Effects of ethanol on calcium channels, potassium channels, and vasopressin release.
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Acetylcholine↗
Biomedical subjects
H Bayley
Publications and source records attributed to H Bayley.
Assaying nanogram amounts of dilute protein.
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Adsorption↗
Cell-free transcription and translation of Na,K-ATPase alpha and beta subunit cDNAs.
Synthetic mRNAs (i.e. cRNA alpha and cRNA beta) were obtained by cell-free transcription of M13 KS(+) (Bluescript) expression vectors which contained the entire coding region of the alpha or beta subunits of lamb kidney Na,K-ATPase. Translation in reticulocyte lysates of cRNA alpha yielded full length alpha polypeptide, as well as a limited array of immunoprecipitable lower molecular weight products. cRNA beta yielded a single immunoprecipitable full length polypeptide. Association of the alpha polypeptide with the microsomal membranes was obtained only co-translationally. Fifteen to 50% of the membrane-associated alpha subunit was resistant to extraction with alkali. The resistance of a 29-kDa fragment to trypsinolysis indicated that the alpha subunit was inserted into microsomal membranes. In the presence of dog pancreatic microsomes, the beta polypeptide was glycosylated as indicated by the appearance of three higher molecular weight polypeptides that were sensitive to endoglycosidase H and bound to Concanavalin A. The beta subunit was predominantly translocated into the lumen of the endoplasmic reticulum since 90% of the mass of the membrane-associated beta polypeptide was resistant to trypsin (i.e. reduced in size from 40 kDa to 37.5 kDa), and 95% of all of the beta chains were resistant to extraction with alkali. Neither the alpha nor the beta subunits have NH2-terminal leader signal sequences, but both may require the signal recognition receptor for membrane insertion, as evidenced by inhibition of incorporation of both subunits into microsomes pretreated with N-ethylmaleimide. Simultaneous translation of cRNA alpha and cRNA beta did not enhance membrane insertion of either the alpha or beta polypeptide.
Acetylglucosaminidase↗
Phototoxic liposomes coupled to an antibody that alone cannot modulate its cell-surface antigen kill selected target cells.
Molecules such as antibodies that bind to cell surfaces can be used to deliver cytotoxic drugs to selected cells. To be effective the drug must usually be taken into the cells by endocytosis. In this study a T-cell line (CCRF-CEM) was effectively killed by liposomes carrying a photosensitizer and bearing the antibody OKT4 (anti-CD4). The unconjugated antibody does not induce antigenic modulation in the target cells, an indication of the absence of endocytosis, and would therefore not normally have been selected as an agent for drug delivery. It cannot, however, be concluded with certainty that the conjugates act at the cell surface and several alternative explanations of their efficacy are offered.
Animals↗
A relative of the catalytic subunit of cyclic AMP-dependent protein kinase in Aplysia spermatozoa.
Transcripts encoding CAPL-B, an apparent member of the cyclic-nucleotide-regulated kinase subfamily in Aplysia californica, are found exclusively in the ovotestis and are concentrated in meiotic and postmeiotic spermatogenic cells. The CAPL-B polypeptide is present in mature spermatozoa, suggesting that the kinase plays a part in regulating events associated with fertilization.
Amino Acid Sequence↗
The RII subunit of cAMP-dependent protein kinase binds to a common amino-terminal domain in microtubule-associated proteins 2A, 2B, and 2C.
Three products of the MAP2 gene are known: MAP2A and MAP2B (Mr approximately 200,000) and MAP2C (Mr 70,000). The structural relationship between these MAPs and the basis for their diversity in size are unknown. Previously, we found that a significant fraction of type II cAMP-dependent protein kinase was associated via its regulatory subunits with MAP2A and MAP2B. We now use an antibody prepared against the microtubule binding domain of MAP2A and MAP2B to identify MAP2C. All three forms of MAP2 bound to cAMP affinity columns and reacted with 32P-labeled RII in a blot overlay assay. By assaying proteolytic fragments of MAP2A and MAP2B as well as segments of MAP2 expressed in E. coli, the binding site for RII was localized to an 83 amino acid stretch at the distal (amino-terminal) end of the MAP2 arm domain. Therefore, the microtubule binding and RII binding domains are located at extreme opposite ends of MAP2A and MAP2B, and both are conserved in the much shorter MAP2C.
