Photoactivatable Analogues of α-Conotoxins GI and MI and Their Interaction with Nicotinic Acetylcholine Receptor

Two photoactivatable analogues of -conotoxin GI with the benzoylphenylalanine residue (Bpa) substituted for His10 or Tyr11 were synthesized using the method of solid-phase peptide synthesis. In addition, -conotoxin MI was chemically modified by placing an azidobenzoyl or a benzoylbenzoyl photo label at N of Gly1 or N of Lys10. All the photoactivatable analogues were purified by HPLC, their structures were confirmed by MALDI MS, and the label positions in their molecules were localized by MS of their trypsinolysis fragments. All the analogues interacted with the nicotinic acetylcholine receptor (AChR) from Torpedo californica as efficiently as the native -conotoxins, with the differences in the inhibition constants being within one order of magnitude under the same conditions. [¹²⁵I] Derivatives prepared from all the analogues retained the ability to be bound by AChR and were used in the photoinduced AChR crosslinking. All the AChR subunits were found to be crosslinked to the photoactivatable analogues, with the linking depending on both the chemical nature of label and its position in the -conotoxin molecule.     

Phosphorylation of the Na,K-ATPase by Ca,phospholipid-dependent andcAMP-dependent protein kinases. Mapping of the region phosphorylated by Ca,phospholipid-dependent protein kinase

Ca,phospholipid-dependent (PKC) andcAMP-dependent (PKA) protein kinases phosphorylate the -subunit of the Na,K-ATPase from duck salt gland with the incorporation of 0.3 and 0.5 mol³²P/mol of -subunit, respectively. PKA (in contrast to PKC) phosphorylates the -subunit only in the presence of detergents. Limited tryptic digestion of the Na,K-ATPase phosphorylated by PKC demonstrates that³²P is incorporated into the N-terminal 41-kDa fragment of the -subunit. Selective chymotrypsin cleavage of phosphorylated enzyme yields a 35-kDa radioactive fragment derived from the central region of the -subunit molecule. These findings suggest that PKC phosphorylates the -subunit of the Na,K-ATPase within the region restricted by C₃ and T₁ cleavage sites.     

The Neuronal Nicotinic Acetylcholine Receptor in Some Hereditary Epilepsies

Recent advances in human genetics and in the neurobiology of neurotransmitter receptors and channels have led to the discovery of specific genes associated with hereditary epileptic phenotypes. All the genes identified to date code for ligand- and voltage-gated ion channels. Some clinically rare idiopathic epilepsies are associated with mutations in genes coding for different neuronal nicotinic acetylcholine receptor (AChR) subunits. Distinct subunits are found in the brain and in the peripheral nervous system, and structural, non- subunits like 2 and 4 confer different properties to neuronal receptors. Thus, the final properties of the oligomeric AChR depend on the different combinations of and subunits. Most mutations found so far occur in the 4 chain, the most abundant subunit in the central nervous system. Specifically, the identification of mutations in the 4 subunit of neuronal AChR in human benign familial neonatal convulsions (BFNC) and autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) raise the possibility that the observed gene defects are linked (causatively) with these two diseases or, alternatively, that AChR 4 mutants increase the probability of epileptic discharges. We discuss testable hypotheses for unraveling the pathophysiology of these two disorders associated with AChR mutations.     

A novel wheat alpha-amylase gene (alpha-Amy3)

Summary A genomic clone of a wheat -amylase gene (Amy3/33) was identified, on the basis of hybridisation properties, as different from -Amy1 and -Amy2 genes which had been characterised previously. The nucleotide sequence revealed that this gene has the normal sequence motifs of an active gene and an open reading frame interrupted by two introns. The protein sequence encoded by this open reading frame is recognisably similar to that of -amylase from the -Amy1 and -Amy2 genes and there is high sequence homology in all three proteins at the putative active sites and Ca⁺⁺ binding region. In addition, the introns are at positions equivalent to the position of introns in the -Amy1 and -Amy2 genes. However, the sequence was less similar to -Amy1 and -Amy2 than these are to each other. Southern blot analysis showed that the Amy3/33 DNA is one of a small multigene family carried on a different chromosome (group 5) from either the -Amy1 or -Amy2 genes. A further difference from the -Amy1 and -Amy2 genes was the pattern of expression. Amy3/33 was expressed only in immature grains and, unlike the -Amy1 and -Amy2 genes, not at all in germinating aleurones. These data suggested therefore that this gene represents a third type of -amylase gene, not described before, which shares a common evolutionary ancestor with the -Amy1 and -Amy2 genes.     

