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P Strittmatter

Publications and source records attributed to P Strittmatter.

At least 37 records · Page 2Linked to original sources
PubMed1983

Isolation and partial characterization of the NH2-terminal membrane-binding domain of NADH-cytochrome b5 reductase.

The amphipathic flavoprotein NADH-cytochrome b5 reductase from steer liver was converted into the membrane-binding and soluble catalytic domains by controlled subtilisin proteolysis of vesicle-bound, externally oriented reductase. Amino acid analysis of the single nonpolar peptide which remained associated with the vesicles showed that it has a molecular weight of about 6400-6500 and that 65-66% of the amino acid residues are hydrophobic. Both the intact reductase and the nonpolar peptide were blocked to sequencing. Carboxypeptidase Y digestion of the holoenzyme and of a chymotryptically generated flavopeptide, which had lost membrane-binding capacity, released 1 residue of alanine and 2 residues of phenylalanine, indicating a common carboxyl terminus. Partial sequence analysis of the reductase placed the nonpolar peptide to the NH2-terminal side of the subtilisin-generated flavopeptide domain. Thus, the membrane-binding domain of cytochrome b5 reductase is localized at the NH2-terminus of the whole protein. The nonpolar peptide bound to vesicles contains a single fluorescent tryptophan with an emission maximum characteristic of a hydrophobic environment.

Amino Acid Sequence↗
PubMed1982

Reduced pyridine nucleotides and cytochrome b5 as electron donors for prostaglandin synthetase reconstituted in dimyristyl phosphatidylcholine vesicles.

Prostaglandin synthetase has been reconstituted in dimyristyl phosphatidylcholine vesicles. These vesicles in either 80 or 2 microM flufenamate utilized NADH or NADPH as electron donor in the reductive peroxidative step of prostaglandin H2 formation. Vesicles containing bound cytochrome b5 reductase and cytochrome b5 in order to complete an NADH cytochrome b5 reductase system also utilized reduced cytochrome b5 as the electron donor for this peroxidative step. In systems containing cytochrome and reductase, the rate of NADH oxidation exceeded that of NADPH oxidation, indicating that reduced cytochrome b5 is an effective electron donor for prostaglandin H2 formation, enhancing both the initial rate and the extent of the reaction. The concentrations of reduced pyridine nucleotides and cytochrome b5 employed in these experiments to provide reductants for prostaglandin synthetase are within the concentration ranges that obtain under physiological conditions.

Chemical Phenomena↗
PubMed1981

The heme-binding properties of prostaglandin synthetase from sheep vesicular gland.

Purified, apoprostaglandin synthetase was prepared from sheep vesicular gland and studied in terms of its heme-binding properties. The enzyme binds a single heme group per enzyme monomer, Mr = 70,000. When reconstituted with heme, the enzyme has an absorption maximum at 412 nm and an absorption coefficient, epsilon 412 nm, of 120 mM-1 cm-1. The binding of heme to the apoenzyme was accompanied by a proportional increase in enzyme activity up to the point of heme-binding saturation. This reconstituted holoenzyme forms prostaglandin H2 from arachidonate. We conclude that prostaglandin synthetase possesses the heme-binding properties of a "typical" heme protein and that a single heme group mediates both the oxygenase and the peroxidase activities of the enzyme.

Animals↗
PubMed1981

Orientation of the carboxyl and NH2 termini of the membrane-binding segment of cytochrome b5 on the same side of phospholipid bilayers.

The present data show that the carboxyl terminal end of the membrane binding segment (nonpolar peptide) of cytochrome b5 is present on the same side of phospholipid bilayers as the hydrophilic, heme-containing, NH2-terminal segment. This orientation was determined by observing rapid ionization of both tyrosyl residues at positions 5 and 8 from the carboxyl terminus upon addition of sodium hydroxide to the outer aqueous phase of vesicle preparations, and the reaction of one of these residues with a polar, impermeant reagent, diazotized sulfanilic acid. The rate of ionization of both aromatic residues occurred at least 1 order of magnitude faster than ionization of indigo trisulfonate trapped in the inner aqueous compartment of the vesicles. These data and consideration of our earlier characterization of cytochrome b5 structure and binding to membranes support a model for the membrane binding segment that is highly structured, penetrates to the middle of the bilayer, and loops back to the outer surface to place both the NH2 and the carboxyl termini on the same surface of the bilayer.

