13Cα and 13Cβ chemical shifts as a tool to delineate β-hairpin structures in peptides

Unravelling the factors that contribute to the formation and the stability of -sheet structure in peptides is a subject of great current interest. A -hairpin, the smallest -sheet motif, consists of two antiparallel hydrogen-bonded -strands linked by a loop region. We have performed a statistical analysis on protein -hairpins showing that the most abundant types of -hairpins, 2:2, 3:5 and 4:4, have characteristic patterns of ¹³C and ¹³C conformational shifts, as expected on the basis of their and angles. This fact strongly supports the potential value of ¹³C and ¹³C conformational shifts as a means to identify -hairpin motifs in peptides. Their usefulness was confirmed by analysing the patterns of ¹³C and ¹³C conformational shifts in 13 short peptides, 10–15 residues long, that adopt -hairpin structures in aqueous solution. Furthermore, we have investigated their potential as a method to quantify -hairpin populations in peptides.     

Food uptake and the fine structure of the dinophytePaulsenella sp. an ectoparasite of marine diatoms

Summary Motile dinospores ofPaulsenella attach to a host diatom frustule, form a feeding tube, drive it between epi- and hypocingulum, pierce the host plasmalemma and suck up host cytoplasm gradually. This mode of endocytosis (myzocytosis) implies that the host plasmalemma is not ingested and that the host cytoplasm within the food vacuole is bounded only by the vacuolar membrane. The feeding tube is formed by the emergence of a preformed microtubular basket consisting of plates of microtubules. At its entrance into the cell body the feeding tube channel is surrounded by an electron-dense ring. Similar sphincters enclose the two exits through which the two flagella emerge. These sphincters are composed of microfibrils which reveal a cross striation when the fixative does not contain calcium ions. The flagellar bases as well as the internal part of the feeding tube are surrounded by a common cavity which is in open connection also with the ampullae of the pusule. The light and electron microscopical observations do not support the assumption that food uptake is driven by a flow of the membrane of the feeding tube channel caused by an interaction with the microtubular basket (as postulated for food uptake inSuctoria) but rather by an hydrostatic gradient which might be caused by rhythmical ion pumping and be based on the existence of the common cavity and the sphincters. Myzocytosis is inhibited by cytochalasin B.—The fine structure of dinospores and trophonts, especially with respect to the cell covering, the amphiesma, and the en- and excystment, is described.     

The carbohydrate structures ofβ-galactosidase from human liver

Acid -galactosidase (EC3.2.1.23) was obtained from human liver in a pure monomeric state (M_r63 000). The carbohydrate content of the enzyme was established to be, 9% by weight; mannose,N-acetylglucosamine, galactose andN-acetylneuraminic acid were found to be the constituent monosaccharides. The carbohydrate structures of the enzyme were studied at the glycopeptide level by employing 500 MHz¹H-NMR spectroscopy, carbohydrate composition analysis and methylation analysis involving GLCMS. Based upon the intensities of relevant signals in the¹H-NMR spectrum, approximately 60% of the chains were found to be of theN-acetyllactosamine type, having the structure The rest appeared to be of the oligomannoside type (Man₅-₆GlcNAc₂Asn). The carbohydrate composition and methylation analysis results sustained these findings, although the calculation of the distribution based upon these techniques indicated a somewhat lower percentage ofN-acetyllactosamine type chains. There are approximately three oligosaccharide chains per molecule. These findings offer an explanation for the abnormal distribution of -galactosidase in tissues and cultured fibroblasts of patients with I-cell disease.     

Distribution and biological activity ofβ-thymosins

Summary -Thymosins, a group of highly homologous peptides consisting of about 40 amino acid residues, were found to be distributed from mammals up to echinoderms. Althogh they have first been isolated from mammalian thymus tissue preparations, their occurrance is not organ-specific and they are present even in different types of cells. For thymosin ₄ several biological activities have been reported, stating that this peptide acts as a thymus peptide hormone and is also involved in the neuroendocrine and immune system. However, it was recently demonstrated that thymosin ₄ has actin-sequestering properties and therefore might play an important role in the regulation of the microfilament system. This fact gives a new outlook on the real biological function of-thymosins.     

