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Biomedical subjects

F M Richards

Publications and source records attributed to F M Richards.

At least 127 records · Page 7Linked to original sources
PubMed1981

The labeling with 8-azido-cyclic adenosine monophosphate of proteins in vesicles of sarcoplasmic reticulum from rabbit skeletal muscle.

Photoinduced labeling with 8-azido-cyclic AMP of proteins in vesicles from sarcoplasmic reticulum rabbit skeletal muscle has been examined. At concentrations of 0.1 microM or less, specific labeling of three or four bands, representing trace components in the SDS gels, was seen. This labeling was prevented by cyclic AMP. Some of the bands correspond approximately in apparent molecular weight to subunits of cyclic AMP-dependent protein kinases reported in other systems. Attempts to use this reagent as a probe for protein position in the membrane have been unsuccessful since 8-azido-cyclic AMP passes into and through the membrane. The reagent also appears to partition with a linear concentration dependence into the vesicular membrane itself, presumably into the lipid portion.

Affinity Labels↗
PubMed1981

Synthesis of carriers of differing Stokes radius with activated acyl groups for use as reagents in labeling membrane proteins.

Compounds with the general formula C(CH2CO--(Pro)n--R1--O--COR2)4 have been synthesized with n = 0, 1, 3, 5. The R1 alcohol group provides an active ester of the acylating function R2CO--. On reaction with a nucleophile the acyl group is attached while the major part of the reagent serves as the leaving group. Even with compounds of relatively low molecular weight the extended structures represent molecules with a substantial Stokes radius. These reagents are intended to probe the accessibility of functional groups in cell organelles, particularly membranes. Subtle differences in labeling patterns have been observed in preliminary experiments with red cell ghosts.U

Acylation↗
PubMed1980

Analysis of membranes photolabeled with lipid analogues. Reaction of phospholipids containing a disulfide group and a nitrene or carbene precursor with lipids and with gramicidin A.

Analogues of fatty acids have been synthesized which contain an S-S bridge in the aliphatic chain and at the omega-carbon the photoactivatable p-(3-trifluoromethyldiazirino)phenyl or p-azidophenyl group as a carbene and nitrene precursor, respectively. These acids were used to acylate 1-palmitoyl phosphatidylcholine by the procedure described by Gupta et al. (Gupta, C.M., Radhakrishnan, R., and Khorana, H.G. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 4315-4319). Multilamellar liposomes prepared from various radioactively labeled lipids and 2 to 10 mol% of one of the photosensitive phospholipid analogues were photolyzed and the extent of cross-linking was determined. The carbene-generating probe labeled dipalmitoyl phosphatidylcholine in yields of 15 to 20%, while labeling with the nitrene probe was about 10 times less efficient. In contrast these probes were found to react with the unsaturated fatty acid of 1-palmitoyl-2-oleoyl phosphatidylcholine with almost equal efficiency. The nitrene was 2 to 3 times more efficient than the carbene in cross-linking to gramicidin A. Amino acid analysis and Edman degradation of the labeled peptide indicated that both probes predominantly attacked tryptophan residues. No geometrical correlation was found between the expected penetration of the reactive group of the probe within the bilayer and the labeled sites of the peptide. This result is discussed in terms of chemical selectivities of the photoactivated intermediates. After photocross-linking, the disulfide bridge can be cleaved to generate a free sulfhydryl group which subsequently can be utilized for preparative or analytical purposes. Photolabeled lipids and peptides can be separated easily from the bulk of the unlabeled material. These phospholipid analogues are substrates for the phospholipid exchange protein isolated from calf liver. Transfer from sonicated dispersions of the analogues into an acceptor membrane was demonstrated. Individual monolayers can thus be labeled separately with these reagents.

Azirines↗
PubMed1980

3-Trifluoromethyl-3-phenyldiazirine. A new carbene generating group for photolabeling reagents.

