Terminal synthesis of xanthommatin in Drosophila melanogaster. II. Enzymatic formation of the phenoxazinone nucleus

Xanthommatin, the brown eye pigment of Drosophila melanogaster is a phenoxazinone compound formed by the bimolecular oxidative condensation of 3-hydroxykynurenine. Evidence is presented in this report for an enzyme system in drosophila which catalyzes the formation of the phenoxazinone chromophore of xanthommatin. This enzyme system, which is separable from the interfering activities of phenol oxidase and cytochrome oxidase, is relatively inactive in crude extracts but can be activated either by heat treatment or by passage through Sephadex G-50. The preliminary characterization of several properties of the enzyme-catalyzed reaction and their biological significance are discussed.Supported by USPHS grant GM12323 and by the Robert A. Welch Foundation, Houston, Texas.Postdoctoral Fellow supported by USPHS (1 FO2 GM44, 562-01) and the Rosalie B. Hite Memorial Fund.     

Terminal synthesis of xanthommatin in Drosophila melanogaster. I. Roles of phenol oxidase and substrate availability

Eye color mutants of Drosophila melanogaster are known which block the conversion of 3-hydroxykynurenine to xanthommatin. It has been proposed that this reaction depends on the presence of 3-hydroxykynurenine and a redox system maintained by phenol oxidase activity. The mutants st and ltd lack throughout development detectable amounts of 3-hydroxykynurenine or its metabolic derivatives. When the substrate is fed or injected, these mutants fail to form xanthommatin even though phenol oxidase activity is normal. The mutant cd accummulates excessive amounts of 3-hydroxykynurenine, has normal phenol oxidase activity, but is also deficient in xanthommatin formation. Mutants are also known which lack phenol oxidase activity but nevertheless form xanthommatin. It is concluded that the proposed relationship between 3-hydroxy-kynurenine and phenol oxidase activity is not sufficient to explain the in vivo synthesis and regulation of synthesis of xanthommatin in Drosophila. The bearing of these findings on the actual mode of synthesis is discussed.Supported by PHS 1029 and NSF GB-4539.     

Multiple actins in drosophila melanogaster

The tissue and developmental specificities of the three Drosophila isoactins, originally identified in primary myogenic cultures and in the permanent Schneider L-2 cell line, have been investigated. Of these three isoactins (I, II, and III), actins I and II are stable and actin III is unstable. Two-dimensional polyacrylamide gel electrophoretic analyses of total cellular extracts after 1-h [(35)S]methionine pulses were performed on a large variety of embryonic, larval, and adult muscle and nonmuscle tissues. The results suggest that isoactins II and III are generalized cellular actins found in all drosophila cell types. Actin I, on the other hand, is muscle-associated and is found exclusively in supercontractile muscle (such as larval body wall and larval and adult viscera) including primary myogenic cell cultures. Although actin I synthesis is not detectable during very early embryogenesis, it is detectable by 25 h and actin I is a major stable actin in all larval muscle tissues. Actin I is synthesized in reduced amounts relative to the other actins in late third instar larvae but is again a major product of actin synthesis in the adult abdomen. A stable actin species with the same pI as actin III has been identified in the adult thorax and appears to be unique to flight muscle tissue. This new stable form of thoracic actin may be the result of a stabilization of the actin III found in other tissues or may be an entirely separate gene product.     

Enzymatic and nonenzymatic cross-linking of collagen and elastin.

Knowledge regarding the steps and mechanisms related to the intra- and interchain cross-linking of collagen and elastin has evolved steadily during the past 30 years. Recently, effort has been directed at identifying the location and types of cross-links that are found in collagen and elastin. There are two major groups of cross-links: those initiated by the enzyme lysyl oxidase and those derived from nonenzymatically glycated lysine and hydroxylysine residues. The formation of enzymatic cross-links depends on specific enzymes, amino acid sequences, and quaternary structural arrangements. The cross-links that are derived nonenzymatically occur more adventitiously and are important to pathobiological processes. Considerable progress has been made in elucidating the pathways of synthesis for several of the enzymatically mediated cross-links, as well as possible mechanisms regulating the specificity of cross-linking. Although less is known about the chemistry of cross-links arising from nonenzymatically glycated residues, recent progress has also been made in understanding possible biosynthetic pathways and control mechanisms. This review focuses on such progress and hopes to underscore the biological importance of collagen and elastin cross-linking.     

