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J Szasz

Publications and source records attributed to J Szasz.

11 recordsLinked to original sources
PubMed1997

Thermo Sequenase DNA polymerase and T. acidophilum pyrophosphatase: new thermostable enzymes for DNA sequencing.

A combination of thermostable enzymes has been developed that produces higher quality cycle sequences. Thermo Sequenase DNA polymerase is a thermostable enzyme engineered to catalyze the incorporation of ddNTPs with an efficiency several thousandfold better than other thermostable DNA polymerases. Since the enzyme also catalyzes pyrophosphorolysis at dideoxy termini, a thermostable inorganic pyrophosphatase is needed to remove the pyrophosphate produced during sequencing reactions. Thermoplasma acidophilum inorganic pyrophosphatase (TAP) is thermostable and effective for converting pyrophosphate to orthophosphate. The use of the combination of Thermo Sequenase polymerase and TAP for cycle sequencing yields sequence data with uniform band intensities, allowing the determination of longer, more accurate sequence reads. Uniform band intensities also facilitate interpretation of sequence anomalies and the presence of mixed templates. Sequencing PCR products of DNA amplified from heterozygous diploid individuals results in signals of equal intensity from each allele.

Cloning, Molecular↗
PubMed1996

Cogan's syndrome.

Explore the source record for details and available documents.

Cyclophosphamide↗
PubMed1993

Site-directed mutagenesis of alpha-tubulin. Reductive methylation studies of the Lys 394 region.

Previous studies have implicated at least two regions in alpha-tubulin that are important for the regulation of microtubule assembly. These regions include a cluster of basic residues consisting of Arg 390, His 393, and Lys 394 and the highly acidic carboxyl terminus. Lys 394 is highly reactive to HCHO and NaCNBH3. The reductive methylation of Lys 394 by these reagents is thought to be responsible for the profound inhibitory effects of low concentrations of HCHO on microtubule assembly (cf. Szasz J., M. B. Yaffe, M. Elzinga, G. S. Blank, and H. Sternlicht. 1986. Biochemistry. 25:4572-4582). In this study we reexamined the basis for this inhibition. Lys 394 in a human keratinocyte alpha-tubulin (k alpha 1) was replaced by a glutamic acid residue using site-directed mutagenesis. The mutant K394E was synthesized in vitro using rabbit reticulocyte lysates, and its ability to coassemble with bovine brain microtubule protein (MTP) before and after reaction with HCHO and NaCNBH3 was compared with that of wild-type. No differences in the coassemblies of the unmethylated proteins were detected suggesting that Lys 394 is not essential for microtubule assembly. However, methylated K394E prepared at low HCHO concentrations (< 1 mM) incorporated into microtubules to a greater extent (approximately 30-40%) than methylated wild-type. This result is consistent with the hypothesis that methylation of Lys 394 interferes with microtubule assembly. However, the extent of protection afforded by the replacement of Lys 394 with Glu 394 was less than half as large as that predicted from the earlier studies. We tentatively conclude that another residue(s) besides Lys 394 contributes significantly to the assembly-inhibition observed with low concentrations of HCHO. Since this residue(s) is less reactive than Lys 394, it would have to inhibit assembly substoichiometrically when methylated. Potential candidates for this residue include bulk lysyl residue(s), a lysyl residue(s) with intermediate reactivity toward HCHO, and the NH2-termini. The NH2-termini are especially attractive candidates since they appear to have a structural role in microtubule assembly.

Animals↗
PubMed1989

Ethoxyformylation of tubulin with [3H]diethyl pyrocarbonate: a reexamination of the mechanism of assembly inhibition.

