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D Safer

Publications and source records attributed to D Safer.

32 records · Page 2Linked to original sources
PubMed1991

Thymosin beta 4 and Fx, an actin-sequestering peptide, are indistinguishable.

At least 50% of the actin in resting human platelets is unpolymerized, and the bulk of this unpolymerized actin is complexed with a recently identified acidic, heat-stable 5-kDa peptide, named "Fx." Purified Fx binds stoichiometrically to muscle G-actin, forming a complex identifiable by nondenaturing polyacrylamide gel electrophoresis. Formation of the complex inhibits salt-induced polymerization of G-actin. Here we report that Fx is indistinguishable from thymosin beta 4, an acidic, heat-stable 5-kDa peptide first isolated from calf thymus and thought to be a thymic hormone. The complete amino acid sequence of Fx was determined and was found to be identical with that of thymosin beta 4. Authentic thymosin beta 4 is functionally equivalent to Fx, forming a 1:1 complex with actin monomers and inhibiting polymerization. The widespread distribution and high intracellular concentration of thymosin beta 4 (Fx) strongly suggest that it plays a significant role in regulating actin polymerization in many cell types.

Actins↗
PubMed1990

Molecular structure of F-actin and location of surface binding sites.

Comparisons of three-dimensional maps of vertebrate muscle thin filaments obtained by cryo-electron microscopy and image analysis, reveal the molecular structure of F-actin, the location of the C terminus of the monomer and the positions of the binding sites of tropomyosin, the myosin head and the N-terminal portion of the myosin A1 light chain on the filament. These data provide strong constraints for evaluating models built from the atomic structure of the monomer and the subsequent identification of molecular contacts.

Actins↗
PubMed1990

Affinity labeling of bovine liver glutamate dehydrogenase with 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-diphosphate and 5'-triphosphate.

Bovine liver glutamate dehydrogenase reacts with 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-diphosphate (8-BDB-TA-5'-DP) and 5'-triphosphate (8-BDB-TA-5'-TP) to yield enzyme with about 1 mol of reagent incorporated/mol of enzyme subunit. The modified enzyme is catalytically active but has decreased sensitivity to inhibition by GTP, reduced extent of activation by ADP, and diminished inhibition by high concentrations of NADH. Since modified enzyme, like native glutamate dehydrogenase, reversibly binds more than 1 mol each of ADP and GTP, it is unlikely that 8-BDB-TA-5'-TP reacts directly within either the ADP or GTP regulatory sites. The rate constant for reaction of enzyme exhibits a nonlinear dependence on reagent concentration with KD = 89 microM for 8-BDB-TA-5'-TP and 240 microM for 8-BDB-TA-5'-DP. The ligands ADP and GTP alone and NADH alone produce only small decreases in the rate constant for the reaction of enzyme with 8-BDB-TA-5'-TP, but the combined addition of 5 mM NADH + 200 microM GTP reduces the reaction rate constant more than 10-fold and the reagent incorporation to about 0.1 mol/mol of enzyme subunit. These results suggest that 8-BDB-TA-5'-TP reacts as a nucleotide affinity label in the region of the GTP-dependent NADH regulatory site of bovine liver glutamate dehydrogenase.

Adenosine Diphosphate↗
PubMed1990

Isolation of a 5-kilodalton actin-sequestering peptide from human blood platelets.

Resting human platelets contain approximately 0.3 mM unpolymerized actin. When freshly drawn and washed platelets are treated with saponin, 85-90% of the unpolymerized actin diffuses out. Analysis by polyacrylamide gel electrophoresis under nondenaturing conditions shows that the bulk of this unpolymerized actin migrates with a higher mobility than does pure G-actin, profilactin, or actin-gelsolin complex. When muscle G-actin is added to fresh or boiled saponin extract, the added muscle actin is shifted to the high-mobility form. The saponin extract contains an acidic peptide having a molecular mass in the range of 5 kDa, which has been purified to homogeneity by reverse-phase HPLC. This peptide also shifts muscle actin to the high-mobility form. Addition of either boiled saponin extract or the purified peptide to muscle G-actin also strongly and stoichiometrically inhibits salt-induced polymerization, as assayed by falling-ball viscometry and by sedimentation. We conclude that this peptide binds to the bulk of the unpolymerized actin in platelets and prevents it from polymerizing.

