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J M Tomich

Publications and source records attributed to J M Tomich.

At least 37 records · Page 2Linked to original sources
PubMed1994

Key of induced tolerance to ischaemia in gerbil hippocampal CA1 is not at transcriptional level of hsp70 gene: in situ hybridization of hsp70 mRNA.

Hippocampal neurons, when pretreated with sublethal ischaemia, acquired tolerance to future treatments of normally lethal ischaemia. The level of transcription of the stress protein hsp70 was studied by in situ hybridization in control, ischaemic and tolerance-induced-ischaemic hippocampal neurons. Mongolian gerbils were subjected to single forebrain ischaemia for 2 min (2-min ischaemia group: sublethal) or 5 min (5-min ischaemia group: lethal). Other animals, were exposed to sublethal ischaemia for 2 min and were subjected subsequently to ischaemia for 5 min, 2 days later. Animals were sacrificed at 6, 12, 24 and 48 h following ischaemia and in situ hybridization was performed with thin cross sections including the hippocampus. Hybridization of the hsp70 probe in the control group (no ischaemia) was barely visible in the brain. In the 2-min ischaemia group, intense hybridization was seen in the CA1 sector from 6 through 24 h of recirculation but hybridization almost disappeared at 48 h of recirculation. In the 5-min and the double ischaemic groups, the intense induction in the CA1 sector occurred during the period from 6 through 48 h of recirculation. Our present study by in situ hybridization reveals high levels of transcription of hsp70 message following 5-min of ischaemia, however no protein was detectable based on our previous experiments employing immunocytochemistry. In tolerance-induced hippocampal neurons, both hsp70 message and protein are present. These results suggest that tolerance-induced neurons are modified such that the hsp70 gene is both transcribed and translated. These changes most likely give rise to the tolerance observed in the double ischaemia group.

Animals↗
PubMed1994

Chemical synthesis and characterization of peptides and oligomeric proteins designed to form transmembrane ion channels.

A strategy for the synthesis of peptides and oligomeric proteins designed to form transmembrane ion channels is described. A folding motif that exhibits a functional ionic pore encompasses amphipathic alpha-helices organized as a four-helix bundle around a central hydrophilic pore. The channel-forming activity of monomeric amphipathic peptides may be examined after reconstitution in lipid bilayers in which peptides self-assemble into conductive oligomers. The covalent attachment of channel-forming peptides to the lysine epsilon-amino groups of a template molecule (KKKPGKEKG) specifies oligomeric number and facilitates the study of ionic permeation and channel blockade. Here we describe detailed protocols for the total synthesis of peptides and template-assembled four-helix bundle proteins, exemplified with the sequence of M2 delta (EKM-STAISVLLAQAVFLLLTSQR), considered involved in lining the pore of the nicotinic acetylcholine receptor channel. For comparison, the synthesis of a second four-helix bundle, T4CaIVS3 with the sequence of predicted transmembrane segment S3 (DPWNVFDFLIVIGSIIDVILSE) of the fourth repeat of the L-type voltage-gated calcium channel, is included. Peptides and proteins are synthesized step-wise by solid-phase methods, purified by reversed-phase HPLC, and homogeneity ascertained by analytical HPLC, capillary zone electrophoresis, SDS/PAGE, amino acid analysis and sequencing. Optimization of synthetic procedures for hydrophobic molecules include reducing resin substitution to avoid steric hindrance and aggregation of the final product. Protocols for the preparation of the samples prior to HPLC purification as well as the conditions and columns required for successful purification are presented. The methods developed are generally applicable for the chemical synthesis, purification and characterization of amphipathic peptides and template directed helical bundle proteins.

Amino Acid Sequence↗
PubMed1993

Synthetic peptides and four-helix bundle proteins as model systems for the pore-forming structure of channel proteins. I. Transmembrane segment M2 of the nicotinic cholinergic receptor channel is a key pore-lining structure.

