Solution conformations of proline rings in proteins studied by NMR spectroscopy

Summary Three different conformations of proline rings in a protein in solution, Up, Down and Twist, have been distinguished, and stereospecific assignments of the pyrrolidine -, - and -hydrogens have been made on the basis of ¹H-¹H vicinal coupling constant patterns and intraresidue NOEs. For all three conformations, interhydrogen distances in the pairs -³, ³-³, ²-², ²-², and ³-³ (2.3 Å) are shorter than those in the pairs -², ²-³, ³-², ²-³, and ³-² (2.7–3.0 Å), resulting in stronger NOESY cross peaks. For the Up conformation, the ³-² and ²-³ spin-spin coupling constants are small (<3 Hz), and weak cross peaks are obtained in a short-mixing-time (10 ms) TOCSY spectrum; all other vicinal coupling constants are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. For the Down form, the -², ²-³, and ³-² vicinal coupling constants are small, leading to weak TOCSY cross peaks; all other couplings again are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. In the case of a Twist conformation, dynamically averaged coupling constants are anticipated. The procedure has been applied to bovine pancreatic trypsin inhibitor and Cucurbita maxima trypsin inhibitor-V, and ring conformations of all prolines in the two proteins have been determined.     

Intron III-Specific Markers for Screening of β-amylase Alleles in Barley Cultivars

In modern malting barley breeding it is important to increase the level of -amylase activity level in barley. The aim of this study was to investigate if a PCR method for screening -amy1 alleles can be used as an indicator for -amylase activity level in barley. Activity was assayed from 24 cultivars, 7 lines, and a Hordeum spontaneum PI 296897 strain grown in the same field. The -amy1 alleles were identified by amplifying the intron III-specific region of the gene using PCR. No new alleles were detected in addition to the three alleles found earlier: cv Adorra-like, cv Haruna Nijo-like and PI 296897-like -amy1 allele. Samples were grouped according to the nature of their -amy1 locus and enzyme activities were compared between the groups. Cultivars carrying a cv Haruna Nijo-like -amy1 allele had 1.3 times and lines carrying a PI 296897-like -amy1 allele had 2.1 times higher -amylase activity than cultivars carrying a cv Adorra-like -amy1 allele. The mean activities are significantly different in the allele groups (Kruskal–Wallis: for protein H= 11.54, P< 0.01; for meal H= 12.74, P< 0.01). PCR fragments can be used as allele specific markers to predict the level of -amylase activity in breeding when such variation of the intron III is concerned.     

Adrenergic and muscarinic receptor regulation and therapeutic implications in heart failure

In end-stage heart failure the expression of different myocardial regulatory proteins involved in the -adrenergic cAMP signalling pathway is altered. The downregulation of -adrenoceptors and their uncoupling from the effector as well as an increased expression of the inhibitory GTP-binding protein seem to be the most important alterations. Since catecholamine levels are elevated in these patients and since some alterations can be restored after treatment with -adrenoceptor antagonists it was hypothesized that excessive -adrenergic stimulation could be involved in these alterations.In this article the changes of -adrenergic receptors, GTP-binding proteins, sarcoplasmic reticulum Ca²⁺-ATPase and of phospholamban found in heart failure are addressed with its possible therapeutic implications.     

Sites of interactions of surfactants with beta-adrenergic responses in trout (Salmo gairdneri) gills

Summary The aims of theis study were to investigate the effects of surfactants on the -adrenergic responses of the arterio-arterial (lamellar) vascular pathway of isolated gills from the troutSalmo gairdneri. The resistance of the arterio-arterial vasculature was monitored by measuring the input pressure of isolated gills perfused at a constant flow and at constant efferent pressure. Specific -adrenergic stimulation with isoprenaline produced a dose-dependant reduction in vascular resistance which was mimicked by the presence of cyclic 8-(4-chlorophenylthio) adenosine-3: 5-monophosphate (8-cpt cyclic AMP). These results suggest that cyclic AMP is involved as an intracellular second messenger of -adrenergic regulatory responses of branchial haemodynamics.Surfactants in the perfusate at nominal concentrations of 1.0 mol l^–1 (0.3–0.5 mg l^–1) inhibited the response of the branchial tissue to isoprenaline but did not affect the response to 8-cpt cyclic AMP. This suggests that the surfactant effects are the result of events occurring within the cell membrane. Furthermore because the surfactants used affected the efficacy, rather than the affinity, of the isoprenaline response then the mechanism of action is unlikely to be an effect on agonist/receptor interaction but may be further along the stimulus response chain, perhaps affecting receptor/regulatory nucleotide/adenylate cyclase interaction or via direct inhibition of the cyclase itself.     

