Subunit size and genetic variation of enzymes in natural populations of Drosophila

The relationships between various measures of enzyme polymorphism and subunit molecular weight of 11 dimeric Drosophila enzymes were analyzed with data from the current literature. All correlations were positive and highly significant. Eighteen percent of the variance of the numbers of alleles per sample for each locus and 60% of the variance of the average numbers of alleles per locus were due to variations of subunit weight. Identical results were obtained from analyses of heterozygosity. Small subunit dimers exhibit a characteristic skewed frequency spectrum of alleles, while the alleles of larger subunit enzymes are more equal in frequency. These relationships suggest that enzyme polymorphism is related to the enzyme structure, particularly weight (size), of constituent subunits.     

Relationship between subunit size and number of rare electrophoretic alleles in human enzymes

Data from published sources were used to compare the numbers of different electrophoretic alleles of 29 monomeric and dimeric human enzymes to their respective subunit molecular weights. Only those human enzymes were considered for which the total sample sizes were in excess of 2000 individuals. Correlations between these two variables were determined within sample size ranges of 2000≤n≤3000 and 4000≤n≤5000 individuals, and separately by quaternary class. There was no statistically significant correlation observed for the smaller sample size range in monomers; however, the correlations for the larger sample size range in monomers and both ranges in dimers were significant. Since there is no relationship between subunit size and heterozygosity, the relationships are due primarily to the incidence of rare alleles. These findings demonstrate the effect of locus-specific mutation rates, expected as a consequence of variation of cistron sizes, and imply that other forces are responsible for the relative frequencies of common alleles at some of the loci.     

Biochemical correlates of genetic variation in marine lower invertebrates

In this paper extensive data on enzyme variation in 23 species of coelenterates and sponges were used to investigate the possible correlation of levels of genetic variation with various parameters of enzyme molecular structure and function. The data provide an opportunity not only to look for such correlations for the first time in lower invertebrates, but also to study organisms with far higher average levels of genetic variability than those used in any previous work. A clear inverse relationship was found between enzyme subunit number and levels of polymorphism, with monomers being more variable than dimers or tetramers. No significant difference in polymorphism could be found in enzymes of the functional groups I and II of Gillespie and Langley (1974). Regulatory enzymes appeared to be significantly more polymorphic than nonregulatory enzymes, but a significant relationship was observed between regulatory power and subunit structure which could bias this result. The results suggest that both neutralist and selectionist ideas may have a useful role to play in the understanding of the factors which can influence or limit levels of genetic variation.A. M. Sole-Cava was supported by grants from CAPES and FAPERJ (Brazil).     

Enzyme function and polymorphism: A test in two Anolis lizard species

Electrophoretic surveys of two lizard species were used to test hypotheses that relate levels of enzyme variability to enzyme function (single-substrate versus multiple-substrate enzymes, regulatory versus nonregulatory enzymes). Anolis roquet behaviorally regulates its body temperature, but its congener A. gundlachi is passive to variations in the thermal environment. As a result, populations of A. gundlachi probably experience the thermal environment as temporally coarse-grained, whereas populations of A. roquet do not. We therefore predicted that A. gundlachi would exhibit greater enzyme heterozygosity than A. roquet and that different enzyme classes would contribute disproportionately to this interspecific difference. The data show (1) that A. gundlachi does have a higher heterozygosity and (2) that this difference appears to result from high levels of heterozygosity at loci coding for multiple-substrate enzymes. The dichotomy between regulatory and nonregulatory enzymes offers no explanation for the variability in heterozygosity among enzyme loci in these species.E.Z. was supported by a grant from the Natural Sciences and Engineering Council of Canada. The study was accomplished while P.E.H. held a postdoctoral fellowship from the Killam Trust of Dalhousie University and a grant from the Research Development Fund of Dalhousie University. The collection of material was made possible by grants (to P.E.H.) from National Science Foundation (DEB 75-16334), the Explorers Club of New York, Sigma Xi, and the Richmond and Anderson Funds of Harvard University. We thank Dr. D. W. Foltz for his help with the calculations.     

Enzyme variation in the brook lamprey,Lampetra planeri (Bloch), a member of the vertebrate group Agnatha

A natural population of the brook lamprey, Lampetra planeri, was assayed for electrophoretically detectable variation at 30 enzyme loci. The mean heterozygosity per locus of this primitive vertebrate, a member of the Agnatha, was found to be 0.076 ± 0.031, a value similar to those recorded for other vertebrates. The high chromosome numbers recorded for this and related species have been attributed to polyploidy, but our studies do not indicate the existence of large numbers of duplicated loci. Indeed, several enzymes that are encoded by duplicate loci in other vertebrate species appear to be encoded by single loci in the lamprey. It is suggested that studies on the biology and taxonomy of lampreys will benefit greatly from an electrophoretic approach.     

