Biomedical subjects
Publications and source records attributed to K E Ebner.
The binding of cyclosporin A and ovine prolactin to rabbit mammary gland membranes was determined. CsA bound with a Kd of 2.2 X 10(-6)M whereas prolactin bound with a Kd of 2 X 10(-10)M. The binding of each ligand was an independent event and neither ligand influenced the binding of the other ligand showing that CsA does not inhibit the binding of prolactin to its specific receptor in this system.
The Cuisinart Model DLC-7 PRO food processor efficiently disaggregates plant and animal tissues resulting in time savings and often increased yields in the isolated material.
The disulfide bonds of two lactogenic hormones, ovine prolactin (oPRL) and human growth hormone (hGH), were reduced with dithiothreitol under denaturing conditions and alkylated with iodoacetic acid. The modified hormones were assayed for their ability to bind the plasma membrane-bound receptor for lactogenic hormone found in the rabbit mammary gland. S-Carboxymethylated ovine prolactin (SCM-oPRL) with all six cysteine residues modified had a nearly 300-fold decrease in binding as compared to native oPRL in a competitive binding assay using [125I]ovine prolactin. The S-carboxymethylated human growth hormone (SCM-hGH) had all four of its cysteine residues modified. It showed only a slightly reduced ability to bind the rabbit mammary gland prolactin receptor in a competitive binding assay with [125I]ovine prolactin. The two modified hormones were assayed for their ability to stimulate proliferation of the lactogen-dependent Nb 2 lymphoma cell line. SCM-oPRL required concentrations greater than 1 X 10(5) that of native oPRL to stimulate 50% of the maximum cell growth. SCM-hGH retained a significant amount of its ability to stimulate the Nb 2 lymphoma cells.
Membrane-bound prolactin receptors from the mammary gland of 6-7-day postpartum lactating rabbits were solubilized using the zwitterionic detergent, Zwittergent 3-12 (3- dodecyldimethylammonio )-1-propanesulfonate). The solubilized receptor from one rabbit was bound to an ovine prolactin-agarose affinity gel and eluted at pH 4.2. The receptor appeared as a single band upon staining sodium dodecyl sulfate-polyacrylamide gels with Coomassie blue. The protein yield from one rabbit was about 8 micrograms and the overall yield of receptor was over 50%. The apparent molecular weight was 42,000 on sodium dodecyl sulfate gels but varied on molecular weight columns due to the type of detergent. Receptor inactivated by iodination had an apparent molecular weight of 21,000 on sodium dodecyl sulfate gels. The purified receptor did not bind to concanavalin A-agarose or Lens culinaris-agarose.
The reversible unfolding of rat alpha-lactalbumin, which, in contrast to other alpha-lactalbumins, has a 17-amino-acid extension at the carboxyl terminus and a carbohydrate unit at Asn-45, was studied by circular dichroism between 193 and 310 nm as a function of pH, heat and guanidine hydrochloride ( GdnHCl ). The native structure of rat alpha-lactalbumin was similar to that of bovine alpha-lactalbumin. Acidification changes rat alpha-lactalbumin to a state similar to the 'A state' of bovine alpha- lactalbumin . The heat-reduced unfolding of the tertiary structure of rat alpha-lactalbumin is highly cooperative. The GdnHCl -induced unfolding of rat alpha-lactalbumin could not be expressed by a two-state mechanism as in the case of bovine alpha-lactalbumin. However, the midpoints of transitions suggested lower stabilities of secondary and tertiary structures in rat alpha-lactalbumin than in bovine alpha-lactalbumin consistent with the differences in the local structure between rat and bovine alpha-lactalbumins.