Hormonal regulation of hepatic glycogenolysis inAmphibolurus nuchalis,the western netted dragon: an in vitro study

Summary In mammals hepatic glycogenolysis is controlled by several hormones using cyclicAMP, Ca²⁺ and/or diacylglycerol as intracellular messengers. In contrast, in teleost fish, lungfish and amphibians fewer hormones promote hepatic glycogenolysis, and cyclicAMP is the sole intra-cellular messenger. This suggests that the -adrenergic mechanism became associated with the liver after amphibians separated from the vertebrate line. Reptiles separated later, and the aim of this study is to elucidate the hormonal control of hepatic glycogenolysis in a reptile,Amphibolurus nuchalis, and especially to determine which adrenergic receptor system is operative.InA. nuchalis liver pieces cultured in vitro, adrenaline and glucagon stimulated glycogen breakdown and glucose release, glycogen phosphorylase activity and accumulation of cyclicAMP in the tissue. Neurohypophysial peptides did not affect these parameters. These actions of adrenaline were completely blocked by the -adrenergic antagonist, propranolol and slightly reduced by the -adrenergic antagonist, phentolamine. Removal of Ca²⁺ from the medium and addition of the Ca²⁺ chelator, EGTA, did not block the actions of adrenaline, and the Ca²⁺ ionophore A23187 did not mimic these actions.The -adrenegic ligand [¹²⁵I]-iodocyanopindolol (ICP) bound specifically to an isolated membrane preparation fromA. nuchalis liver with a calculated K_D of 100 pM and a Bmax of 37.6 fmol·mg protein^–1. The adrenergic ligands propranolol, isoprenaline, adrenaline, noradrenaline, phenylephrine and phentolamine displaced ICP with K_D's of 20 nM, 1 M, 4.5 M, 32 M, 35 M and 500 M, respectively. The ₂-adrenergic ligand yohimbine did not bind specifically to the membrane, but at 1 nM and 100 pM, specific binding of the ₁-adrenergic ligand prazosin was 45% of total with a mean of 11.3 fmoles·mg protein^–1 specifically bound.These findings indicate that the glycogenolytic actions of adrenaline are mediated primarily via -adrenergic receptors inA. nuchalis, but that -adrenergic receptors may also play some role in the control of hepatic metabolism.     

Evidence of β-carotene 7,8(7′,8′) oxygenase (β-cyclocitral,crocetindial generating) in Microcystis

A -carotene oxygenase is described which occurs in the Cyanobacterium Microcystis. It cleaves -carotene and zeaxanthin specifically at the positions 7,8 and 7,8, while echinenone and myxoxanthophyll are not affected. The oxidative cleavage of -carotene leads to the formation of -cyclocitral and crocetindial and that of zeaxanthin to hydroxy--cyclocitral and crocetindial in nearly stoichiometric amounts. Oxidant is dioxygen as has been demonstrated by high incroporation (86%) of ¹⁸O₂ into -cyclocitral. -Carotene oxygenase is membrane bound, sensitive to sulfhydryl reagents, antioxidants and chelating agents. Iron seems to be an essential part of the enzyme activity. Cofactors necessary for the reaction could not be detected.Abbreviations TLC thin layer-chromatography - PIPES piperazine-N,N-bis-(2-ethanesulfonate) Na - TES 2{[tris-(hydroxymethyl)-methyl]-amino} ethanesulfonic acid Dedicated to Professor G. Drews on occasion of his 60th birthday     

Amyloid Beta Peptide Membrane Perturbation Is the Basis for its Biological Effects

Experimental studies have indicated that the mechanisms offered for explaining the neurotoxicity of amyloid beta peptide (AP) are diverse, and include altered enzyme activities, disrupted calcium homeostasis, and increased free radical formation. AP appears to interact at the cell membrane with a multitude of receptor sites and also inserts physically into the membrane matrix. This membrane insertion affects the membrane fluidity and potentially influences the function of resident membrane proteins. We propose a unifying hypothesis to explain the experimental observations of the diverse cellular responses to AP. The indiscriminate physical insertion of AP into the cell membrane unspecifically activates a host of membrane processes by perturbation of the membrane proteins. This recurrent activation of membrane processes eventually culminates in neuronal cell death. We recommend that successful therapeutic interventions should be directed at reducing or preventing the interaction of AP with neuronal cell membranes.     