The temperature influences the ratio of glucosidase and galactosidase activities of β-glycosidases

The selectivity for the glycon part of a donor substrate of -glycosidases from almond, a mesophilic (Kluyveromyces fragilis) and three highly thermophilic organisms (Caldocellum saccharolyticum, Sulfolobus solfataricus and Pyrococcus furiosus) was investigated at various temperatures (25–90 °C). On the basis of kinetic constants, the selectivity was calculated as the specificity constant (V _max /K _m ) ratio or V _max ratio of glucoside to galactoside donor. In the almond -glucosidase and the mesostable enzyme one enzyme activity dominated whereas the thermostable enzymes expressed both high -glucosidase and high -galactosidase activities. Surprisingly, for -glycosidases from almond, K. fragilis, and C. saccharolyticum the donor selectivity decreased as the temperature increased. In contrast, two of the highly thermostable enzymes (from S. solfataricus and P. furiosus) had constant donor selectivity as the temperature increased. The results thus showed -glycosidases of differing origins to differ markedly in their substrate specificity and in the extent to which their selectivity for the glycon part of the donor substrate is influenced by the temperature.     

The TF1-ATPase and ATPase activities of assembled α3β3γ, α3β3γδ, and α3β3γε complexes are stimulated by low and inhibited by high concentrations of rhodamine 6G whereas the dye only inhibits the α3β3, and α3β3δ complexes

The ATPase activity of the F₁-ATPase from the thermophilic bacterium PS3 is stimulated at concentrations of rhodamine 6G up to about 10 µM where 70% stimulation is observed at 36°C. Half maximal stimulation is observed at about 3 µM dye. At rhodamine 6G concentrations greater than 10 µM, ATPase activity declines with 50% inhibition observed at about 75 µM dye. The ATPase activities of the ₃₃ and ₃₃ complexes assembled from isolated subunits of TF₁ expressed inE. coli deleted of theunc operon respond to increasing concentrations of rhodamine 6G nearly identically to the response of TF₁. In contrast, the ATPase activities of the ₃₃ and ₃₃ complexes are only inhibited by rhodamine 6G with 50% inhibition observed, respectively, at 35 and 75 µM dye at 36°C. The ATPase activity of TF₁ is stimulated up to 4-fold by the neutral detergent, LDAO. In the presence of stimulating concentrations of LDAO, the ATPase activity of TF₁ is no longer stimulated by rhodamine 6G, but rather, it is inhibited with 50% inhibition observed at about 30 µM dye at 30°C. One interpretation of these results is that binding of rhodamine 6G to a high-affinity site on TF₁ stimulates ATPase activity and unmasks a low-affinity, inhibitory site for the dye which is also exposed by LDAO.     

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.     

Purification and characterization of two intracellular β-glucosidases from the Neurospora crassa mutant cell-1

Two intracellular -glucosidases (E.C. 3.2.1.21) were purified from the filamentous fungus Neurospora crassa, mutant cell-1 (FGSC no. 4335) and characterized. The extent of purification were 2.55- and 28.89-fold for -glucosidase A and -glucosidase B, respectively. -Glucosidase A was a dimeric protein, and B a monomeric protein, with molecular masses of 178 and 106 kDa, respectively. Both isoenzymes were glycoproteins with relatively high carbohydrate contents (-glucosidase A, 29.2%; -glucosidase B, 34.2%). The isoelectric points determined by IEF were 6.27 and 4.72, respectively. pH optima for activity were determined to be 5.0 and 5.5, and temperature optima to be 55 and 60 °C, for -glucosidases A and B, respectively. Both purified -glucosidases. especially -glucosidase B, showed relatively high stability against pH and temperature. Both enzymes were stable in the pH range of 5.0–9.0. The activities were completely retained up to 48 h at temperatures below 40 °C. At higher temperatures, enzymes were relatively unstable and lost their activities at 60 °C after 24 h. Both -glucosidases were highly activated by CuCl₂, and inhibited by SnCl₂ and KMnO₄. Hg²⁺ and Ag⁺ also inhibited severely -glucosidase B. The K _m and V _max values of the isoenzymes against cellobiose as substrate were 1.50 mM and 12.2mol min^–1 mg^–1 for -glucosidase A and 2.76 mM and 143.5 mol min^–1 mg^–1 for -glucosidase B.     