Monomeric peptides and four-helix bundle proteins with amino acid sequences of the predicted transmembrane segment M2 of nicotinic acetylcholine receptors (AChR) were designed, synthesized, and used as probes to elucidate the pore-forming structure of the authentic AChR channel. Peptides M2 delta and M2 alpha 4 with sequences of M2 from muscle-like Torpedo californica AChR delta subunit and from rat neuronal AChR alpha 4 subunit form cation-selective channels in lipid bilayers with predominant single-channel conductances in 0.5 M KCl of 20 pS and 27 pS, respectively. Corresponding analogs with presumed pore-lining residues serine 8 or phenylalanine 16 specifically substituted by alanine retain the ability to self-assemble into conductive oligomers and form channels with primary conductances of 16 pS and 22 pS for M2 delta analogs and of 14 pS and 26 pS for M2 alpha 4 analogs. In contrast, peptides with randomized sequences and the same amino acid composition as M2 delta do not form channels. Four-helix bundle proteins T4M2 delta and T4M2 alpha 4 exhibit conductances in 0.5 M KCl of 20 pS and 26 pS. Analogs of T4M2 delta with selective replacement of serine 8 for alanine exhibit lower conductances, whereas substitution of phenylalanine 16 for alanine increases the single-channel conductance. T4M2 delta channels are blocked by open channel blockers such as the quaternary derivative of lidocaine QX-222 and by chlorpromazine. Channel open probability is reduced, and open time is abbreviated. Conduction properties of T4M2 delta and analogs are in accord with several properties of authentic AChRs. These comprehensive studies provide insights into the components of the pore-forming structure of nicotinic AChRs.

Amino Acid Sequence↗
PubMed1993

Synthetic peptides and four-helix bundle proteins as model systems for the pore-forming structure of channel proteins. II. Transmembrane segment M2 of the brain glycine receptor is a plausible candidate for the pore-lining structure.

A synthetic 23-mer peptide (M2GlyR) with the amino acid sequence of the putative transmembrane segment M2 of the strychnine-binding alpha subunit of the inhibitory glycine receptor forms anion-selective channels in phospholipid bilayers. The most frequent events show single-channel conductances, gamma, of 25 pS and 49 pS in symmetric 0.5 M KCl with channel open lifetimes, tau o, in the millisecond time range. These properties match those of authentic glycine receptors studied in inside-out patches of cultured rat spinal cord neurons, namely gamma = 27 pS and gamma = 45 pS, and tau o in the millisecond time range. The channel activity of M2GlyR is sequence-specific: 1) a synthetic peptide with the sequence of putative transmembrane segment M1 (M1GlyR), not considered to contribute to the channel lining, does not form channels; 2) an analog of M2GlyR with site-specific substitutions displays distinct channel properties: 2 arginine residues at the N and C termini of M2, postulated to contribute to the anion selectivity of the channel, are substituted by glutamic acids, and the analog peptide ([Glu3,22]M2GlyR) forms cation-selective channels. Further, a four-helix bundle protein (T4M2GlyR) formed by tethering four identical M2GlyR modules to a carrier template forms homogeneous anion-selective channels with gamma = 25 pS in 0.5 M KCl. These channels are blocked by picrotoxin and by the anion channel blockers 9-anthracene carboxylic acid and niflumic acid, but not by an analog of the local anesthetic lidocaine (QX-222), a cation channel blocker. Observed single-channel properties suggest that a pentameric assembly of alpha and beta subunits with a central pore lined by M2 segments would account for conductance properties of the authentic glycine receptor and the 2 arginines at either end of M2 could confer anion specificity to the receptor channel.

Amino Acid Sequence↗
PubMed1993

Design, synthesis and functional characterization of a pentameric channel protein that mimics the presumed pore structure of the nicotinic cholinergic receptor.

Nicotinic cholinergic receptors are membrane proteins composed of five subunits organized around a central aqueous pore. A pentameric channel protein, T5M2 delta, that emulates the presumed pore-forming structure of this receptor was generated by assembling five helix-forming peptide modules at the lysine epsilon-amino groups of the 11-residue template [K*AK*KK*PGK*EK*G], where * indicates attachment sites. Helical modules represent the sequence of the M2 segment of the Torpedo californica acetylcholine receptor (AChR) delta subunit; M2 segments are considered involved in pore-lining. Purified T5M2 delta migrates in SDS-PAGE with an apparent M(r) approximately 14,000, concordant with a protein of 126 residues. T5M2 delta forms cation-selective channels when reconstituted in planar lipid bilayers. The single channel conductance in symmetric 0.5 M KCl is 40 pS. This value approximates the 45 pS single channel conductance characteristic of authentic purified Torpedo AChR, recorded under otherwise identical conditions. These results, together with conformational energy calculations, support the notion that a bundle of five amphipathic alpha-helices is a plausible structural motif underlying the inner bundle that forms the pore of the pentameric AChR channel.

Amino Acid Sequence↗
PubMed1993

Characterization of the functional domain of tissue inhibitor of metalloproteinases-2 (TIMP-2).

