scholarly journals Developmental changes of chicken liver AMP deaminase

1985 ◽  
Vol 231 (2) ◽  
pp. 329-333 ◽  
Author(s):  
J Spychała ◽  
K Kaletha ◽  
W Makarewicz
Keyword(s):  
Developmental Stages ◽  
Liver Extract ◽  
Kinetic Studies ◽  
Chicken Liver ◽  
Amp Deaminase ◽  
Enzyme Preparations ◽  
Enzyme Form ◽  
Substrate Saturation ◽  
Two Peaks ◽  
Regulatory Properties

The AMP deaminase activity measured in crude chicken liver extract did not change significantly during development. The livers of 10- and 14-day chick embryos, 1-day, 5-, 10- and 16-week-old chickens and adult hens were examined for the existence of multiple forms of AMP deaminase. Phosphocellulose column chromatography revealed the existence of two peaks of enzyme activity in the liver of 10- and 16-week-old chickens and adult hens. Kinetic studies with the preparations of AMP deaminase revealed sigmoid-shaped substrate-saturation curves at all developmental stages and hyperbolic-shaped saturation curves for the enzyme form appearing in 10-week-old chickens. All AMP deaminases investigated were susceptible to activation by ATP and inhibition by Pi. Kinetic and regulatory properties as well as pH optima of all the enzyme preparations tested indicate that AMP deaminase isolated from the embryos and from 1-day-old chicks was similar to the form I isolated from adult hens and differed significantly from the form II of this enzyme.

scholarly journals Oestradiol-17α dehydrogenase from chicken liver

1984 ◽  
Vol 222 (3) ◽  
pp. 761-768 ◽  
Author(s):  
J Johnston ◽  
A G C Renwick
Keyword(s):  
Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Kinetic Studies ◽  
Limited Range ◽  
Chicken Liver ◽  
Temperature Dependent ◽  
Enzyme Preparations ◽  
Dependent Inhibition ◽  
Two Peaks

The NADP+-linked oestradiol-17 alpha dehydrogenase (EC 1.1.1.148) present in cell-free extracts of chicken liver was investigated with the aim of separating it from a closely related oestradiol-17 beta dehydrogenase (EC 1.1.1.62) found in the same subcellular fraction. However, its chromatographic behaviour on CM-cellulose and DEAE-cellulose was almost identical with that previously reported for the latter enzyme, including resolution into two peaks on the anion-exchanger. Both peaks contained oestradiol-17 alpha dehydrogenase and oestradiol-17 beta dehydrogenase activity. Further attempts to separate the putative enzymes by dye-ligand chromatography with the use of the dyes Procion Yellow, Reactive Red and Cibachron Blue linked to Sepharose were unsuccessful, and they behaved identically on affinity columns of adenosine 2′,5′-bisphosphate-agarose and 17 beta-oestradiol 3-hemisuccinate bound to Sepharose. A previous report of partial separation on Sephadex G-200 was not confirmed. Slab gel electrophoresis of enzyme preparations after affinity chromatography on adenosine 2′,5′-bisphosphate-agarose revealed multiple bands in systems containing sodium dodecyl sulphate, whereas analysis by rod gel electrophoresis gave two major and one minor bands that stained coincidently for oestradiol-17 alpha dehydrogenase, oestradiol-17 beta dehydrogenase, epitestosterone dehydrogenase and testosterone dehydrogenase activities. Isoelectric focusing gave four enzymically active peaks that each oxidized oestradiol-17 alpha and −17 beta. Apparent Km values for the two forms of oestradiol-17 alpha dehydrogenase obtained by DEAE-cellulose chromatography were 17 and 23 microM for oestradiol-17 alpha, and 8.7 and 11.0 microM for NADP+. Limited kinetic studies with oestradiol-17 alpha and −17 beta with the use of the mixed-substrate method showed that the total velocity was equal to the sum of the separate velocities. The active-site inhibitor-alkylating agent 17 beta-(1-oxoprop-2-ynyl)androst-4-en-3-one did not cause time- or temperature-dependent inhibition, in contrast with the reported case of the oestradiol-17 beta dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase activities of the human placental oestradiol dehydrogenase. NADP+ appeared to afford some protection against inhibition. Investigation of substrate specificity with a limited range of steroids suggests that the enzyme(s) from chicken liver differs substantially from the oestradiol-17 beta dehydrogenase from human placenta, and although the evidence is not conclusive it suggests the existence of one enzyme.

scholarly journals Developmental forms of human skeletal-muscle AMP deaminase. The kinetic and regulatory properties of the enzyme