Protein variation in the marine bivalve Scrobicularia plana

Seventeen enzyme loci have been assayed for electrophoretically detectable variation in a population of the marine bivalve Scrobicularia plana . Mean heterozygosity is 0.120 ± 0.033. In a comparison involving thirteen enzymes there is a significant correlation between heterozygosity in S. plana and Mytilus edulis and a suggestion of lower mean heterozygosity in S. plana . These findings are discussed in relation to current theories concerning the selective significance of protein variation.     

Fused dimerization increases expression,solubility, and activity of bacterial dehydratase enzymes

FabA and FabZ are the two dehydratase enzymes in Escherichia coli that catalyze the dehydration of acyl intermediates in the biosynthesis of fatty acids. Both enzymes form obligate dimers in which the active site contains key amino acids from both subunits. While FabA is a soluble protein that has been relatively straightforward to express and to purify from cultured E. coli, FabZ has shown to be mostly insoluble and only partially active. In an effort to increase the solubility and activity of both dehydratases, we made constructs consisting of two identical subunits of FabA or FabZ fused with a naturally occurring peptide linker, so as to force their dimerization. The fused dimer of FabZ (FabZ‐FabZ) was expressed as a soluble enzyme with an ninefold higher activity in vitro than the unfused FabZ. This construct exemplifies a strategy for the improvement of enzymes from the fatty acid biosynthesis pathways, many of which function as dimers, catalyzing critical steps for the production of fatty acids.     

Physical and functional characterisation of monomeric and dimeric eukaryotic cytochrome c oxidases

Cytochrome c oxidases were isolated from heart tissue of beef (Bos tauros), sheep (Ovis aries), horse (Equus caballus), pig (Sus scrofa) (native dimers) and hammerhead shark (Sphyrna lewinii) (native monomer). Limited proteolysis of dimeric enzymes selectively depleted subunit III, resulting in monomerisation and a blue shift (2nm) of the reduced α band to the same wavelength maximum (603nm) as that of the hammerhead shark enzyme. Monomeric enzymes retain the ability to accept electrons rapidly from cytochrome c, and the second-order rate constants for electron transfer between cytochromes c and a are reported. The steady-state kinetics of both native and subunit III-depleted cytochrome c oxidases were biphasic, thus ruling out any explanation for this behaviour that depends on cooperation between functional units (monomers) within a dimer. Functional integrity of the subunit III-depleted enzyme prepared by proteolysis was maintained during multiple turnover, in contrast to reports elsewhere of loss of activity caused by subunit III removal by other means. A model is proposed to explain this difference, in which removal of a hydrophobic membrane-spanning segment of subunit III leads to monomerisation but a residual extra-membrane segment is retained, preserving the functional integrity of the enzyme.     

Variabilité et différenciation génétiques chez cing espèces d'ecrevisses Astacidae

Analysis of fifteen loci encoding enzymes has shown an important differentiation between five European and North American species of the Astacidae. Evaluation of the genetic distances revealed that closely related species are on the one hand, Orconectes limosus and Procambarus clarkii and, on the other, Astacus astacus and Astacus leptodactylus. This analysis brings no genetic justification for the separation of these five species into two sub-families. However, the distinction between the genera Astacus and Austropotamobius seems confirmed. The possibility of a correlation between heterozygosity and oxygen consumption is evocated.     

An examination of structural interactions presumed to be of importance in the stabilization of phospholipase A2 dimers based upon comparative protein sequence analysis of a monomeric and dimeric enzyme from the venom ofAgkistrodon p. piscivorus

