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J M Tomich

Publications and source records attributed to J M Tomich.

At least 55 records · Page 3Linked to original sources
PubMed1991

Characterization of human glucocerebrosidase from different mutant alleles.

Human cDNA was mutagenized to duplicate six naturally occurring mutations in the gene for glucocere-brosidase. The mutant genes were expressed in NIH 3T3 cells. The abnormal human enzymes were purified by immunoaffinity chromatography and characterized. The Asn370----Ser mutant protein differed from normal enzyme in its inhibition by both conduritol B epoxide and glucosphingosine demonstrating that the 370 mutant enzyme has an abnormal catalytic site. In addition, the 370 mutant enzyme is less activated by saposin C, but more stimulated by phosphatidylserine than the wild type enzyme. The Arg463----Cys mutant protein was normal with respect to conduritol B epoxide and glucosphingosine inhibition, but was less activated by both saposin C and phosphatidylserine. The Arg120----Gln mutant protein was catalytically inactive. The Leu444----Pro, the pseudopattern, and the Pro415----Arg mutants appear to have reduced amounts of enzyme protein in cells. The studies demonstrated that mutations in the gene for glucocerebrosidase have different effects on the catalytic activity and stability of the enzyme.

Alleles↗
PubMed1991

Identification of a peptide inhibitor of the cardiac sarcolemmal Na(+)-Ca2+ exchanger.

The deduced amino acid sequence of the cardiac sarcolemmal Na(+)-Ca2+ exchanger has a region which could represent a calmodulin binding site. As calmodulin binding regions of proteins often have an autoinhibitory role, a synthetic peptide with this sequence was tested for functional effects on Na(+)-Ca2+ exchange activity. The peptide inhibits the Na(+)-dependent Ca2+ uptake (KI approximately 1.5 microM) and the Nao(+)-dependent Ca2+ efflux of sarcolemmal vesicles in a noncompetitive manner with respect to both Na+ and Ca2+. The peptide is also a potent inhibitor (KI approximately 0.1 microM) of the Na(+)-Ca2+ exchange current of excised sarcolemmal patches. The binding site for the peptide on the exchanger is on the cytoplasmic surface of the membrane. The exchanger inhibitory peptide binds calmodulin with a moderately high affinity. From the characteristics of the inhibition of the exchange of sarcolemmal vesicles, we deduce that only inside-out sarcolemmal vesicles participate in the usual Na(+)-Ca2+ exchange assay. This contrasts with the common assumption that both inside-out and right-side-out vesicles exhibit exchange activity.

Amino Acid Sequence↗
PubMed1991

Nucleotide sequence of the Rhodobacter capsulatus fruK gene, which encodes fructose-1-phosphate kinase: evidence for a kinase superfamily including both phosphofructokinases of Escherichia coli.

The fruK gene encoding fructose-1-phosphate kinase (FruK), located within the fructose (fru)-catabolic operon of Rhodobacter capsulatus, was sequenced. FruK of R. capsulatus (316 amino acids; molecular weight = 31,232) is the same size as and is homologous to FruK of Escherichia coli, phosphofructokinase B (PfkB) of E. coli, phosphotagatokinase of Staphylococcus aureus, and ribokinase of E. coli. These proteins therefore make up a family of homologous proteins, termed the PfkB family. A phylogenetic tree for this new family was constructed. Sequence comparisons plus chemical inactivation studies suggested the lack of involvement of specific residues in catalysis. Although the Rhodobacter FruK differed markedly from the other enzymes within the PfkB family with respect to amino acid composition, these enzymes exhibited similar predicted secondary structural features. A large internal segment of the Rhodobacter FruK was found to be similar in sequence to the domain bearing the sugar bisphosphate-binding region of the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase of plants and bacteria. Proteins of the PfkB family did not exhibit statistically significant sequence identity with PfkA of E. coli. PfkA, however, is homologous to other prokaryotic and eukaryotic ATP- and PPi-dependent Pfks (the PfkA family). These eukaryotic, ATP-dependent enzymes each consist of a homotetramer (mammalian) or a heterooctamer (yeasts), with each subunit containing an internal duplication of the size of the entire PfkA protein of E. coli. In some of these enzymes, additional domains are present. A phylogenetic tree was constructed for the PfkA family and revealed that the bacterial enzymes closely resemble the N-terminal domains of the eukaryotic enzyme subunits whereas the C-terminal domains have diverged more extensively. The PPi-dependent Pfk of potato is only distantly related to the ATP-dependent enzymes. On the basis of their similar functions, sizes, predicted secondary structures, and sequences, we suggest that the PfkA and PfkB families share a common evolutionary origin.