Analysis of the functional domain of tissue inhibitor of metallo-proteinases-2 (TIMP-2) was performed using limited proteolytic degradation with trypsin. This treatment generated a 13.5 kDa fragment which was purified and shown to consist of an uncleaved N-terminal region extending from residue 1 to residue 132. The fragment retains the ability to inhibit activated interstitial collagenase and to block the autocatalytic activation of procollagenase.

Amino Acid Sequence↗
PubMed1993

Design of a functional calcium channel protein: inferences about an ion channel-forming motif derived from the primary structure of voltage-gated calcium channels.

To identify sequence-specific motifs associated with the formation of an ionic pore, we systematically evaluated the channel-forming activity of synthetic peptides with sequence of predicted transmembrane segments of the voltage-gated calcium channel. The amino acid sequence of voltage-gated, dihydropyridine (DHP)-sensitive calcium channels suggests the presence in each of four homologous repeats (I-IV) of six segments (S1-S6) predicted to form membrane-spanning, alpha-helical structures. Only peptides representing amphipathic segments S2 or S3 form channels in lipid bilayers. To generate a functional calcium channel based on a four-helix bundle motif, four-helix bundle proteins representing IVS2 (T4CaIVS2) or IVS3 (T4CaIVS3) were synthesized. Both proteins form cation-selective channels, but with distinct characteristics: the single-channel conductance in 50 mM BaCl2 is 3 pS and 10 pS. For T4CaIVS3, the conductance saturates with increasing concentration of divalent cation. The dissociation constants for Ba2+, Ca2+, and Sr2+ are 13.6 mM, 17.7 mM, and 15.0 mM, respectively. The conductance of T4CaIVS2 does not saturate up to 150 mM salt. Whereas T4CaIVS3 is blocked by microM Ca2+ and Cd2+, T4CaIVS2 is not blocked by divalent cations. Only T4CaIVS3 is modulated by enantiomers of the DHP derivative BayK 8644, demonstrating sequence requirement for specific drug action. Thus, only T4CaIVS3 exhibits pore properties characteristic also of authentic calcium channels. The designed functional calcium channel may provide insights into fundamental mechanisms of ionic permeation and drug action, information that may in turn further our understanding of molecular determinants underlying authentic pore structures.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1993

Synthesis and characterization of a bioactive 82-residue sphingolipid activator protein, saposin C.

The sphingolipid activator protein, saposin C (also termed SAP 2), was chemically synthesized, purified, and characterized. The fully protected 82-residue protein was synthesized by automated solid-phase methods, with multiple recoupling steps resulting in a high average coupling efficiency of 98.8%. The overall yield was estimated to be approx 40%. Deprotection and cleavage of the peptide from the resin was followed by folding in the absence of chaotropic agents at pH 8.5. The protein was purified by reversed-phase high pressure liquid chromatography (HPLC) and its purity determined by capillary electrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The composition of the synthetic saposin C was determined by amino acid analysis. Its sequence was verified by Edman sequence analysis of overlapping peptide fragments generated by chymotryptic and Staphylococcus aureus V8 digestions. The sequence at the C-terminus was determined by digestion with carboxypeptidase P, followed by phenylthiohydantoin (PTH) derivitization and HPLC analysis of the released amino acid residues. Deglycosylated native saposin C appeared as a lower molecular-weight species than synthetic saposin C on SDS-PAGE. This has been explained by amino acid and C-terminal analysis showing native saposin C to be two amino acids shorter at the C terminus than a deduced sequence (from cDNA) previously published. Synthetic saposin C displayed 85% of full biological activity as determined by its ability to stimulate glucocerebrosidase activity in vitro: Synthetic and native saposin C increased glucocerebrosidase catalyzed hydrolysis of 4-methylumbelliferyl beta-D-glucoside by factors of 6.0 and 7.1, respectively. Furthermore, synthetic and native saposin C share similar K(act) values (0.5 and 1.5 microM respectively) indicating that they bind to glucocerebrosidase with similar affinities.

Amino Acid Sequence↗
PubMed1992

Identification of an ion channel-forming motif in the primary structure of tetanus and botulinum neurotoxins.

Synthetic peptides with amino acid sequences corresponding to predicted transmembrane segments of tetanus toxin were used as probes to identify a channel-forming motif. A peptide denoted TeTx II, with sequence GVVLLLEYIPEITLPVIAALSIA, forms cation-selective channels when reconstituted in planar lipid bilayers. The single channel conductance in 0.5 M NaCl or KCl is 28 +/- 3 and 24 +/- 2 pS, respectively. In contrast, a peptide with sequence NFIGALETTGVVLLLEYIPEIT, denoted as TeTx I, or a peptide with the same amino acid composition as TeTx II but with a randomized sequence, do not form channels. Conformational energy calculations show that a bundle of four amphipathic alpha-helices is a plausible structural motif underlying observable pore properties. The identified functional module may account for the channel-forming activity of both tetanus toxin and the homologous botulinum toxin A.