Animals↗
Purification and sequencing of neuropeptides contained in neuron R15 of Aplysia californica.
R15 is a large identified neuron present in the abdominal ganglion of the mollusc Aplysia. Previous studies have indicated that this neuron may play a role in water balance and possibly renovascular functions. A peptidic factor contained in the neuron R15 has been shown to increase the water content of Aplysia. To determine the structure of the peptides contained in R15, we purified the extracts of 820 R15 cells by means of two steps of reverse-phase HPLC. The purification yielded a number of peptides, only one of which, R15 alpha 1, resulted in water uptake when injected into animals. Determination of the amino acid content and sequence analysis of the R15 alpha 1 peptide demonstrated that this peptide contains 38 residues, including two cysteines. The peptide failed to react with iodoacetate, indicating that the two cysteines are connected by a disulfide bridge. To confirm the assigned structure, the peptide was synthesized with a disulfide bridge. The chromatographic properties and bioactivity of the synthetic material were identical to those of the native peptide. Several other R15 peptides were inactive in the bioassay for water uptake. The sequence of one of these peptides (R15 beta) was determined, and it was established that the peptide contains 28 residues. Amino acid analysis of three other peaks was performed. One of these peaks contained a peptide (R15 beta f) whose amino acid composition suggests that it is a fragment of the R15 beta peptide. The other two peaks contained peptides with identical amino acid compositions, suggesting that they are variants of a single peptide (R15 gamma). The amino acid sequences of all the peptides identified in neuron R15 correspond to stretches of a polyprotein encoded by a recently sequenced R15 cDNA.
Amino Acid Sequence↗
Two catalytic subunits of cAMP-dependent protein kinase generated by alternative RNA splicing are expressed in Aplysia neurons.
The amino acid sequences of two catalytic (C) subunits of Aplysia cAMP-dependent protein kinase (cAPK) have been deduced from the nucleotide sequences of cDNAs generated from neuronal poly(A)+ RNA. Both subunits contain 352 residues and are identical except for amino acids 142-183, which differ at 10 out of 42 positions. They derive from alternatively spliced transcripts of a single gene (CAPL) containing two mutually exclusive exon cassettes. CAPL transcripts are present in several classes of identified neurons containing transmitter-sensitive adenylate cyclase, including sensory cells, bag cells, and the left pleural giant cell. Combinatorial expression of the various regulatory (R) and C subunits might produce kinase isoforms with distinct roles in neuronal modulation. Alternatively, holoenzymes with overlapping properties together might contribute to the definition of individual cell types and physiological states.
Amino Acid Sequence↗
Interaction of blood coagulation factor Va with phospholipid vesicles examined by using lipophilic photoreagents.
Two different lipophilic photoreagents, [3H]adamantane diazirine and 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine (TID), have been utilized to examine the interactions of blood coagulation factor Va with calcium, prothrombin, factor Xa, and, in particular, phospholipid vesicles. With each of these structurally dissimilar reagents, the extent of photolabeling of factor Va was greater when the protein was bound to a membrane surface than when it was free in solution. Specifically, the covalent photoreaction with Vl, the smaller subunit of factor Va, was 2-fold higher in the presence of phosphatidylcholine/phosphatidylserine (PC/PS, 3:1) vesicles, to which factor Va binds, than in the presence of 100% PC vesicles, to which the protein does not bind. However, the magnitude of the PC/PS-dependent photolabeling was much less than has been observed previously with integral membrane proteins. It therefore appears that the binding of factor Va to the membrane surface exposes Vl to the lipid core of the bilayer, but that only a small portion of the Vl polypeptide is exposed to, or embedded in, the bilayer core. Addition of either prothrombin or active-site-blocked factor Xa to PC/PS-bound factor Va had little effect on the photolabeling of Vl with TID, but reduced substantially the covalent labeling of Vh, the larger subunit of factor Va. This indicates that prothrombin and factor Xa each cover nonpolar surfaces on Vh when the macromolecules associate on the PC/PS surface. It therefore seems likely that the formation of the prothrombinase complex involves a direct interaction between Vh and factor Xa and between Vh and prothrombin.(ABSTRACT TRUNCATED AT 250 WORDS)
Adamantane↗
Selective killing of T lymphocytes by phototoxic liposomes.
