 ◽  
Vol 136 (2) ◽  
pp. 429-431
Author(s):  
Cynthia A. Walker ◽  
Norman N. Durham
Keyword(s):  
Pseudomonas Fluorescens ◽  
Metal Ion ◽  
Short Communication ◽  
Enzymic Activity ◽  
Membrane Stabilization ◽  
Oxidation Of Glucose

The effects of Mg2+, Mn2+, Ca2+ and Fe3+ on the uptake and oxidation of glucose in induced and non-induced Pseudomonas fluorescens cells were studied. Mg2+ is the most effective single metal ion in the uptake of glucose by the cell, and it functions in membrane stabilization and enzymic activity. Ca2+ will substitute for Mg2+, but Mn2+ and Fe3+ will not.

scholarly journals Biosynthesis of peptides containing α-aminoadipic acid and cysteine in extracts of a Cephalosporium sp

1971 ◽  
Vol 123 (3) ◽  
pp. 477-482 ◽  
Author(s):  
P. Bronwen Loder ◽  
E. P. Abraham
Keyword(s):  
Adipic Acid ◽  
Cell System ◽  
Particulate Fraction ◽  
Supernatant Fraction ◽  
Cephalosporin C

1. Three intracellular peptides found in small amount in a Cephalosporium sp. were rapidly labelled when dl-[14C]valine was added to a shaken suspension of the organism. More 14C was incorporated into peptide P3, δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine, than into peptide P2 (containing α-aminoadipic acid, cysteine, valine and glycine) or peptide P1 (containing β-hydroxyvaline in place of the valine in peptide P2). 2. Peptides P3 and P2, but not peptide P1 were formed in a broken-cell system from the Cephalosporium sp. in the presence of δ-(l-α-aminoadipyl)-l-cysteine and dl-[14C]valine. No synthesis was observed in the presence of δ-(d-α-aminoadipyl)-l-cysteine or of dl-α-amino[14C]adipic acid and l-cysteinyl-l-valine or l-cysteinyl-d-valine. 3. The biosynthesis of these peptides was catalysed by the particulate fraction of the broken-cell system, whereas that of glutathione was catalysed by the supernatant fraction. 4. These results are discussed in relation to penicillin N and cephalosporin C biosynthesis.

Immunogenicity of potassium thiocyanate extracted and electrofocused Pasteurella multocida X-73I antigens in chickens and mice

1982 ◽  
Vol 28 (11) ◽  
pp. 1219-1225 ◽  
Author(s):  
Kevin L. McKinney ◽  
Paul A. Rebers ◽  
Richard B. Rimler
Keyword(s):  
Antibody Response ◽  
Pasteurella Multocida ◽  
Potassium Thiocyanate ◽  
Particulate Fraction ◽  
Supernatant Fraction ◽  
Chicken Embryos ◽  
Isoelectric Points ◽  
Serotype 1 ◽  
Soluble Supernatant

The immunogenicity of antigenic fractions obtained by the extraction of Pasteurella multocida strain X-73 (serotype 1) with potassium thiocyanate (KSCN) was determined in chickens and mice. The initial KSCN extract was centrifuged at 105 000 × g, and the antigens were separated into a particulate fraction (40p) and a soluble supernatant fraction (40s). The ultracentrifuged fractions were further resolved by preparative electrofocusing. The 40p fraction was resolved into two subgroups having isoelectric points of 3.5–3.9 and 5.5–6.0; the 40s fraction was resolved into five subgroups ranging in isoelectric points from 4.4 to 9.0. The 40p fractions were antigenically similar and contained lipopolysaccharide (LPS) and protein. The 40s fractions were antigenically distinct from the 40p fractions and from each other; they contained proteins and polysaccharides but no LPS. The 40p antigens were strongly immunogenic in mice and chickens, whereas the 40s antigens were weakly immunogenic in chickens and not immunogenic in mice. The incorporation of Freund's complete adjuvant increased the immunogenicity of the 40s antigens in chickens. The 40p antigens induced greater frequencies of serological responses in chickens than the 40s antigens as detected by counterimmunoelectrophoresis and immunodiffusion. This suggested that the increased protection associated with the 40p antigens may have been the result of better antibody response. The toxicity of all the fractions was evaluated by determination of lethality for 10-day-old chicken embryos because of the sensitivity and reliability of the test. The 40p fraction had an LD50 = 0.38 μg, and the 40s fraction had an LD50 = 2.5 μg. Since the 40s fraction contained no detectable LPS, it is likely that two toxins are present, one which contains LPS and one which does not.

