scholarly journals Adenylate metabolism in the heart. Regulatory properties of rabbit cardiac adenylate deaminase

1979 ◽  
Vol 182 (2) ◽  
pp. 361-366 ◽  
Author(s):  
R Barsacchi ◽  
M Ranieri-Raggi ◽  
C Bergamini ◽  
A Raggi
Keyword(s):  
Skeletal Muscle ◽  
Kinetic Properties ◽  
Amp Deaminase ◽  
Alkaline Ph ◽  
Muscle Enzyme ◽  
Adenylate Deaminase ◽  
Activated State ◽  
Maximal Activation ◽  
Regulatory Properties

The kinetic properties of a 300-fold purified cardiac AMP deaminase were studied and compared with those of the corresponding enzyme from skeletal muscle. The heart enzyme is activated by ATP and less efficiently by ADP, and is inhibited by Pi, phosphocreatine and GTP. ATP, even at micromolar concentrations, is able to abolish the effects of the inhibitors. The affinity of the enzyme for AMP is low in the absence of activators (Km 3.1 mM), but, in the presence of ATP, becomes as high as that of skeletal-muscle AMP deaminase (Km 0.4 mM). The maximal activation by ATP is observed at alkaline pH (pH 7.5-8.0). Under the same conditions ATP is maximally inhibitory for skeletal-muscle enzyme. These results suggest that AMP deaminase in the heart is always in the activated state, whereas in skeletal muscle the enzyme is active only during exhaustive contractions.

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 Expression, purification and kinetic properties of human recombinant phospholipase C delta 3.

1997 ◽  
Vol 44 (2) ◽  
pp. 221-229 ◽  
Author(s):  
T Pawełczyk ◽  
A Matecki
Keyword(s):  
Phospholipase C ◽  
Kinetic Studies ◽  
Exchange Chromatography ◽  
Kinetic Properties ◽  
Cation Exchange Chromatography ◽  
E Coli ◽  
His Tag ◽  
Absolute Requirement ◽  
Maximal Activation ◽  
Regulatory Properties

To obtain sufficient quantities of pure phospholipase C delta 3 (PLC delta 3) necessary for structural and kinetic studies, cDNA of human fibroblast PLC delta 3 was cloned in the pPROEX-1 vector, expressed in E. coli cells as a (6 x His) fusion protein and purified to homogeneity. From 1 L of E. coli culture 8 mg of pure PLC delta 3 was obtained by a two step purification procedure, which includes phosphocellulose and Mono S cation exchange chromatography. The presence of His tag did not affect the catalytic and regulatory properties of PLC delta 3. The K(app) for PIP2 was 142 +/- 11 and 156 +/- 12 microM for His.PLC delta 3 and PLC delta 3, respectively. Recombinant PLC delta 3 showed an absolute requirement for Ca2+. Increasing the free Ca2+ concentration from 0.2 to 0.5 microM resulted in a sharp increase in enzyme activity. In comparison with human recombinant PLC delta 1 the delta 3 isoenzyme was more sensitive to low Ca2+ concentration. The Ca2+ concentration yielding maximal activation of PLC delta 1 and PLC delta 3 was 10 and 1 microM, respectively. The activity of PLC delta 3 was stimulated by polyamines and by basic proteins such as protamine, histone and mellitin. PLC delta 3 was activated most effectively by spermine and histone but the extent of this activation was lower than for PLC delta 1. The data presented indicate that the expression of PLC delta 3 in E. coli cells permits to obtain active enzyme. The catalytic and regulatory properties of PLC delta 3 are similar to those of PLC delta 1.

scholarly journals Properties and reaction with iodoacetamide of adenosine 5′-triphosphate–creatinine phosphotransferase from human skeletal muscle. Further evidence about the role of the essential thiol group in relation to the mechanism of action

1970 ◽  
Vol 117 (3) ◽  
pp. 513-523 ◽  
Author(s):  
I. Kumudavalli ◽  
B. H. Moreland ◽  
D. C. Watts
Keyword(s):  
Skeletal Muscle ◽  
Transition State ◽  
Thiol Group ◽  
Equilibrium Mixture ◽  
Common Mechanism ◽  
Kinetic Properties ◽  
Rabbit Muscle ◽  
Muscle Enzyme ◽  
Similar Chemical ◽  
High Concentrations

