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L A Holladay

Publications and source records attributed to L A Holladay.

At least 37 records · Page 2Linked to original sources
PubMed1977

Circular dichroism of human urinary Tamm-Horsfall glycoprotein.

Human urinary Tamm-Horsfall glycoprotein, which contains 28% carbohydrate, has a monomeric molecular weight of about 80,000 but is isolated from urine in the form of intertwining helical suprastructures with molecular weights greater than 10(7). The native glycoprotein was dissociated and denatured with 6 M guanidinium chloride and was subsequently renatured by dialysis against a Tris-HCl buffer. Using sedimetation equilibrium, the renatured glycoprotein was characterized by a Mw cell of 256,800 and a Mz cell of 356,000. The ratio, Mz/Mw, of 1.39 indicates some polydispersity with regard to molecular size. There was no evidence of helical suprastructures in the renatured glycoprotein as judged by electron microscopy. Ca2+ concentrations of up to 50 mM failed to precipitate the renatured glycoprotein; in contrast, the native glycoprotein is precipitated by Ca2+ concentrations between 5-10 mM. The circular dichroic spectrum of renatured Tamm-Horsfall glycoprotein was obtained, resolved, and tentative band assignments made. The spectrum, which is quite similar to that of native Tamm-Horsfall glycoprotein, exhibited negative extrema at 269 nm (due in large part to disulfides and tyrosines) and at 215 nm (due to protein beta-structure and the N-acetylated hexosamines). The alpha-helical content of the glycoprotein was estimated to be no more than 10% and the amount of beta-structure to be about 33%; these values were not affected by the presence of Ca2+ (1 mM). A glcopeptide fraction (ca. 90% carbohydrate), prepared by extensive pronase digestion of the reduced, S-carboxymethylated glycoprotein, exhibited an ellipticity extremum at 212 nm of + 4,750 deg-cm2/dmole, referred to the concentration of (N-acetylated) hexosamines and neuraminic acid.

Carbohydrates↗
PubMed1977

Circular dichroism of glycopeptide fractions from alpha1-acid glycoprotein, thyroglobulin, and ovalbumin.

Using sequential pronase digestions, glycopeptide fractions were prepared from human alpha1-acid glycoprotein, hen egg ovalbumin, and bovine thyroglobulin, two types of glycopeptides being obtained from the latter. The fractions were characterized on the basis of hexose, hexosamine, sialic acid, and peptide content. The glycopeptide fraction from alpha1-acid glycoprotein is complex (i.e., the carbohydrate moiety contains mannose, galactose, N-acetylglucosamine, and sailic acid), as is one of the glycopeptide fractions from thyroglobulin (type I). The glycopeptide fraction from ovalbumin and the type II glycopeptide fractions from thyroglobulin are simple (i.e., the carbohydrate moiety contains only mannose and N-acetylglucosamine). The circular dichroic spectra of the two complex glycopeptide fractions and the ovalbumin glycopeptide fraction were similar and were characterized by a negative extremum between 207.5 nm and 211 nm with magnitudes in the range of --6400 deg-cm2-dmol-1 to --7200 deg-cm2-dmol-1 (referred to the molar concentration of N-acetylated sugars). The thyroglubulin type II glycopeptide fraction exhibited a circular dichroic spectrum with an extremum of --29 200 deg-cm2-dmol-1 at 205 nm. Removal of sialic acid from the complex glycopeptide fractions greatly increased the (negative) magnitude of ellipticity at the extremum. The circular dichroic spectra of the complex of glycopeptide fractions were reasonably additive using the spectra of monomeric sialic acid and the asialo-derivatives. This demonstrates that the contributions of sialic acid to the circular dichroic spectrum are nearly additive. The implications of this observation are that covalent attachment of these terminal residues to the oligosaccharides does not lead to strong interactions with other chromophores nor to positioning in particularly asymmetric environment. In contrast, the magnitudes of the observed ellipticity extrema in the circular dichroic spectra of the asialo-derivatives, in which N-acetylglucosamine is the major chromophore, are much greater than can be accounted for on the basis of monomeric contributions (i.e., free N-acetylglucosamine). This finding shows that the optical activity of N-acetyl-glucosamine is greatly influenced by the formation of the carbohydrate core in glycoproteins and suggests the possible formation of secondary structure in the carbohydrate moiety.

Amino Acids↗
PubMed1977

Circular dichroism of insulin A chain in water: trifluoroethanol mixtures. Use of linear and nonlinear least squares analysis.