Isolation of α and β Brain Tubulin Subunits after Alkaline Treatment of the Protein

We demonstrate here that brain purified tubulin can be dissociated into and subunits at pH > 10 and that the subunits can be separated by using the Triton X-114 phase separation system. After phase partition at pH > 10, tubulin but not tubulin behaves as a hydrophobic compound appearing in the detergent rich phase. After three extractions of the alkaline aqueous phase with Triton X-114, about 90% of the tubulin was recovered in the detergent rich phase. The hydrophobic behavior observed for tubulin after its dissociation at pH 11.5 was not due to an irreversible change of the protein, because when the detergent rich phase containing tubulin was diluted with a buffer solution at pH 7.3 and the solution allowed to partition again, -tubulin is recovered in the aqueous phase. The detergent in the aqueous phase of the and tubulin preparations can be removed up to 90% by 12 h dialysis. The and subunits of tubulin from kidney and liver behave, in this phase separation system, like those of brain tubulin.     

Analysis of exposed regions on the main extracellular domain of mouse acetylcholine receptor α subunit in live muscle cells by binding profiles of antipeptide antibodies

To study the structural organization of the main extracellular domain of the nicotinic acetylcholine receptor (AChR) subunit in live muscle cells, we examined the native membrane-bound receptors in cultured mouse skeletal muscle cells for their ability to bind a panel of antibodies against uniform-sized overlapping synthetic peptides which collectively represent this entire domain. The binding profile indicated that the regions 23–49,78–126,146–174, and182–210 are accessible to binding with antibody. Residues23–49,78–126, and194–210 contain binding regions for-neurotoxin and some myasthenia gravis autoantibodies. A comparison of this binding profile with the profile obtained for membrane-boundTorpedo californica AChR in isolated membrane fractions showed some similarities as well as significant differences between the subunit organization in the isolated membrane fraction and that in the membrane of live muscle cells. Regions89–104 and158–174, which are exposed in the isolated membrane fraction, are also exposed in the live cell. On the other hand, regions23–49, and182–210, which are exposed in the live cell, are not accessible in the isolated membrane and, furthermore, the region1–16, which has marginal accessibility in the cell, becomes highly accessible in the membrane isolates. The exposed regions defined by this study may be the primary targets for the initial autoimmune attack on the receptors in experimental autoimmune myasthenia gravis.     

Erratum: Cloning and sequencing of the phycocyanin gene from Spirulina maxima and its evolutionary analysis

The genes were cloned for the two apoprotein subunits, and ,of phycocyanin from the cyanobacterium Spirulina maxima = Arthrospiramaxima) strain F3. The - and -subunit gene-coding regionscontain 489 bp and 519 bp, respectively. The -subunit gene is upstreamfrom the -subunit gene, with a 111-bp segment separating them.Similarities between the -subunits of S. maxima and nine othercyanobacteria were between 58% and 99%, as were those between the -subunits. The maximum similarity between the - and -subunits from S. maxima was 27%.     

