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D N Luck

Publications and source records attributed to D N Luck.

12 recordsLinked to original sources
PubMed1992

Analysis of disulphide bridge function in recombinant bovine prolactin using site-specific mutagenesis and renaturation under mild alkaline conditions: a crucial role for the central disulphide bridge in the mitogenic activity of the hormone.

We have previously described a method for isolating Escherichia coli-produced methionyl bovine prolactin (Met-bPRL) and its renaturation using thioredoxin. This report describes an alternative renaturation procedure in which extracted Met-bPRL is incubated in air at pH 10 and 20 degrees C. Within 1 h of such treatment essentially all of the reduced Met-bPRL was converted to the oxidized form; this was accompanied by an increase to full mitogenic activity in the Nb2 cell bioassay. It was also found that, to minimize contamination by high mol. wt Met-bPRL derivatives, it is essential to have a reducing agent (dithiothreitol) present during disruption of the bacteria and to extract the protein at neutral pH. The contribution of each of the three disulphide bridges in bPRL to its bioactivity was studied with Met-bPRL variants, prepared via site-specific mutagenesis, in which cysteines were replaced by serines to prevent disulphide bond formation. Variants lacking the C4-C11 bridge, the C191-C199 bridge or both these terminal bridges were as mitogenic as authentic bPRL. (Variants lacking the C191-C199 bridge had markedly increased solubility in the presence of deoxycholate.) In contrast, variants lacking the C58-C174 bridge had greatly reduced bioactivity, indicating that integrity of the large disulphide loop is crucial to the hormone's mitogenic activity.

Animals↗
PubMed1992

Epitope mapping of monoclonal antibodies to bovine prolactin.

The epitopes recognized by three monoclonal antibodies generated to sheep prolactin were determined by evaluating their cross-reactivities by immunodot analysis with 14 mutants of bovine prolactin, in which individual amino acids had been deleted or substituted. Mutations were made throughout the molecule and included disruption of the amino-terminal, carboxyl-terminal, and central disulfide loops. Lack of immunoreactivity was taken as an indication that the site of mutation was part of the epitope. Antibody 6F11 reacted with all bovine prolactin mutants tested, except those in which the carboxyl-terminal cysteine (position 199) was substituted by a serine. Antibodies 5G2 and 4C10 reacted with all of the bovine prolactin mutants, except those in which the amino-terminal cysteine (position 4) was substituted by a serine. Western blot analysis of sheep, squirrel monkey, and rat prolactins with the monoclonal antibodies revealed that 5G2 and 4C10 were specific for sheep prolactin, whereas antibody 6F11 cross-reacted with prolactins from all three species. The mitogenic activity of sheep or rat prolactin in the Nb2 bioassay was determined in the presence of the antibodies to determine whether the epitopes were part of the functional domains of these prolactins. The bioactivity of sheep prolactin (0.4 ng/ml) was unaffected by the monoclonal antibodies [0.01-1 microgram immunoglobulin G (IgG)/ml], whereas the bioactivity of rat prolactin (1.25 ng/ml) was inhibited by 6F11 with an apparent 50% inhibitory concentration of 0.25 microgram IgG/ml. These results indicate that monoclonal antibodies 5G2 and 4C10 cross-react with a species-specific region of the amino-terminal disulfide loop of bovine prolactin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Single amino acid substitutions in recombinant bovine prolactin that markedly reduce its mitogenic activity in Nb2 cell cultures.

Three amino acid residues of bovine PRL (bPRL) have been examined for their roles in the mitogenic activity of the hormone in Nb2 lymphoma cell cultures. The residues of interest, R21, R177, and K187, are conserved in eight pituitary PRLs, but not in the related, nonlactogenic bGH. Using site-specific mutagenesis, a number of recombinant methionyl bPRL variants have been prepared, each of which contained a single amino acid substitution of one of the three residues; a variety of amino acids was used for substitution. Twelve exchanges of R177 (to A, L, N, K, D, E, Y, G, S, Q, H, and F) all led to marked decreases in mitogenic activity. Even the conservative change, R177K, led to a decrease in mitogenic activity of about 90%; all the other R177 substitutions led to even more marked decreases; there was essentially complete loss of activity when the positively charged R177 was replaced by the negatively charged aspartate. Exchanges of R21 (to A, L, N, and K) were less dramatic, with the greatest decrease (79%) occurring in the case of R21A. Exchanges of K187 (to A, L, N, and R) had a relatively minor effect on the mitogenic activity of the hormone. Residues R21 and R177 in bPRL are located in putative helices 1 and 4, respectively; in the three-dimensional structure of the hormone these residues are predicted to be quite closely apposed. The results suggest that R177 and, to a lesser degree, R21 have important roles in the mitogenic activity of bPRL.

Amino Acid Sequence↗
PubMed1990

Recombinant methionyl bovine prolactin: loss of bioactivity after single amino acid deletions from putative helical regions.