Circular dichroic spectra obtained for S-carboxymethylated insulin A chain in water: trifluoroethanol mixtures show that a marked conformational transition occurs as the concentration of trifluoroethanol is increased to (v/v) 83% to give a polypeptide containing about 43% of the residues in the alph-helical conformation. Several proposed methods of analysis, including both linear and two nonlinear lest squares methods, were unable to quantitate the amount of beta-structure present in the polypeptide in 83% trifluoroethanol. Examination of the methods of analysis lead to the conclusion that the current models for far UV CD analysis are not adequare for the data obtained in this study and shows that nonlinear least squares procedures may lead to erroneous conclusions.

Circular Dichroism↗
PubMed1977

The effects of denaturant and temperature on the circular dichroic spectrum of ovine lutropin-alpha.

Circular dichroic (CD) spectra of the glycoprotein hormone subunit, lutropin-alpha, have been obtained under denaturing conditions (6 M guanidinium chloride, GdmCl) with the disulfides intact, reduced, and reduced and S-carboxymethylated. Above 260 nm, these spectra are similar (although not identical), showing that the disulfide chromophores do not contribute appreciably to the near ultraviolet CD spectrum under denaturing conditions. However, these spectra differ considerably from that of native lutropin-alpha over this spectral region demonstrating that the tertiary structure of lutropin-alpha has a major influence on the near ultraviolet CD spectrum. The magnitude of the 232.5 nm positive CD extremum of lutropin-alpha was found to increase with increasing GdmCl concentration and with decreasing temperature. Between 5 degrees C and 45 degrees C, the ellipticity [theta] at 232.5 nm changed in a linear fashion with temperature; at other wavelengths the thermal changes in [theta] were quite small. [theta] at 232.5 nm increased by about 80% over a 3 M GdmCl range; in contrast, [theta] at 216 nm (indicative of beta-structure) exhibited maximal changes over a 0.5 M GdmCl range. These results indicate some conformational flexibility in lutropin-alpha. The positive 232.5 nm CD extremum has been assigned to one or more tyrosyls which are influenced by an intact disulfide. The latter was demonstrated by showing that reduced and S-carboxymethylated lutropin-alpha failed to exhibit the positive extremum.

Animals↗
PubMed1977

Comparative conformational analysis of human choriomammotropin and somatotropin from several species.

The conformations of porcine somatotropin and human choriomammotropin have been studied using circular dichroism (CD) and the results compared with spectra of human, murine, ovine, and bovine somatotropin. The far ultraviolet CD spectra of the six proteins were similar, and each spectrum was analyzed using constrained linear least squares. The following average percentages of alpha-helicity, beta-structure, and aperiodic (nonhelical) conformation were obtained: 57, 6, and 37, respectively, based on a standard protein reference set, and 42, 22, and 36, respectively, based on poly-L-lysine as reference. Thus, the estimated secondary structure is strongly dependent upon the reference data used. Interestingly for these similar proteins, it appears that over 60% of the residues are part of ordered secondary structure and less that 40% are in an aperiodic conformation. The near ultraviolet CD spectra of these hormones were similar in many respects, although certain significant differences were observed, particularly in the sign of various extrema. These spectral differences probably reflect non-identical microenvironments of the aromatics and disulfides, arising from differences both in amino acid sequence and local conformation.

Amino Acid Sequence↗
PubMed1977

Tissue-specific stimulation of ornithine decarboxylase activity by pituitary factors immunologically related to growth hormone.

Ornithine decarboxylase (L-ornithine carboxylase, EC 4.1.1.17) is an important enzyme in polyamine synthesis. Its activity is influenced by several peptides hormones, including growth hormones, which have physiological significance in various growth situations. A crude ovine pituitary growth hormone preparation (NIH-GH-S10) was subjected to gel exclusion chromatography (Sephadex G-100) and two major fractions were obtained. One of these corresponded to dimeric growth hormone (GH). The other fraction was excluded by the gel matrix, suggesting a material of higher molecular weight than GH. This was confirmed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Analysis of a high molecular weight fraction by radioimmunoassay (antisera prepared against GH) and by bioassay (weight gain in hypophysectomized rats) gave apparent GH contents of 19% and 6%, respectively. On a weight basis, the high molecular weight fraction was more effective than GH in stimulating the activity of hepatic and adrenal ornithine decarboxylase, but GH was more effective in stimulating renal ornithine decarboxylase activity. Subfractionation of the high molecular weight fraction using a high porosity gel (Sephadex G-200) gave four fractions, which were shown by amino acid analysis and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate to be distinct from GH and heterogenous. These subfractions had different potencies for stimulating renal and hepatic ornithine decarboxylase activity. The ability of crude growth hormone preparations to stimulate ornithine decarboxylase activity in some tissues may be a function of pituitary factors, in addition to GH, which have minimal growth promoting activity.

