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Biomedical subjects

P Horowitz

Publications and source records attributed to P Horowitz.

At least 37 records · Page 2Linked to original sources
PubMed1984

Bis(1,8-anilinonaphthalenesulfonate). A novel and potent inhibitor of microtubule assembly.

Two related compounds, 1,8-anilinonaphthalenesulfonate (1,8-ANS) and bis(1,8-anilinonaphthalenesulfonate) (Bis-ANS), are useful fluorescent probes for hydrophobic areas on protein molecules. Using fluorescence, we examined the binding of these compounds to bovine brain tubulin and found that Bis-ANS and 1,8-ANS bound to tubulin with Ki values of 2 and 25 microM, respectively. Bis-ANS potently inhibited the polymerization of tubulin into microtubules in vitro. In the presence of microtubule-associated protein 2, half-maximal inhibition of assembly was obtained at 3 microM Bis-ANS. In the presence of tau protein, half-maximal inhibition was obtained at 15 microM Bis-ANS. Surprisingly, 1,8-ANS, even at 200 microM, did not inhibit assembly. Scatchard analysis indicated one binding site for Bis-ANS on tubulin. Previous reports of 1,8-ANS binding to tubulin may have been influenced by the presence of Bis-ANS which until recently was a common contaminant of commercial supplies. Because of its intense fluorescence in addition to its potent inhibitory effects, Bis-ANS appears to be a useful probe to study microtubule assembly and other interactions involving tubulin.

Anilino Naphthalenesulfonates↗
PubMed1983

The use of tritium exchange to detect conformational differences between intermediates in catalysis by the enzyme rhodanese.

Tritium-exchange experiments have been performed on enzyme forms related to obligatory catalytic intermediates in the rhodanese reaction: the free enzyme, E, and the sulfur-substituted enzyme, ES. Under the experimental conditions used, each form displayed tritium-exchange behavior that can be interpreted in terms of at least three classes of exchangeable sites. Class I with a t 1/2 approximately equal to 180 min and Class II with a t 1/2 approximately equal to 15 min were present in both E and ES. The conversion of ES to E has the effect of changing approx. 50 protons from the slow Class I behavior to exchange at a rate too fast to measure (Class III). These results are consistent with a conformational change on conversion of rhodanese from ES to E. The fact that a large number of protons are involved together with the very large change in exchange rate might indicate that extended areas of the protein change contact with the solvent, and would be compatible with a model for rhodanese catalysis that includes a coupled conformational change.

Animals↗
PubMed1983

The use of intrinsic protein fluorescence to quantitate enzyme-bound persulfide and to measure equilibria between intermediates in rhodanese catalysis.

The intrinsic fluorescence of the enzyme rhodanese is quenched by as much as 30% when sulfur is transferred to the free enzyme form, E, giving the sulfur-substituted enzyme, ES. This fluorescence change (lambda ex = 295 nm and lambda em = 335 nm) has been used to quantitate the E and ES forms which are isolatable, obligatory intermediates in rhodanese catalysis. Fluorescence titration was performed using cyanide to irreversibly remove sulfur from ES. The results show a stoichiometry corresponding to 1 bound sulfur/molecule of the ES form of rhodanese (Mr = 33,000). The fluorescence changes were used to measure the concentrations of E and ES when these were in reversible equilibria induced by interactions with the substrates S2O3(2-) and SO3(2-). These results were compared with an equilibrium constant derived from published kinetic studies for the reaction (formula; see text) The very close agreement between the physical and kinetic methods indicate that there are no significant concentrations of intermediates other than E and ES. Overall, the results are compatible with the formation of a persulfide intermediate in rhodanese catalysis and are consistent with conclusions from x-ray crystallography and absorption spectroscopy. In addition, these procedures offer a facile method to measure equilibria between catalytic intermediates in the rhodanese reaction using functionally relevant concentrations.

Kinetics↗
PubMed1983

Proteolytic interconversion of electrophoretic variants of the enzyme rhodanese.