Two-fold specificity in drug delivery obtained through the localized activation of drugs by physical means and the attachment of drugs to proteins that bind to target cells might be used for highly selective cancer chemotherapy or for immunosuppression. Toward this end, a monoclonal antibody against an antigen on the surface of T lymphocytes was covalently attached to liposomes containing a phototoxic drug, pyrene, bound to the lipid bilayer. When unfractionated peripheral blood lymphocytes, or B- and T-cell lines, were irradiated after treatment with these liposomes, T cells were killed while B cells were spared, demonstrating the validity of the approach in a simple in vitro assay.
Antibodies, Monoclonal↗
Molecular cloning and primary structure of myelin-associated glycoprotein.
Myelin-associated glycoprotein (MAG) may play a role in the cellular interactions leading to myelination. Using monoclonal antibodies and conventional antisera against MAG, we have isolated a cDNA clone from an expression library prepared from rat brain mRNA. The identity of the clone was confirmed by the exact match between its nucleotide sequence and two peptide sequences of 13 and 9 amino acids that we obtained by Edman degradation of two CNBr fragments of MAG. The cDNA clone hybridized to two size species of mRNA in rat approximately 3.5 kilobases in length. These mRNAs were present in brain but not liver and were expressed most abundantly at the time of active myelination (day 14). The mRNA for MAG was present at barely detectable levels in hypomyelinating jimpy mice compared to normal littermate controls. Therefore the MAG cDNA clone is both brain and myelin specific. DNA sequence analysis revealed that our MAG cDNA was derived from the same mRNA as clone p1B236, a randomly selected, brain-specific, partial cDNA isolated by Sutcliffe et al. [Sutcliffe, J. G., Milner, R. J., Shinnick, T. M. & Bloom, F. E. (1983) Cell 33, 671-682]. Analysis of the predicted protein sequence suggests that MAG has a long extracellular domain (499 amino acids), followed by a short transmembrane segment (20 amino acids) and an intracellular carboxyl-terminal domain (90 amino acids). The molecule has several glycosylation sites, three internal repeats homologous to a repeat in the neural cell adhesion molecule (N-CAM), and sites for phosphorylation near the carboxyl terminus. The primary structure reported here provides a molecular framework for further investigations into the function of the MAG molecule.
Amino Acid Sequence↗
Structural studies on a family of cAMP-binding proteins in the nervous system of Aplysia.
Five major cAMP-binding proteins that differ in size and charge have been identified in neurons of Aplysia californica by photoaffinity labeling with [32P]8-N3cAMP. These proteins, which we believe are regulatory subunits of cAMP-dependent protein kinase, all differ from the major cAMP-binding protein of buccal muscle. We have compared the structures of these proteins by peptide mapping after chemical and proteolytic cleavage. These analyses indicate that the five binding proteins from nervous tissue and the major muscle protein are closely related to each other. For example, the three neuronal proteins that are most alike and the cAMP-binding protein from muscle have a similar, if not identical, Mr 20,000 domain that contains the 8-N3cAMP-binding site; beyond this domain they diverge. All six proteins appear to belong to a family in which homologous regions have been conserved to maintain common functions. We suggest that the regions of the molecules that differ mediate special functions such as ticketing to particular compartments of the cell. Evidence for regional assortment of the cAMP-dependent protein kinases according to structural type was afforded by subcellular fractionation of Aplysia nervous tissue; photoaffinity labeling of cytoplasm, cytoskeleton, and membrane fractions demonstrated a differential distribution of the five neuronal cAMP-binding proteins. Selective phosphorylation of specific substrates could be a consequence of the compartmentation of diverse cAMP-dependent kinases.
Animals↗
Secondary structure and assembly mechanism of an oligomeric channel protein.
The alpha-toxin of Staphylococcus aureus is secreted as a water-soluble, monomeric polypeptide (Mr 33 182) that can assemble into an oligomeric membrane channel. By chemical cross-linking, we have confirmed that the major form of the channel is a hexamer. The circular dichroism spectrum of this hexamer in detergent revealed that it contains a high proportion of beta-sheet that we deduce must lie within the lipid bilayer when the protein is associated with membranes. The circular dichroism spectrum of the monomeric toxin in the presence or absence of detergent was closely similar to the spectrum of the hexamer, suggesting that the secondary structure of the polypeptide is little changed on assembly. Results of experiments involving limited proteolysis of the monomer and hexamer are consistent with the idea that assembly involves the movement of two rigid domains about a hinge located near the midpoint of the polypeptide chain. The hydrophilic monomer is thereby converted to an amphipathic rod that becomes a subunit of the hexamer.
