Brodie / Bressler Minireviews of the Neurosciences from Life Sciences

1. Auflage 2013
ISBN: 978-1-4831-5478-7
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: Adobe DRM (»Systemvoraussetzungen)

E-Book, Englisch, 504 Seiten

Minireviews of the Neurosciences from Life Sciences is a collection of minireviews of research in the neurosciences and originally published by the Journal of Life Sciences. These minireviews cover a wide range of topics such as the function and organization of the chromaffin vesicle; taste receptor proteins; the role of cyclic nucleotides in visual excitation; and regulation of tryptophan and tyrosine hydroxylase. Comprised of 34 chapters, this volume begins with a discussion on Na,K-ATPase, followed by an analysis of the function and organization of the chromaffin vesicles of the adrenal medulla. Subsequent chapters focus on the nerve growth factor and the acetylcholine receptor; sympathetic regulation of thyroid hormone secretion; the role of cyclic AMP in the action of antidiuretic hormone on the kidney; and neurochemical correlates of synaptically active amino acids. The role of calcium in the central effects of biogenic amines is also examined, along with the brain mechanisms underlying motor control, molecular coding of memory, and opiate receptors. This book should be of value to teachers, researchers, and students.

Autoren/Hrsg.

Brodie, Bernard B.

Bressler, R.