Amino Acid Sequence↗
PubMed1992

Insertion and folding of the amino-terminal amphiphilic signal sequences of the mannitol and glucitol permeases of Escherichia coli.

Peptides which correspond to the NH2-terminal 23 or 22 residues of the mannitol and glucitol permeases (enzymes IImtl and IIgut of the bacterial phosphotransferase system; mtl-23 and gut-22) and which are believed to function in envelope targeting were synthesized chemically, and their interactions with lipid model membranes were studied. Both wild-type peptides penetrated phospholipid monolayers up to high surface pressures, and partition constants of 8.0 x 10(4) M-1 and 4.2 x 10(4) M-1, respectively, were derived from the incorporation isotherms of mtl-23 and gut-22 with monolayers of 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine at 32 mN/m or bilayers of the same lipid. The mtl-23 peptide was highly alpha-helical in trifluoroethanol, sodium dodecyl sulfate, lysolecithin, or vesicles of 1-palmitoyl-2-oleoyl-3-sn-phosphatidylglycerol, with estimated percentages of alpha-helix ranging between 60 and 85%. The interactions with model membranes of several single site mutants (S3P, D4P, and D4K) of mtl-23 which were defective in properly assembling the mannitol permease in the cytoplasmic membrane of Escherichia coli were also studied. The contents of alpha-helix of these peptides in detergent micelles or phospholipid bilayers were not significantly changed compared with those of the wild type, suggesting that the amphiphilic NH2-terminal membrane-targeting domain could still be formed in these mutants. However, the mutants which contained a proline in positions 3 or 4, i.e. NH2-terminal to the proposed amphiphilic alpha-helix, partitioned into phospholipid monolayers with partition constants that were 2 or 4 times smaller than those of the wild type. Based on these data, a model of the amphiphilic structure of the NH2-terminal domain of the mannitol permease is discussed. This domain may interact physiologically with amphiphilic interfaces of lipids and/or proteins during membrane insertion.

Amino Acid Sequence↗
PubMed1991

A totally synthetic histidine-2 ferredoxin: thermal stability and redox properties.

The entire polypeptide of Clostridium pasteurianum ferredoxin (Fd) with a site-substituted tyrosine-2----histidine-2 was synthesized using standard t-Boc procedures, reconstituted to the 2[4Fe-4S] holoprotein, and compared to synthetic C. pasteurianum and native Fds. Although histidine-2 is commonly found in thermostable clostridial Fds, the histidine-2 substitution into synthetic C. pasteurianum Fd did not significantly increase its thermostability. The reduction potential of synthetic histidine-2 Fd was -343 and -394 mV at pH 6.4 and 8.7, respectively, versus standard hydrogen electrode. Similarly, Clostridium thermosaccharolyticum Fd which naturally contains histidine-2 was previously determined to have a pH-dependent reduction potential [Smith, E.T., & Feinberg, B.A. (1990) J. Biol. Chem. 265, 14371-14376]. An electrostatic model was used to calculate the observed change in reduction potential with pH for a homologous ferredoxin with a known X-ray crystal structure containing a hypothetical histidine-2. In contrast, the reduction potential of both native C. pasteurianum Fd and synthetic Fd with the C. pasteurianum sequence was -400 mV versus standard hydrogen electrode and was pH-independent [Smith, E.T., Feinberg, B.A., Richards, J.H., & Tomich, J.M. (1991) J. Am. Chem. Soc. 113, 688-689]. On the basis of the above results, we conclude that the observed pH-dependent reduction potential for both synthetic and native ferredoxins that contain histidine-2 is attributable to the electrostatic interaction between histidine-2 and iron-sulfur cluster II which is approximately 6 A away.

Amino Acid Sequence↗
PubMed1991

Insertion of the mannitol permease into the membrane of Escherichia coli. Possible involvement of an N-terminal amphiphilic sequence.

