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Biomedical subjects

D Puett

Publications and source records attributed to D Puett.

At least 181 records · Page 10Linked to original sources
PubMed1976

Biotransformations of pituitary luteinizing hormone in serum and urine. I. Association with serum components.

In the effort to elucidate the nature of luteinizing hormone (LH) in the circulation, studies in adult male rats have been conducted using a highly purified and well-characterized tritiated and methylated ovine pituitary LH, a derivative which retains full biological activity. Following an intravenous injection of the radioactive hormone, serum chromatograms (molecular exclusion chromatography) are characterized by three radioactive components. The first one, a rapidly formed high molecular weight heterogeneous fraction, involves the non-covalent interaction of the hormone with one or more circulating proteins. These high molecular weight complexes are cleared rather slowly from the circulation, relative to the non-associated hormone. A second component co-chromatographs with control hormone and is rapidly cleared from the circulation. These two fractions retain full biological activity judged by their ability to stimulate testosterone production in Leydig cell suspensions. A third component is observed in the circulation about 15 minutes after injection and was shown to represent tritiated amino acids. The lag period for the appearance of the labeled amino acids in the circulation correlates with the kinetics of hepatic and renal uptake and subsequent lysosomal catabolism of [3H]methylated-LH. The high molecular weight fraction may represent a physiologically important circulatory storage form for LH by conferring a relatively long circulatory half-life. This appears to be brought about mainly by reduced urinary excretion, presumably due to its high molecular weight.

Animals↗
PubMed1976

Biotransformations of pituitary luteinizing hormone in serum and urine. II. Evidence for reduced potency following urinary excretion.

The renal clearance of tritiated and methylated ovine pituitary luteinizing hormone (LH) has been determined in mature male rats, and the urinary product has been characterized with regard to molecular size, charge, and heterogeneity. [3H]Methylated-LH was found to have a renal clearance of 0.17+/-0.03 ml/min/100 g body weight, compared to a value of 0.99+/-0.18 for tritiated inulin. This indicates that LH is not secreted and that much of the filtered hormone is reabsorbed from the tubular lumen. Evidence is presented which shows that urinary ovine [3H]methylated-LH is not extensively degraded; yet, the biological potency is only 10-20% that of pituitary and serum LH. The large reduction in potency is a result of excretion since the in vitro incubation of LH with urine does not produce the same effect. The biotransformation occurring during excretion results in charge heterogeneity of LH; this may arise from either inhibitor binding or enzymic alterations.

Animals↗
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

Tetanus toxin. The effect of chemical modifications on toxicity, immunogenicity, and conformation.

Tetanus toxin has been isolated from the extract of Clostridium tetani and analyzed for purity using various methods, e.g. sedimentation velocity, gel filtration, polyacrylamide gel electrophoresis, immunoelectrophoresis, and immunodiffusion. The homogeneous toxin, characterized by a minimum lethal dose of 10 pg (18- to 20-g mouse), was judged to be of high purity. The amino acid composition was determined and found to be in good agreement with reported values for both filtrate and extract toxin. The corrected sedimentation coefficient, so20,w, was found to be 7.5, and the molecular weight was estimated to be 150,000. These values agree closely with those reported by others. The toxin was modified using the conventional formaldehyde reaction to produce toxoid and both reductive methylation and carbamylation, which are highly specific for lysyl residues. Under certain reaction conditions, carbamylation of the toxin completely eliminated toxicity. Whereas reductive methylation yielded a high degree of conversion of lysine to dimethyllysine and monomethyllysine, the toxicity, albeit greatly reduced, was never completely eliminated. The circular dichroic spectrum of each chemically modified toxin was obtained, resolved into Gaussian components, and compared with that of native toxin, which is estimated to contain about 20% alpha helix and 23% beta structure. The far ultraviolet circular dichroic spectra of toxin and toxoid were characterized by negative extrema at 208 nm and 217 nm attributable to ordered secondary structure, and toxin also exhibited a distinct shoulder at 223 nm. Carbamylated toxin and methylated toxin were characterized by negative extrema at 210 nm and 206 nm, respectively, and both exhibited shoulders at 216 to 217 nm and 223 nm. The toxin and derivatives exhibited multiple negative extrema above 250 nm which were assigned to the various aromatic residues. There were differences in the spectra of the toxin and derivatives over the entire wavelength region, thus suggesting changes in the local environment of various chromophores. In particular, the rotational strengths of many of the bands assigned to tryptophan, tyrosine, and phenylalanine were altered in the derivatives. Also, in the far ultraviolet region of the circular dichroic spectrum, the data were suggestive of some reduction in the amount of both alpha helix and beta structure in the derivatives. However, there was no evidence of extensive conformational changes, e.g. unfolding, in the modified toxins. Presently, it is not known if the small conformational differences between toxin and toxoid are important in the loss of toxicity with the retention of immunogenicity in the derivative. The modification data are consistent with the hypothesis that separate amino acid residues are involved in toxicity and immunogenicity.