A pictorial map of the lactose synthase (galactosyl transferase) acceptor binding site has been formulated from this and published studies on substrate analogs and inhibitors. The basic requirements are a pyranose, thiopyranose or inositol ring structure and equatorial substituents (if any) at C-2, C-3, C-4, and C-5. The aglycone (at C-1) may be either alpha or beta-, but alpha- is somewhat preferred. In the absence of alpha-lactalbumin galactosyl transferase will accept long chain 2-N-acyl substituents on the glucosamine (GlcNH2) structure. An equatorial amino or N-acetyl substituent (e.g. mannosamine, N-acetylmannosamine) is also a suitable acceptor in the absence of alpha-lactalbumin since both N-acetylglucosamine and N-acetylmannosamine have complementary binding loci for the N-acyl moiety. The aglycone moiety must be equatorial (beta-configuration). However, upon alpha-lactalbumin binding the aglycone specificity allows for axial (alpha-configuration) as well as equatorial substituents. Furthermore, the 2-N-acyl substituent binding locus is blocked beyond a 2-N-hexanoyl group. It is suggested that alpha-lactalbumin binds to a hydrophobic site some distance from the C-2 group.
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Ovine prolactin specifically bound to rabbit mammary membrane prolactin receptor was rapidly dissociated in a pH dependent manner with 0.1M ammonium acetate. Up to 75% of the bound hormone was dissociated at pH 4.0 or lower in less than 5 minutes. The pK of the dissociation was 4.7, implicating one or more critical carboxyl groups. Exposure of the membrane bound receptor to the dissociating buffer for up to one hour did not reduce its ability to bind hormone. The dissociated hormone was characterized as intact ovine prolactin by Bio-Gel P-150 gel chromatography and by its ability to bind to fresh rabbit prolactin receptor with the same binding affinity as native hormone.
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The amino acid sequence of rat alpha-lactalbumin has been determined. Unlike other alpha-lactalbumins which contain 122 or 123 amino acids, rat alpha-lactalbumin is unique in that it contains 140 amino acids. The extra amino acids are a 17 amino acid extension at the carboxyl terminus. The amino acid sequence of this extension is Gly124-Ala-Pro-Ala-Leu-Val-Val130-Pro-Ala-Leu-Asp-Gly135-Glu-Thr-Pro-Val-Pro140 . The extension is proline rich, which may contribute to the anomalous structural properties of rat alpha-lactalbumin. The amino acid sequence from residues 1 to 123 is similar to that of other alpha-lactalbumins. One possible explanation for the 17 amino acid extension is a mutation at the termination codon.
The interactions of Mn2+, nucleotides, monosaccharide substrates, and the modifier protein alpha-lactalbumin with galactosyltransferase have been studied by difference spectroscopy. MnCl2, required for significant binding of UDP-galactose or UDP to the enzyme, does not exhibit specific interactions with tyrosine or tryptophan residues. The interaction of UDP-galactose or UDP with galactosyltransferase in 2 mM MnCl2 produces difference spectra with a major positive peak at 284 nm, a second positive peak at 298 nm, and a large negative trough at 254 nm, suggesting the involvement of tyrosine and tryptophan residues in the interaction. The interaction of GlcNAc with the enzyme in 2 mM MnCl2 produces only small nonspecific difference spectra. However, the addition of 100 mM GlcNAc markedly increases the difference extinction coefficient at 284 nm of the UDP-bound enzyme-ligand complex, while the coefficient at 254 nm which arises from UDP remains constant. The results suggest that a conformational change involving tyrosine residues, which does not affect UDP, occurs in the process, enzyme.Mn + UDP + GlcNAc leads to enzyme.Mn.UDP.GlcNAc. Glucose does not show a similar effect. The interaction of galactosyltransferase and alpha-lactalbumin produces difference spectra characteristic of tryptophan and does not affect the difference spectra of galactosyltransferase produced by the interaction with UDP and GlcNAc. This implies that the interaction of the two proteins does not involve the bound UDP on galactosyltransferase and does not affect the conformational change induced by UDP and GlcNAc.