Bacterial Toxins↗
A carbene-yielding amino acid for incorporation into peptide photoaffinity reagents.
3-[p-[3-(Trifluoromethyl)-3H-diazirin-3-yl]phenyl]alanine has been prepared in 10 steps from p-bromobenzyl alcohol. The alcohol was converted to 3-(alpha-iodo-p-tolyl)-3-(trifluoromethyl)-3H-diazirine, which was used to alkylate N-(diphenylmethylene)glycine ethyl ester. After deprotection the amino acid was obtained with an overall yield of 18%. The D- and L-isomers of 3-[p-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenyl]alanine have also been resolved, and 3-[p-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenyl]alanine labeled with tritium has been prepared. The advantages of using this amino acid as a building block for peptide photoaffinity reagents is discussed.
Affinity Labels↗
The delivery of phototoxic drugs to selected cells.
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Antibodies↗
Orientation of retinal in bacteriorhodopsin as studied by cross-linking using a photosensitive analog of retinal.
The photosensitive m-diazirinophenyl analog of retinal (Fig. 1, II) bound to bacterio-opsin at Lys-216 and regenerated a chromophore with lambda max at 470 nm. Photolysis of the complex at 365 nm resulted in covalent cross-linking of the retinal analog to the bacterio-opsin in greater 30% yield. Investigation of the sites of cross-linking between the 3H-labeled retinal analog and the protein showed the peptide fragment (amino acid residues 190-248) to be the main radioactively labeled product. Stepwise Edman degradation showed Ser-193 and Glu-194 to be the predominant sites of cross-linking. These results show that the chromophore in bacteriorhodopsin is inclined towards helix 6 and towards the exterior of the cell. These data also provide information on the approximate angle that the chromophore makes with the plane of the membrane and they require a modification of the current secondary structure model for bacteriorhodopsin.
Amino Acid Sequence↗
The site of attachment of retinal in bacteriorhodopsin. A resonance Raman study.
The retinal chromophore of bacteriorhodopsin is attached as a Schiff's base with the epsilon-amino group of a lysine residue. The site of attachment has now been investigated by the use of resonance Raman spectroscopy which has previously been shown to be sensitive to 15N isotope substitution at the Schiff's base. Bacteriorhodopsin samples obtained from bacteria grown in a medium containing either [epsilon-14N]- or [epsilon-15N]lysine were cleaved with chymotrypsin to give, in each case, the two fragments C-1 (amino acids 72-248) and C-2 (amino acids 1-71). The fragments were recombined in different combinations into lipid/detergent mixtures and retinal was added to regenerate the chromophore. Resonance Raman spectroscopy showed that, in both the light-adapted (BR 570) and the M 412 intermediate forms, the chromophore is attached to the large C-1 fragment. This result eliminates Lys-41 as the attachment site in these forms of bacteriorhodopsin. Together with the accompanying report, which demonstrates that the epsilon-amino group in Lys-41 is not required for regeneration of the native chromophore or for proton translocation, these results provide strong evidence that the chromophore remains attached as a Schiff's base to Lys-216 during the entire photocycle.
Bacteriorhodopsins↗
Light-driven proton translocation by bacteriorhodopsin reconstituted with the phenyl analog of retinal.
The properties of bacteriorhodopsin in which the natural cofactor, retinal (Fig. 1, I), has been replaced by the synthetic analog, phenyl retinal (Fig. 1, II), have been studied. Phenyl retinal binds at the same site as retinal and supports light-dependent transmembrane proton translocation in phospholipid vesicles that contain bacterio-opsin. This result allows us to rule out proton abstraction from the beta-ionone ring of retinal as a step in the catalytic cycle. Furthermore, phenyl retinal, a planar molecule, binds to the apomembrane more rapidly than retinal, which suggests that the rate-determining step in the binding of retinal itself may be the attainment of a planar molecular configuration. The new chromophore undergoes a pH-dependent spectral shift that is not observed in the native membrane.
Bacteriorhodopsins↗