Enzymatic and nonenzymatic ADP-ribosylation of cysteine

Mono-ADP-ribosylation is a protein modification that occurs at a number of different amino acids, dictated by the specificity of the individual ADP-ribosyltransferases. A specific cysteine in several guanine nucleotide-binding regulatory proteins is ADP-ribosylated by the bacterial protein pertussis toxin. Recent purification of an ADP-ribosylcysteine hydrolase and NAD:cysteine ADP-ribosyltransferase, and detection of ADP-ribose-cysteine linkages in tissue samples has raised hope that an endogenous regulatory cysteine-specific ADP-ribosylation pathway exists. A current goal is the identification of such a pathway for ADP-ribosylation of cysteine within animal cells. Interpretation of the data in this field has been complicated by recent reports that revealed several unforeseen chemical reactions of NAD and its metabolites with free cysteine and cysteine in proteins. This mini-review covers the latest understanding of the ADP-ribosylation reactions associated with cysteine, and provides a set of criteria for future research to establish positively the existence of an endogenous cysteine-specific mono-ADP-ribosyltransferase.     

Striated visceral muscle of drosophila melanogaster

Striated visceral muscle cells are scattered singly or in small groups at the base of the intestinal cells of the mid-gut of Drosophila melanogaster larvae. Fibers less than 1 μ in diameter, designated as small, contain a single myofibril, few, if any, dyads and few mitochondria. Fibers of somewhat larger diameter contain dyads and more mitochondria. Both types of fiber have a perforate Z band which appears as discontinuous bodies in longitudinal sections and as a perforate sheet of dense rims and clear perforations in transverse sections. The Z rims contain filaments, 30 to 50 Å, oriented in the transverse direction. The number and arrangement of myofilaments and the ultrastructure of the Z band are consistent with the function of these muscles.     

Enzymatic and nonenzymatic dehydration reactions of L-arogenate

L-Arogenate, an immediate precursor of either L-tyrosine, L-phenylalanine, or both in many microorganisms and plants, may undergo two types of dehydration reactions that yield products of increased stability. Under acidic conditions, a facile aromatization attended by loss of the C-4 hydroxyl and the C-1 carboxyl moieties results in quantitative conversion to L-phenylalanine. When aromatization was largely prevented by maintaining pH in the range of 7.5-12, a second dehydration reaction occurred in which the alanyl side chain and the carboxyl group at C-1 formed a lactam ring to yield spiro-arogenate. The latter reaction occurs at 100 degrees C, roughly 50% conversion being obtained in 2 h. The product formed from L-arogenate was authentic spiro-arogenate, as demonstrated by high-performance liquid chromatography and thin-layer chromatography identification procedures. Further confirmation was obtained by 1H nuclear magnetic resonance, ultraviolet spectroscopy, and mass spectrometry. Thus far, the conversion of L-arogenate to spiro-arogenate is not known to be enzyme catalyzed. The other dehydratase reaction, however, is catalyzed in nature by an enzyme denoted arogenate dehydratase. An improved assay is described for this in which [3H]dansyl derivatives of L-arogenate (substrate) and L-phenylalanine (product) are separated by using bidimensional thin-layer chromatography. The radioactive reaction product is then quantitated. This assay was used to study partially purified arogenate dehydratase from Pseudomonas diminuta, an organism that depends upon the arogenate pathway for L-phenylalanine biosynthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

The ommochrome biosynthetic pathway in Drosophila melanogaster: The head particulate phenoxazinone synthase and the developmental onset of xanthommatin synthesis

Particulate fractions from the heads of Drosophila melanogaster catalyze the conversion of o-aminophenols to phenoxazinones. This particulate enzyme is stimulated by Mn²⁺. It has a number of features which distinguish it clearly from the Mn²⁺-dependent activity found in the soluble fraction. The particulate enzyme has a characteristic developmental pattern, showing a marked increase in activity at about the time of onset of xanthommatin synthesis. In addition, it is much reduced in activity in a number of xanthommatin-deficient mutants (v, cn, st, cd, and w). We believe that the head particulate enzyme is involved in xanthommatin biosynthesis and that the developmental onset of synthesis of this pigment is brought about by the synthesis or activation of this enzyme.     