In this study we reexamined the basis for the profound inhibitory effects of low concentrations of diethyl pyrocarbonate (DEP) on tubulin's ability to assemble into microtubules [cf. Lee, Y. C., Houston, L. I., & Himes, R. H. (1976) Biochem. Biophys. Res. Commun. 70, 50-56]. Assembly inhibition at low DEP concentrations can be resolved into two components: a component reversible with hydroxylamine (attributed to monoethoxyformylation of histidyl residues) that contributes approximately 40% of the inhibition and a hydroxylamine-resistant component (attributed to ethoxyformylation of non-histidyl residues) that contributes approximately 60% of the inhibition. Comparisons between the extent of assembly inhibition associated with each component and the degree of residue modification argue for the involvement of a small number of highly reactive residues in the inhibition process. To identify these residues, tubulin was reacted with limiting concentrations of [3H]DEP and subjected to tryptic digestion and HPLC analysis. Only one moderately reactive histidyl residue was detected. This residue (approximately 2-3-fold more reactive than the bulk histidyl residues) eluted in an apparently large, hydrophobic fragment. We failed to detect any non-histidyl residues that were exceptionally reactive to [3H]DEP. However, we did observe that the N-terminal methionyl residues in native protein were ethoxyformylated at rates comparable to that of the bulk histidyl residues. In denatured protein these methionyl residues were ethoxyformylated to a much larger extent (approximately 3-4-fold) than the bulk histidyl residues. We suggest that the N-terminal methionyl residues in tubulin are partly buried or are in a salt-bridge interaction in native protein and that ethoxyformylation of these residues disrupts tubulin structure and interferes with microtubule assembly.

Amino Acid Sequence↗
PubMed1988

Expression of a human alpha-tubulin: properties of the isolated subunit.

We examined the in vitro expression and biochemical properties of the isolated alpha subunit of tubulin both in rabbit reticulocyte lysates and in Escherichia coli extracts. Both systems produce soluble, full-length human alpha-tubulin polypeptide. When alpha-tubulin mRNA is translated in rabbit reticulocyte lysates, the isolated alpha subunit is fully functional as assayed by coassembly with bovine brain tubulin using temperature-dependent or taxol/salt assembly procedures. The conformation of the isolated alpha subunit was probed by limited proteolytic digestion with chymotrypsin and by reductive methylation. Limited proteolysis studies indicated that the "monomeric" alpha subunit is highly susceptible to chymotrypsin digestion and becomes resistant to chymotrypsin cleavage following incorporation into the heterodimer. Reductive methylation indicated that the unassociated alpha subunit has a highly reactive lysyl residue essential for microtubule assembly similar to that observed in the heterodimer. In contrast, alpha-tubulin expressed in E. coli lysates was incapable of coassemblying with bovine brain tubulin. Differences in assembly competence of the two alpha-tubulin products appear to be related to formylation of the N-terminal methionine in the procaryotic synthesized subunit. These findings suggest that the amino-terminal methionine of alpha-tubulin plays an essential role in the isolated subunit and/or in the heterodimer, a hypothesis supported by chemical reactivity studies [Sherman, G., Rosenberry, T.L., & Sternlicht, H. (1983) J. Biol. Chem. 258, 2148-2156] which imply that this residue is in a salt-bridge interaction in the dimer.

Alkaloids↗
PubMed1986

Microtubule assembly is dependent on a cluster of basic residues in alpha-tubulin.

Previous studies have shown that tubulin, a major protein component of the microtubule, is rendered assembly incompetent when a highly reactive lysine residue (HRL) in the alpha polypeptide of tubulin dimer is reductively methylated [cf. Sherman, G., Rosenberry, T. L., & Sternlicht, H. (1983) J. Biol. Chem. 258, 2148-2156]. In this study we demonstrate that the HRL in bovine brain tubulin is Lys-394, a residue proximal in the alpha-tubulin sequence to the highly negatively charged carboxy-terminus region (residues 412-450) previously implicated in assembly. pH studies were undertaken to probe the local environment of Lys-394. These studies indicated that Lys-394 reactivity toward HCHO is sensitive to the titration of a pKa 6.3 group presumed to be a histidine residue. This assignment is supported by our finding that histidine modification via diethyl pyrocarbonate strongly affects Lys-394 reactivity toward HCHO as well as microtubule assembly. We propose on the basis of secondary structure considerations and published sequence data for a variety of tubulins that Lys-394 is part of an evolutionarily conserved cluster of basic residues (effective charge: 2+ to 2.5+ at neutral pH) composed of Lys-394, His-393, and Arg-390, which is important for tubulin function and which renders Lys-394 reactive as a nucleophile.

Amino Acid Sequence↗
PubMed1986

Pneumatic rupture of the posterior pharyngeal wall.

A fourth case of pharyngeal injury after an attempt to open a bottle of 'home brew' is presented. For the first time the potential barotrauma causing this injury is recognized. The principles of diagnosis, investigation and treatment are described. Early surgical intervention will minimize contamination and allow adequate drainage of the contaminated space. Early institution of antibiotics will minimize local and generalized toxic phenomena. Alternate routes of alimentation allow local healing and rapid recovery of the patient.