Amino Acid Sequence↗
PubMed1991

A simple nonisotopic in vitro bioassay for LT and TNF employing sodium fluoride-treated L-929 target cells that detects picogram quantities of LT and TNF and is as sensitive as TNF assays done with ELISA methodology.

We have developed a highly sensitive in vitro bioassay for lymphotoxin (LT) and tumor necrosis factor (TNF). This system employs a monolayer of 20,000 L-929 target cells in each well of 96-well microplates that are incubated in dilutions of LT and TNF at 37 degrees C for 20-24 h, after which the viability of the L-929 target cells is assessed by spectrophotometric techniques. We find that incorporation of 2 mM NaF increases the sensitivity of L-929 cells to lysis by 10(3)-10(4) x for LT and 10(4)-10(5) x for TNF. This method allows the regular detection of less than 1-5 pg of bioactive protein. ELISA assays did not detect synthetic TNF and TNF fragments that were not biologically active in this assay. The L-929 bioassay requires no isotopes, is very reproducible, is as sensitive as ELISA techniques, and detects only biologically active LT and TNF.

Animals↗
PubMed1990

Structure and evolution of a multidomain multiphosphoryl transfer protein. Nucleotide sequence of the fruB(HI) gene in Rhodobacter capsulatus and comparisons with homologous genes from other organisms.

The gene order of the fructose (fru) operon and nucleotide sequence of the first gene (fruB(HI) of Rhodobacter capsulatus are reported, analyzed and compared with homologous genes from other bacteria, and the gene products are identified. Included within the region reported is a gene encoding a multiphosphoryl transfer protein (MTP) of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). MTP consists of three moieties: a fructose-specific enzyme III (IIIfru)-like N-terminal moiety (residues 1 to 143) followed by an FPr(HPr)-like moiety (residues 157 to 245) and an enzyme I-like moiety (residues 273 to 827). The enzyme III-like moiety closely resembles the N-terminal 143 residues of the IIIfru-FPR fusion protein from Salmonella typhimurium (40.6% identity throughout its length) and the C-terminal 145 residues of the mannitol-specific enzyme II (IImtl) (37.8% identity throughout its length with the IIImtl moiety of IImtl). The FPr-like domain of MTP resembles the S. typhimurium FPr (42.4% identity) and the Escherichia coli or S. typhimurium HPr (38.8% identity). The enzyme I-like moiety resembles the E. coli enzyme I (38.9% identity). Predicted phosphorylation sites within the three functional units of MTP (His62 in the IIIfru-like moiety; His171 in the FPr-like moiety and His457 in the enzyme I-like moiety) were identified on the basis of sequence comparisons with the homologous proteins from enteric bacteria. The three functional domains of MTP are joined by two flexible "linkage" regions, rich in alanine, glycine and proline, which show 47% sequence identity with each other. They also exhibit a high degree of sequence identity with the linkage region of the mannose-specific enzyme III (IIIman) of the E. coli PTS as well as several other proteins of bacterial, eukaryotic and viral origin. At the RNA level, these linker regions formed hairpin structures with high (90%) G + C content. Analyses of the IIIfru-FPr fusion protein of S. typhimurium revealed that between the IIIfru and FPr moieties of this protein is a stretch of 142 amino acids that do not show homology to known PTS proteins. This region and the adjacent FPr-like region contain a sequence of 110 residues exhibiting 59% similarity to the receiver consensus motif defined by Kofoid and Parkinson. Because the Salmonella IIIfru-FPr fusion protein has been implicated in transcriptional regulation, this region of the Salmonella protein may prove to have regulatory significance.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1990

Synporins--synthetic proteins that emulate the pore structure of biological ionic channels.