It has been confirmed that the enzyme rhodanese, although a homogeneous single polypeptide chain protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is separable by electrophoresis under nondenaturing conditions into four species which differ in net surface charge (I-IV in the order of increasing positive charge). Limited proteolysis can interconvert these species. Chymotrypsin converts IV and III to II and forms a small amount of I. Carboxypeptidase B converts IV to III. The total protein among the species remains constant, and two-dimensional gels show that the change induced is below the resolution of the sodium dodecyl sulfate-polyacrylamide gel system. The suggestion that the products can be produced in the order IV, III, and II is supported by the results of sequential treatment of rhodanese first with carboxypeptidase B and then with chymotrypsin. It is concluded that there are covalent differences among the rhodanese species identified to date and an interconversion of forms can be triggered by proteolysis at the COOH-terminal end of the Mr = 33,000 single polypeptide chain which comprises the enzyme. This conclusion is strengthened by the close similarity between the amino acid composition of the peptide released by chymotrypsin and the composition expected on the basis of the known sequence. Furthermore, it appears that form IV is the primary in vivo product and the other species arise from it.

Animals↗
PubMed1983

Molecular heterogeneity of the concanavalin A tetramer: effects on binding to human red blood cells.

We have found that the distribution of the three main monomer species found in tetrameric concanavalin A was approximately 73% type A monomer (27,000 MW); 4% type B monomer (14,000 MW); and 23% type C monomer (12,000 MW). When this tetrameric concanavalin A was bound to human erythrocytes and the monomer distribution of the bound concanavalin A was examined, we found that it resembled that of the concanavalin A used in the binding reaction. However, when competing sugars were used, either to inhibit the binding of concanavalin A or to remove previously-bound lectin, examination of cell-bound monomer distribution revealed that there was a significant increase in type C monomers and a simultaneous decrease in type A monomers. The shifts in monomer distribution varied depending on experimental conditions and the particular competing inhibitor employed. These findings were taken to indicate that not all concanavalin A cell surface interactions are identical and that quantitative methods are available for studying this phenomenon.

Concanavalin A↗
PubMed1982

The specificity of active-site alkylation by iodoacetic acid in the enzyme thiosulfate sulfurtransferase.

The active-site sulfhydryl group in the enzyme thiosulfate sulfurtransferase (rhodanese; thiosulfate:cyanide sulfurtransferase; EC 2.8.1.1) is alkylated rapidly by iodoacetic acid in the free enzyme form, E, with complete loss of sulfurtransferase activity. Iodoacetic acid is completely ineffective with the sulfur-substituted form of the enzyme, ES. Iodoacetamide, on the other hand, has no effect on either enzyme form. The competitive enzyme inhibitor, toluenesulfonic acid, protects against inactivation in a strictly competitive way and analysis gives an apparent binding constant for toluenesulfonic acid of 12.5 mM, which is in agreement with studies of its effect on the catalyzed reaction. These results are taken to indicate that iodoacetic acid is an affinity analog for the substrate, thiosulfate, and inactivates because it can use the specific thiosulfate binding interactions, correctly orient its reactive center and displace intraprotein interactions which appear to protect the active-site sulfhydryl group in the E form.

Alkylation↗
PubMed1979

The ultrasonographic and radiologic features of a histologically proven case of alveolar soft-part sarcoma of the orbit.

An 18-year-old woman had a rapidly progressive proptosis, vascular congestion, and orbital pain. Ultrasonographic and radiologic tests supported the diagnosis of a solid vascularized mass that proved histologically to be an alveolar soft-part sarcoma of the orbit. Uncertainty as to the histogenesis of alveolar soft-part sarcoma still exists, but prognosis for patient survival is poor.

Adolescent↗
PubMed1977

Studies of the N-bromosuccinimide inactivation of the enzyme rhodanese.

