Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-09-10
2004-07-13
Saucier, Sandra E. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S015000, C435S016000, C435S021000
Reexamination Certificate
active
06762026
ABSTRACT:
TECHNICAL FIELD
The present invention provides a method of determining cAMP content or an adenylate cyclase activity in a biological sample containing cAMP (cyclic adenosine-3′,5′-monophosphate) produced from ATP by endogenous adenylate cyclase and non-cyclic adenine nucleotides selected from the group consisting of ATP (adenosine-triphosphate), ADP (adenosine-diphosphate), AMP (adenosine-3′,5′-monophosphate) and a mixture thereof without the use of radioactive agents. More particularly, the invention relates to a method which comprises: (1) combining a biological sample with effective amounts of apyrase, adenosine deaminase and alkaline phosphatase to enzymatically remove non-cyclic adenine nucleotides other than cAMP, and glucose-6-phosphate in the sample; (2) enzymatically converting cAMP into AMP; (3) determining an amount of AMP without the use of radioactive agents.
Adenylate cyclase (adenylyl cyclase, adenylate cyclase, EC4,6.1.1) is an enzyme catalyzing the conversion:
ATP→cAMP
in the presence of Mg
2+
or Mn
2+
.
Adenylate cyclase exists locally on cell membranes and plays a critical role as a signal transduction cascade of a number of fundamental hormones and neurotransmitters.
For example, measurement of adenylate cyclase activity has been employed to study the altered physiology exhibited by transplanted human hearts and in congestive heart failure. See M. R. Bristow et al.,
New Engl. J. Med
., 307, 205 (1982); K. G. Lurie et al.,
J. Thorac. Cardiovasc. Surg
., 86, 195 (1983).
Adenylate cyclase activity can be determined by monitoring the changes of CAMP content synthesized from ATP by the catalytic action of adenylate cyclase.
However, a more clear elucidation of the biological role of adenylate cyclase has been limited by the difficulty in monitoring accurately changes in the tissue level of cAMP.
cAMP (cyclic adenosine-3′,5′-monophosphate) was found as a factor which intermediates blood sugar rising action of adrenaline and glucagon in liver cells. [E. W. Sutherland et al.,
J. Am. Chem. Soc
., 79, 3608 (1957)]. And also cAMP was found to intermediate actions of hormones such as adrenocorticotropin (ACTH), luteinizing hormone (LH), tyrosine stimulating hormone (TSH) and parathyroid hormone (PTH) or physiological active substances such as prostaglandin. Thus, when peptide hormones or active amines have been secreted and have reached at target cells, cAMP transfers information for them to proceed enzymatic reactions, that is, plays a role of a second messenger.
cAMP is synthesized from ATP by adenylate cyclase located on membranes in the living body and decomposed by phosphodiesterase into 5′-AMP. cAMP is present widely in bacteria or animals but the concentration of cAMP is extremely low (a stationary concentration is 0.1-1 nmol/g wet weight). As an assay of cAMP, an assay using cAMP binding protein or radioimmunoassay is conveniently employed. The cAMP content depends on eutrophy, proliferation, differentiation, adaptation of cells and changes in sensitivity.
Measurement of cAMP in a wide variety of mammalian and non-mammalian tissue and fluids provides a useful way to assess cell viability, endocrine hormonal axis function, adenylate cyclase activity and phosphodiesterase activity. In addtion, measurement of cAMP can be used to evaluate the activity of a number of signal transproduction proteins, including, but not limited to, the family of G proteins (guanine-nucleotide binding protein) which play a major role in signal transduction, ribosomal protein synthesis, translocation of nascent proteins and other important cellular functions. Bourne et al.,
Nature
, 348, 125 (1990).
Furthermore, measurement of cAMP may be used in evaluating other endogenous and exogenous compounds (for example, nitrous oxide) which may alter the level of cyclic nucleotides in a particular cell, tissue, organ or body fluid.
Many hormones use cAMP as a second messenger including, but not limited to: epinephrine, norepinephrine, adrenocorticotropin (ACTH), vasopression, glucagon, thyroxine, and thyroid-stimulating and melanocyte-stimulating hormones which are some of the principle regulatory hormones/proteins in the living organism. The activity of all of these hormones and regulators can be measured in tissues, serum, body fluids, and in all cell cultures (cells and medium) using the method for cAMP of the present invention. Measurement of these hormones is performed in a wide variety of disease states where hormonal imbalance may lead to specific pathology.
Once a hormone or regulatory protein interacts with a specific receptor, the second messenger, in this case, cAMP, is produced through a cascade of biochemical events. The production of cAMP can also be specifically inhibited in some cases by hormones which use a decrease in cAMP as part of the specific hormonal signal-transduction pathway. The result of this regulatory protein or hormone and receptor interaction can be, but is not limited to, (1) an alteration in cell permeability secondary, for example, to changes in ion channels, (2) and alteration in the rate of enzyme catalyzed reactions sensitive to the concentration of cAMP, and (3) an alteration in the rate of protein synthesis including the synthesis and degradation of other enzymes. a content of cAMP can be used to directly and indirectly monitor the consequences after interacting a hormone or regulatory protein with a receptor.
Specifically, cAMP can be measured in urine or blood for use as a marker for drug levels, like aminophylline or theophylline which stimulate the adrenergic nervous system by preventing the breakdown of endogenous cAMP. Measurement of cAMP in cell cultures can be used to-assess specific hormones, regulatory protein and drugs where cAMP represents a vital link in the signal transduction process.
cAMP can also be used to assess cell viability and stability by studying cells in the absence or presence of a specific hormone or regulatory protein. For example, measurement of cAMP in liver cells (hepatocyte) by glucagon, can be used to assess hepatocyte viability. This may be useful, for example, in organ and/or cell transplantation, for example heart, liver, lung, kidney, pancreas, skin and brain cell transplantation.
Measurement of the responsiveness of cells from biopsy samples after activation by a wide variety of hormones, regulatory proteins and drug which either increase or decrease cellular cAMP levels, can be used as a way to specifically assess cell function.
A specific clinical example is the use of cAMP measurement in cardiac biopsies to assess the responsiveness of myocardium. Cardiomyopathic heart cells do not respond with the same rise in cAMP content after &bgr;-adrenergic stimulation as normal heart cells. The diagnosis of the severity of the heart disease and the efficacy of some drugs, such as &bgr;-adrenergic blockers and angiotensin converting enzyme inhibitors, can be made comparing the responsiveness of biopsy samples from normal hearts to cardiomyopathic hearts. Measurement of basal and/or stimulated levels of adenylate cyclase activity or cAMP in blood cells can be used to guide therapy in such patients. In addition, release of cAMP either intracellarly or into the arterial or venous circulation can be used as an indicator of the response of an organ and/or tissue to a variety of different physiologic and nonphysiologic stresses such as ischemia, hypoxia, or drug or hormonal stimulation. Tissue or body fluid levels of cAMP can be measured in nearly ever mammalian cell or body fluid, including blood cells and platelets, with this approach. In some tissues, cAMP levels can be measured in response to specific stimulators as an index on oncogenicity and/or invasiveness, in the case of samples of potentially tumorous cells. In other cases, measurement of cAMP can be used to determine the effectiveness of specific therapies which may alter cAMP synthesis or degradation.
As described above, cAMP plays an important role as a second messenger in information transfer in
Fuso Pharmaceutical Industries Ltd.
Saucier Sandra E.
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