1988 ◽  
Vol 249 (1) ◽  
pp. 255-261 ◽  
Author(s):  
K Kaletha ◽  
G Nowak
Keyword(s):  
Skeletal Muscle ◽  
Human Fetus ◽  
Human Skeletal Muscle ◽  
Fetal Tissue ◽  
Amp Deaminase ◽  
Half Saturation Constant ◽  
Substrate Saturation ◽  
Regulatory Ligands ◽  
Regulatory Properties ◽  
Similar Kinetic

AMP deaminase isoforms from human skeletal muscle can be separated chromatographically [Kaletha, Spychała & Nowak (1987) Experientia 43, 440-443]. In adult tissue nearly all the AMP deaminase activity was eluted from phosphocellulose with 0.75 M-KCl (‘adult’ isoform), and the remaining activity could be eluted with 2.0 M-KCl. Conversely, most of the AMP deaminase activity from 11-week-old fetal tissue was eluted from phosphocellulose with 2.0 M-KCl (‘fetal’ isoform). In the present paper the kinetic and regulatory properties of AMP deaminase extracted from 11- and 16-week-old fetal skeletal muscle are reported. The two isoforms from 11-week-old human fetus differed distinctly in these properties. The ‘fetal’ isoform had about 5-fold higher half-saturation constant (S0.5) value than the ‘adult’ form. It was also more sensitive to the influence of some important regulatory ligands (ADP, ATP and Pi), and exhibited a different pH/activity profile. The ‘adult’ isoform of AMP deaminase from fetal muscle and the enzyme from mature muscle possessed similar kinetic and regulatory properties. This isoform seems not to be subject to any major modifications during further ontogenesis. This is not true, however, for the ‘fetal’ isoform. In the muscle of 16-week-old human fetus, the ‘fetal’ isoform showed a peculiar, biphasic, type of substrate-saturation kinetics. This phenomenon may reflect appearance of the next, developmentally programmed, isoform of human skeletal-muscle AMP deaminase.

scholarly journals Platelet AMP deaminase. Purification and kinetic studies.

1981 ◽  
Vol 256 (20) ◽  
pp. 10519-10523
Author(s):  
B. Ashby ◽  
H. Holmsen
Keyword(s):  
Kinetic Studies ◽  
Amp Deaminase

Intracellular distribution of α-galactosidase in leaves of Cucurbita pepo

1979 ◽  
Vol 57 (18) ◽  
pp. 1904-1911 ◽  
Author(s):  
Brian Thomas ◽  
John A. Webb
Keyword(s):  
Cucurbita Pepo ◽  
Developmental Stages ◽  
Molecular Size ◽  
Total Activity ◽  
Intracellular Distribution ◽  
Molecular Forms ◽  
Enzyme Release ◽  
Enzyme Preparations ◽  
Isolated Protoplasts

The intracellular distribution of α-galactosidase in leaves of Cucurbita pepo was studied at different developmental stages using tissue strips, homogenates, and isolated protoplasts. About 85% of the total activity was found in the 500 g supernatant after tissues were homogenized either in water, in buffer at pH 5.6 or at pH 7.0, or in buffer containing 0.8 M KCl. Isolated protoplasts contained less than 10% of the total activity which was confined to the 20 000 g supernatant after lysis. p-Nitrophenyl-α-D-galactoside was readily hydrolysed when incubated with leaf strips but less than 3% of α-galactosidase could be leached from strips held for 4 h in 100 mM phosophate buffer or in buffer containing either 0.8 M KCl, 1 mM EDTA, or 1 mM dithioerythritol. It is concluded that at all stages of leaf development a high proportion of α-galactosidase is located in the exocellular region, not strongly bound either to the outer surface of the plasmalemma or to the cell wall but prevented from diffusing through the wall matrix by some physical attribute such as molecular size. Enzyme release occurred only following breakage or enzymatic digestion of the wall. The in vivo properties of the exocellular enzyme in leaf strips were compared with those of three molecular forms of α-galactosidase (LI, LII, and LIII) which were partially purified from mature leaves. The exocellular enzyme was active over a broad pH range with optima at pH 3.0 and pH 6.0; this resembles a combination of pH optima for LI and LIII. Inhibition by Cu2+ and p-chloromercuribenzoate resembled that for LIII and LII, respectively. Galactose and galactinol at a 5 mM concentration were 25–30% inhibitory for all enzyme preparations; melibiose, raffinose, and stachyose were very weakly inhibitory. The function of an exocellular α-galactosidase and its bearing on the transport of galactosylsucrose oligosaccharides to and from the minor veins of C. pepo are discussed.

scholarly journals Acetyl-CoA:4-Hydroxybutinylbithiophene O-Acetyltransferase Isoenzymes from Tagetes patula Seedlings