Comparative amino acid sequence analysis of Thermotoga maritima β-glucosidase (BglA) deduced from the nucleotide sequence of the gene indicates distant relationship between β-glucosidases of the BGA family and other families of β-1,4-glycosyl hydrolases

The primary structure of the bglA gene region encoding a -glucosidase of Thermotoga maritima strain MSB8 was determined. The bglA gene has the potential to code for a polypeptide of 446 amino acids with a predicted molecular mass of 51545 Da. The T, maritima -glucosidase (BglA) was overexpressed in E. coli at a level comprising approximately 15–20% of soluble cellular protein. Based on its amino acid sequence, as deduced from the nucleotide sequence of the gene, BglA can be classified as a broad-specificity -glucosidase and as a member of the -glucosidase family BGA, in agreement with the results of enzymatic characterization of the recombinant protein. Comparative sequence analysis revealed distant amino acid sequence similarities between BGA family -glucosidases, a -xylosidase, -1,4-glycanases of the enzyme family F (mostly xylanases), and other families of -1,4-glycosyl hydrolases. This result indicates that BGA -glucosidases may comprise one enzyme family within a large enzyme order of retaining -glycosyl hydrolases, and that the members of these enzyme groups may be inter-related at the level of active site architecture and perhaps even on the level of overall three-dimensional fold.     

Thrombin Specificity: Further Evidence for the Importance of the β-Insertion Loop and Trp96. Implications of the Hydrophobic Interaction Between Trp96 and Pro60B Pro60C for the Activity of Thrombin

A number of thrombin mutants have been constructed to investigate the role of Trp⁹⁶ and the -insertion loop for the specificity of thrombin. Thrombin(60D) consists of the replacement of the -insertion loop (14 amino acid residues from 59 to 63, including a 9-residue insertion at position 60) with the corresponding four residues in trypsin, Tyr-Lys-Ser-Gly; thrombin(GGG) is a smaller loop mutation in which the residues Tyr^60APro^60BPro^60CTrp^60D Asp^60ELys^60F of the -insertion loop were replaced by Gly-Gly-Gly; thrombin(96S) consists of a point mutation Trp⁹⁶Ser; and thrombin(GGG/96S) is the double mutant incorporating both changes. Thrombin(96S) clots fibrinogen ~3 times more slowly than thrombin, with the two -insertion loop mutants, thrombin(GGG) and thrombin(GGG/96S), reacting ~3000- and 1300-fold more slowly, respectively. The specificity constant k _cat/K _m for the cleavage of fibrinopeptide A and fibrinopeptide B by thrombin(96S) was 2.6 and 0.35 M^–1 s^–1 respectively, compared to 10 and 2.5 M^–1 s^–1 for wild-type recombinant thrombin, respectively. Kinetic constants were determined for the hydrolysis of H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroaniline. The Michaelis constant K _m increased ~6-fold for thrombin(96S) and >200-fold for thrombin(GGG) and thrombin(GGG/96S) when compared to wild-type recombinant thrombin, while the catalytic constant k _cat remained approximately the same. All mutants were more susceptible to inhibition by BPTI than wild-type recombinant thrombin. Clearly, the -insertion loop is important for thrombin activity. But the mutation of Trp⁹⁶Ser can compensate somewhat for the loss of binding at the -insertion loop. The deletion of the hydrophobic interaction between Trp⁹⁶ and Pro^60BPro^60C appears to decrease the stability of the -insertion loop, thereby causing a decrease in binding efficiency.     