Phospholipases A₂ may exist in solution both as monomers and dimers, but enzymes that form strong dimers (K _D approximately 10^–9 M) have been found, thus far, only in venoms of the snake family Crotilidae. The complete amino acid sequences of a basic monomeric and an acidic dimeric phospholipase A₂ fromAgkistrodon piscivorus piscivorus (American cotton-mouth water moccasin) venom have been determined by protein sequencing methods as part of a search for aspects of structure contributing to formation of stable dimers. Both the monomeric and dimeric phospholipases A₂ are highly homologous to the dimeric phospholipases A₂ fromCrotalus atrox andCrotalus adamanteus venoms, and both have the seven residue carboxy-terminal extension characteristic of the crotalid and viperid enzymes. Thus, it is clear that the extension is not a prerequisite for dimerization. Studies to date have revealed two characteristic features of phosphilipases A₂ that exist in solution as strong dimers. One is the presence in the dimers of a Pro-Pro sequence at position 112 and 113 which just precedes the seven residue carboxy-terminal extension (residues 116–122). The other is a low isoelectric point; only the acidic phospholipases A₂ have been observed, thus far, to form stable dimers. These, alone or together, may be necessary, though not sufficient conditions for phospholipase A₂ dimer formation. Ideas regarding subunit interactions based upon crystallographic data are evaluated relative to the new sequence information on the monomeric and dimeric phospholipases A₂ fromA. p. piscivorus venom.     

Brownian dynamics simulations of glycolytic enzyme subsets with F-actin

Previous Brownian dynamics (BD) simulations identified specific basic residues on fructose-1,6-bisphophate aldolase (aldolase) (I. V. Ouporov et al., Biophysical Journal, 1999, Vol. 76, pp. 17-27) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (I. V. Ouporov et al., Journal of Molecular Recognition, 2001, Vol. 14, pp. 29-41) involved in binding F-actin, and suggested that the quaternary structure of the enzymes may be important. Herein, BD simulations of F-actin binding by enzyme dimers or peptides matching particular sequences of the enzyme and the intact enzyme triose phosphate isomerase (TIM) are compared. BD confirms the experimental observation that TIM has little affinity for F-actin. For aldolase, the critical residues identified by BD are found in surface grooves, formed by subunits A/D and B/C, where they face like residues of the neighboring subunit enhancing their electrostatic potentials. BD simulations between F-actin and aldolase A/D dimers give results similar to the native tetramer. Aldolase A/B dimers form complexes involving residues that are buried in the native structure and are energetically weaker; these results support the importance of quaternary structure for aldolase. GAPDH, however, placed the critical residues on the corners of the tetramer so there is no enhancement of the electrostatic potential between the subunits. Simulations using GAPDH dimers composed of either S/H or G/H subunits show reduced binding energetics compared to the tetramer, but for both dimers, the sets of residues involved in binding are similar to those found for the native tetramer. BD simulations using either aldolase or GAPDH peptides that bind F-actin experimentally show complex formation. The GAPDH peptide bound to the same F-actin domain as did the intact tetramer; however, unlike the tetramer, the aldolase peptide lacked specificity for binding a single F-actin domain.     

Mechanism of transducin activation of frog rod photoreceptor phosphodiesterase. Allosteric interactiona between the inhibitory gamma subunit and the noncatalytic cGMP-binding sites

The rod photoreceptor phosphodiesterase (PDE) is unique among all known vertebrate PDE families for several reasons. It is a catalytic heterodimer (alphabeta); it is directly activated by a G-protein, transducin; and its active sites are regulated by inhibitory gamma subunits. Rod PDE binds cGMP at two noncatalytic sites on the alphabeta dimer, but their function is unclear. We show that transducin activation of frog rod PDE introduces functional heterogeneity to both the noncatalytic and catalytic sites. Upon PDE activation, one noncatalytic site is converted from a high affinity to low affinity state, whereas the second binding site undergoes modest decreases in binding. Addition of gamma to transducin-activated PDE can restore high affinity binding as well as reducing cGMP exchange kinetics at both sites. A strong correlation exists between cGMP binding and gamma binding to activated PDE; dissociation of bound cGMP accompanies gamma dissociation from PDE, whereas addition of either cGMP or gamma to alphabeta dimers can restore high affinity binding of the other molecule. At the active site, transducin can activate PDE to about one-half the turnover number for catalytic alphabeta dimers completely lacking bound gamma subunit. These results suggest a mechanism in which transducin interacts primarily with one PDE catalytic subunit, releasing its full catalytic activity as well as inducing rapid cGMP dissociation from one noncatalytic site. The state of occupancy of the noncatalytic sites on PDE determines whether gamma remains bound to activated PDE or dissociates from the holoenzyme, and may be relevant to light adaptation in photoreceptor cells.     

