Spectroscopic analysis of acylated and deacylated myelin proteolipid protein

The effect of covalently bound fatty acid on the conformation of the myelin proteolipid protein has been studied by ultraviolet and intrinsic fluorescence spectroscopy. With dimethyl sulfoxide used as a perturbant, the exposure of Trp and Tyr residues in various mixtures of chloroform-methanol was evaluated by difference spectroscopy of the proteolipid protein (APL) and its chemically deacylated form (d-APL). The fraction of chromophoric groups exposed increased with the proportion of chloroform with 25% of the groups exposed in 1:2 chloroform-methanol and 98% in 3:1 chloroform-methanol. These conformational changes correlate well with changes in intrinsic viscosity. Values for the deacylated form were indistinguishable from those of the acylated protein, suggesting that fatty acids do not affect protein conformation in organic solvents. In water, UV difference spectroscopy indicated that the number of Tyr and Trp groups exposed in both APL and d-APL was relatively small and was independent of the molecular size of the perturbant. However, differences in the environment of the Trp groups in the two forms of the protein could be demonstrated by intrinsic fluorescence. When the protein was excited at 295 nm, the maximum emission wavelength for the acylated protein was 330 nm, whereas it was 335 nm for the deacylated form. Furthermore, the Trp groups in d-APL were more easily quenched by acrylamide than in APL, indicating that they were more exposed, or in a more hydrophilic environment, following deacylation. Protein aggregation appears to be independent of the presence of fatty acids, suggesting that the fluorescence differences between APL and d-APL are related to factors other than aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of the alkali light-chain-20-kilodalton fragment complex from skeletal myosin heads

We have developed a rapid and reproducible procedure widely applicable to the preparation of pure aqueous solutions of the complex between an alkali light chain and the COOH-terminal heavy-chain fragments of skeletal myosin chymotryptic subfragment 1 (S-1) split by various proteases. It was founded on the remarkable ethanol solubility of these complexes. A systematic study of the ethanol fractionation of the tryptic (27K-50K-20K)-S-1 (A2) showed the NH2-terminal 27K fragment to behave like a specific protein entity being quantitatively precipitated at a relatively low ethanol concentration. Only the 20K peptide-A2 complex remained in solution when the S-1 derivative was treated with exactly 4 volumes of ethanol in the presence of 6 M guanidinium chloride. At a lower ethanol concentration, a soluble mixture of 50K and 20K peptides together with the light chain was obtained. The isolated 20K fragment-A2 system containing a 1:1 molar ratio of each component was investigated by biochemical and 1H nuclear magnetic resonance (NMR) techniques to highlight its structure and the interaction of the 20K heavy-chain segment with F-actin and with the light chain. During the treatment of the complex with alpha-chymotrypsin, only the 20K peptide was fragmented in contrast to its stability within the whole S-1. The binding of F-actin to the complex led, however, to a strong inhibition of its chymotryptic degradation. 1-Ethyl-3-[3-(dimethylamino)propyl]carbodiimide cross-linking of F-actin to the complex produced covalent actin-20K peptide only, the amount of which was lower relative to that observed with the entire split S-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Demonstration of the GC-rich common arm in yeast ribosomal 5.8S RNA via 500-MHz proton nuclear magnetic resonance and Overhauser enhancements

In this paper we report the first 1H NMR study of the base-paired secondary structure of yeast 5.8S RNA. On the basis of a combination of homonuclear Overhauser enhancements and temperature dependence of the proton 500-MHz NMR spectrum, we are able to identify and assign eight of the nine base pairs in the most thermally stable helical arm: G116.C137-C117.G136-C118.G135- C119.G134-C120.G133-U121.G132- U122.A131-G123.C130. This arm contains an unusually temperature-stable (to 71 degrees C) segment of four consecutive G.C base pairs. This work constitutes the most direct evidence to date for the existence and base-pair sequence of the GC-rich helix, which is common to most currently popular secondary structural models for eukaryotic 5.8S ribosomal RNA.     

Proflavin binding to poly[d(A-T)] and poly[d(A-br5U)]: triplet state and temperature-jump kinetics