Patterns and conformations of commonly occurring supersecondary structures (basic motifs) in protein data bank

Short peptides connecting-helices and-strands have been analyzed in 240 proteins refined at resolutions of 0.25 nm or better. Connecting peptides of lengths between one and five residues have been classified as part of supersecondary motifs of four types:, , , and. Careful consideration has been given to the definition of secondary structures on the basis of hydrogen bonds and main-chain conformational angles. Using five classes of residue conformation—a, b, e, l, t—in the nonregular structure regions of, space, 34 classes of supersecondary motifs occurring at least five times have been identified. Among these 34 classes, 11 classes that occur more than 25 times are commonly occurring supersecondary structure motifs. The patterns and conformations of the 11 commonly occurring supersecondary structure motifs have been characterized, demonstrating that patterns and conformations adopted by supersecondary structure motifs are limited. The results have relevance to structure prediction, comparative modeling, and protein folding.     

The structure of two distinct pancreatic amylase genes in mouse strain YBR

The amylase complex on mouse chromosome 3 encodes both salivary and pancreatic amylase. It appears that one active gene is present for salivary amylase, whereas pancreatic amylase in some strains is coded by at least 4, and perhaps by more than 10, genes. Strain YBR is different from other strains in that it produces twice as much salivary amylase. Pancreatic amylase in YBR is present as two different protein forms, A and B, the sum of which amounts to only one-third of that in, for instance, strain A/J. YBR chromosomal DNA was cloned in phage , followed by restriction and heteroduplex analysis of recombinant phages carrying amylase genes. Among 32 phage isolates, 5 carried parts of the salivary amylase sequence. The remaining phage isolates contained pancreatic amylase-like sequences and represented three nonoverlapping genomic regions, i.e., one of 34 kb containing a complete gene, PAN-II; another of 41 kb with a complete but different gene, PAN-I, plus a truncated gene, PAN-1; and finally, one of 23 kb with another truncated gene, PAN-2. Parts of the amino acid sequence of A and B have previously been determined, and we report here the sequencing of a 4-kb DNA fragment from Pan-II which establishes that this gene codes for B.This work was supported by the Danish Natural Science Research Council.     

A fluorometric assay of ceramide glycanase with 4-methylumbelliferyl β-d-lactoside derivatives

4-Methylumbelliferyl 6-O-benzyl--d-lactoside (6Bn-MU-Lac) and some related compounds were synthesizedvia different selective reactions including phase-transfer glycosylation. Their suitability as substrates for a fluorometric assay of ceramide glycanase (CGase) was evaluated. Among others, the 6Bn-MU-Lac, which is resistant to exogalactosidase, was found to be a suitable substrate for routine assay of the CGase activity. For American leech CGase, theK _m value is 0.232 mM at pH 5. Abbreviations: CGase, ceramide glycanase; Gal, galactose; Glc, Glucose; Lac, lactose; MU, 4-methylumbelliferone; MU-Lac, 4-methylumbelliferyl -d-lactoside; bBn-Lac, 6-O-benzyl-lactose; 6Bn-MU-Lac, 4-methylumbelliferyl 6-Obenzyl--d-lactoside; 46Bd-MU-Lac, 4-methylumbelliferyl 4,6-O-benzylidene--d-lactoside; MU-Cel, 4-methylumbellifery -d-cellobioside; 46Bd-MU-Cel, 4-methylumbelliferyl 4,6-O-benzylidene--d-cellobioside; TLC, thin layer chromatography;¹H-NMR, proton nuclear magnetic resonance; GSL, glycosphingolipids; CSA, 10-camphorsulfonic acid. See Scheme 1 for chemical structures.     