Structural and functional properties of proteasome activator PA28

The proteasome activator PA28 or 11S regulator is a protein complex composed of two different but homologous polypeptides, termed PA28 and PA28. The purified activator protein (_200 kDa) is a ring-shaped heteromultimer containing the two polypeptides, possibly with an _{3 3} stoichiometry. The activator, which by itself shows no hydrolytic activity elicits activation of the proteasome's multiple peptidase activities by binding to the terminal rings of the proteinase. In vitro, active PA28 can be reconstituted from isolated and subunits, yielding two different oligomers: with the single subunit, PA28 homomultimers with moderate stimulatory activity toward 20S proteasomes are obtained whereas isolated -subunits are unable to form oligomers and are devoid of stimulatory activity. However, in the presence of both subunits, heteromultimers form, concomitant with restoration of full stimulatory activity. The recent finding that PA28 modulates the proteasome-catalyzed production of antigenic peptides presented to the immune system on MHC class I molecules indicates a cellular function of the activator in antigen processing. Abbreviations: IFN – interferon; LMP – low molecular weight peptide; MHC – major histocompatibility complex.     

Sensitive Nonradioactive Method for Screening Compounds Interacting with Neuronal α7 Cholinoreceptor

A sensitive nonradioactive method for detection of substances interacting with the neuronal 7-type nicotinic acetylcholine receptor (AChR) was proposed. The method uses biotinylated -cobratoxin (Bt-CTX) and is based on the ability of the N-terminal ligand-binding extracellular domain (LBED) of AChR to interact with -cobratoxin (CTX) as does the whole receptor. LBED produced by heterologous expression of a gene fragment of the 7 subunit of AChR from the rat brain in Escherichia coli cells was sorbed in wells of a 96-well plate and incubated with Bt-CTX. The specifically bound Bt-CTX was determined by staining with streptavidin–peroxidase complex. The ability of other compounds to interact with 7-AChR was checked according to the degree with which they inhibit the Bt-CTX binding to LBED. Nicotine, carbamylcholine, d-tubocurarin, anabaseine, conotoxin ImI, and neurotoxin II were used as model compounds. The sensitivity of this method was comparable with that of the radioligand method (up to 10 pmol).     

The quaternary structure of four crustacean two-hexameric hemocyanins: immunocorrelation,stoichiometry, reassembly and topology of individual subunits

Summary Two-hexameric (2×6) hemocyanins from the brachyuran crabsCancer pagurus andCallinectes sapidus, the freshwater crayfishAstacus leptodactylus and the lobsterHomarus americanus were isolated and dissociated into native subunits.The subunits of each hemocyanin were analyzed by electrophoresis and immunology. Three immunologically distinct subunit types, which were termed, and, could be identified in each case. They were isolated preparatively, and interspecifically correlated. Subunit is subdivided into several electrophoretically distinct isoforms which are immunologically closely related (Astacus) or identical (other species). InAstacus andCancer one of these isoforms was shown to dimerize and to act as inter-hexamer bridge. It represents a fourth subunit type termed. A fifth, diffuse component, which in PAGE migrated at the position of a dimer, was identified in the crossed immunoelectrophoretic patterns as denatured hemocyanin.A common feature of the four hemocyanins is the presence of 4 copies of and 8 copies of/ within the 2×6 particles. The: ratio is 4:4 in the two Astacidea and 6:2 in the two Brachyura. exists in 2 copies inAstacus andCancer which means that a single dimer- is present in a two-hexamer. This leaves 2 monomeric copies inAstacus and 4 inCancer.Every subunit from the four species except ofAstacus - was capable to form hexamers in reassembly experiments. If subunit combinations were tested, hetero-hexamers were formed preferentially. Two-hexamers were reconstituted only in the presence of all subunit types and the native subunit stoichiometry was required to obtain twohexamers in considerable yields. Factors limiting 2×6 reassembly are discussed.Authentic 2×6 molecules ofAstacus, Homarus andCancer hemocyanin were immunolabeled with subunit-specific antibody fragments (F_ab) or IgG molecules, and the resulting immuno complexes were studied in the electron microscope. A topological model of the quaternary structure of decapod 2×6 hemocyanins is derived, showing the position of each copy of the four subunit types. In this model, the inter-hexamer bridge- is surrounded by two and two subunits forming the central core of the dodecamer. Two additional and two additional subunits form the periphery together with one subunit occupying the peripheral short edges of each hexameric half structure. The model is discussed with respect to the current literature.Abbreviations PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate This paper is decicated to Professor Dr. Bernt LinzenPreliminary accounts of this work have been presented in the proceedings of a symposium at Tutzing 1985. Linzen B (ed) (1986) Invertebrate oxygen carriers. Springer, Berlin Heidelberg New York. This also includes: Stöcker et al. 1986; Markl et al. 1986) and in a review article (Markl 1986)     