β-Adrenoceptors in the Tree Shrew Brain. I. Distribution and Characterization of [125I]Iodocyanopindolol Binding Sites

1. The number and distribution pattern of -adrenergic receptors in the brain have been reported to be species specific. The aim of the present study was to describe binding of the -adrenoceptor ligand [¹²⁵I]iodocyanopindolol in the brain of the tree shrew (Tupaia belangeri), a species which provides an appropriate model for studies of psychosocial stress and its consequences on central nervous processes.2. ¹²⁵I-Iodocyanopindolol (¹²⁵ICYP) labeling revealed a high degree of nonspecific binding, which was due mainly to interactions of this ligand with serotonin binding sites. For a quantitative evaluation of ₁- and ₂-adrenoceptors, serotonin binding sites had to be blocked by 100 M 5HT.3. Binding of the radioligand to ₁- and ₂-adrenoceptors was characterized using the ₁-specific antagonist CGP20712A and the ₂-specific antagonist ICI118.551. ₁-adrenoceptor binding is present in the whole brain, revealing low receptor numbers in most brain regions (up to 1.5 to 2.7 fmol/mg). A slight enrichment was observed in cortical areas (lateral orbital cortex: 4.0±0.7 fmol/mg) and in the cerebellar molecular layer (8.7±1.0 fmol/mg).4. Competition experiments demonstrated high- and low-affinity binding sites with considerable variations in K _i values for CGP20712A, showing that various affinity states of ₁-adrenoceptors are present in the brain (K _i: 0.61 nM to 67.1 M). In the hippocampus, only low-affinity ₁-adrenoceptors were detected (K _i: 1.3±0.2 M). Since it is known that ¹²⁵ICYP labels not only membrane bound but also internalized -adrenoceptors, it can be assumed that the large population of the low-affinity sites represents internalized receptors which may be abundant due to a high sequestration rate.5. High numbers of ₂-adrenoceptors are present in only a few brain structures of tree shrews (external layer of the olfactory bulb, 15.8±2.0 fmol/mg; claustrum, 19.3±1.5 fmol/mg; anteroventral thalamic nucleus, 19.4±1.5 fmol/mg; cerebellar molecular layer, 55.0±4.3 fmol/mg). Also for this class of -adrenoceptors, high- and low-affinity binding sites for the ₂-selective antagonist ICI118.551 were observed, indicating that ¹²⁵ICYP labels membrane bound and internalized ₂-adrenoceptors. Only in the cerebellar molecular layer was a high percentage of high-affinity ₂-adrenoceptors detected (K _i for ICI118.551 was 1.8±0.3 nM for 90% of the receptors).6. In conclusion, ₁- and ₂-adrenoceptor binding can be localized and quantified by in vitro receptor autoradiography in the brains of tree shrews when serotonergic binding sites are blocked. Modulatory effects of long-term psychosocial conflict on the central nervous -adrenoceptor system in male tree shrews are described in the following paper.     

Expression and characterization of Kluyveromyces lactis β-galactosidase in Escherichia coli

Kluyveromyces lactis -galactosidase gene, LAC4, was expressed in Escherichia coli as a soluble His-tagged recombinant enzyme under the optimized culture conditions. The expressed protein was multimeric with a subunit molecular mass of 118 kDa. The dimeric form of the -galactosidase was the major fraction but had a lower activity than those of the multimeric forms. The purified enzyme required Mn²⁺ for activity and was inactivated irreversibly by imidazole above 50 mM. The activity was optimal at 37 and 40 °C for o-nitrophenyl--d-galactopyranoside (oNPG) and lactose, respectively. The optimum pH value is 7. The K _m and V _max values of the purified enzyme for oNPG were 1.5 mM and 560 mol min^–1 mg^–1, and for lactose 20 mM and 570 mol min^–1 mg^–1, respectively.     