1987 ◽  
Vol 42 (7-8) ◽  
pp. 885-890 ◽  
Author(s):  
Gernot Metschulat ◽  
Rainer Sütfeld
Keyword(s):  
Coenzyme A ◽  
Developmental Stages ◽  
Complete Separation ◽  
Tagetes Patula ◽  
Acetyl Coa ◽  
Ph Optimum ◽  
Acyltransferase Activity ◽  
Enzyme Preparations ◽  
Naturally Occurring ◽  
Acyl Coenzyme A

Naturally-occurring hydroxybutinylbithiophene derivatives were acylated by enzyme preparations of Tagetes patula seedlings in the presence of distinct acyl-Coenzyme A esters. The O-acyltransferase activity could only be detected after almost complete separation of the enzyme from counter-currently acting esterases which were present in the same extracts. This was achieved by affinity chromatography on Cibachron Blue A. During this procedure, the O-acyl-transferase was split, yielding two active fractions. Both had a Mr of 37,000 (±5,000), equal isoelectric properties, a pH optimum of pH 7.0, and were considerably inhibited in the presence of free Coenzyme A. Small differences existed in their affinities for their thiophenic substrates (3,4-dihydroxybutinylbithiophene and 4-hydroxybutinylbithiophene, respectively), as well as for various acyl-CoA esters as cosubstrates. With the preferred cosubstrate, acetyl-CoA, acylation took place at the 4-position of the butinyl side chain of the molecules, forming the naturally- occurring 4-acetoxybutinylbithiophene and 3-OH,4-OAc-butinylbithiophene, respectively. From the other acyl-CoA esters employed, only propionyl-CoA was likewise converted, forming the corresponding O-propionyl esters. The reactions observed are suggested to be catalyzed by two acetyl-CoA: 4-hydroxybutinylbithiophene O-acetyltransferase isoenzymes which exhibit different affinities for particular substrates and cosubstrates. The activities of both the isoenzymes changed drastically if plant material from different developmental stages was used as enzyme source. Therefore, it may be suggested that these isoenzymes play an important regulatory role in the metabolism of naturally-occurring hydroxy- and acetoxybutinylbithiophenes and their derivatives.

Mechanisms of inhibition of adenine phosphoribosyltransferase by adenine nucleosides and nucleotides

1970 ◽  
Vol 48 (5) ◽  
pp. 573-579 ◽  
Author(s):  
J. Frank Henderson ◽  
R. E. A. Gadd ◽  
H. M. Palser ◽  
M. Hori
Keyword(s):  
Adenosine Triphosphate ◽  
Kinetic Studies ◽  
Adenosine Diphosphate ◽  
Free Enzyme ◽  
Adenine Phosphoribosyltransferase ◽  
Enzyme Form

Kinetic studies of the inhibition of adenine phosphoribosyltransferase by adenine 6′-deoxyallofuranoside and 2′-deoxyadenylate indicate that both compounds bind to free enzyme and to the enzyme–phosphoribosylpyrophosphate complex, although they bind with different relative affinities to each enzyme form. The sites to which these inhibitors bind appear to be different from those to which substrates and products bind. Kinetic and physical studies show that adenosine diphosphate and adenosine triphosphate also bind to several enzyme forms, and that their mechanisms of inhibition of this enzyme are complex.

STUDIES ON A LIPOXIDASE SYSTEM FROM SUNFLOWER SEEDS AND SEEDLINGS

1958 ◽  
Vol 36 (11) ◽  
pp. 1149-1157 ◽  
Author(s):  
Oluf L. Gamborg ◽  
Saul Zalik
Keyword(s):  
Kinetic Studies ◽  
Maximum Activity ◽  
Sunflower Seeds ◽  
Reaction Products ◽  
Total Oxidation ◽  
Ph Optimum ◽  
Potassium Oleate ◽  
Enzyme Preparations ◽  
Cytoplasmic Proteins ◽  
Total Fat

Lipoxidase activity was obtained in enzyme preparations from sunflower seeds and seedlings. A partly purified preparation from seedlings was used for enzyme kinetic studies. The pH optimum was 6.8 and 100% oxygen was required for maximum activity. The Michaelis constant, with potassium linoleate as substrate, was 1.64 × 10−3 M. The reaction products were conjugated dienes. Enzyme activity was not affected by various metal and sulphydryl inhibitors nor by α-tocopherol, but catechol, α-naphthol, ethanol, and potassium oleate were inhibitory. Oil from flax, rape, and sunflower seeds reduced total oxidation of linoleate by the enzyme. Copper sulphate increased the rate and total oxidation of the linoleate–lipoxidase system, but iron, manganese, magnesium, and calcium were without effect. Lipoxidase activity was associated with mitochondrial (15,000 × g), intermediate (25,000 × g), and microsomal (100,000 × g) fractions, as well as with the soluble cytoplasmic proteins. Lipoxidase activity in seedlings increased during initial stages of germination, then decreased. The most rapid depletion of total fat in the seedlings coincided with maximum lipoxidase activity.

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