A comparative assessment of TLC overlay technique and microwell adsorption assay in the examination of influenza A and Sendai virus specificities towards oligosaccharides and sialic acid linkages of gangliosides

Influenza A and Sendai viruses bind toneolacto-series gangliosides isolated from human granulocytes. Differences in receptor specificity of influenza viruses A/PR/8/34 (H1N1), A/X-31 (H3N2), and parainfluenza Sendai virus (HNF1, Z-strain) were determined by two direct solid phase binding assays: the overlay technique, which combines high-resolution in the separation of gangliosides on thin-layer chromatograms with direct binding; and the microwell adsorption assay as a convenient binding assay which is performed in microtitre wells to estimate the avidity of binding to an isolated ganglioside. Both methods were applied for comparative binding studies. Viruses were found to exhibit specificity for oligosaccharides and sialic acids as well as for chain length of the neutral carbohydrate backbone, whereas differing fatty acids (C_24:1 and C_16:0) in the ceramide portion had no impact on virus adsorption. Terminal sialyloligosaccharides Neu5Ac2-3Gal1-4Glc-R of G_M3, and Neu5Ac2-3Gal1-4GlcNAc-R as well as Neu5Ac2-6Gal1-4GlcNAc-R ofneolacto-series gangliosides with nLcOse₄Cer and nLcOse₆Cer backbone, exhibited significant specific receptor activity towards the different viruses. To compare the data revealed from both test systems, values of virus binding were ascertained by a non-parametric statistical approach based on rank correlation. The rank correlation coefficientr _s was calculated according to Spearman from each virus binding towards G_M3, IV³Neu5Ac-nLcOse₄Cer, IV⁶Neu5Ac-nLcOse₄Cer and VI³Neu5Ac-nLcOse₆SCer. The rank correlation coefficients 0.74, 0.95 and 0.92, which were determined for A/PR/8/34 (H1N1), A/X-31 (H3N2) and Sendai virus (HNF1, Z-strain), respectively, indicated that both assays generate highly correlated experimental data. Based on these results, analyses of virus binding on thin-layer chromatograms as well as in microwells were found equivalent tools for ganglioside receptor studies. Abbreviations: BSA, bovine serum albumin; GSL(s), glycosphingolipids; HPTLC, high performance thin-layer chromatography; PBS, phosphate buffered saline; Neu5Ac,N-acetylneuraminic acid [35];r _s = rank correlation coefficient according to Spearman. The designation of the glycosphingolipids follows the IUPAC-IUB recommendations [36]. LacCer or lactosylceramide, Gal1-4Glc1-1Cer; lacto-N-neotetraosylceramide or nLcOse₄Cer, Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer; lacto-N-norhexaosylceramide or nLcOse₆Cer, Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer; G_M3 (according to Svennerholm [37]) or II³Neu5AcLacCer.     

Theβ-subunit of human chorionic gonadotropin containsN-glycosidic trisialo tri- and tri′-antennary carbohydrate chains

TheN-linked carbohydrate chains of the-subunit of highly purified urinary human chorionic gonadotropin have been re-investigated. The oligosaccharides were released enzymatically by peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F, and fractionated by a combination of FPLC and HPLC. As a result of the application of improved fractionation methods, apart from the earlier reported carbohydrate chains, also small amounts of trisialo tri- and tri-antennary oligosaccharides were found. The primary structures of the latter carbohydrate chains have been determined by 500-MHz¹H-NMR spectroscopy to beAbbreviations hCG human chorionic gonadotropin - hCG- -subunit - hCG- -subunit - PNGase-F peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F (E.C. 3.5.1.52) - endo-F endo--N-acetylglucosaminidase-F (E.C. 3.2.1.96) - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - CBB coomassie brilliant blue R 250 - GlcNAc N-acetylglucosamine - NeuAc N-acetylneuraminic acid - Man mannose - Gal galactose - Fuc fucose     

Genetic characterization of a new splice variant of the β2 subunit of the voltage-dependent calcium channel

This study reports a novel splice variant form of the voltage-dependent calcium channel ₂ subunit (_2g). This variant is composed of the conserved amino-terminal sequences of the _2a subunit, but lacks the -subunit interaction domain (BID), which is thought essential for interactions with the ₁ subunit. Gene structure analysis revealed that this gene was composed of 13 translated exons spread over 107 kb of the genome. The gene structure of the ₂ subunit was similar in exon-intron organization to the murine ₃ and human ₄ subunits. Electrophysiological evaluation revealed that _2a and _2g affected channel properties in different ways. The _2a subunit increased the peak amplitude, but failed to increase channel inactivation, while _2g had no significant effects on either the peak current amplitude or channel inactivation. Other subunits, such as ₃ and ₄, significantly increased the peak current and accelerated current inactivation.     