A class of proteins that mimic the fundamental pore structure of authentic ionic channels has been designed, synthesized, and characterized. The design is based on our earlier result that a 23-mer peptide with the sequence of the M2 segment of the Torpedo californica acetylcholine receptor delta subunit--Glu-Lys-Met-Ser-Thr-Ala-Ile-Ser-Val-Leu-Leu-Ala-Gln-Ala-Val-Phe -Leu- Leu-Leu-Thr-Ser-Gln-Arg--forms cation-selective channels in lipid bilayers, presumably by self-assembly of conductive oligomers. Accordingly, a tethered parallel tetramer was synthesized with four M2 delta peptides attached to a carrier template--a 9-amino acid backbone with four attachment sites. As expected, the complete 101-residue protein does form channels in lipid bilayers reproducing several features that are characteristic of authentic acetylcholine receptor channels, such as single-channel conductance, cation selectivity, transitions between closed and open states in the millisecond time range, and sensitivity to local anesthetic channel blockers. An analogue protein, in which the serine residue in position 8 is replaced with alanine in each of the four M2 delta 23-mer peptides ([Ala8]M2 delta), also forms channels that, however, exhibit lower single-channel conductance. By contrast, a similar tethered tetramer with M1 delta peptides does not form channels, in accord with expectations. The general validity of this strategy to other channel sequences and oligomer numbers is anticipated. Thus, synporins--a term coined to identify this class of synthetic pore proteins--enrich our armamentarium directed toward the elucidation of structure-function relationships.

Amino Acid Sequence↗
PubMed1989

Human deoxycytidine kinase. Sequence of cDNA clones and analysis of expression in cell lines with and without enzyme activity.

Deoxycytidine kinase (dC kinase) is the rate-limiting enzyme in the anabolism of important anticancer and retroviral nucleoside derivatives. Its activity is often decreased in resistance to these drugs. To analyze the structure, function, and control of this clinically important enzyme we isolated 15 cDNA clones for human deoxycytidine kinase from lambda gt11 thymus and Molt 4 libraries. Four clones were sequenced. The largest clone is 2.9 kilobases and codes for a 626-amino acid open reading frame. The DNA and deduced amino acid sequence of the human dC kinase clones are homologous with a previously unidentified murine cDNA clone p3.4J (EMBL:MM34j) reported to be related to granulocyte-macrophage colony-stimulating factor. Deoxycytidine kinase also has cysteine-rich regions that are homologous with thioredoxin, the beta subunit of prolyl 4-hydroxylase, phosphoinositide-specific phospholipase C, thyroid hormone-binding protein, and protein disulfide isomerase. No differences were seen in the amount and size of deoxycytidine kinase protein and mRNA between CCRF/CEM and L1210 leukemic cell lines that express and do not express enzyme activity. Genomic restriction fragments were similar between the active and inactive CCRF/CEM cell lines. These data suggest that the cells deficient in dC kinase activity have a small defect in the structural gene.

Amino Acid Sequence↗
PubMed1989

The M2 delta transmembrane domain of the nicotinic cholinergic receptor forms ion channels in human erythrocyte membranes.

A synthetic peptide with the sequence of the M2 delta segment of the nicotinic acetylcholine receptor from Torpedo californica forms pores in human erythrocyte membranes as determined by hemoglobin and potassium release. This peptide forms a permeability pathway with an apparent cross-sectional diameter of 7-9 A. The M2 delta pore is oligomeric and a pentamer is the species that accounts for the properties of the permeation path. Peptides that mimic other identifiable segments of the Torpedo acetylcholine receptor, M1 delta and MIR, do not form channels in erythrocytes under the same conditions.

Amino Acid Sequence↗
PubMed1989

Membrane incorporation and induction of secondary structure of synthetic peptides corresponding to the N-terminal signal sequences of the glucitol and mannitol permeases of Escherichia coli.