Identification of a locality in snake venomα-neurotoxins with a singificant compositional similarity to marine snailα-conotoxins: Implications for evolution and structure/activity

Summary -neurotoxins from elapid snake venoms and-conotoxins from marine snails bind specifically and with high affinity to nicotinic cholinoceptors. Although both types of toxin are polypeptides, there is more than a fourfold difference in size between the two and no clear sequence homology is evident. A systematic computer search of the three-dimensional structure of erabutoxin b (an-neurotoxin from the false sea snakeLaticauda semifasciata) was performed to identify the locality that most closely matched the amino acid compositions of the smaller-conotoxins (from the marine snailsConus magus andConus geographus). The area of greatest similarity centered on residue position 25 of erabutoxin b, a locale that is conserved throughout the snake-neurotoxins and their homologues. Six Proteins unrelated to erabutoxin b were compared to the-conotoxins to show that the extent of the erabutoxin b/-conotoxin match was too high to be coincidental. Homologues of erabutoxin b, namely-cobratoxin fromNaja naja siamensis and cytotoxin V^II4 fromNaja mossambica mossambica, were also analyzed. The extent of the matching with the-conotoxins decreased in the series erabutoxin b>-cobratoxin>cytotoxin V^II4, and this also relates the order of similarity to the pharmacological properties of the-conotoxins.The-conotoxin-like area of the snake-neurotoxins is peripheral to the site previously considered important for binding to the cholinoceptor, even though it seems to represent the focus of evolutionary convergence between the two types of neurotoxin. The area of resemblance does, however, have strong associations with the conformational behavior of the snake toxins. Hence, the outcome of this study has important consequences for the current ideas on snake-neurotoxin structure/activity relationships and the evolutionary origins of neurotoxicity.     

Enhancement of α-amylase production by integrating and amplifying the α-amylase gene of Bacillus amyloliquefaciens in the genome of Bacillus subtilis

Summary The -amylase gene of Bacillus amyloliquefaciens was integrated into the genome of Bacillus subtilis by homologous recombination. In the first transformation step, several strains were obtained carrying the -amylase gene as two randomly located copies. These strains produced -amylase in the quantities comparable with that of the multicopy plasmid pKTH10, carrying the same -amylase gene. With the plasmid system, however, the rate of the -amylase synthesis was faster and the production phase shorter than those of the chromosomally encoded -amylase. The two chromosomal gene copies were further multiplied either by amplification using increasing antibiotic concentration as the selective pressure or by performing a second transformation step, identical to the first integration procedure. Both methods resulted in integration strains carrying up to eight -amylase gene copies per one genome and producing up to eightfold higher -amylase activity than the parental strains. Six out of seven transformants, studied in more detail, were stable after growth of 42 h even without antibiotic selection. The number of the DNA and mRNA copies of the -amylase gene was quantitavely determined by sandwich hybridization techniques, directly from culture medium.     

Two Novel α-Neurotoxins Isolated from Taiwan Cobra: Sequence Characterization and Phylogenetic Comparison of Homologous Neurotoxins