Effect of transmembrane Ca2+ gradient on the coupling of β-adrenergic receptors and adenylyl cyclase

In order to investigate the effect of transmembrane Ca²⁺ gradient on G_s mediated coupling of -AR and adenylyl cyclase, -AR from duck erythrocytes and G_s and adenylyl cyclase from bovine brain cortices were co-reconstituted into asolectin liposomes with different transmembrane Ca²⁺ gradient. These proteoliposomes were proven to be impermeable to water-soluble substances. The results obtained indicate that a physiological transmembrane Ca^2– gradient (1000-fold) is essential for higher stimulation of adenylyl cyclase by hormone-activated -AR via coupling to G_s and can be further enhanced by the decrease of such Ca²⁺ gradient within certain range (100 fold) following Ca²⁺ influx into cells during signal transduction. Fluorescence polarization of DPH revealed that transmembrane Ca²⁺ gradient modulates adenylyl cyclase and its stimulation by hormones through mediating a change in lipid fluidity. Correspondent conformational changes of -AR were also detected from the fluorescence spectra and quenching of Acrylodan-labelled -AR in those proteoliposomes. It is suggested that a proper transmembrane Ca²⁺ gradient is essential for the optimal fluidity of the phospholipid bilayer in the proteoliposomes, which favors the formation of a suitable conformation of the reconstituted -AR and thus promotes the stimulation of adenylyl cyclase activities by hormone-activated -AR via Gs.Abbreviations ATP adenosine triphosphate - -AR -adrenergic receptors - AC adenylyl cyclase - DHA dihydroalprenolol - DPH diphenylhexatriene - [Ca²⁺]_i Ca²⁺ concentration inside proteoliposomes - [Ca²⁺]_o Ca²⁺ concentration outside proteoliposomes - cAMP cyclic adenosine monophosphate - DTT Dithiothreitol - FS fluorescein sulfonate - G_s Stimulatory GTP-binding protein - GTP guanosine triphosphate - GTPS guanosine 5-O-(3-thiotriphosphate) - kDa kilodalton - SDS sodium dodecyl sulfate - Tris N-tris(hydroxymethyl)aminomethane     

Primary structures of alpha- and beta-subunits of alpha-amylase inhibitors from seeds of three cultivars of Phaseolus beans

The primary structures of three -amylase inhibitors (TAI, DAI, and MAI-2) consisting of glycoprotein subunits and from the respective seeds of three cultivars of Phaseolus beans, Toramame (Phaseolus vulgaris L.), Daifukumame (Phaseolus vulgaris L.), and Murasakihanamame (Phaseolus coccineus L.) were determined by sequencing the peptide fragments derived from their enzymatic digestions. Major sugar chains of the inhibitors were also assessed by analyzing glycopeptides in the enzymatic digests. The subunits, and , were shown to be composed of 76 and 139 amino acid residues, respectively, in each inhibitor. The overall amino acid sequences of the inhibitors were slightly different from one another. Furthermore, the sequence of TAI was the same as that deduced from a cDNA clone encording -amylase inhibitor-1 from the common bean (Phaseolus vulgaris L.). It was also revealed that there were two N-glycosylation sites in each -subunit: PA-derivatives of the major N-glycans were estimated to be M6B at Asn(12) and M9A at Asn(65). Each -subunit of TAI and MAI-2 had two N-glycosylation sites, while the -subunit of DAI had only one site. The major N-glycans pyridylaminated were estimated to be M3X at Asn(63) in each -subunit and M3FX at Asn(83) in -subunits of TAI and MAI-2.     

