Leslie A. Holladay

Gonadotropin and subunit conformation

Abstract Circular dichroic spectra have been obtained and resolved for the gonadotropins, ovine pituitary luteinizing hormone and human chorionic (urinary) gonadotropin, their subunits and glycopeptides. Much of the gonadotropin ellipticity above 250 nm can be attributed to the disulfide chromophore, although there are discernible contributions from tyrosyl and phenylalanyl residues as well. Of the two dissimilar subunits, the β-subunit makes the greatest contribution to the near-ultraviolet circular dichroic spectrum of the gonadotropins. From the position of the 0-0 tyrosyl band, i.e., 286–287 nm, one can ascertain that at least some of the tyrosyl residues of the gonadotropins are located in a hydrophobic environment. A positive circular dichroic extremum at 232.5 nm, present in luteinizing hormone but not in chorionic gonadotropin, can be ascribed to the α-subunit and probably results from tyrosines 21 and/or 30 in luteinizing hormone. An analysis of the circular dichroic difference spectrum above 230 nm, generated by subtracting the sum of the molecular ellipticities of the respective subunits from the molecular ellipticities of each gonadotropin, indicates that the local environment of disulfides and of tyrosyl residues is altered when gonadotropins dissociate. Circular dichroic difference spectra between the two α-subunits and between the two β-subunits indicated major contributions from- tyrosyl residues, presumably arising from tyrosyl substitutions. Between 200 and 230 nm, both gonadotropins exhibit negative circular dichroic extrema. The extremum occurs at 210 nm for luteinizing hormone and at 207.5 nm for chorionic gonadotropin. Each extremum can be described by two negative resolved bands, one at 215 nm and the other between 207 and 208.5 nm. The 215-nm resolved band is assigned to the peptide chromophore in a β-pleated sheet conformation and there is no evidence of α-helicity. The lower-wavelength resolved band is believed to have a significant contribution from the N -acetyl groups of glucosamine, galactosamine, and sialic acid, particularly since the glycopeptide fractions, prepared from each gonadotropin by digestion of the S -carboxymethyl derivatives with Pronase, exhibited a negative circular dichroic extremum at about 207 nm. The extent of β-structure in both gonadotropins is estimated to be about 28% whereas the separated subunits contain less β-structure, e.g., about 21 and 13% for the α- and β-subunits, respectively. The sum of the subunit β-structure, corrected for the respective molecular weight of each subunit, is about 17%. This is substantially less than that of the native hormone, thus indicating that significant conformational changes occur during gonadotropin dissociation to the biologically inactive subunits. Also, part of the gonadotropin β-structure may arise from intermolecular hydrogen bonding involving a pleated sheet arrangement between the subunits.

On the conformation of the acetylcholine receptor protein from Torpedo nobiliana.

It is well established that acetylcholine acts as a regulatory ligand controlling selective increase in membrane permeability to cations [ 11. This permeability change is thought to be mediated by a minimum of two distinct structural elements [2] : a ‘receptor’ protein which zzcognizes cholinergic agonists and an ‘ionophore’ that accounts for the selective transition of ions. The latter could be a region of the receptor protein or may constitute a separate but tightly coupled entity [2]. AChRT from several species has been extensively purified by several laboratories [3-91 and its molecular properties are currently under investigation. However, no information is available on the receptor conformation. This report describes for the first time the far ultraviolet CD spectrum of AChR. A detailed analysis of the resolved spectrum indicates that the Torpedo nobiliana AChR contains a particularly high content of ordered secondary structure, e.g., about 34% a-helix and 29% p-structure. The amino acid composition of the receptor is also reported and the overall polarity and hydrophobicity is typical of soluble proteins.

An approximate solution to the Lamm equation.

An approximate solution to the Lamm equation subject to the initial and boundary conditions for conventional sedimentation velocity experiments is derived and compared with the approximate solution of Fujita and MacCosham. Calculations with this solution demonstrate that the half-height method of estimating sedimentation coefficients yields correct values for epsilon < 0.02.

Rapid and large-scale purification and characterization of renin from mouse submaxillary gland

Abstract Little is known about the structural basis for the highly restricted substrate specificity of renin, whose only function known to date is to hydrolyze the unique leucyl peptide bond in the prohormone angiotensinogen to form angiotensin I. Our lack of knowledge is due to our inability to purify this enzyme in a large quantity sufficient for structural studies. A two-step column chromatographic method for rapid and large-scale purification of renin from mouse submaxillary gland has been developed. It allows isolation of the enzyme in a hundreds-of-milligrams quantity at an overall yield of 60%. Single bands obtained by polyacrylamide gel electrophoresis, isoelectric focusing, and the result of ultracentrifugal studies indicated homogeneity of the product. The moelcular weight of renin was estimated to be 36,000 by sedimentation equilibrium studies in 6 m guanidine · HCl. Sedimentation velocity study gave a single sedimenting boundary with an s 20,w of 2.58 × 10 −13 s. A Stokes radius of 27 A was obtained by gel filtration. The far ultraviolet-circular dichroism spectrum indicated a high content of β-structure (46%). In contrast to renal renin, submaxillary gland renin does not contain amino sugars or neutral sugars. No renin activity was retained by concanavalin Aagarose gels. Results of amino acid analysis, isoelectric focusing, and determination of amino-terminal residues by the dansylchloride reaction, together indicated that this protein is identical with renin A isolated previously in a much smaller quantity.