Adrenal Glands↗
PubMed1976

Conformation and unfolding thermodynamics of epidermal growth factor and derivatives.

Mouse submaxillary epidermal growth factor (EGF) is a 53-residue single chain peptide hormone of known amino acid sequence which contains three disulfides, five tyrosines, and two tryptophans. Circular dichroic (CD) spectra have been obtained and resolved for EGF, several well-characterized chemical and enzymic derivatives, and related low molecular weight model compounds. Assignments have been made to most of the resolved bands; these include the peptide, aromatic, and disulfide chromophores. From a comparison of the rotational strength of the 213-nm resolved CD band in native EGF with that of standard proteins, EGF is estimated to contain about 22% beta structure and no alpha helicity. A derivative of EGF lacking the five carboxyl-terminal residues (prepared by limited trypsin digestion) and the cyanogen bromide derivative, in which there is a single main-chain cleavage at residue 21, have spectra properties indicative of approximately 10 and 12% beta structure, respectively. The near-ultraviolet CD spectra of the derivatives are similar to, albeit not identical with, that of EGF. The rotational strengths characteristic of the side-chain chromophores in EGF and these derivatives are several-fold higher than the corresponding values in low molecular weight model compounds. Thus, it appears that EGF and these modified forms contain a stable (and similar) tertiary structure. In contrast, the S-aminoethylated derivative of EGF exhibits a drastically altered CD spectrum relative to EGF indicating a different conformation(s). Equilibrium studies on the guanidinium hydrochloride (GdmCl) mediated reversible unfolding of EGF showed that the transition midpoint is quite high (i.e., 6.89 M GdmCl at 25.0 degrees C), thus, indicating considerable stability. From these data a rough estimate of 16 kcal/mol can be made for the unfolding free energy (delta G degrees) of EGF in the absence of denaturant. Interestingly, EGF exhibits greater stability characteristics than several proteins two to four times its size. The cyanogen bromide derivative of EGF exhibited greatly reduced stability characteristics, e.g., the transition midpoint occurred at 4.19 M GdmCl (25.0 degrees C) and delta G degrees was estimated to be approximately 4 kcal/mol. Thus, a single main-chain cleavage reduced the stability of EGF by about 70%. Thermal transitions of EGF and the cyanogen bromide derivative in the presence of concentrated GdmCl are characterized by a relatively high enthalpy of about 25 kcal/mol at 40 degrees C and a low (probably zero) heat capacity. From these thermodynamic parameters one can calculate that the large reduction in delta G degrees due to scission of the single peptide bond between residues 21 and 22 can be attributed almost completely to a change in entropy; e.g., at 40 degrees C the apparent entropy of unfolding of EGF is 20.4 cal mol-1 deg-1 while that of the cyanogen bromide derivative is 66.4 cal mol-1 deg-1.

Amino Acid Sequence↗
PubMed1976

Purification and characterization of the catalytic subunit of adenosine 3':5'-cyclic monophosphate-dependent protein kinase from bovine liver.