Effect of propranolol on cardiac cytokine expression after myocardial infarction in rats

The pro-inflammatory cytokines interleukin (IL)-1 and IL-6 have been shown to be upregulated in the myocardium after injury and after adrenergic receptor stimulation. Together with other cytokines, such as the transforming growth factor (TGF)-, the pro-inflammatory cytokines have been implicated in the initiation of tissue repair and wound healing after myocardial infarction (MI). In the present study, the effect of -adrenergic receptor blockade with propranolol (2 mg/kg·h s.c. by miniosmotic pumps) on cardiac cytokine expression and on wound healing was analyzed in rats from 6–72 h after MI. IL-1 and IL-6 gene expression strongly increased in the infarcted myocardium 6 h after MI and peaked after 12 h, while TGF-, progressively increased from 12 h onwards. Also, TGF-₂ increased after 12 h, peaked after 24 h and declined thereafter, while TGF-, was only elevated after 72 h. Treatment with propranolol had a negative chronotropic effect throughout the observation period of 72 h. It attenuated the initial elevation in LVEDP and increased cardiac output ultimately. Furthermore, propranolol attenuated IL-1 mRNA expression, but had not effect on the other cytokines. Moreover, MMP-9 gelatinolytic activity was markedly attenuated by propranolol indicating a delayed resorption of the necrotic tissue and, possibly, collagen turnover. Replacement by scar tissue, however, was not affected as indicated by normal collagen expression.     

Effects of lactation on the regulation of hepatic metabolism in the rat and sheep: adrenergic receptors and cyclic AMP responses

The number and coupling efficiency of -adrenoceptors in liver membranes and intact hepatocytes of lactating and non-lactating female rats were compared to assess whether or not alterations in this signalling system could contribute towards the changed pattern of hepatic metabolism during lactation. In view of the different adaptations of hepatic metabolism to lactation in ruminants, the adrenergic receptor profile of sheep liver membranes was also determined. Post-receptor responses at two stages down-stream of cyclic AMP generation were also evaluated in rat hepatocytes in response to the -adrenergic agonist isoprenaline. No changes in the number or affinity of hepatic -adrenoceptors were found in sheep or rats when lactating and non-lactating individuals were compared. Sheep liver was found to have a much greater concentration of -adrenoceptors than rat liver, and a much higher ratio of :₁. The sensitivity and responsiveness of cyclic AMP generation in response to isoprenaline were similar in hepatocytes prepared from lactating and non-lactating rats, although the response to saturating concentrations of glucagon was diminished in hepatocytes from lactating rats. The activity ratio of cyclic AMP-dependent protein kinase (PK-A) also reacted similarly (in respect of both responsiveness and sensitivity) to isoprenaline in these two groups of hepatocytes. Contrastingly, the sensitivity of rat hepatocyte phosphorylase activity to -adrenergic stimulation was greatly diminished during lactation.     

Identification of beta subunit of the rhesus monkey chorionic gonadotropin (rmCG)

Chorionic gonadotropin (CG) is a placental derived hormone that plays a crucial role in successful implantation and establishment of early pregnancy in the primates. The rhesus monkey was chosen as a model to understand the feasibility of developing human DNA immuno-contraceptive. The coding region of rhesus monkey CG -subunit (rmCG) was isolated by the TDRT-PCR method. The nucleotide sequence including the leader peptide was 499 nucleotide long and encoded 166 amino acids. In comparing with the previous known primates CG -subunits, the rmCG was the highest degree of homology with baboon CG -subunit at the deduced amino acid sequence (94%), 79.5% homology with human CG -subunit and 70.4% homology with marmoset monkey CG -subunit. The eukaryotic expression vector pCMV4-rmCG inserted full-coding cDNA sequence of rmCG was constructed, and the expression of rmCG -subunit in HeLa cells transient expressing system in vitro and BALB/c mice in vivo was determined. The results demonstrated that the recombinant PCMV4-rmCG eukaryotic expression vector could express rmCG -subunit in vitro and in vivo.     

An Analysis of the Amino Acid Clustering Pattern in (α/β)8 Barrel Proteins

The (/)₈ barrel proteins, in spite of having a common fold, do not show any sequence similarity. In order to understand the factors which are responsible for maintaining the common fold, the three-dimensional structures of 36 (/)₈ barrel proteins are analyzed for the presence of identical amino acid clusters or physicochemically similar clusters. The results reveal 14 identical amino acid clusters and a large number of physicochemically similar clusters. Further analysis of the similar clusters points to the conservation of secondary structures, the presence of pairs of residues occupying topologically equivalent secondary structures, and the presence of certain key residues which may play a vital role in directing and stabilizing the (/)₈ barrel fold.