Two novel postsynaptic neurotoxins (-neurotoxins) isolated and purified from the Taiwan cobra venom (Naja naja atra) possess distinct primary sequences and different neurotoxicities as compared with the most abundant and lethal component in the venom, i.e., cobrotoxin characterized before from the same venom. The complete sequences of two neurotoxin analogues were determined by N-terminal Edman degradation and comparison of amino acid compositions of proteolytic toxin fragments with other homologous toxins of known sequences. The short-chain neurotoxin consists of 61 amino acid residues with eight conserved cysteine residues and is found to show 78% sequence identity with cobrotoxin. The other toxin, consisting of 65 residues with ten cysteines, belongs to the family of long-chain neurotoxins. It is the first long-chain -neurotoxin reported from the Taiwan cobra. The lethal toxicities of these two novel neurotoxins were much lower than cobrotoxin, albeit with close structural homology among the three toxins in terms of their primary sequences and tertiary structure predicted by homology modeling. Multiple sequence alignment and comparison coupled with construction of a phylogenetic tree for various -neurotoxins of Naja and closely related genuses have established that all nicotinic -neurotoxins present in the snake family of Elapidae are closely related to each other, presumably derived from an ancestral polypeptide by gene duplication and subsequent multiple mutational substitutions.     

Proteolytic digestion of α-lactalbumin: Physiological implications

The kinetics of the partial digestion of bovine -lactalbumin (-LA) by trypsin, -chymotrypsin, and pepsin was monitored by lactose synthase activity, HPLC, and difference spectrophotometry. The relative stabilities of the various metal-bound states of -LA to trypsin and chymotrypsin at 37 and 5°C decrease in the following order: Ca(II)--LA>Zn(II), Ca(II)--LA>apo--LA. The HPLC digestion patterns of Ca(II)--LA and Zn(II), Ca(II)--LA at 5 and 37°C were similar, while the corresponding digestion patterns for apo--LA were quite different, reflecting the existence of the thermally induced denaturation states of apo--LA within this temperature region. Occupation of the first Zn(II)-binding site in Ca(II)-loaded -LA slightly alters the HPLC digestion patterns at both temperatures and accelerates the digestion at 37°C due to Zn(II)-induced shift of the thermal transition of -LA, exposing some portion of thermally denatured protein. The results suggest that the binding of Zn(II) to the first Zn(II)- (or Cu(II))-specific site does not cause any drastic changes in the overall structure of -LA. The acidic form of -LA (atpH 2.2 and 37°C) was digested by pepsin at rates similar to that for the apo- or Cu(II), Ca(II)-loaded forms by trypsin or -chymotrypsin at neutralpH. Complexation of -LA with bis-ANS affords protection against pepsin cleavage. It is suggested that the protective effects of similar small lipophilic compounds to -LA may have physiological significance (e.g., for nutritional transport).On leave from the Institute of Biological Physics, USSR Academy of Sciences, Pushchino, Moscow Region, 142292, USSR.     

Models of the three-dimensional structures of echidna, horse, and pigeon lysozymes: Calcium-binding lysozymes and their relationship with α-lactalbumins

Similarities in amino acid sequences, three-dimensional structures, and the exon-intron patterns of their genes have indicated thatc-type lysozymes and-lactalbumins are homologous proteins, i.e., descended by divergent evolution from a common ancestor. Like the-lactalbumins, echidna milk, horse milk, and pigeon eggwhite lysozymes all bind Ca(II). Models of their three-dimensional structures, based on their amino acid sequences and the known crystal structures of domestic hen eggwhite and human lysozymes and baboon and human-lactalbumins, have been built. The several structures have been compared and their relationships discussed.     

Induction of extracellular arabinases on monomeric substrates in Aspergillus niger

The induction of extracellular arabinases by pentose sugars and polyols generated by the metabolic pathway of l-arabinose and d-xylose catabolism in Aspergillus niger was investigated. Induction occurred with l-arabinose and l-arabitol but not with d-xylose or xylitol. l-arabitol in particular was found to be a good inducer for -l-arabinofuranosidase and endo-arabinase activities. Western blotting analysis showed both -l-arabinofuranosidase A and B to be present. No induction was observed using d-arabitol. Unlike the wild type A. niger N402 strain, the A. niger xylulose kinase negative mutant N572 also showed induction of -l-arabinofuranosidases A and B and endo-arabinase activity on d-xylose and xylitol. This is due to metabolic conversion of these compounds leading to the accumulation of both xylitol and l-arabitol in this mutant, the latter of which then acts as inducer. The induction of the two -l-arabinofuranosidases and endo-arabinase is under the control of two regulatory systems namely pathway specific induction and carbon catabolite repression. Under derepressing conditions in the wild type only -l-arabinofuranosidase B could be detected by Western blotting analysis. This indicates that -l-arabinofuranosidase B is of importance in the initiation of specific induction of the various arabinose activities in A. niger grown on arabinose containing structural polysaccharides.Abbreviations PNA p-nitrophenyl--l-arabinofuranoside     