Subcellular site of porphyrin biosynthesis in nucleated erythrocytes

1965 ◽  
Vol 208 (6) ◽  
pp. 1270-1274 ◽  
Author(s):  
Vishwanath M. Sardesai ◽  
Sally A. Doehr ◽  
James M. Orten
Keyword(s):  
Liver Cell ◽  
Aminolevulinic Acid ◽  
Subcellular Fractions ◽  
Particulate Fraction ◽  
Supernatant Fraction ◽  
Intact Cells ◽  
Cellular Fraction ◽  
Chicken Erythrocytes

The activity of hemolysates, prepared by several different methods from nucleated (chicken) erythrocytes, and of their subcellular fractions in forming porphyrins from glycine plus acetate or succinate or from δ-aminolevulinic acid (ALA) was studied. In general, hemolysates were less active than intact cells in porphyrin biosynthesis from glycine and acetate but were more active with ALA as the substrate. The supernatant fraction formed coproporphyrin from ALA but not from glycine and acetate. Protoporphyrin was not formed from either precursor by the supernatant fraction. The particulate fraction alone was devoid of porphyrin-forming activity, and also of ALA- and porphobilinogen- (PBG) forming activity. The combined particulate-supernatant fractions, however, synthesized both copro- and protoporphyrins from either glycine and acetate or ALA, but of course more from the latter. Dialysis or heating to 60 C destroyed the activity of the supernatant. These results indicate that "compartmentation" of porphyrin biosynthesis occurs in the nucleated erythrocyte much as in the liver cell, the step from ALA to coproporphyrinogen being carried out in the soluble, "nonparticulate" cellular fraction and the remaining steps in the "particulate" fraction.

Further studies on the inactivation of thyrotrophin releasing hormone (TRH) by enzymes in the rat hypothalamus

1980 ◽  
Vol 93 (4) ◽  
pp. 385-391 ◽  
Author(s):  
E. C. Griffiths ◽  
J. A. Kelly ◽  
N. White ◽  
S. L. Jeffcoate
Keyword(s):  
Enzyme Activity ◽  
Biologically Active ◽  
Particulate Fraction ◽  
Supernatant Fraction ◽  
Hypothalamic Area ◽  
Thyrotrophin Releasing Hormone ◽  
Releasing Hormone ◽  
Rat Hypothalamus ◽  
Polypeptide Antibiotic ◽  
Bacitracin A

Abstract. Thyrotrophin-releasing hormone (TRH) is known to be inactivated by enzymes present in the rat hypothalamus. To make a further study of the enzymes' action on the tripeptide, synthetic TRH was incubated with two hypothalamic subcellular fractions. By using a direct radioimmunoassay for TRH, the tripeptide was shown to be rapidly degraded by both supernatant and particulate fractions, with higher enzyme activity in the particulate fraction. Of several biologically-active peptides tested, only luteinizing hormone-releasing hormone was found to inhibit TRH inactivation; bacitracin, a polypeptide antibiotic, was also effective in inhibiting inactivation. Enzyme activity was highest in the middle hypothalamic area and lowest in the posterior hypothalamic area. Thin layer chromatography of the products of enzyme cleavage revealed the formation of only deamidated TRH in the supernatant fraction and the constituent amino acids (pyroGlu, His, ProNH2) and histidylproline-diketopiperazine by the particulate fraction, suggesting the presence of an amidase in the supernatant and two peptidases in the particulate fractions. These properties of the enzymes inactivating TRH may indicate that the enzymes could be of importance in regulating the endocrine and other functions attributed to this hypothalamic regulatory hormone.

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