The 22-residue synthetic signal peptide of the glucitol permease (Enzyme IIgut of the bacterial phosphotransferase system; gut22), which in the intact protein is believed to function in envelope targeting, was found to insert into phospholipid monolayers of various phospholipid compositions up to high limiting pressures (36-41 milliNewton/m). The partition coefficient, derived from monolayer area expansion experiments, was greatest for the negatively charged gut22 when partitioning into monolayers of the zwitterionic lipid 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (about 1.1 X 10(5] as compared with that obtained with a mixture of 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine and the negatively charged lipids 1-palmitoyl-2-oleoyl-3-sn-phosphatidylglycerol and cardiolipin. Gut22 contains a titratable histidyl residue (pKa = 6.8), and its protonation decreased the relative monolayer area increase 3-fold. Circular dichroism spectra showed that gut22 formed an amphiphilic alpha-helix when incorporated into lipid membranes (estimated percent helix = 65%). Fluorescence measurements indicated that tryptophan 11 is in a more hydrophobic environment in the presence of lipid than in its absence, with the environment being more hydrophobic at pH 5 than at pH 8. The more hydrophilic 15-residue signal peptide of the mannitol permease (mtl15) also incorporated into monolayers and detergent micelles (although to a lesser extent) with induction of secondary structure. Based on these results and a parallel with mitochondrial targeting in eucaryotes, we suggest that the induction of N-terminal amphiphilic structures and their association with a hydrophobic-hydrophilic interface are important for envelope targeting and the initiation of the membrane insertion of bacterial phosphoenol-pyruvate-dependent phosphotransferase system permeases.

Amino Acid Sequence↗
PubMed1988

Amphiphilicity is essential for mitochondrial presequence function.

We have shown earlier that a mitochondrial presequence peptide can form an amphiphilic helix. However, the importance of amphiphilicity for mitochondrial presequence function became doubtful when an artificial presequence, designed to be non-amphiphilic, proved to be active as a mitochondrial import signal. We now show experimentally that this 'non-amphiphilic' presequence peptide is, in fact, highly amphiphilic as measured by its ability to insert into phospholipid monolayers and to disrupt phospholipid vesicles. This result, and similar tests on three additional artificial presequences (two functionally active and one inactive), revealed that all active presequences were amphiphilic whereas the inactive presequence was non-amphiphilic. One of the active presequence peptides was non-helical in solution and in the presence of detergent micelles. We conclude that amphiphilicity is necessary for mitochondrial presequence function whereas a helical structure may not be essential.

Circular Dichroism↗
PubMed1988

Prevention of aggregation of synthetic membrane-spanning peptides by addition of detergent.

In our initial attempts to solubilize and purify a chemically synthesized 22-amino acid, membrane-spanning peptide, we encountered numerous difficulties. The peptide was not soluble in dilute acids, organic solvents, or chaotropic agents (+/- detergent) following standard HF cleavage protocols. The insolubility was a direct result of the formation of peptide-(peptide)n aggregates that occurred during the initial phase of isolation, i.e., during the HF cleavage. Eliminating the ether precipitation and subsequent washes did not decrease the degree of aggregation of the product. Inclusion of nonionic detergents in the HF-cleavage reactions displayed little ability in preventing aggregation. Cleavage in the presence of sodium dodecyl sulfate, however, dramatically reduced the degree of aggregation, even after washing with organic solvents. The cleaved peptide was purified to homogeneity using a detergent-based HPLC protocol. This column procedure also permits the quantitative exchange of the sodium dodecyl sulfate for n-octyl-beta-D-glucopyranoside. Combined use of the two protocols results in high-yield isolations for a class of peptides that is generally difficult to handle.

Amino Acid Sequence↗
PubMed1987

Subcutaneous island pedicle flaps.

The subcutaneous island pedicle flap is a useful and reliably successful means of closing small- to medium-sized cutaneous excisional defects. It is especially useful in areas where primary closure could result in distortion of critical features. The technique is conceptually straightforward and offers advantages over skin grafting and transposition flaps. We describe our experience with 60 consecutive, successful subcutaneous island pedicle flaps.

Face↗
PubMed1986

A chemically synthesized pre-sequence of an imported mitochondrial protein can form an amphiphilic helix and perturb natural and artificial phospholipid bilayers.

Subunit IV of yeast cytochrome oxidase is made in the cytoplasm with a transient pre-sequence of 25 amino acids which is removed upon import of the protein into mitochondria. To study the function of this cleavable pre-sequence in mitochondrial protein import, three peptides representing 15, 25 or 33 amino-terminal residues of the subunit IV precursor were chemically synthesized. All three peptides were freely soluble in aqueous buffers, yet inserted spontaneously from an aqueous subphase into phospholipid monolayers up to an extrapolated limiting monolayer pressure of 40-50 mN/m. The two longer peptides also caused disruption of unilamellar liposomes. This effect was increased by a diffusion potential, negative inside the liposomes, and decreased by a diffusion potential of opposite polarity. The peptides, particularly the two longer ones, also uncoupled respiratory control of isolated yeast mitochondria. The 25-residue peptide had little secondary structure in aqueous buffer but became partly alpha-helical in the presence of detergent micelles. Based on the amino acid sequence of the peptides, a helical structure would have a highly asymmetric distribution of charged and apolar residues and would be surface active. Amphiphilic helicity appears to be a general feature of mitochondrial pre-sequences. We suggest that this feature plays a crucial role in transporting proteins into mitochondria.