Bounded water kinetic model of β-galactosidase in reverse micelles

In the present investigation, -galactosidase was solubilized into Aerosol OT (AOT)/isooctane reverse micelles. Kinetic data for the hydrolysis of o-nitrophenyl--D-galactopyranoside (ONPG) at different pH values and molar ratios of water to AOT (W_o) were collected. It was observed that the usual kinetic model used for -galactosidase catalysis in aqueous systems failed to represent the experimental data. A bounded water model, however, showed a better correlation between enzymatic activity and W_o. In contrast to the aqueous system, controlling the water concentration in the reverse micelles allows the rate constants for the reaction between water molecules and glycosyl-enzyme complexes to be evaluated.     

Fromβ-lactams toα- andβ-amino acid derived peptides

Summary The potential of-lactams as intermediates for the access to- and-amino acid-derived peptides is shortly reviewed, with major focus on the technologies developed in our group. The two general strategies lie, on one side, in the oxidative ring expansion of 3-hydroxy-lactams toN-carboxy-amino acid anhydrides or Leuch's anhydrides and subsequent coupling with-amino acid esters and, on the other side, in the nucleophilic ring opening ofN-Boc--lactams. Both approaches have been successfully applied to the synthesis of,-diamino acid,-amino--hydroxy acid, polyhydroxylated-amino acid,,-disubstituted-amino acid,-amino acid,-amino--hydroxy acid and,-disubstituted-amino acid derived peptides. Because of the mild reaction conditions needed for the above transformations and the highly stereoselective procedures employed for the construction of the starting-lactam ring, the whole process allows the production of optically pure final products.     

Heterogeneity of caprine beta-casein elucidated by RP-HPLC/MS: genetic variants and phosphorylations

Casein variants occurring in milks from goats homozygous at the _s1-Cn locus were separated and identified by an RP-HPLC/ESI-MS method. Preferential haplotypes arose as well as some particularities in posttranslational modifications. A new variant of caprine -Cn, named C, as well as the phosphorylations pattern of the protein were characterized by the combined use of peptide mass fingerprinting and sequencing by tandem mass spectrometry. The molecular mass of the new variant in its 6P form was measured as 23854 Da and it differs in a mono amino acid substitution, A₁₇₇ V₁₇₇, from the variant A. The phosphorylation pattern of caprine -Cn is homologous to bovine -Cn concerning the 5P located on Ser_{15, 17, 18, 19, 35} but it presents a specific additional phosphorylation site on Thr₁₂ that is comparable to human -Cn phosphorylation located on Thr₃.     

Determination of Hα,Hβ and Hβ,C′ coupling constants in13C-labeled proteins

Summary A sensitive method to assign H protons stereospecifically as well as to determine rotamer populations about ₁, in two 3D experiments is presented. The SOFT-HCCH-COSY experiment allowed us to measure the³J(H,C) couplings, using constant time evolution of C in t₂ and C_aliphatic-selective decoupling during t₃. The SOFT-HCCH-E.COSY experiment allowed us to measure the³J(H,H) couplings, using constant time evolution of C in t₂, a small flip angle¹H excitation pulse in the second mixing time, and double-band-selective decoupling (aliphatic and carbonyl carbons) during t₃. The method was applied to ribonuclease T₁.     

Role of mitogen-activated protein kinase pathway in reactive oxygen species-mediated endothelin-1-induced β-myosin heavy chain gene expression and cardiomyocyte hypertrophy