Conformational stability and self-association of zinc-free bovine insulin at neutral pH

Abstract Zinc-free bovine insulin has been studied by sedimentation equilibrium and circular dichroism. At pH 7.4, ionic strength 0.368, and a temperature of 20 °C, the peptide hormone appears to form a monomer-dimer-hexamer system characterized by a second virial coefficient of 8·10 -6 mol·dl·g -2 , and association constants of 2.2·10 4 M -1 and 8.6·10 9 M -2 for monomer-dimer and dimer-hexamer formation, respectively. The dimerization free energy is —5.8 kcal/mol and the free energy for hexamer formation per (dimer) contact site is —4.4 kcal/mol. Guanidinium chloride induces marked changes in the self-association, the far ultraviolet circular dichroic spectrum, and the near ultraviolet circular dichroic spectrum. In the presence of 1.5 M guanidinium chloride Zn 2+ -free insulin exists mainly as a monomer-dimer system, and above 3.5 M guanidinium chloride the hormone forms a non-ideal monodisperse system. A broad transition, indicated by changes in the far ultraviolet circular dichroic spectrum between 2 and 6 M guanidinium chloride, probably results from a loss of secondary structure with increasing denaturant concentration. Under the conditions used, the near ultraviolet circular dichroic spectrum of native Zn 2+ -free insulin was not dependent on hormone concentration, although an apparent transition midpoint, measured as a decrease in the ellipticity at 270 nm with increasing guanidinium chloride concentration between 0 and 2 M, was dependent on insulin concentration. These results suggest that major changes in insulin secondary and tertiary structure occur over a range of denaturant concentrations in which insulin exists principally in the monomeric form. Thus, these data suggest that the maintenance of the insulin quarternary structure is not a prerequisite for the preservation of a considerable degree of secondary structure in the insulin molecule.

A temperature conversion nomogram for glycosylated hemoglobin analysis

Analysis of glycosylated hemoglobins by commercial mini-column methods was shown to vary linearly with the ambient temperature. However, the slopes of the temperature dependence lines for diabetic and healthy individuals was different. A method for temperature correction and a nomogram is proposed.

Evidence for structural dissociation of two biologic actions of growth hormone

The effects of purified growth hormone and its CNBr fragments on somatomedin induction and on the stimulation of hepatic and renal ornithine decarboxylase (L-ornithine carboxylase, EC 4.1.1.17) activity in rats have been investigated. At the doses tested, none of the CNBr fragments induced somatomedin as evidenced by lack of an effect on sulfate, leucine, and thymidine incorporation into cartilage of hypophysectomized rats. However, the largest fragment, consisting of two peptides corresponding to Residues 6-124 and 150-179 linked by a disulfide bridge, stimulated both renal and hepatic ornithine decarboxylase activity in hypophysectomized rats and the activity of the hepatic enzyme in intact animals. A smaller CNBr fragment corresponding to Residues 125-149 slightly stimulated the activity of renal ornithine decarboxylase but failed to increase activity of the hepatic enzyme. A similar slight stimulation of the activity of the renal, but not the hepatic, enzyme was produced by a large carboxyl-terminal fragment (molecular weight 8000) prepared by proteolytic cleavage of partially purified ovine growth hormone. Circular dichroic spectra of the CNBr fragments demonstrated that the largest fragment retained much of the ordered secondary structure of intact growth hormone while two smaller CNBr fragments were devoid of ordered secondary structure. These observations indicate that different biological activities of growth hormone may be dissociated by fragmentation of the parent molecule.

Simultaneous rapid estimation of sedimentation coefficient and molecular weight.

Data obtained from the early portion of sedimentation velocity experiments may be analyzed to simultaneously estimate both s and s/D. The C versus r data obtained are analyzed using a nonlinear least squares algorithm and an approximate solution to the Lamm equation. This procedure was tested both with simulated noisy data and with experimental data obtained using ribonuclease, ovalbumin, and somatostatin.dodecylsulfate. The procedure assumes that both s and D are independent of concentration. The results suggest that optimal estimation of both s and s/D is obtained at values of (= 2D/(s.omega(2).r(a)(2))) in the range of 0.002 to 0.01 and values of tau(= 2 somega(2)t) less than 0.04. Appropriate selection of rotor speed allows the estimation of both s and s/D for nearly globular macromolecules in the range of 10(4) to 10(6) daltons with data obtained during the first 3000-5000 seconds of a sedimentation velocity experiment.

Physical and chemical properties of moloney murine leukemia virus p30 protein: A major core structural component exhibiting high helicity and self-association

The major internal structural protein of the Moloney murine leukemia oncornavirus is a single polypeptide chain of 28,300 molecular weight in dilute solution. Self-association into a multimeric form occurs at increasing concentrations. The molecule is composed of 50% α-helical regions and approximately 12% β-structure which is not altered by self-association. The near ultraviolet circular dichroism spectrum has been resolved into its aromatic and disulfide contributions and is consistent with quantitative amino acid analysis. The hydrodynamic frictional ratio indicates that the molecule is moderately asymmetric.

Tetanus toxin conformation

SummaryThe circular dichroic spectrum of highly purified tetanus toxin has been determined between 200–310 nm. A comparison of the ellipticity between 207–243 nm and of the rotational strengths of the major resolved bands between 200–250 nm with the corresponding values from proteins of known conformation indicates that tetanus toxin contains about 20% α-helix and 23% β-structure. Above 250 nm the resolved spectrum showed contributions from tryptophanyl, tyrosyl, and phenylalanyl groups. The rotational strengths of the major near ultraviolet circular dichroic bands were significantly higher in the toxin than in low molecular weight peptides containing aromatic residues. This indicates that tetanus toxin has a stable tertiary structure.