Leseproben

FUNCTION AND ORGANIZATION OF CHROMAFFIN VESICLE
Norman Kirshner,     Department of Biochemistry, Duke University Medical Center, Durham, N. C. 27710 (Received in final form 26 December 1973) Publisher Summary
This chapter describes the function and organization of chromaffin vesicle. The vesicles contain the highest concentration of adenine nucleotides, about 0.12 M, known to occur in any tissue or organelle. ATP is the major nucleotide component accompanied by smaller amounts of ADP and AMP, but the relative amounts vary in different species. The presence of small amounts of GTP, UTP, and mucopolysaccharides has also been reported. The concentration of CA within the vesicles is estimated to be 0.55 M. The molar ratio of CA: adenosine phosphates are close to 4:1 providing electrical equivalence at physiological pH within the vesicle. Disk-gel chromatography of the soluble proteins shows seven or eight major components and a similar number of minor components. Dopamine-3-hydroxylase is present both as a constituent of the membrane and as a constituent of the soluble interior milieu of the vesicles. In bovine and rabbit vesicles, 40 to 45% of the enzyme activity remains with the membrane after several washes, while in rat 80 to 85% of the activity remains with the vesicles. Chromaffin vesicles of the adrenal medulla were first isolated more than twenty years ago and shown to contain most of the adrenaline and noradrenaline present in the gland (1,2). Subsequent studies have indicated that very little of the catecholamines exist in the cytoplasm (3). During the past twenty years it has become apparent that chromaffin vesicles are not merely passive storage organelles but are structures highly organized to take up, synthesize, store, and secrete catecholamines (CA). It is the purpose of this discussion to examine each of these functions in relation to the current status of our knowledge of the composition and organization of the chromaffin vesicle. Composition of Chromaffin Vesicles
Highly purified chromaffin vesicles of the adrenal medulla can be isolated by a combination of differential and density gradient centrifugations. The overall composition of the vesicle is shown in Table 1. The vesicles are readily lysed upon suspension in distilled water or dilute buffer and after several washes practically all of the CA, adenosine phosphates, other small molecules and 75 to 80% of the total protein are solubilized. It is convenient to consider the soluble and particulate components separately. TABLE 1 Composition of Bovine Adrenal Storage Vesicles A Soluble Components The vesicles contain the highest concentration of adenine nucleotides, about 0.12 M, known to occur in any tissue or organelle. ATP is the major nucleotide component accompanied by smaller amounts of ADP and AMP but the relative amounts vary in different species. The presence of small amounts of GTP, UTP (4) and mucopolysaccharides (5,6) have also been reported. The concentration of CA within the vesicles is estimated to be 0.55 M. The molar ratio of CA:adenosine phosphates is close to 4:1 providing electrical equivalence at physiological pH within the vesicle. Disc-gel chromatography of the soluble proteins shows 7 or 8 major components and a similar number of minor components. Two of the proteins have been characterized – dopamine-ß-hydroxylase [EC 1.14.2.1] (DBH) which accounts for 5 to 7% of the protein and chromogranin A which represents 30 to 35% of the protein. The water soluble proteins have been named chromogranins but DBH should be excluded from this classification since it appears to be entirely different from the other proteins. Although a considerable amount of work has been done on chromogranin A there are some puzzling aspects of its structure and behavior in solution which require additional studies to understand (7). The remaining chromogranins have been characterized only to the extent that they are acidic proteins having an isoelectric point around pH 4.5, and that the amino acid composition of the mixture of chromogranins is very similar to the amino acid composition of chromogranin A. B Membrane Components The membrane of the chromaffin vesicle contains approximately 20% of the total vesicle protein and practically all of the lipid. On a dry weight basis the membrane of bovine adrenal vesicles contain 36% protein and 64% lipid. Approximately 23% of the total lipid is cholesterol and the remainder is a mixture of phospholipids containing an unusually high (15%) content of lysolecithin. Proteins which have been identified as constituents of the vesicle membrane are DBH, ATPase, cytochrome b 561 and a cytochrome b:NADH reductase. Gel electrophoresis of the membrane proteins after solubilization in sodium dodecyl sulfate (SDS) and mercaptoethanol shows the presence of 15 peptides with a pattern quite distinct from that of mitochondria and microsomes (7). Winkler and co-workers (8) have preposed the generic name chromomembrins for the protein constituents of the vesicle membranes. In their studies in which the membranes were solubilized in either phenol-acetic acid-urea or in SDS without the addition of mercaptoethanol they identified two major peptide components – chromomembrin A and chromomembrin B. Chromomembrin A appears to be identical with DBH while immunological studies with chromomembrin B suggest that this protein is localized on the exterior of the vesicle membrane (8). Chromogranin A has been reported to be a major constituent of the vesicle membrane (9) but other studies (7,8) indicate that it represents less than 10% of the total membrane protein and may be present as a contaminant. Dopamine-ß-hydroxylase is present both as a constituent of the membrane and as a constituent of the soluble interior milieu of the vesicles. In bovine and rabbit vesicles 40 to 45% of the enzyme activity remains with the membrane after several washes while in the rat 80 to 85% of the activity remains with the vesicles. This of course raises questions. Are the soluble and membrane bound enzymes different in structure? Is the soluble form of the enzyme derived from the membrane enzyme or vice versa? What structural properties of the enzyme enable it to become firmly associated with the membrane? What is the physiological significance of the distribution? Gel electrophoresis studies of the membrane bound and soluble form of the enzyme indicate no differences between the two. The amino acid composition of the soluble and membrane bound enzymes have been reported to be similar by two laboratories (10,11). The data agree well within each of the laboratories but there are several large discrepancies in the reported amino acid compositions between the groups. In addition a third study (12) of the amino acid composition of DBH shows ever more significant differences but it is not clear whether the purified DBH used in the latter study was the soluble form only or a mixture of the soluble and membrane bound enzymes. The data indicate that significant contamination occurred in one or more of the preparations and further work is necessary to determine whether the amino acid composition of the enzymes are identical. The subunit structure of DBH has been reported from several laboratories (8,12,13). The native enzyme has a molecular weight of 290,000 determined by sedimentation equilibrium in 0.1 M sodium chloride. In 6 M quanidine HCl the molecular weight is 150,000 to 160,000 and in 6 M quanidine HCl containing mercaptoethanol or dithiothreitol the molecular weight is 77,000. The subunit molecular weight of 77,000 has also been confirmed for both the soluble and membrane bound enzyme by gel electrophoresis after treatment with 1% SDS – 0.1% mercaptoethanol. The enzyme thus consists of subunits joined in pairs by disulfide bonds and two such units held together by non-covalent forces. Recently Wallace et al. (13) have shown DBH to be a glycoprotein containing about 4% carbohydrate which consists of residues of mannose, glucosamine, galactose, fucose and sialic acid. Role of ATP and Chromogranins in the Storage of Catecholamines
Studies of isolated chromaffin vesicles led to the concept that CA were maintained within the vesicle in a non-diffusible complex consisting of catecholamines, ATP and chromogranins (14). Physical chemical studies utilizing NMR spectroscopy and titrimetric methods indicated that only weak interactions between CA and ATP occurred in solutions and binding of CA to the chromogranins also could not account for the amount or stability of the amines within the vesicles. However other types of physical chemical studies have yielded more positive results. High speed centrifugation of mixtures of catecholamines and ATP results in the formation of sedimentable high molecular weight complexes of CA and ATP. Addition of low concentration of bivalent cations increases the degree of aggregation and upon dilution of solutions of CA and ATP (17% w/v CA and molar ratio of CA:ATP of 3:5) phase separation occurs in the presence of Ca++. Chromogranins and gelatin sediment with the multimolecular complexes of CA and ATP but serum albumin does not. Osmolality and...