Adult↗
PubMed1983

Theory for modeling the copolymerization of tubulin and tubulin-colchicine complex.

Substoichiometric concentrations of tubulin-colchicine complex (TC) inhibits microtubule assembly through a copolymerization reaction between tubulin and TC. We have determined the rates and extent of TC incorporation into bovine brain microtubules and developed a theory that models copolymerization. Our analysis suggests that while the apparent association rate constants for tubulin and TC are similar, the apparent dissociation rate constants for TC are a factor of five or more larger than those of tubulin. Copolymer composition showed only slight changes during assembly despite changes in the solution phase and showed little dependence at high TC upon the initial tubulin concentration. The theory was based on coupled Oosawa-Kasai equations that allow for the co-assembly of two components, tubulin and TC. An expression was derived that relates copolymer composition to reaction mixture composition and to the affinity of microtubule ends for tubulin and TC. This expression predicts copolymer composition at TC concentrations less than 10 microM and correlates composition with assembly inhibition. We perceive copolymerization as a facilitated incorporation of TC requiring the presence of tubulin. TC incorporation was dependent on the ratio of total tubulin to the dissociation constant for TC bound to microtubule ends. The copolymerization reaction is thus characterized by an interplay of two effects (a) where tubulin facilitates the incorporation of TC into the microtubule, and (b) where TC inhibits the assembly of tubulin into microtubules.

Animals↗
PubMed1982

Effects of reductive methylation on microtubule assembly. Evidence for an essential amino group in the alpha-chain.

Microtubule protein from bovine brain was reacted at pH 6.7 with formaldehyde and NaCNBH3. These reagents react specifically with protein amino groups, causing their conversion to mono- and dimethylated forms (Jentoft, N., and Dearborn, D. (1979) J. Biol. Chem. 254, 4359-4365). Reductive methylation both inhibited microtubule assembly and induced extensive depolymerization in assembled microtubules. These effects occurred at low levels of methylation (10%) and appeared to arise from an alteration in tubulin which rendered tubulin assembly incompetent. This alteration had no significant effect on colchicine or GTP binding or on the critical tubulin concentration required for assembly. Comparative methylation studies over a range of formaldehyde concentrations involving microtubule polymer, microtubule protein, and several test proteins suggested that assembly inhibition results from the methylation of one or two highly reactive amino groups in the alpha-chain. The reactivities of these amino group(s) were reduced in the polymerized and denatured states.

Animals↗
PubMed1980

The co-polymerization of tubulin and tubulin chochicine complex in the absence and presence of associated proteins.

Tubulin x colchicine complex (TC) is a potent inhibitor of microtubule assembly whereas microtubule-associated proteins (MAPs) facilitate assembly. Previous studies done under MAP-depleted conditions (Sternlicht, H., and Ringel, I. (1979) J. Biol. Chem. 254, 10540) suggested that tubulin binds to the ends of the microtubule with an apparent dissociation constant (affinity T-1) which increases as the TC/tubulin ratio in the microtubule increases. Affinity T-1 was identified with the critical, i.e. minimum tubulin concentrations required for assembly. In this study, we examined the TC-tubulin co-polymerization reaction as a function of MAP availability. Critical tubulin concentrations, which increases in the presence of TC and decreases in the presence of MAP, could be approximated as a sum of contributions from MAPs and TC. An expression was derived which successfully predicted the per cent inhibition observed for a variety of TC, tubulin, and MAP concentrations. In all cases, increases in critical tubulin concentrations correlated with increases in the TC/tubulin ratio in the microtubules. Our data suggest that substoichiometric inhibition by TC is not a consequence of impaired MAP function, nor is it a consequence of a marked increase in the apparent free energy of assembly in the presence of TC. Rather, the large per cent inhibition values observed at low TC concentrations (TC less than or equal to 5 microM) appear to be a consequence of the small concentrations of tubulin typically used in assembly studies (less than or equal to 2 to 4 mg/ml active tubulin) and the constraint that assembly in the presence of TC requires a minimum concentration of tubulin equal to affinity T-1. The molecular processes by means of which TC and MAPs affect affinity T-1 remain to be established.

Animals↗