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Iodine is readily incorporated from ICl into galactosyltransferase with total loss of enzymatic activity. The extent of modification was determined by separation of 125I-labeled proteins on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Tyrosines are the only amino acids modified by iodination of galactosyltransferase, a maximum of five, and alpha-lactalbumin, a maximum of three. The major product of the iodination was 3,5-diiodo-L-tyrosine and the remainder was 3-monoiodo-L-tyrosine. Inactivation of galactosyltransferase was dependent on the degree of modification. Substrates of galactosyltransferase are capable of partial protection against enzymatic inactivation. alpha-Lactalbumin, by itself, was also capable of protecting galactosyltransferase against inactivation. This represents evidence for a specific interaction of galactosyltransferase with alpha-lactalbumin in the absence of carbohydrate. The protection of galactosyltransferase by alpha-lactalbumin was enhanced by the presence of the substrates, Mn2+, N-acetylglucosamine, and UDP. Under conditions of either substrate protection or in the absence of substrates, the inactivation reaction of galactosyltransferase by ICl is apparent third order, apparent first order in galactosyltransferase, and apparent second order in galactosyltransferase, and apparent second order in ICl. The apparent order, with respect to ICl, suggests the involvement of 2 mol of ICl either both at one site or one each at two different sites.
Galactosyltransferase (UDPgalactose:D-glucose 4-beta-D-galactosyltransferase, EC 2.4.1.22) was totally inactivated by iodination with lactoperoxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7). Substrates protected against inactivation. The presence of 10 mM Mn2+ and 1 mM UDPgalactose gave partial protection which was enhanced by the addition of 10 mM N-acetylglucosamine, but not by glucose. These results are consistent with a conformational change upon binding of UDPgalactose. Only monoiodotyrosine and diiodotyrosine were identified in the pronase digest of iodinated galactosyltransferase. Galactosyltransferase was also inactivated with N-acetylimidazole and partial activity was restored by treating acetylated galactosyltransferase with hydroxylamine. These results suggest that tyrosine(s) is essential for galactosyltransferase activity.
alpha-Lactalbumin was purified from the milk of Wistar rats and was compared to that obtained from Fisher 344 rats. alpha-Lactalbumin isolated from the Wistar rat exists as two forms which differ in their sialic acid content, as the desialyated forms migrate to one identical position on polyacrylamide gels. These two charge forms are identical with the charge Forms II and III previously characterized from Fisher rat alpha-lactalbumin. No evidence was found to verify previous reports that one form of the Wistar rat alpha-lactalbumin had a higher molecular weight than the other form. Indeed, the molecular weights and the amino acid compositions of the two forms of Wistar rat alpha-lactalbumin are identical. In addition, the partial amino acid sequence at the NH2-terminal end and the amino acid composition of the COOH-terminal cyanogen bromide peptide of the two forms are identical. The results in this study contradict those reported previously and show that rat alpha-lactalbumin exists as a single molecular weight species.
Rat alpha-lactalbumin is unique in contrast to alpha-lactalbumin isolated from other species in that it exists in three charge forms. Each form contains carbohydrate and is active in the lactose synthetase reaction. Form II comprises about 80% of the total alpha-lactalbumin and contains a single heteropolysaccharide unit which is attached to the polypeptide chain at Asn45. The detailed structure of this unit was ascertained using specific exoglycosidases, endoglycosidases, and methylation analysis. The following structure is proposed for the heteropolysaccharide unit: (formula: see text).
alpha-Lactalbumin has been purified from mouse milk by the use of ammonium sulfate precipitation, Bio-Gel P-100 chromatography and DEAE-cellulose chromatography. Mouse alpha-lactalbumin exists as two major charge forms with the same molecular weight. Both charge forms have been used in the development of a radioimmunoassay to mouse alpha-lactalbumin. The assay is specific for mouse alpha-lactalbumin, reacting identically with both forms, and sensitive with a minimal detectable concentration of 0.35 ng. alpha-Lactalbumin induced in mouse mammary gland explants has been measured with the use of the radioimmunoassay and is detectable in both the explants and media of the cultures.
Three charge forms of rat alpha-lactalbumin were separated by ion exchange chromatography on DEAE-cellulose. The amino acid composition of each form was similar but they differed in carbohydrate composition. Each form contained a tryptic glycopeptide having a common polypeptide and heteropolysaccharide unit. The tryptic glycopeptide was sequenced and positioned in rat alpha-lactalbumin, which was partially sequenced from residues 1 to 50. The carbohydrate attachment site was at Asn45. Secondary structure calculations predicted that Asn45 is in a beta bend conformation whereas Asn45 in bovine alpha-lactalbumin, a poorly glycosylated protein, is not in a bend conformation.