Actins↗
PubMed1989

An electrophoretic procedure for detecting proteins that bind actin monomers.

The electrophoretic mobility of fluorescently labeled G-actin in polyacrylamide gels under nondenaturing conditions is altered by the formation of complexes with actin-binding proteins. This effect offers a convenient method for detecting and quantitating such proteins in tissue fractions and for monitoring their purification. When followed by second-dimension electrophoresis in the presence of sodium dodecyl sulfate, the method also gives the apparent molecular weights of the actin-binding components and the stoichiometry of the complexes. The method has also been used to identify actin-binding fragments in digests of actin-binding proteins, to investigate the formation of multicomponent complexes, and to determine the calcium-sensitivity of complexes.

Actins↗
PubMed1989

Hyperactivity and inattentiveness. School assessment of stimulant treatment.

The school health folders of all students receiving stimulant medication for hyperactivity/inattentiveness from five public elementary and two middle schools in Baltimore County were evaluated to 1) record pre-treatment teacher ratings of those on stimulant medication; 2) assess, using the rating scales, the initial and subsequent effectiveness of the medication; and 3) to identify treatment subgroups. Major findings were 1) three-fourths of the 176 students on stimulant medication in 1987 had pre-treatment ratings consistent with moderate to severe hyperactivity/inattentiveness; 2) over 90 percent of the students evidenced at least 50 percent improvement initially following stimulant treatment by teacher ratings; 3) 76 percent of the medicated students continued to show this level of improvement at the end of the 1987 school year; 4) medication improvement and compliance declined in middle school; 5) 15 percent of the students on stimulant medication in 1987 were inattentive but not hyperactive on baseline teacher ratings, but their degree of improvement with stimulants was equivalent to that of hyperactives; 6) in the county-run hyperkinetic clinic, the subpopulation of inattentive/non-hyperactive students on stimulants rose from 7 percent to 18 percent of the total between 1976 and 1987.

Adolescent↗
PubMed1983

Applications of energy filtered imaging in biology.

We describe the use of a magnetic sector spectrometer positioned below the projection chamber of an electron microscope for energy filtered transmission imaging. The spectrometer used has circular pole face edges and is corrected for second order aberrations. A round EM lens is placed after the sector to form a real image of the virtual achromatic image produced by the spectrometer. A slit placed in the dispersion plane allows the passage of electrons in a selected energy range. The filtered image is projected onto a transmission phosphor and acquired with a silicon intensified TV camera and stored in digital form on computer disk. Filtered images are taken at two energies, one immediately preceding (pre-edge) and one on the characteristic energy loss (edge). To obtain images showing the distribution of elements, background subtraction is performed by either subtraction or division of edge and pre-edge images. The optical properties of the imaging system are described and the results are illustrated by energy filtered images of single ferritin molecules (Fe M2,3 and C k), the phosphorus distribution in ribosomes (PL2,3) and the localization of calcium in muscle (Ca L2, 3). The major advantage of the system, compared to other energy filtered imaging methods, is that it can be readily adapted to existing high vacuum microscopes without the necessity of modifying the column to insert a spectrometer.

Animals↗
PubMed1982

Biospecific labeling with undecagold: visualization of the biotin-binding site on avidin.

The biotin-binding site on avidin has been labeled with biotin conjugated to undecagold, an organometallic cluster compound containing 11 gold atoms in a core angestroms in diameter. Examination of unstained specimens by scanning transmission electron microscopy reveals the labeled sites directly, without computational averaging or filtering of the images. This approach should be widely applicable for determining the locations of subunits and functional site in biological macromolecules at a resolution at a resolution in range of 15 angstroms.

Avidin↗