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Biomedical subjects

L D Burtnick

Publications and source records attributed to L D Burtnick.

At least 37 records · Page 2Linked to original sources
PubMed1989

Interaction of iodoacetamidofluorescein-labelled tropomyosin with deoxyribonuclease I.

5-Iodoacetamidofluorescein (IAF) reacted with rabbit cardiac muscle tropomyosin (TM) to yield a highly fluorescent product, IAF-TM. The extent of labelling reached one fluorescein group per TM molecule in solutions at pH 8.5. While fluorescence polarization values for IAF-TM solutions were unaffected by the presence or absence of KCl, addition of pancreatic deoxyribonuclease I (DNase I) resulted in a 10% drop, suggestive of a greater freedom of motion of the fluorescein label in the presence of DNase I. Furthermore, a 15% increase in slopes of Stern-Volmer plots for IAF-TM in the presence of DNase I demonstrated a greater susceptibility of the fluorescein group to dynamic quenching by iodide. These results suggest that interaction between DNase I and TM produces a localized unfolding of the coiled coil near the IAF reactive site on TM.

Animals↗
PubMed1988

Fluorescence from pyrene-labeled native and reconstituted chicken gizzard tropomyosins.

Sulfhydryl groups at Cys-36 on the beta chain and at Cys-190 on the gamma chain of chicken gizzard tropomyosin were reacted with the pyrene-containing sulfhydryl-specific reagents N-(1-pyrenyl)iodoacetamide and N-(1-pyrenyl)maleimide. Tropomyosin prepared and labeled under nondenaturing conditions displayed significant pyrene monomer emission but low levels of pyrene excimer fluorescence. In contrast, tropomyosin subjected to denaturation and renaturation prior to labeling, or labeled in the denatured state prior to renaturation, displayed considerable excimer emission. Furthermore, labeling of isolated beta or gamma chains in denaturant, followed by reconstitution, gave separate samples of beta beta- and gamma gamma-tropomyosin that exhibited even greater pyrene excimer to monomer emission ratios. As pyrene excimers can form only when an excited pyrene is immediately adjacent to a ground state pyrene, i.e., when the labeled Cys residues on the two chains in a tropomyosin coiled coil share the same cross section, these results support conclusions based upon chemical crosslinking studies [C. Sanders, L. D. Burtnick, and L. B. Smillie (1986) J. Biol. Chem. 261, 12774-12778] that native gizzard tropomyosin exists predominantly as a beta gamma-heterodimer. In addition, the low degree of labeling of native gizzard tropomyosin and the differences in degrees of labeling of beta beta- and gamma gamma-tropomyosins in the absence of denaturants reflect on the accessibilities of the sulfhydryl groups in these tropomyosin isoforms. Circular dichroism measurements indicate that the labeled proteins form stable coiled coil structures that have thermal stabilities comparable to that of the native protein.

Affinity Labels↗
PubMed1988

Fluorescence of equine platelet tropomyosin labeled with acrylodan.

Equine platelet tropomyosin was labeled with the sulfhydryl-specific fluorescent reagent 6-acryloyl-2-dimethylaminonaphthalene (acrylodan). The extent of labeling at 4 degrees C could be regulated between 0.5 and 1.3 acrylodans per tropomyosin chain by varying the reaction time from 1 to 4.5 h. Acrylodan-labeled platelet tropomyosin, AD-P-TM, was highly fluorescent, having an emission maximum near 518 nm on excitation at 365 nm. Steady-state measurements of polarization of the fluorescence of AD-P-TM in both low and high ionic strength solutions gave Perrin plots that exhibited sharp changes in slope near 50 degrees C, indicative of a sharp increase in mobility of the label at that temperature. This correlates with the melting temperature of the platelet tropomyosin coiled coil observed by circular dichroism [G. P. Côté, W. G. Lewis, M. D. Pato, and L. B. Smillie, (1978) FEBS Lett. 91, 237-241]. Perrin plots of carboxypeptidase A-treated platelet tropomyosin that was labeled with acrylodan after digestion resembled more closely those of acrylodan-labeled cardiac tropomyosin rather than those of AD-P-TM, suggesting that the observed emission arose from label at Cys-153 on each truncated platelet tropomyosin chain. In solutions containing 150 mM KCl and 5 mM MgCl2, addition of actin at up to a sixfold molar excess over AD-P-TM caused both the fluorescence emission intensities and fluorescence polarization values of samples to increase. In the presence of actin, the wavelength of maximal emission was shifted to shorter values by about 5 to 7 nm. These changes indicate that actin does bind to AD-P-TM and that the binding affects the environment of the label, both by making it more hydrophobic and by reducing the freedom of the label to tumble in solution.

2-Naphthylamine↗
PubMed1987

Quenching of the fluorescence of actin modified at lysine-61.