Amino Acid Sequence↗
PubMed1981

The role of COOH-terminal anionic residues in binding cytochrome b5 to phospholipid vesicles and biological membranes.

The COOH-terminal, anionic residues of the membrane binding segment of cytochrome b5 were examined to determine their possible significance in stabilizing the "tight" binding of the cytochrome to phospholipid vesicles. The removal of the 6 COOH-terminal residues, which include the carboxyl groups of Glu 132 and Asn 133, by carboxypeptidase digestion resulted in the loss of the characteristic "tight" binding to either synthetic phospholipid vesicles or isolated microsomes. Chemical modification of the four carboxyl groups of the nonpolar peptide of cytochrome b5 with carbodiimide and methylamine to produce a derivative with no anionic charged residues also resulted in a loss of this type of stable membrane interaction. These results suggest that the short polar COOH-terminal segment, containing two of the four carboxyl groups of the membrane binding domain of cytochrome b5, plays a crucial role in lipid-protein interactions that lead to the normal "tight" binding both in situ and in reconstituted phospholipid bilayer systems.

Amino Acid Sequence↗
PubMed1979

Intramembrane positions of membrane-bound chromophores determined by excitation energy transfer.

A detailed theory has been derived to evaluate the efficiency of nonradiative transfer of electronic excitation energy between nonassociated membrane-bound chromophores. Two different approaches are presented and shown to lead to identical numerical results. In the first of these the efficiency of transfer is computed from the decay with time of the donor excited state. In the second approach, the efficiency is calculated directly, demonstrating that to a high degree of accuracy the array of acceptors can be represented as consisting of a single nearest acceptor plus a continuum of secondary acceptors. A general expression is derived for the dipole-dipole orientation factor as a function of the position of an acceptor. It is shown that, by invoking the range of orientations that must be present at the very least in a particular case, the expected values of transfer efficiency may be limited to a relatively narrow band of uncertainty about those predicted for total randomization. In the limit of total randomization, the theory reduces to functions of but two dimensionless parameters: an effective number of acceptors and a normalized distance of closest approach, a parameter which in turn is a function of an excluded surface area and the depth in the membrane of a donor relative to that of an acceptor. Finally, data analysis procedures are presented whereby one can determine the surface density of acceptors for a known geometry or, alternatively, determine the distance of closest approach for known surface densities.

Energy Transfer↗
PubMed1979

Intramembrane position of the fluorescent tryptophanyl residue in membrane-bound cytochrome b5.

We have developed a method to measure the intramembrane position of the fluorescent tryptophanyl residue in whole cytochrome b5 and the nonpolar membrane binding segment when these molecules are bound to phospholipid vesicles [Koppel, D.E., Fleming, P., & Strittmatter, P. (1979) Biochemistry (preceding paper in this issue)]. The method utilizes excitation energy transfer from the donor tryptophanyl residue in the protein to trinitrophenyl or danysl acceptor groups on the surface of the phospholipid bilayer. It was determined that that single fluorescent tryptophanyl residue in vesicle-bound cytochrome b5 and the nonpolar segment is located approximately 20-22 A below the surface of the bilayer. This position represents a minimum depth of penetration of this portion of the cytochrome in the membrane.

Animals↗
PubMed1979

Formation and properties of 1000-A-diameter, single-bilayer phospholipid vesicles.