1. The purification of creatine kinase from human and monkey skeletal muscle by horizontal electrophoresis on Sephadex blocks is described. 2. The purified enzymes are shown to have similar chemical and kinetic properties to the rabbit muscle enzyme and a common mechanism is inferred. 3. Iodoacetamide has a similar apparent second-order inhibition constant with the human and rabbit enzymes, but the inhibition does not go to completion with the former. This is even more marked with the monkey enzyme, which has more reactive thiol groups, but inhibition is only about 50%. 4. Single substrates have little effect on the inhibition by iodoacetamide, but with the primate enzymes, in contrast with the rabbit enzyme, high concentrations of ADP–Mg2+ plus creatine convert the essential thiol group from being pH-independent into one with a normal ionization. Low concentrations of ADP–Mg2+ plus creatine first enhance the rate of inactivation, but cause protection as the reaction proceeds. These results are interpreted to indicate an activation of the thiol group on the subunit to which the substrates bind and a co-operatively induced decrease in the activity of the thiol group on the other subunit which lacks substrates. 5. The effects of a substrate equilibrium mixture on the rate of inhibition are essentially those of ADP–Mg2+ plus creatine. 6. Since no substrate combination affords significant protection to the thiol group associated with the catalytic site to which the substrates are bound, it is concluded that any mechanism involving the thiol group in a direct participation in the transition-state complex of the catalytic reaction must be abandoned unless the transition state is only a small part of the time taken for one catalytic cycle.

scholarly journals Regulatory properties of AMP deaminase isoenzymes from rabbit red muscle

1987 ◽  
Vol 242 (3) ◽  
pp. 875-879 ◽  
Author(s):  
A Raggi ◽  
M Ranieri-Raggi
Keyword(s):  
Skeletal Muscle ◽  
Inhibitory Action ◽  
Ph Value ◽  
Velocity Curve ◽  
Amp Deaminase ◽  
Work Intensity ◽  
Red Muscle ◽  
Regulatory Properties ◽  
Optimal Ph

We examined the kinetic and regulatory properties of the two isoenzymes of red muscle AMP deaminase, forms A and B, corresponding respectively to the single isoenzymes present in the heart and white skeletal muscle. At the optimal pH value, 6.5, both enzymes show hyperbolic substrate-velocity curves and are inhibited by GTP, inducing sigmoid kinetics. An effect similar to that of GTP is exerted on form B by ATP, whereas form A is almost insensitive to this nucleotide. At pH 7.1 both enzymes follow sigmoid kinetics. ATP enhances the sigmoidicity of the substrate-velocity curve of form B, but it stimulates form A, reverting sigmoidal to hyperbolic kinetics shown by the enzyme at optimal pH. At pH 7.1, form A is also less sensitive to the inhibitory action of Pi and GTP. These results suggest that, owing to the presence of form A, AMP deamination occurs in red muscle also at moderate work intensity. A possible role of this process in counteracting the production of adenosine by 5′-nucleotidase is hypothesized.

scholarly journals AMP deaminase histochemical activity and immunofluorescent isozyme localization in rat skeletal muscle.

1992 ◽  
Vol 40 (7) ◽  
pp. 931-946 ◽  
Author(s):  
J L Thompson ◽  
R L Sabina ◽  
N Ogasawara ◽  
D A Riley
Keyword(s):  
Skeletal Muscle ◽  
Vascular Tissue ◽  
Immunofluorescence Microscopy ◽  
Capillary Endothelium ◽  
Kinetic Properties ◽  
Amp Deaminase ◽  
Rat Skeletal Muscle ◽  
Connective Tissues ◽  
Functional Roles ◽  
Liver And Kidney

The cellular distribution of AMP deaminase (AMPda) isozymes was documented for rat soleus and plantaris muscles, utilizing immunofluorescence microscopy and immunoprecipitation methods. AMPda is a ubiquitous enzyme existing as three distinct isozymes, A, B and C, which were initially purified from skeletal muscle, liver (and kidney), and heart, respectively. AMPda-A is primarily concentrated subsarcolemmally and intermyofibrillarly within muscle cells, while isozymes B and C are concentrated within non-myofiber elements of muscle tissue. AMPda-B is principally associated with connective tissues surrounding neural elements and the muscle spindle capsule, and AMPda-C is predominantly associated with circulatory elements, such as arterial and venous walls, capillary endothelium, and red blood cells. These specific localizations, combined with documented differences in kinetic properties, suggest multiple functional roles for the AMPda isozymes or temporal segregation of similar AMPda functions. Linkage of the AMPda substrate with adenosine production pathways at the AMP level and the localization of isozyme-C in vascular tissue suggest a regulatory role in the microcirculation.

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