The delayed fluorescence properties of proflavin have been exploited in studies of the excited-state binding kinetics of the dye to poly[d(A-T)] and its brominated analogue poly[d(A-br5U)] at room temperature and pH 7. The two analyzed luminescence decay times of the DNA-dye complex are dependent on the total nucleic acid concentration. This dependence is shown to reflect a temporal coupling of the intrinsic delayed emission decay rates with the dynamic chemical kinetic binding processes in the excited state. Temperature-jump kinetic studies conducted on the brominated polymer and corresponding information on poly[d(A-T)] from a previous study [Ramstein, J., Ehrenberg, M., & Rigler, R. (1980) Biochemistry 19, 3938-3948] provide complementary information about the ground state. In the ground state, the poly[d(A-T)]-proflavin complex has one chemical relaxation time, which reaches a plateau at high DNA concentrations. The brominated DNA-dye complex exhibits two relaxation times: a faster relaxation mode that behaves similarly to that for the unhalogenated DNA and a slower relaxation mode that is apparent at high DNA concentrations. The ground-state kinetic data are analyzed in terms of two alternative models incorporating series and parallel reaction schemes. The former consists of two sequential binding steps--a fast bimolecular process followed by a monomolecular step--while the latter consists of two coupled bimolecular steps. A similar analysis for the excited-state data yields reasonable kinetic constants only for the series model, which, in accordance with previous proposals for acridine intercalators, consists of a fast outside binding step followed by intercalation of the dye. A comparison of the ground- and excited-state kinetic parameters reveals that the external binding process is much stronger and the intercalation is much weaker in the excited state. That the excited-state data are only consistent with the series model suggests that delayed luminescence studies may provide a general tool for distinguishing between the two kinetic mechanisms. In particular, we demonstrate the use of delayed luminescence spectroscopy as a tool for probing dynamic DNA-ligand interactions in solution.     

Affinity-purified tetanus neurotoxin interaction with synaptic membranes: properties of a protease-sensitive receptor component

The pharmacokinetic interaction of an affinity-purified 125I-labeled tetanotoxin fraction with guinea pig brain synaptosomal preparations was investigated. Binding of tetanotoxin was time- and temperature-dependent, was proportional to protein concentration, and was saturable at about 8 X 10(-9) M as estimated by a solid-surface binding assay. Binding was optimal at pH 6.5 under low ionic strength buffer and was almost entirely blocked by gangliosides or antitoxin. In analogy to intact nerve cells, binding of toxin to membranes resulted in a tight association operationally defined as sequestration. Binding and sequestration were abolished after membrane pretreatment with sialidase. The enzyme could not dissociate the membrane-bound toxin formed at 4 or 37 degrees C under low ionic strength conditions, which is in part compatible with internalization as defined in nerve cell cultures. In the latter system the toxin could be removed at 4 degrees C but not at 37 degrees C. Binding was significantly reduced upon pretreatment of guinea pig brain membranes by a variety of hydrolytic enzymes. Trypsin and chymotrypsin inhibited binding between 55% and 68% while bacterial protease abolished it by 91-95%. The effect was species-specific as it was not seen in rat or bovine synaptosomes. Collagenase and hyaluronidase had little or no inhibitory effect when applied to synaptosomes (27% and 9%) but inhibited binding to synaptic vesicles by 56% and 49%, respectively. Phospholipases A2 and C caused 42-43% inhibition of binding in vesicles and less than 22% in synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Response of Peyer's patch lymphocyte subsets to Giardia muris infection in BALB/c mice. II. B-cell subsets: enteric antigen exposure is associated with immunoglobulin isotype switching by Peyer's patch B cells

The aim of this study was to quantify the response of Peyer's patch B cells, surface IgA-bearing (sIgA) B cells, and surface IgM-bearing (sIgM) B cells to Giardia muris infection. Following infection of a cohort of immunocompetent BALB/c mice with G. muris cysts, Peyer's patch cell suspensions were prepared at serial time points during the infection, incubated with fluorescein-conjugated monoclonal antibodies directed against murine leukocytes, B cells, sIgA B cells, sIgM B cells, or T cells, and analyzed by flow cytometry. Of total Peyer's patch leukocytes, the percentages of B cells, sIgA B cells, and sIgM B cells in uninfected BALB/c mice were 64.7 +/- 2.0% (mean +/- SEM), 30.3 +/- 1.5%, and 52.5 +/- 2.4%, respectively. The total number of Peyer's patch leukocytes increased significantly (1.8 X) during G. muris infection, and returned to control levels as the infection was cleared. The percentages of Peyer's patch T and total B cells did not change significantly during Giardia infection. However, sequential changes were observed in the percentages and numbers of sIgM and sIgA B cells during the infection. Peyer's patch sIgM B cells rapidly increased in percentage and number, reaching maximum levels 1 week after cyst inoculation. After remaining constant the first week, the number of Peyer's patch sIgA B cells increased during the second week of G. muris infection, reaching a maximum level 11-14 days after cyst inoculation. The data support the hypothesis that immunoglobulin isotype switching in Peyer's patches is induced by antigen exposure.     