Amino Acid Sequence↗
PubMed1986

Late recurrent basal cell carcinoma presenting as a unilateral exophthalmos.

A late recurrent basal cell carcinoma which presented as a unilateral exophthalmos is described. Published surgical protocols of the 1960's advocated deferring further treatment for incompletely excised cutaneous basal cell carcinoma unless there was clinical evidence of tumor recurrence. Our patient demonstrates that this approach may result in late, unusual, and fatal patterns of recurrence. Complete excision of primary basal cell carcinoma is advocated.

Aged↗
PubMed1985

Reaction of 5'-p-fluorosulfonylbenzoyl-1,N6-ethenoadenosine with histidine and cysteine residues in the active site of rabbit muscle pyruvate kinase.

The inactivation of rabbit muscle pyruvate kinase by 0.3 mM 5'-p-fluorosulfonylbenzoyl-1,N6-ethenoadenosine at pH 7.8 is biphasic. The first phase proceeds rapidly to yield a partially active enzyme (46% residual activity) followed by a slower rate which leads to total inactivation. The inactivation of the first phase can be reversed by addition of 20 mM dithiothreitol, whereas the second phase is unaffected. These two phases have second-order rate constants of 250 M-1 X min-1 (dithiothreitol-sensitive reaction) and 52 M-1 X min-1 (dithiothreitol-insensitive reaction), respectively. Marked protection against inactivation is afforded by phosphoenolpyruvate and by metal-nucleotide complexes in the presence of free metal, indicating that reaction occurs in the region of the active site. Loss of approximately two sulfhydryls per enzyme subunit correlates well with the dithiothreitol-sensitive inactivation, suggesting that this phase of the inactivation may be attributable to disulfide formation. Incorporation of about one mole of fluorescent reagent per enzyme subunit correlates closely with the dithiothreitol-insensitive phase of inactivation, yielding a modified histidine residue. The quantum yield of the fluorescent sulfonylbenzoyl-1,N6-ethenoadenosine-pyruvate kinase is only 0.007, as compared to 0.54 for the parent nucleoside 1,N6-ethenoadenosine. The quenched fluorescence is consistent with stacking of the sulfonylbenzoyl moiety on the purine ring in the modified enzyme, which suggests that the altered histidine may be located in the adenine region of the metal-nucleotide binding site.

Adenosine↗
PubMed1985

Electrolysis and thermolysis for permanent hair removal.

The historical, legal, and theoretical aspects and clinical technics of both electrolysis and thermolysis are critically reviewed. The pitfalls of electronic tweezers and the dangers of self-electrolysis are discussed. Complications of electrolysis and thermolysis and the pathophysiology of hair regrowth are presented. In the United States, the lack of uniform training requirements and standards for electrologists may pose an unrecognized risk to public health. It is suggested that more responsible state legislation be enacted in order to decrease the present potential threat to the public health and safety.

Animals↗
PubMed1985

Diagnoses of skin disease: dermatologists vs. nondermatologists.

Health care professionals (medical students, practicing physician's assistants, and residents in the specialties of internal medicine, surgery, and dermatology) were evaluated for their ability to diagnose malignant and benign skin disease. It was found that dermatology residents did significantly better in the diagnosis of malignant and benign skin disease than all other groups, and there was no significant difference among medical students, physician's assistants, and residents in internal medicine or surgery. Findings suggest that the current ability of nondermatologists to correctly diagnose malignant and benign skin disease is not at a comparable level to that of dermatologists and that a 1-month training period in the specialty of clinical dermatology during the primary care residency training period may be inadequate for training nondermatologists to distinguish malignant from benign skin disease. In some specialty areas, such as dermatology, specialization may be preferable to a primary care approach for initially evaluating skin disease.

Dermatology↗