The enzyme rhodanese (Thiosulfate: cyanide sulphurtransferase, EC 2.8.1.1) is rapidly inactivated by treatment with N-bromosuccinimide. Spectrophotometric titration and sodium dodecyl sulfate polyacrylamide gel electrophoresis show that neither tryptophan oxidation nor polypeptide chain cleavage can account for the inactivation. Sulfhydryl group assays using the colormetric reagent 5,5'-dithiobis(2-nitrobenzoic acid) after destruction of excess N-bromosuccinmide, indicate that approximately 2 sulfhydryl groups per enzyme molecule are lost. Further, rhodanese inactivated by N-bromosuccinimide can be reactivated (approximately 95%) by incubation with the substrate thiosulfate. It is postulated that N-bromosuccinimide inactivates rhodanese by inducing the formation of a disulfide bond involving the active site sulfhydryl group of the enzyme and a second sulfhydryl group which can be brought close to the active site in the flexible native structure.

Binding Sites↗
PubMed1976

Elemental analysis of biological specimens in air with a proton microprobe.

The unique capabilities of the proton microprobe in an atmospheric environment as a biological tool are illustrated in studies of arsenic and mercury distributions in siingle strands of hair from poisoning victims and of the distributions of several abundant elements in frozen hydrated eye and kidney specimens from rats.

Animals↗
PubMed1976

Fluorimetric studies of tryptophyl exposure in concanavalin A.

Studies of the iodide ion quenching of the intrinsic fluorescence of Concanavalin A indicate that 50% of the tryptophyl fluorescence originates from exposed residues. This agrees with the X-ray crystallographic determination that two of the four tryptophan residues in a Concanavalin A monomer are on the surface. Previous studies have indicated that conformational changes induced by sugar binding alter the environment of aromatic residues. The present investigation finds that neither the specific binding of alpha-methyl-D-mannoside nor alteration of the Concanavalin A quaternary structure changes the number or accessibility of the solvent-exposed tryptophan residues. It therefore appears that the major conformational transitions in Concanavalin A do not affect steric access to the surface tryptophans and the effects previously observed may be ascribed to structurally internal tryptophan residues.

Binding Sites↗
PubMed1972

A scanning x-ray microscope using synchrotron radiation.

Focused synchrotron radiation collimated by means of a pinhole has been used to construct a scanning x-ray microscope capable of making stereoscopic element-discriminating pictures of relatively thick specimens in an atmospheric environment.

Microscopy, Electron, Scanning↗
PubMed1970

Absolute time-of-arrival measurements of optical pulses from the Crab pulsar.

An attempt is made to understand the characteristics of pulsar slowdown in terms of various possible emission mechanisms. Though magnetic-dipole radiation seems to be dominant, the deviations from a pure dipolar decay are large compared with errors in measurement and indicate the presence of more complicated braking mechanisms, small fluctuations, and major "jumps", or period discontinuities. Some rapid-relaxation effect is evident following the discontinuous jump in period in September 1969, but there is no evidence of the quasi-sinusoidal behaviour reported before the jump by Arecibo. An alternative interpretation of the Arecibo data is given.

Journal Article↗
PubMedDate not supplied

Synchronization of the acoustic evidence in the assassination of President Kennedy.

We have revisited the acoustic evidence in the Kennedy assassination--recordings of the two Dallas police radio channels upon which our original NRC report (Ramsey NF et al., Report of the Committee on Ballistic Acoustics. National Research Council (US). Washington: National Academy Press, 1982. Posted at http://www.nap.edu/catalog/10264.html) was based--in response to the assertion by DB Thomas (Echo correlation analysis and the acoustic evidence in the Kennedy assassination revisited. Science and Justice 2001; 41: 21-32) that alleged gunshot sounds (on Channel 1), apparently recorded from a motorcycle officer's stuck-open microphone, occur at the exact time of the assassination (as established by emergency communications on Channel 2). We have critically reviewed these two publications, and have performed additional analyses. In particular we have used recorded 60 Hz hum and correlation methods to obtain accurate speed calibrations for recordings made on both channels, cepstral analysis to seek instances of repeated segments during playback of Channel 2 (which could result from groove jumping), and spectrographic and correlation methods to analyze instances of putative crosstalk used to synchronize the two channels. This paper identifies serious errors in the Thomas paper and corrects errors in the NRC report. We reaffirm the earlier conclusion of the NRC report that the alleged "shot" sounds were recorded approximately one minute after the assassination.

Acoustics↗