The fluorescence of actin modified with fluorescein isothiocyanate at Lys-61 is quenched in the presence of iodide. The approximate second order rate constant for quenching is 9 X 10(-8) M-1 s-1, which approaches that for a diffusion-limited process. However, a measurable degree of protection is provided to the fluorescein group by the protein structure around it. A tryptic digest of the modified actin is more susceptible to quenching than is the native protein. Free fluorescein is even more vulnerable. The addition of deoxyribonuclease I to the modified actin reduces the susceptibility of the fluorescence to quenching by only a small degree.

Actins↗
PubMed1986

Native chicken gizzard tropomyosin is predominantly a beta gamma-heterodimer.

Unmodified chicken gizzard tropomyosin (TM) has been fractionated into its two major isoforms beta and gamma, by chromatofocussing in the presence of 9 M urea and dithiothrieitol. Treatment of the native protein with several bifunctional N-hydroxysuccinimide esters gave the beta gamma-heterodimer as the major cross-linked product. A comparison of the thermal transition profiles of the two homodimers and of the native unfractionated TM also indicated the predominance of the beta gamma-heterodimer in the native protein. This conclusion is consistent with the absence of excimer fluorescence in pyrene-labeled gizzard TM and the relative resistance of the molecule to intramolecular disulfide formation (Lehrer, S.S., Betteridge, D.R., Graceffa, P., Wong, S., and Seidel, J. C. (1984) Biochemistry 23, 1591-1595) since the single cysteines on each of the two isoforms are widely separated. We conclude that further experimental evidence is required to assess the possibility that the gizzard TM is more rigid in its conformation than are those of the skeletal and cardiac proteins.

Animals↗
PubMed1986

Excimer fluorescence of equine platelet tropomyosin labeled with N-(1-pyrenyl)iodoacetamide.

Tropomyosin from equine platelets was reacted with N-(1-pyrenyl)iodoacetamide, a sulfhydryl-specific fluorescent reagent, to give an average extent of incorporation of 1.12 pyrene (Py) groups per platelet tropomyosin (P-TM) chain. The predominant site of reaction on P-TM was the penultimate COOH-terminal residue, Cys-246. The high proportion of the total emission that is due to pyrene ecximers and the pretransition observed in thermal denaturation of Py-P-TM point to a rather loose structure for the COOH-terminal amino acid residues of P-TM. The label on Cys-246 also reports on end-to-end overlap interactions that occur between two different tropomyosin molecules. Additions to a Py-P-TM solution at low ionic strength of unlabeled P-TM, rabbit cardiac tropomyosin (C-TM), or a carboxypeptidase A treated, nonpolymerizable derivative of C-TM all reduce the extent of excimer fluorescence from the sample. Addition of salt greatly reduces the effects of the unlabeled TM species on the Py-P-TM emission spectrum. Circular dichroism measurements indicate Py-P-TM still to be greater than 95% helical. However, analysis of excimer fluorescence levels in samples that contained a constant protein concentration but different mole ratios of labeled to unlabeled P-TM suggests that the bulky pyrene group may diminish the tendency of Py-P-TM to polymerize in an end-to-end manner.

Animals↗
PubMed1986

Interaction of actin with dansyl-tropomyosin.

5-[Dimethylamino]naphthalene-1-sulfonyl chloride (dansyl chloride) reacts with rabbit skeletal muscle tropomyosin (TM) to yield a highly fluorescent product, DNS-TM. The extent of modification of TM can be regulated over a wide range, 0.3-15.5 dansyl groups per TM, depending upon the temperature and duration of the reaction. However, under all conditions employed, about 15 different fluorescent tryptic peptides of TM were produced. DNS-TM undergoes end-to-end polymerization at low ionic strengths, but to a somewhat lesser extent than unlabelled TM does. DNS-TM also binds muscle F-actin. This interaction may be followed fluorimetrically by observing a blue-shift in emission maximum, an increase in fluorescence intensity or an increase in fluorescence polarization of the DNS-TM complex with F-actin.

Actins↗
PubMed1984

Modification of actin with fluorescein isothiocyanate.

Reaction of rabbit skeletal muscle G-actin at pH 8.5 with fluorescein isothiocyanate (FITC) resulted in incorporation of up to 1.20 mol FITC/mol actin. At pH 8.8, the level of incorporation was raised to 1.98 mol FITC/mol actin. When excited with ultraviolet light, the FITC-actin samples fluoresced strongly with an emission maximum near 517 nm. Tryptic digests of FITC-actin containing about 1.0 mol FITC/mol actin could be separated into a nonfluorescent 33.5 kDa trypsin-resistant core protein and a fluorescent pool of small peptides. Chromatography on DEAE-Bio-Gel or two-dimensional separation on cellulose TLC plates of the peptide pool revealed that FITC was highly selective in the site of its reaction with actin, resulting in a single highly fluorescent peptide after tryptic digestion. NH2-terminal and amino acid analyses demonstrated this peptide to be derived from residues 51 to 62, with Lys-61 proposed as the major FITC-sensitive site on actin. FITC-actin is similar to G-actin in gross conformation; circular dichroism spectra of actin before and after labelling are identical. FITC-actin is also able to interact strongly with deoxyribonuclease I. However, FITC-actin solution viscosities and fluorescence properties are not altered by the addition of KCl or MgCl2. Therefore, either a localized conformational change near Lys-61 or steric hindrance due to the FITC attached to Lys-61 blocks the polymerization of actin.