Enzyme heterozygosity,metabolism, and developmental stability

Developmental homeostasis, measured as either fluctuating asymmetry or variance of morphological characters, increases with enzyme heterozygosity in many, but not all, natural populations. These results have been reported forDrosophila, monarch butterflies, honeybees, blue mussels, side-blotched lizards, killifish, salmonid fishes, guppies, Sonoran topminnows, herring, rufous-collared sparrows, house sparrows, brown hares, white-tailed deer, and humans. Because heterozygosity at a few loci can not predict heterozygosity of the entiry genome, these loci must be detecting localized zones that influence the developmental environment. Studies of malate dehydrogenase in honeybees,Apis mellifera, and lactate dehydrogenase in killifish,Fundulus heteroclitus, revealed that developmental homeostasis varied with heterozygosity of individual loci. Heterozygotes differed from homozygotes in fluctuating asymmetry, morphological variance, and in correlations between morphological characters. The protein loci in these studies code for enzymes, and therefore do not directly influence morphological characters. However, some enzymatic loci substantially influence metabolism, and contribute to variation in the amount of energy available for development and growth. This argument can be made most convincingly for the LDH polymorphism in killifish. LDH genotypes differ in enzyme kinetic properties that measure differences in physiological efficiency, and these differences produce measurable and predictable differences in physiology and development. Under environmental conditions which impose a stress upon development, genotypes at these loci may have different amounts of energy available for development, and consequently exhibit different levels of developmental homeostasis.     

The stromal processing peptidase activities from Chlamydomonas reinhardtii and Pisum sativum: unexpected similarities in reaction specificity

We have partially purified the stromal processing peptidase from Chlamydomonas reinhardtii and compared the properties of this activity with those of the pea counterpart. Whereas previous studies have suggested that the two enzymes may have significantly different reaction specificities, we find that they are in fact very similar. Both enzymes process precursors of two higher-plant thylakoid lumen proteins, and one C. reinhardtii lumenal protein, to similar intermediate-size forms. However, whereas the algal enzyme processes the precursor of C. reinhardtii Rubisco small subunit to the correct mature size, this precursor is cleaved only to an intermediate size by the pea enzyme. The small subunit precursor from pea appears to be cleaved by both enzymes in a similar manner. In terms of sensitivity to inhibitors, the two activities are notably different; the pea enzyme has previously been shown to be inhibited by several types of heavy-metal chelator, but we have found that none of these compounds affect the algal activity.     

Invertebrates yield a plethora of atypical guanylyl cyclases

Invertebrate model systems have a long history of generating new insights into neuronal signaling systems. This review focuses on cyclic GMP signaling and describes recent advances in understanding the properties and functions of guanylyl cyclases in invertebrates. The sequencing of three invertebrate genomes has provided a complete catalog of the guanylyl cyclases in C. elegans, Drosophila, and the mosquito Anopheles gambiae. Using this data and that from cloned guanylyl cyclases in Manduca sexta, C. elegans, and Drosophila, plus predictions and models from vertebrate guanylyl cyclases, evidence is presented that there is a much broader array of properties for these enzymes than previously realized. In addition to the classic homodimeric receptor guanylyl cyclases, C. elegans has at least two receptor guanylyl cyclases that are predicted to require heterodimer formation for activity. Soluble guanylyl cyclases are generally recognized as being obligate heterodimers that are activated by nitric oxide (NO). Some of the soluble guanylyl cyclases in C. elegans may heterodimeric, but all appear to be insensitive to NO. The β2 soluble guanylyl cyclase subunit in mammals and similar ones in Manduca and Drosophila are active in the absence of additional subunits and there is evidence that Drosophila and Anopheles also express an additional subunit that enhances this activity.     

Genetic tags in the New Zealand hoki Macruronus novaezelandiae1

Twelve enzymes and liver proteins were examined by starch and cellulose acetate electrophoresis in two samples of hoki Macruronus novaezelandiae from New Zealand. Two polymorphic enzymes, a-glycerophosphate dehydrogenase and sorbitol dehydrogenase, were found giving a mean heterozygosity of 0.027 over 12 enzyme loci or 0.016 over 15 protein loci. No differences in gene frequency were detected in nine regional samples collected from the New Zealand 200-mile exclusive economic zone. Together with existing biological information on spawning grounds these data suggest that hoki in the New Zealand fishery form one stock.     