Endothelin-1 (ET-1) has been found to increase cardiac -myosin heavy chain (-MyHC) gene expression and induce hypertrophy in cardiomyocytes. ET-1 has been demonstrated to increase intracellular reactive oxygen species (ROS) in cardiomyocytes. The exact molecular mechanism by which ROS regulate ET-1-induced -MyHC gene expression and hypertrophy in cardiomyocytes, however, has not yet been fully described. We aim to elucidate the molecular regulatory mechanism of ROS on ET-1-induced -MyHC gene expression and hypertrophic signaling in neonatal rat cardiomyocytes. Following stimulation with ET-1, cultured neonatal rat cardiomyocytes were examined for ³H-leucine incorporation and -MyHC promoter activities. The effects of antioxidant pretreatment on ET-1-induced cardiac hypertrophy and mitogen-activated protein kinase (MAPKs) phosphorylation were studied to elucidate the redox-sensitive pathway in cardiomyocyte hypertrophy and -MyHC gene expression. ET-1 increased ³H-leucine incorporation and -MyHC promoter activities, which were blocked by the specific ET_A receptor antagonist BQ-485. Antioxidants significantly reduced ET-1-induced ³H-leucine incorporation, -MyHC gene promoter activities and MAPK (extracellular signal-regulated kinase, p38, and c-Jun NH₂ -terminal kinase) phosphorylation. Both PD98059 and SB203580 inhibited ET-1-increased ³H-leucine incorporation and -MyHC promoter activities. Co-transfection of the dominant negative mutant of Ras, Raf, and MEK1 decreased the ET-1-induced -MyHC promoter activities, suggesting that the Ras-Raf-MAPK pathway is required for ET-1 action. Truncation analysis of the -MyHC gene promoter showed that the activator protein-2 (AP-2)/specificity protein-1 (SP-1) binding site(s) were(was) important cis-element(s) in ET-1-induced -MyHC gene expression. Moreover, ET-1-induced AP-2 and SP-1 binding activities were also inhibited by antioxidant. These data demonstrate the involvement of ROS in ET-1-induced hypertrophic responses and -MyHC expression. ROS mediate ET-1-induced activation of MAPK pathways, which culminates in hypertrophic responses and -MyHC expression. Tzu-Hurng Cheng, Neng-Lang Shih: These authors have equally contributed to this work     

The inhibitory effect of trilinolein on norepinephrine-induced β-myosin heavy chain promoter activity,reactive oxygen species generation,and extracellular signal-regulated kinase phosphorylation in neonatal rat cardiomyocytes

The myocardial protective effects of trilinolein, isolated from the traditional Chinese herbSanchi (Panax notoginseng), are thought to be related to its antioxidant activity. However, the intracellular mechanism underlying the protective effect of trilinolein in the heart remains unclear. In the present study, we investigated the effect of trilinolein on norepinephrine (NE)-induced protein synthesis in cardiomyocytes. Cultured neonatal rat cardiomyocytes were stimulated with NE, then protein content, [³H]-leucine incorporation, and -myosin heavy chain (-MyHC) promoter activity were examined. The effect of trilinolein on NE-induced intracellular reactive oxygen species (ROS) generation was measured with a redoxsensitive fluorescent dye (2,7-dichlorofluorescin diacetate) and extracellular signal-regulated kinase (ERK) phosphorylation by Western blotting. Trilinolein inhibited NE-increased protein synthesis, -MyHC promoter activity, and intracellular ROS. Both trilinolein and the antioxidant, N-acetyl-cysteine, decreased NE- and H₂O₂-induced protein synthesis, -MyHC promoter activity, and ERK phosphorylation. These data indicate that trilinolein inhibits NE-induced protein synthesis via attenuation of ROS generation in cardiomyocytes.     

Enhancement of β-glucosidase stability and cellobiose-usage using surface-engineered recombinant Saccharomyces cerevisiae in ethanol production

To enhance the use of cellobiose by a recombinant Sachharomyces cerevisiae, the expressed -glucosidase that hydrolyzes cellobiose was stabilized using a surface-display system. The C-terminal half of -agglutinin was used as surface-display motif for the expression of -glucosidase in the cell wall. The surface-displayed -glucosidase had a half-life time (t _1/2) of 100 h in acidic culture broth conditions, while secreted -glucosidase had a t _1/2 of 60 h. With such stabilization of -glucosidase, the surface-engineered S. cerevisiae utilized 7.5 g cellobiose l^–1 over 60 h, while S. cerevisiae secreting -glucosidase into culture broth used 5.8 g cellobiose l^–1 over the same period.