Differential subcellular localization of ACTH and α-MSH in corticotropes of the rat anterior pituitary

Summary Specific antisera to -melanotropin (-MSH) and corticotropin (ACTH 1-39) were used to obtain immunocytochemical evidence for the differential localization of -MSH and ACTH in the secretory granules of corticotropes of rat anterior pituitary. The specificity of the antisera was established by binding ¹³¹I-labeled -MSH and ACTH 1-39 to their respective antisera. Double-labeling immunocytochemistry (for -MSH, ferritin; for ACTH, colloidal gold) was performed. Some secretory granules were labeled with ferritin particles (-MSH), whereas others contained gold particles (ACTH). Only a few granules showed both ACTH and -MSH. In typical corticotropes (stellate in form with a small number of secretory granules aligned along the cell periphery) only some of the secretory granules that were labeled with anti-ACTH serum were also immunoreactive to anti--MSH. In atypical corticotropes (polygonal in shape and containing a large number of secretory granules) almost all of the immunoreactive ACTH secretory granules were also positive to anti--MSH serum. An intermediate type of corticotrope was observed containing a small number of secretory granules, almost all of which were labeled with anti--MSH. Thus, rat anterior pituitary corticotropes may be classified into three types according to the distribution and content of -MSH. The light-microscopic immuncytochemistry provided similar results.     

Confirmation of assignment of the human α1-crystallin gene (CRYA1) to chromosome 21 with regional localization to q22.3

Summary The crystallins are highly conserved structural proteins universally found in the eye lens of all vertebrate species. In mammals, three immunologically distinct classes are present, -, -, and -crystallins, and each class represents a multigene family. The -crystallin gene family consists of 1-crystallin (CRYA1) and 2-crystallin (CRYA2) genes (previously designated A-and B-crystallin, respectively), which show extensive sequence homology. We constructed a synthetic oligonucleotide probe of 25 bases corresponding to a specific region of the human 1-crystallin gene sequence. This 25-mer probe bears little sequence homology to human 2-crystallin gene and does not cross-hybridize to 2-crystallin sequences in Southern blot analysis. Using this unique synthetic probe, we have demonstrated the identity of the 1-crystallin gene in human genomic DNA. In addition, we have also confirmed its chromosomal location on human chromosome 21. Finally, we have regionally localized the gene to q22.3 by using both Southern blot analysis of a panel of cell hybrids containing different parts of human chromosome 21, and in situ hybridization to metaphase chromosomes. The use of synthetic oligonucleotide probes specific for individual genes should be useful in identifying and mapping members of multigene families.     

Genetics of α-amylases from rye endosperm

Summary Fifteen inbred lines of rye, F₁ and F₂ progenies from crosses between lines were studied using polyacrylamide gel electrophoresis. Conventional genetic analysis of -amylase zymograms showed that the 19 bands detected in the endosperm of germinating caryopses were controlled by three linked structural loci and one independent modifying locus, which influenced the electrophoretic mobility of isozymes. Two codominant alleles were found at the -Amy1, -Amy2 structural loci and the M--Amy modifying locus while the -Amy3 locus had three alleles. Double-banded expression of the -amylase alleles was probably due to the simultaneous presence of modified and unmodified forms of isozymes on the zymogram.This work was supported by Polish Academy of Sciences under project MR-II/7 and was also a part of the author's PhD Thesis