Pigment patterns in mutants affecting the biosynthesis of pteridines and xanthommatin in Drosophila melanogaster

Eye-color mutants of Drosophila melanogaster have been analyzed for their pigment content and related metabolites. Xanthommatin and dihydroxanthommatin (pigments causing brown eye color) were measured after selective extraction in acidified butanol. Pteridines (pigments causing red eye color) were quantitated after separation of 28 spots by thin-layer chromatography, most of which are pteridines and a few of which are fluorescent metabolites from the xanthommatin pathway. Pigment patterns have been studied in 45 loci. The pteridine pathway ramifies into two double branches giving rise to isoxanthopterin, drosopterins, and biopterin as final products. The regulatory relationship among the branches and the metabolic blockage of the mutants are discussed. The Hn locus is proposed to regulate pteridine synthesis in a step between pyruvoyltetrahydropterin and dihydropterin. The results also indicate that the synthesis and accumulation of xanthommatin in the eyes might be related to the synthesis of pteridines.Support for this work was provided to J.F. in part by a grant from the Ministerio de Universidades e Investigación (Spain) and to F.J.S. by a grant from the Ministerio de Educación y Ciencia (Spain).     

Enzymatic and nonenzymatic degradation of myelin basic protein

A procedure for large scale isolation of myelin basic protein (BP) has been modified to insure BP preparations free of neutral proteinase activity. Fractions were monitored by electrophoretic analysis of BP solutions incubated under various conditions of temperature and pH. Maximum degradation of human BP prepared by the old batch procedure occurs at pH 7, 47°C. BP preparations obtained by the new procedure, as well as BP preparations purified by CM-cellulose chromatography, are stable under these conditions. The latter, however, do undergo significant breakdown at pH 9, 100°C. The results suggest that the degradation observed under these conditions is non-enzymatic in nature.Special Issue dedicated to Dr. Elizabeth Roboz-Einstein.     

Functional analysis of iodotyrosine deiodinase from drosophila melanogaster

The flavoprotein iodotyrosine deiodinase (IYD) was first discovered in mammals through its ability to salvage iodide from mono‐ and diiodotyrosine, the by‐products of thyroid hormone synthesis. Genomic information indicates that invertebrates contain homologous enzymes although their iodide requirements are unknown. The catalytic domain of IYD from Drosophila melanogaster has now been cloned, expressed and characterized to determine the scope of its potential catalytic function as a model for organisms that are not associated with thyroid hormone production. Little discrimination between iodo‐, bromo‐, and chlorotyrosine was detected. Their affinity for IYD ranges from 0.46 to 0.62 μM (K_d) and their efficiency of dehalogenation ranges from 2.4 – 9 x 10³ M^?1 s^?1 (k_cat/K_m). These values fall within the variations described for IYDs from other organisms for which a physiological function has been confirmed. The relative contribution of three active site residues that coordinate to the amino acid substrates was subsequently determined by mutagenesis of IYD from Drosophila to refine future annotations of genomic and meta‐genomic data for dehalogenation of halotyrosines. Substitution of the active site glutamate to glutamine was most detrimental to catalysis. Alternative substitution of an active site lysine to glutamine affected substrate affinity to the greatest extent but only moderately affected catalytic turnover. Substitution of phenylalanine for an active site tyrosine was least perturbing for binding and catalysis.     

非水相体系酶催化反应研究进展

非水相酶催化反应是酶催化反应中的一个重要方面。非水相溶剂通常可增加底物溶解度,减少水相中的副反应,加快生物催化的速率和效率,在药物及药物中间体和食品等方面具有较大的应用价值。以下探讨了非水相体系对酶活力及酶促反应速率的影响因素,并阐述酶的化学修饰、固定化及定点突变对酶活力的影响,进一步分析无溶剂系统、反胶束、超临界流体及离子液体的不同溶剂体系对酶反应速率及催化效率的影响。此外,还列举一些非水相酶催化反应的应用实例。