Amino Acids↗
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↗
PubMed1975

The metabolism of luteinizing hormone. Plasma clearance, urinary excretion, and tissue uptake.

The kinetics of plasma clearance, tissue uptake, and urinary excretion of tritiated ovine pituitary luteinizing hormone in adult male rats are reported. Most of the intravenously injected tritiated gonadotropin is cleared from circulation with a half-life of five minutes, and this is independent of the injected amount of hormone over a wide dose range. It was found that the hormone is rapidly removed from circulation by the kidneys, probably by glomerular filtration, and excreted in the urine. The radioactivity present in the urine is associated with material of the same molecular size as the native hormone and, moreover, the urinary hormone retains a significant amount of biological activity. A small amount of the hormone is catabolized by the kidney and liver, and our data suggest that this occurs in the cortex and hepatocytes, respectively.

Animals↗
PubMed1973

Collagen-mediated platelet aggregation. Effects of collagen modification involving the protein and carbohydrate moieties.

In an effort to elucidate the nature of the collagen-platelet interaction, the effects of collagen modification on platelet aggregation have been studied. We have shown that purified rat skin (salt) soluble collagen is effective at about 20 nM in mediating platelet aggregation in human platelet-rich plasma. This concentration is somewhat greater than that required of several skin insoluble collagens (ca. 10 nM). Both the alpha1(I) and alpha2 chains from rat skin soluble collagen produced platelet aggregation, but only at concentrations of about 13 muM and 55 muM, respectively. In contrast, heat-denatured collagen and chains (e.g., 65 muM alpha1(I) and 160 muM alpha2) failed to induce platelet aggregation and to inhibit platelet aggregation by native collagen. Glycopeptides were prepared from human skin insoluble collagen by extended digestion with bacterial collagenase and trypsin, and were purified by gel filtration into two classes. One class of higher molecular weight contained sialic acid, glucosamine, galactosamine, fucose, mannose, galactose, and glucose, and the other of lower molecular weight consisted primarily of a mixture of galactose and galactosyl-glucose units O-glycosidically linked to hydroxylysine-containing peptides. We found that, after the residual tryptic activity contaminating the higher molecular weight fraction was inhibited, neither of the glycopeptide classes produced nor inhibited native human skin insoluble collagen-mediated platelet aggregation at the highest concentration examined (ca. 1-2 mg glycopeptide per ml of platelet-rich plasma). Highly purified samples of the hydroxylysyl glycosides, hydroxylysylgalactose and hydroxylysylgalactosylglucose (Hyl-Gal and Hyl-Gal-Glc, respectively), were prepared from human urine and labeled at galactose using galactose oxidase followed by reduction with tritiated borohydride. Binding studies with platelet-rich plasma showed that, at concentrations greater than 50 nM, Hyl-Gal gives apparent binding to platelets, but there was no evidence of Hyl-Gal-Glc binding to platelets at concentrations up to 250 nM. At concentrations several hundredfold higher than the equivalents present in the minimum concentration of rat skin soluble collagen required for platelet aggregation, neither Hyl-Gal (at 29 muM) nor Hyl-Gal-Glc (at 18 muM) caused platelet aggregation or inhibited platelet aggregation by native collagen. Also, at a concentration of 85 muM (which represents a concentration about two thousandfold higher than the equivalents in the minimum concentration of soluble collagen required for platelet aggregation) the Gal-Glc-containing 36 residue rat skin soluble collagen alpha1(I)cyanogen bromide #5 peptide had no platelet aggregating or inhibiting activity. Modification of at least 90% of the rat skin soluble collagen carbohydrate by mild periodate oxidation had no effect on the platelet aggregating activity. Human skin insoluble collagen was reacted with periodate under the same conditions, and this had no demonstrable effect on its ability to induce platelet aggregation. This indicates that the normal carbohydrate side chains of these collagens are not required for the platelet interaction that produces the release of ADP and other metabolic constituents and leads to aggregation.Thus, collagen-platelet interactions appear to involve at least two distinct binding sites on the platelet plasma membrane. One is a protein binding site that activates platelet aggregation and has high specificity and affinity for the collagen triple-helical fold or perhaps even for a particular amino acid sequence in the triple helix.

Amino Acids↗