The synthesis of 3-trifluoromethyl-3-phenyldiazirine (TPD) is reported in an overall yield of 60% based on 2,2,2-trifluoroacetophenone as starting material. TPD is rapidly photolyzed on irradiation near 350 nm to yield 35% of the diazoisomer and 65% of the corresponding carbene. No internal rearrangement of the latter compound by fluorine migration was detected. Photolysis in methanol yielded the product of a formal OH insertion in greater than 95% yield. Photolysis in cyclohexane gave at least a 50% yield of the CH insertion product at a diazirine concentration of 15 mM. The products were identified by gas chromatographic mass spectra and by 19F NMR spectra. In the dark the diazirine is stable in 1 M acid or base and at temperatures as high as 75 degrees C for at least 30 min. The diazoisomer is much less photolabile and is stable in 0.1 M acetic acid for at least 12 h. The synthesis of a derivative of TPD containing the 2-hydroxyethyl[O-tosylate] group on the para position of the phenyl ring is described. This compound permits the easy attachment of the TPD function to other molecules. It is suggested that the ease of synthesis, dark stability, rapid photolysis, and reactivity of the carbene may make this group useful in the preparation of various photochemical probes.

Affinity Labels↗
PubMed1977

Solvents, interfaces and protein structure.

Mean packing densities in protein interiors are comparable to those of most organic solids but the variations between small regions may be substantial. Packing defects may be related to allowed structural fluctuations. Molecular surface areas can be correlated with free energies of transfer between different solvents. The proportionality factor will depend, in general, on the nature of the solute and both the solvents. The changes in solvent-protein interfacial area on chain folding are large and the implied changes in free energy from this solvent-squeezing effect are correspondingly large. The strong tendency to minimize surface area is reflected in the globular shape of most protein molecules or domains in larger structures. The formation of isolated units of secondary structure from an extended chain represents about one half of the eventual total area change. The tendencies of amino acids to form beta-sheets correlate well with the rank-ordered list based on non-polar area change for each residue type. The calculated area changes for helix and sheet formation are not identical in rank order. The rank-ordered list for alpha-helix formation correlates satisfactorily with the probability list prepared from actual structures if glutamic acid and tyrosine are removed. What special characteristics unrelated to surface area these two amino acids might have is not clear. Tertiary structure formation from preformed secondary structural units can be rank ordered on area change and possible nucleation sites can be identified. A prediction scheme for helix-helix interactions is proposed. The hydrophobic force begins to be felt when two helices are about 0.6 nm (6 A) from their final contact positions. Interfacial surface tension is a logical parameter to relate free energy and solvent contact area, but this macroscopic parameter must be used with great caution. It is suggested that water in the deep grooves, characteristic of the active sites of many enzymes, may have a substantially higher fugacity than bulk water as indicated, at least qualitatively, by the Kelvin equation based on surface curvature. Such water would be more easily displaced than its plane surface counterpart and could contribute significantly to ligand-binding energy. This factor would be in addition to the usual solvent entropic effects associated with surface area reduction on association.

Amino Acids↗
PubMed1976

Reversible inhibition of glucose transport in human erythrocytes by a series of pyridine derivatives.

A series of pyridine derivatives was chosen on the basis of structural similarity to glucose and to the hydrogen bonding capacity of this sugar. These compounds were found to reversibly inhibit glucose transport in a systematic fashion. The inhibition data can be rationalized by assuming that both competitive and noncompetitive binding sites exist on the cell membrane. The competitive site appears to have a much lower dissociation constant than the noncompetitive site for most derivatives. The effects of changes in substituents on the pyridine ring closely match, both in ring position and in the magnitude of the change in binding constant, those reported for derivatives of glucose. The stimulation of fluorodinitrobenzene inactivation of glucose transport by 2-amino-5-chloropyridine is in accord with this analysis. The strongest inhibitors bind about 100-fold more tightly than glucose itself. This series of pyridine compounds would appear to present a useful starting point for the development of site-specific irreversible inhibitors.

Biological Transport, Active↗
PubMed1976

Proteolytic and chemical modification of colicin E3 activity.

Proteolyses of colicin E3 by both trypsin and subtilisin yield fragments of various molecular weights. On the basis of sodium dodecyl sulfate gel electrophoresis, tryptic cleavage yields peptides of molecular weight about 42 000 and 18 000, while the comparable pieces in a subtilisin digest have apparent weights of about 36 000 and 24 000. The digests lose almost all of their in vivo cell killing activity but the in vitro activity leading to ribosomal inactivation is augmented. Trypsin-treated colicin E3 shows a 20-30-fold increase in its ability to release the 52 nucleotide fragment from the 16S ribosomal ribonucleic acid (rRNA) and this activity is associated with the smaller fragment. Subtilisin-treated colicin E3 is only about two to three fold more active than the native protein in vitro, and the peptides obtained upon cleavage cannot be separated by gel filtration or polyacrylamide gel electrophoresis without sodium dodecyl sulfate. However, in the presence of 0.1% sodium dodecyl sulfate, subtilisin-treated E3 shows a 20-30-fold augmentation in in vitro activity which is again associated with the smaller fragment extracted from the sodium dodecyl sulfate gel. Amino terminal end-group studies showed that the two larger fragments and intact E3 have the same N-terminal residue, valine. These fragments presumably originate from the amino end of the native protein. The smaller tryptic fragment has an N-terminal alanine, while the smaller subtilisin piece has an N-terminal leucine. In addition, modification of a single carbosyl group in intact colicin E3 abolishes more than 90% of the in vivo activity with a simultaneous increase in in vitro activity. This carboxyl group is located in the larger fragments obtained in both trypsin and subtilisin cleavage. Binding of E3 to sensitive cells is drastically reduced or eliniated by this chemical modification and by both of the limited proteolytic cleavages.