The expression and anaerobic induction of alcohol dehydrogenase in cotton

The alcohol dehydrogenase (ADH) system in cotton is characterized, with an emphasis on the cultivated allotetraploid speciesGossypium hirsutum cv. Siokra. A high level of ADH activity is present in seed of Siokra but quickly declines during germination. When exposed to anaerobic stress the level of ADH activity can be induced several fold in both roots and shoots of seedlings. Unlike maize andArabidopsis, ADH activity can be anaerobically induced in mature green leaves. Three major ADH isozymes were resolved in Siokra, and it is proposed that two genes,Adh1 andAdh2, are coding for these three isozymes. The genes are differentially expressed. ADH1 is predominant in seed and aerobically grown roots, while ADH2 is prominent in roots only after anaerobic stress. Biochemical analysis demonstrated that the ADH enzyme has a native molecular weight of approximately 81 kD and a subunit molecular weight of approximately 42 kD, thus establishing that ADH in cotton is able to form and is active as dimers. Comparisons of ADH activity levels and isozyme patterns between Siokra and other allotetraploid cottons showed that the ADH system is highly conserved among these varieties. In contrast, the diploid species of cotton all had unique isozyme patterns.This work was generously supported by an Australian Cotton Research Council Postgraduate Studentship.     

Investigations on the mechanism of action of crotoxin

Crotoxin, the major toxic protein of Crotalus durissus terrificus, is composed of a basic phospholipase, component-B, and of an acidic subunit, component-A. The crotoxin complex is insensitive to an active site directed reagent, p-bromophenacyl bromide, while its isolated enzymatic component-B is rapidly and irreversibly inactivated. We observed that crotoxin possesses an intrinsic phospholipase A2 activity on monodispersed substrates, indicating that the active site of component-B is not masked by component-A in the complex. The inactivation of component-B by p-bromophenacyl bromide follows pseudo-first order kinetics, with a rate constant proportional to the concentration of phospholipase, as expected for a reaction of second order with respect to the protein. On the basis of a detailed kinetic analysis of this reaction, and of physico-chemical studies of component-B in various experimental conditions, we propose that (1) an equilibrium exists between reactive dimers of component-B and preponderant but non-reactive monomer; (2) component-A protects component-B against inactivation by p-bromophenacyl bromide by preventing the formation of reactive dimers. When comparing the reactivity of component-B with that of other phospholipases, we observed that the enzymes which have not been shown to produce dimers all react with p-bromophenacyl bromide with similar low rates of reaction, while phospholipases which have been reported to form dimers react much more rapidly.     

Structural aspects of aldehyde dehydrogenase that influence dimer-tetramer formation

Aldehyde dehydrogenases are isolated as dimers or tetramers but have essentially identical structures. The homotetramer (ALDH1 or ALDH2) is a dimer of dimers (A-B + C-D). In the tetrameric enzyme, Ser500 from subunit "D" interacts with Arg84, a conserved residue, from subunit "A". In the dimeric ALDH3 form, the interaction cannot exist. It has been proposed that the formation of the tetramer is prevented by the presence of a C-terminal tail in ALDH3 that is not present in ALDH1 or 2. To understand the forces that maintain the tetramer, deletion of the tail in ALDH3, addition of different tails in ALDH1, and mutations of different residues located in the dimer-dimer interface were made. Gel filtration of the recombinantly expressed enzymes demonstrated that no change in their oligomerization occurred. Urea denaturation showed a diminution to the stability of the ALDH1 mutants. The K(m) for propionaldehyde was similar to that of the wild-type enzyme, but the K(m) for NAD was altered. A double mutant of D80G and S82A produced an enzyme with the ability to form dimers and tetramers in a protein-concentration-dependent manner. Though stable, this dimeric form was inactive. The tetramer exhibited 10% activity compared with the wild type. Sequence alignment demonstrated that the hydrophobic surface area is increased in the tetrameric enzymes. The hydrophobic surface seems to be the main force that drives the formation of tetramers. The results indicated that residues 80 and 82 are involved in maintaining the tetramer after its assembly but the C-terminal extension contributes to the overall stability of the assembled protein.     

Quaternary Structure of Nucleoside Diphosphate Kinases

Nucleoside (NDP) diphosphate kinases are oligomeric enzymes. Most are hexameric, but somebacterial enzymes are tetrameric. Hexamers and tetramers are constructed by assemblingidentical dimers. The hexameric structure is important for protein stability, as demonstratedby studies with natural mutants (the Killer-of-prune mutant ofDrosophila NDP kinase andthe S120G mutant of the human NDP kinase A in neuroblastomas) and with mutants obtainedby site-directed mutagenesis. It is also essential for enzymic activity. The function of the tetrameric structure is unclear.