Two methods are reported for the formation of large, uniform-sized phospholipid vesicles. The methods involve the treatment of phospholipid, in the form of either small, sonicated vesicles or a dry lipid film, at a molar ratio of deoxycholate to phospholipid of 1:2. Subsequent removal of deoxycholate yields a stable preparation of vesicles. These vesicles are bounded by a single bilayer, have an average diameter of 1000 A, and are readily separated from sonicated vesicles (230 A) by gel filtration on Sepharose 4B. Since the 1000-A vesicles are capable of trapping enzymes and other macromolecules, they may prove valuable for the delivery of liposome-entrapped solutes to cells and for the localization of peptide segments of a spectrum of membrane-bound proteins.

Bile Acids and Salts↗
PubMed1978

The nonpolar peptide segment of cytochrome b5. Binding to phospholipid vesicles and identification of the fluorescent tryptophanyl residue.

The nonpolar peptide segment of cytochrome b5 consisting of the COOH-terminal 43 amino acid residues binds rapidly to dimyristyl lecithin vesicles above the transition temperature of the phospholipid. The binding is complete as indicated by density gradient centrifugation and is accompanied by approximately a 2-fold increase in the fluorescence emission of the protein, and insertion in the bilayer is apparently in an orientation indistinguishable from that of the whole cytochrome b5 molecule. Quantitative reaction of the protein with N-bromosuccinimide destroys the fluorescence of the protein with a stoichiometry which indicates that a single tryptophanyl residue is responsbile for the fluorescence. The fluorescent tryptophanyl residue is one of 2 adjacent residues, Trp-108 or Trp-109, as shown by carboxypeptidase digestion of N-bromosuccinimide-reacted nonpolar peptide.

Animals↗
PubMed1978

Structural and functional properties of the membrane binding segment of cytochrome b5.

Derivatives of cytochrome b5 that had been selectively shortened at the COOH-terminal, membrane binding segment of this amphipathic protein were employed to examine the minimum structural requirements for binding to phospholipid vesicles and for catalytic interactions in the stearyl-CoA desaturase system. Three derivatives shortened by 6, 18, and 27 amino acid residues were produced by controlled proteolysis with carboxypeptidases. The two largest derivatives bound to synthetic lipid vesicles and interacted with cytochrome b5. The third derivative neither bound to vesicles nor reacted with the desaturase. Whole nonpolar peptide and the nonpllar peptides of the two largest derivates contain only 29 to 34% polar residues, whereas the nonpolar peptide of the third derivative contains 44% polar residues. The secondary structure of the membrane binding segment was studied by circular dichroism of whole nonpolar peptide and the corresponding peptides of the three derivatives. The data for whole nonpolar peptide are consistent with a structure containing approximately 50% helical and 25% beta sheet structure. The CD of the nonpolar peptides of the two largest derivatives are consistent with structures containing 56% helix and 19% beta sheet structure, and 40% helix and 20% beta sheet structure. These data support a predicted model for secondary structure, proposed previously, based upon the primary structure (Fleming, P. J., Dailey, H. A., Corcoran D., and Strittmatter, P. (1978) J. Biol. Chem. 253, 5369-5372).

Amino Acid Sequence↗
PubMed1978

The primary structure of the nonpolar segment of bovine cytochrome b5.

The primary structure of the membrane bound segment of amphipathic bovine liver microsomal cytochrome b5 has been determined. This 43 residue nonpolar polypeptide is present at the COOH terminus of cytochrome b5. The sequence was obtained by automated sequence analysis and carboxypeptidase digestions. The sequence obtained is: Ile-Thr-Lys-Pro-Ser-Glu-Ser-Ile-Ile-Thr-Ile-Asp-Ser-Asn-Pro-Ser-Trp-Trp-Thr-Asn-Trp-Leu-Ile-Pro-Ala-Ile-Ser-Ala-Leu-Phe-Val-Ala-Leu-Ile-Tyr-His-Leu-Tyr-Thr-Ser-Glu-Asn. Conformational analysis using predictive algorithms is presented along with circular dichroism data on the peptide bound to phospholipid vesicles.

Amino Acid Sequence↗