Amino Acids↗
PubMed1976

Behavior of colicins E1, E2, and E3 attached to sephadex beads.

Colicins E1, E2, and E3 were covalently attached to Sephadex G-25 beads by cyanogen bromide activation. These immobilized colicins were still active in binding to specific receptors on sensitive and tolerant cells but not to resistant cells which lack such receptors. Bound colicin E3 also retained its ability to inhibit protein synthesis in vitro. Leakage of free colicin from these coated beads was negligible. Assays sensitive to free colicin activity of 1 part in 10(7) of the bound toxin failed to detect any soluble activity. The viability of different cell types bound specifically onto these colicin-Sephadex beads was assayed by using autoradiography based on labeled amino acid uptake. Immobilized E1 killed 90% of bound sensitive cells while less than 10% of sensitive cells bound to E2 and E3 were killed in this assay. These observations agree very well with previously suggested mechanisms which propose that E1, whose target site appears to be at the membrane level, can kill sensitive cells by binding to the cell surface, but that for E2 and E3 penetration of part or all of the molecule is necessary for killing action to be observed.

Binding Sites↗
PubMed1975

Photochemical labeling of the cytoplasmic surface of the membranes of intact human erythrocytes.

N-(4-azido-2-nitrophenyl)-2-aminoethyl sulfonate (NAP-taurine), a photolabile nitrene precursor, has been shown to permeate the human erythrocyte membrane at 37degrees but not at 0 degrees. Utilizing this differential permeability, we have loaded intact erythrocytes with NAP-[35S]taurine in the dark at 37degrees, cooled them to 0 degrees, washed them free of external NAP-[35S]taurine in the dark and cold, and photolyzed them, resulting in labeling of hemoglobin and of proteins on the cytoplasmic surface of the membrane. These experiments complement those previously reported on the labeling of the external surface of the membranes with this reagent (Staros, J. V., and Richards, F. M. (1974) Biochemistry 13, 2720-2726).

Binding Sites↗
PubMed1975

Reaction of dimethyl-3,3'-dithiobispropionimidate with intact human erythrocytes. Cross-linking of membrane proteins and hemoglobin.

Dimethyl-3,3'-dithiobispropionimidate penetrates intact human erythrocytes and cross-links many of the membrane proteins to hemoglobin as well as to each other. The cross-linked complexes so produced have been analyzed by both one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis, making use of the easy cleavability of the disulfide-containing reagent. The basic pattern of cross-linked complexes appears identical with that seen with unsealed ghosts. Although subtle relative motions cannot be ruled out, no rearrangement of nearest neighbor peptide chains, on a scale that would alter the cross-linking pattern, occurs during osmotic lysis of erythrocytes. Superimposed on the basic pattern was a series of complexes involving globin chains. Bands 1, 2, 2.2, 2.4, 3, 4.1, 4.2, 6, and 7 (nomenclature of Steck, T.L. (1972) J. Mol. Biol. 66, 295-305) are all cross-linked to hemoglobin. Bands 2.2 and 2.4, recently shown to be accessible to the external surface of the membrane (Staros, J. V., and Richards, F. M. (1974) Biochemistry 13, 2720-2726), may be transmembrane proteins on the basis of the present findings. Band 5 is the only major band to show no detectable complexes with hemoglobin; oligomers of Band 5 itself, however, are seen. The absence of hemoglobin/Band 5 cross-linking in this case could reflect a special, as yet unexplained, environment for the Band 5 peptide. The amount of Band 6 in isolated membranes diminishes with increasing reagent concentration.

Binding Sites↗