1. The catalytic subunit of bovine liver cyclic AMP-dependent protein kinase (EC2.7.1.37) was purified essentially by the method of Reimann & Corbin [(1976) Fed. Proc. Fed. Am. Soc. Exp. Biol. 35, 1384]. 2. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, sedimentation-velocity centrifugation and sedimentation-equilibrium centrifugation showed that the catalytic subunit was monodisperse. Polyacrylamide-gel isoelectric-focusing electrophoresis revealed the presence of at least three isoenzyme forms of catalytic subunit activity with slightly different pI values (6.72, 7.04 and 7.35). 3. Physical properties of the catalytic subunit were determined by several different methods. It had mol.wt. 39000-42000, Stokes radium 2.73-3.08 nm, so20.w 3.14S, f/fo 1.19-1.23 and, assuming a prolate ellipsoid, axial ration 4-5. 4. Amino acid analysis was performed on the catalytic subunit. It had one cysteine residue/molecule which was essential for activity. Inhibition by thiol-specific reagents was partially prevented by the presence of ATP-Mg2+. 5. The circular-dichroic spectrum showed the catalytic subunit contained 29% alpha-helical form, 18% beta-form and 53% aperiodic form. Near-u.v. circular dichroism showed the presence of aromatic residues whose equivalent molar ellipticity was greatly altered by the addition of ATP-Mg2+. 6. Kinetic experiments showed that the catalytic subunit had an apparent Km for ATP of 7 muM. 5'-Adenylyl imidodiphosphate inhibitied competitively with ATP with a Ki of 60 muM. The kinetic plot for histone (Sigma, type II-A) was biphasic showing 'high'-and 'low'-Km segments. Under assay conditions the specific activity of the catalytic subunit was 3 X 10(6) units/mg of protein. Of various metal ions tested, the catalytic subunit was most active with Mg2+.7. When assayed with histone (Sigma, type II-A) as substrate, the activity of the catalytic subunit was increased by non-ionic detergents or urea. No such activation was observed with casein as substrate.

Adenosine Triphosphate↗
PubMed1976

Somatostatin conformation: evidence for a stable intramolecular structure from circular dichroism, diffusion, and sedimentation equilibrium.

Somatostatin is a hypothalamic tetradeca peptide that inhibits the release of growth hormone insulin, and glucagon. The circular dichroism spectrum is characterized by negative extrema at 238 nm and 270 nm, and a positive extremum at 225 nm. The far ultraviolet circular dichroism spectrum is consistent with the presence of ordered secondary structure such as beta-structure, but not alpha-helix. Sedimentation equilibrium results demonstrate that somatostatin exists in its monomeric form (i.e., a molecular weight of 1610 +/- 36 was obtained) and, thus, the structure must arise from intramolecular interactions. The diffusion constant of somatostatin was estimated to be 1.66 X 10(-6) cm2/sec. These data are consistent with an ellipsoidal rather than a spherical shape. The magnitude of the ellipticity at both 225 nm and 238 nm is quite dependent on guanidinium hydrochloride concentration; the midpoint occurs at about 3 M and the transition is cooperative-like. These data strongly suggest that somatostatin has a stable conformation in aqueous solution. A model, consistent with the results of the physicochemical studies and with semi-empirical rules for secondary structure formation, is proposed for somatostatin. The proposed structure consists of a hairpin loop with several residues in an antiparallel beta-pleated sheet, is somewhat elongated, and contains a hydrophobic domain at one end and a hydrophilic domain at the other end.

Circular Dichroism↗
PubMed1976

Circular dichroism of human pituitary luteinizing hormone and its glycopeptides. Curve resolution and band assignments to the peptide chromophore, aromatic residues, disulfides, and N-acetylated amino sugars.

The circular dichroic (CS) spectrum of the glycoprotein hormone, human pituitary luteinizing hormone (hLH), has been determined between 195-320 nm and resolved into gaussian constituents. Below 230 nm the CD spectrum is characterized by a negative extremum at 207 nm with a shoulder at 217 nm. Resolution into gaussian constituents of the 200-230 nm CD spectrum resulted in two resolved negative bands, one at 206 nm and the other at 215 nm. The latter band is assigned to beta-structure which is estimated to be about 25%. The 206 nm resolved band is assigned to the N-acetylated carbohydrate groups (e.g. N-acetyl glucosamine, galactosamine, and neuraminic acid). This is based partly on the evidence that the CD spectrum of the hLH glycopeptide fraction (prepared by a pronase digestion of s-carboxymethylated hLH) exhibited a negative extremum at 207.5 nm, which is close to the resolved 206 nm band in hLH. Above 230 nm the CD spectrum is characterized by a negative extremum at about 275 nm. Most of the ellipticity in this region is attributed to the disulfides in hLH. Both strong acid (0.1 N hcl) and concentrated guanidine hydrochloride (4 M) affect the ellipticity in the vicinity of 275 nm, but only the latter (as well as concentrated urea) has a major effect on the CD spectrum below 230 nm indicating extensive conformational changes. There is, however, some loss of beta-structure in 0.1 N hcl. Thus, it appears that the conformation of the hLH subunits in these subunit-dissociating agents is rather different. There was no dramatic change in the magnitude of the 207 nm extremum of native hLH between 10-50C.

Acetylation↗
PubMed1975

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.

Amino Acid Sequence↗