The in vivo membrane assembly of the mannitol permease, the mannitol Enzyme II (IImtl) of the Escherichia coli phosphotransferase system, has been studied employing molecular genetic approaches. Removal of the N-terminal amphiphilic leader of the permease and replacement with a short hydrophobic sequence resulted in an inactive protein unable to transport mannitol into the cell or catalyze either phosphoenol-pyruvate-dependent or mannitol 1-phosphate-dependent mannitol phosphorylation in vitro. The altered protein (68 kDa) was quantitatively cleaved by an endogenous protease to a membrane-associated 39-kDa fragment and a soluble 28-kDa fragment as revealed by Western blot analyses. Overproduction of the wild-type plasmid-encoded protein also led to cleavage, but repression of the synthesis of the plasmid-encoded enzyme by inclusion of glucose in the growth medium prevented cleavage. Several mtlA-phoA gene fusions encoding fused proteins with N-terminal regions derived from the mannitol permease and C-terminal regions derived from the mature portion of alkaline phosphatase were constructed. In the first fusion protein, F13, the N-terminal 13-aminoacyl residue amphiphilic leader sequence of the mannitol permease replaced the hydrophobic leader sequence of alkaline phosphatase. The resultant fusion protein was inefficiently translocated across the cytoplasmic membrane and became peripherally associated with both the inner and outer membranes, presumably via the noncleavable N-terminal amphiphilic sequence. The second fusion protein, F53, in which the N-terminal 53 residues of the mannitol permease were fused to alkaline phosphatase, was efficiently translocated across the cytoplasmic membrane and was largely found anchored to the inner membrane with the catalytic domain of alkaline phosphatase facing the periplasm. This 53-aminoacyl residue sequence included the amphiphilic leader sequence and a single hydrophobic, potentially transmembrane, segment. Analyses of other MtlA-PhoA fusion proteins led to the suggestion that internal amphiphilic segments may function to facilitate initiation of polypeptide trans-membrane translocation. The dependence of IImtl insertion on the N-terminal amphiphilic leader sequence was substantiated employing site-specific mutagenesis. The N-terminal sequence of the native permease is Met-Ser-Ser-Asp-Ile-Lys-Ile-Lys-Val-Gln-Ser-Phe-Gly.... The following point mutants were isolated, sequenced, and examined regarding the effects of the mutations on insertion of IImtl into the membrane: 1) S3P; 2) D4P; 3) D4L; 4) D4R; 5) D4H; 6) I5N; 7) K6P; and 8) K8P.(ABSTRACT TRUNCATED AT 400 WORDS)

Alkaline Phosphatase↗
PubMed1991

A molecular blueprint for the pore-forming structure of voltage-gated calcium channels.

A protein that imitates the sequence of a highly conserved segment predicted to line the pore of dihydropyridine-sensitive L-type calcium channels was designed and synthesized. Single-channel conductance properties were studied in planar lipid bilayers. The synthetic protein emulates the ionic conductance, ionic selectivity, and pharmacological properties of the authentic calcium channel, including the stereospecific action of agonist and antagonist enantiomers of the dihydropyridine BayK 8644. The identified sequence is identical in L-type calcium channels from skeletal muscle and isoforms of cardiac muscle, brain, and aorta. It is plausible that this structural motif represents the molecular blueprint for the pore-forming structure of voltage-gated calcium channels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1991

Mitochondrial ATP synthase complex. Membrane topography and stoichiometry of the F0 subunits.

The topography of the subunits of the membrane sector F0 of the ATP synthase complex in the bovine mitochondrial inner membrane was studied with the help of subunit-specific antibodies raised to the F0 subunits b, d, 6, F6, A6L, OSCP (oligomycin-sensitivity-conferring protein), and N,N' -dicyclohexylcarbodiimide (DCCD)-binding proteolipid and to the ATPase inhibitor protein (IF1) as an internal control. Exposure of F0 subunits in inverted and right-side-out inner membranes was investigated by direct antibody binding as well as by susceptibility of these subunits to degradation by various proteases as monitored by gel electrophoresis of the membrane digests and immunoblotting with the subunit-specific antibodies. Results show that subunits b, d, F6, A6L (including its C-terminal end) and OSCP were exposed on the matrix side. Sufficient masses of these subunits to recognize antibodies or undergo proteolysis were not exposed on the cytosolic side. This was also the case for subunit 6 and the DCCD-binding proteolipid on either side of the inner membrane. Quantitative immunoblotting in which bound radio-activity from [125I]protein A was employed to estimate the concentration of an antigen in a sample allowed the determination of the stoichiometry of several F0 subunits and IF1 relative to F1-ATPase. Results showed that per mol of F1 there are in bovine heart mitochondria 1 mol each of d, OSCP, and IF1, and 2 mol each of b and F6. Subunit 6 and the DCCD-binding proteolipid could not be quantitated, because the former transferred poorly to nitrocellulose and the latter's antibody did not bind [125I]protein A.

Animals↗