Actins↗
PubMed1984

Modification of actin with 2-(N-methylanilino)naphthalene-6-sulfonyl chloride.

2-(N- Methylanilino )naphthalene-6-sulfonyl chloride (Mns-Cl) was used to label up to eight sites, presumed to be lysine residues, on rabbit skeletal muscle actin. The resultant Mns-actin was highly fluorescent, displaying emission bands near 445 and 480 nm. Incorporation of three Mns groups per actin resulted in impairment of the ability of actin to polymerize. Final relative viscosities attained for salt-induced polymers of actin having no bound Mns groups and three bound Mns groups per actin, respectively, were 1.9 and 1.4. More extensive modification further lowered the ability of actin to polymerize, but the changes were less dramatic. The fluorescence properties of Mns-actin samples that contained up to eight Mns groups per actin were not affected significantly by the addition of salt to the samples. It seems that the modified lysines either lie outside a region of direct intersubunit contact in F-actin or that the modification of a particular lysine residue or residues on a particular actin prevents that actin unit from adding on to a growing F-actin chain. Modification of actin with Mns-Cl does not affect its ability to bind and to inhibit DNase I until more than three Mns groups have been incorporated into each actin. The loss of DNase I inhibitory power only becomes apparent after the ability of actin to polymerize has been reduced substantially.

Actins↗
PubMed1983

Fluorescence of actin-bound hydrophobic molecules.

2-(N-methylanilino)naphthalene-6-sulfonic acid (MANS) binds to G-actin at a single high affinity hydrophobic site (Kd = 41 microM). Salt-induced polymerization of MANS-G-actin results in a general enhancement of sample emission intensities at all wavelengths. At 430 nm, KCl-induced polymerization yields a 2,3-fold enhancement, while MgCl2-induced polymerization gives a 2.0-fold increase. Polymerization of MANS-G-actin in the absence of agitation produces MANS-F-actin samples that have fluorescence polarization values at 430 nm of 0.33. Subsequent mixing or sonication of such MANS-F-actin samples results in a dramatic drop in fluorescence polarization values to 0.14. After cessation of mixing, the polarization values do not recover to their initial levels. Circular polarization of luminescence studies on MANS-actin demonstrate that agitation of MANS-F-actin samples drastically alters emission anisotropy values. 9-Anthroyl choline (9AC) binds to G-actin at a single hydrophobic site (Kd = 68 microM). The fluorescence of 9AC-actin is sensitive to salt-induced polymerization and depends upon the identity of the salt employed. KCl causes a drop in the fluorescence intensity at 490 nm to 70% of the value for 9AC-G-actin, while MgCl2 produces a 30% increase in intensity. Polarization experiments with 9AC-actin produced qualitatively the same results as did those with MANS-actin. Differences in the behaviours of MANS-actin and 9AC-actin in response to polymerization by KCl and MgCl2 and in response to the binding of deoxyribonuclease I suggest that the binding sites on actin for MANS and 9AC do not overlap completely.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1980

Protection of actin against proteolysis by complex formation with deoxyribonuclease I.

G-actin bound to deoxyribonuclease I (DNase I) is resistant to digestion by trypsin and chymotrypsin. In the absence of DNase I, G-actin is cleaved by these proteases to yield a 33 500 molecular weight core protein which is not degraded further. The major sites of proteolytic action in the amino acid sequence of actin have been identified as being adjacent to residues arginine-62 and lysine-68 for trypsin and leucine-57 for chymotrypsin. These residues are rendered inaccessible to proteases in the buffer by complex formation with DNase I. Digestion of G-actin with pronase from Streptomyces griseus yields fragmentation patterns that are similar to those observed with trypsin and chymotrypsin. This is likely to be because the specificities of the major constituents of pronase resemble those of trypsin and chymotrypsin. Again, complex formation with DNase I protects the otherwise vulnerable bonds in actin against proteolysis. Incubation with subtilisin Carlsberg leads to complete digestion of G-actin. No subtilisin-resistant core protein accumulates during the incubation. Protection of G-actin when complexed to DNase I is less than complete in this case but still is significant. This is interpreted in terms of the broad specificity of subtilisin and the observed fragmentation pattern of free G-actin when treated with subtilisin.

Actins↗