Sensitivity of preadipose 3T3 cells to growth hormone

Cultured preadipose 3T3 cells are able to undergo a process of differentiation through which they are converted into adipose cells. Growth hormone induces this conversion in resting cultures but not in growing cultures. It was of interest to determine the period of cell sensitivity to the hormone and the timing of the induction of glycerophosphate dehydrogenase, a key enzyme in lipogenesis. It was found that 3T3-F442A cells became highly sensitive to rat growth hormone at confluence but that high sensitivity remained for only 3 days; thereafter, the responsiveness to the rat growth hormone declined rapidly. Refeeding of the cells with fresh medium did not lead to the recovery of the hormone sensitivity, indicating that the decrease in sensitivity was not due to exhaustion of medium components but that it seemed to be a specific property of F442A cells. As glycerophosphate dehydrogenase activity was detected at nearly the same time as its mRNA was measurable, it is likely that the mRNA is translated immediately after its synthesis.     

Gold labeling of microtubules in cleaved whole mounts of cortical root cells

A method is described for localizing microtubules using gold-labeled antibodies in combination with anti-tubulin. Cortex cells of Equisetum hyemale are broken open while still in buffer, after initially being attached to poly-L-lysine-coated grids. Thus, the cytoplasm becomes accessible to the antibodies. After application of the antibodies, the cleaved cells are post-fixed, stained, dehydrated, and critical point-dried. Different fixation procedures are compared: fixation in paraformaldehyde, in glutaraldehyde, and in glutaraldehyde followed by a sodium borohydride reduction step. All three methods result in good labeling of the microtubules, with low backgrounds. However, organization of the cytoplasm is best preserved in cells fixed in glutaraldehyde without sodium borohydride treatment. The method is highly suitable for studying the membrane-bound cytoskeleton because detergent extraction and/or embedding are avoided.     

Three distinct H-2Ks molecules differing at the carboxy terminus are expressed on a tumor from SJL/J mice

A dimethylbenzanthracene-induced leukemia of H-2s origin expressed at least two class I molecules on the cell surface that were precipitated by anti-H-2.19, an alloantiserum prepared against the private H-2Ks specificity. Mapping studies in recombinant inbred strains along with comparisons of tryptic peptide maps and N-terminal sequences indicated that the proteins were virtually identical and probably encoded by the same class I gene. When cells were labeled in the presence of tunicamycin, the proteins precipitated by anti-H-2.19 were further resolved into three distinct peptides. Experiments were performed to determine which of these various proteins were phosphorylated and which were recognized by an anti-synthetic peptide serum directed against the ultimate C-terminus of H-2K class I molecules. The results indicate that a single class I gene from the H-2Ks region may encode three class I molecules that differ only at the C-terminus due to alternative splicing of pre-mRNA.     

Transforming growth factor beta is an important immunomodulatory protein for human B lymphocytes

The growth and differentiation of B cells to immunoglobulin (Ig)-secreting cells is regulated by a variety of soluble factors. This study presents data that support a role for transforming growth factor (TGF)-beta in this regulatory process. B lymphocytes were shown to have high-affinity receptors for TGF-beta that were increased fivefold to sixfold after in vitro activation. The addition of picogram quantities of TGF-beta to B cell cultures suppressed factor-dependent, interleukin 2 (IL 2) B cell proliferation and markedly suppressed factor-dependent (IL 2 or B cell differentiation factor) B cell Ig secretion. In contrast, the constitutive IgG production by an Epstein Barr virus-transformed B cell line was not modified by the presence of TGF-beta in culture. This cell line was found to lack high-affinity TGF-beta receptors. The degree of inhibition of B cell proliferation observed in in vitro cultures was found to be dependent not only on the concentration of TGF-beta added but also on the concentration of the growth stimulatory substance (IL 2) present. By increasing the IL 2 concentrations in culture, the inhibition of proliferation induced by TGF-beta could be partially overcome. In contrast, the inhibition of Ig secretion induced by TGF-beta could not be overcome by a higher concentration of stimulatory factor, demonstrating that the suppression of B cell differentiation by TGF-beta is not due solely to its effects on proliferation. Furthermore, it was demonstrated that B lymphocytes secrete TGF-beta. Unactivated tonsillar B cells had detectable amounts of TGF-beta mRNA on Northern blot analysis, and B cell activation with Staphylococcus aureus Cowan (SAC) resulted in a twofold to threefold increase in TGF-beta mRNA. Supernatants conditioned by unactivated B cells had small amounts of TGF-beta, SAC activation of the B cells resulted in a sixfold to sevenfold increase in the amount of TGF-beta present in the supernatants. Thus, B lymphocytes synthesize and secrete TGF-beta and express receptors for TGF-beta. The addition of exogenous TGF-beta to cultures of stimulated B cells inhibits subsequent proliferation and Ig secretion. TGF-beta may function as an autocrine growth inhibitor that limits B lymphocyte proliferation and ultimate differentiation.