We have previously described a method for producing recombinant methionyl bovine PRL (Met-bPRL), which is as bioactive as the authentic hormone in the Nb2 cell lactogen bioassay; in contrast, a Met-bPRL variant lacking tyrosine 28 was essentially devoid of bioactivity. In the present study we have investigated this loss of bioactivity at the molecular level by determining the bioactivities of a number of Met-bPRL variants engineered to contain specific changes in their primary structures. It was found that the presence of tyrosine per se at the 28 position in Met-bPRL was not essential for high bioactivity, since Met-bPRL variants prepared by replacing tyrosine 28 with other amino acids (arginine, phenylalanine, alanine, and histidine) still had substantial bioactivity (40-74% that of Met-bPRL). Neither was the loss of bioactivity related to a shift in the relative positions of conserved histidines 27 and 30; in fact, histidine 27 was found not to be essential for the bioactivity of the hormone. The loss of bioactivity after deletion of tyrosine 28 from Met-bPRL appears to be related to the removal of an amino acid from the middle of a putative helix (no. 1) rather than to the loss of a residue specific to lactogen function. This suggestion is supported by the finding that Met-bPRL variants obtained by deletion of selected single amino acids from center domains of putative helix 2, 3, or 4 were also essentially devoid of bioactivity. It is speculated that this lack of bioactivity reflects an inability of the proteins to assume a native conformation.

Amino Acid Sequence↗
PubMed1989

Bioactive recombinant methionyl bovine prolactin: structure-function studies using site-specific mutagenesis.

A method has been developed for the extraction from transformed Escherichia coli cells of methionyl bovine PRL (met-bPRL) in a relatively pure form. While the extracted met-bPRL was as reactive as the native hormone with respect to polyclonal anti-bPRL antibodies, its bioactivity, as measured by the Nb2 lactogen in vitro bioassay, was relatively low. The bioactivity of the met-bPRL could be increased to the same order as that of the native hormone by treatment with a mixture of oxidized and reduced thioredoxin. A number of variant met-bPRLs containing specific amino acid changes have been generated by site-specific mutagenesis. The changes involved the substitution (or deletion) of some of the conserved amino acids in bPRL by the different amino acids present at the corresponding positions in the related, but nonlactogenic bovine GH. Nine mutants containing single amino acid changes had bio- and immunoactivities of the same order as those of met-bPRL. One mutant, which incorporated two of the single amino acid changes (serine 62 to threonine and threonine 65 to alanine), had immunoactivity approximating that of met-bPRL but much lower bioactivity (45%). A further mutant, generated by the deletion of tyrosine 28, had essentially no bioactivity although it could not be distinguished immunologically from met-bPRL or bPRL. The findings are discussed in the light of the putative three-dimensional PRL structure and current hypotheses which seek to relate specific regions of PRL to lactogenic activity. It appears that the first putative alpha-helix of bPRL is important for the binding and mitogenic activity of the hormone.

Amino Acid Sequence↗
PubMed1986

Synthesis of bovine prolactin in Escherichia coli.

Transformation of Escherichia coli cells with a recombinant plasmid (pESP4) containing a modified bovine prolactin cDNA clone in a pEMBL vector resulted in efficient expression of prolactin. The cDNA was modified by removal of a 5' nontranslated sequence as well as the sequence that specified the signal peptide of preprolactin. To achieve a high level of synthesis, a sequence of 30 nucleotides in the cDNA, which included the ATG initiation codon and the first 7 codons of mature bovine prolactin, was replaced by a chemically synthesized oligonucleotide duplex. The sequence of this duplex was chosen from the consensus sequence around the initiation codon of E. coli genes and by the amino acid sequence of the protein. Prolactin, a single-chain polypeptide of molecular weight 24,000, was identified by Coomassie Blue staining of NaDodSO4-polyacrylamide gels of total protein from transformed E. coli cells, and by reaction with specific antibody. Increased levels of expression of the hormone, corresponding to the form secreted from the pituitary, were observed in the presence of isopropyl-beta-D-thiogalactopyranoside (IPTG).

Animals↗
PubMed1984

Characterization of the bovine prolactin gene.

The gene coding for bovine prolactin was shown to exist as a single copy per haploid genome. Three restriction fragment polymorphisms were detected in the prolactin gene by Southern blot analysis of DNA obtained from the semen of pedigreed bulls representing eight breeds. The organization of the bovine prolactin gene was determined by restriction mapping of a clone isolated from a genomic library and by genomic blots. The 5'-flanking region and two exons were sequenced and the transcription start site mapped by primer extension. Comparison of the bovine prolactin sequence reported here with the published sequence of the rat prolactin gene revealed extensive homology (79%), extending 360 nucleotides upstream from the cap site, after which the sequences diverge. The homology exceeds that of the coding regions. A possible alternate intron-exon splice site was noted within the sequence coding for the signal peptide.

Amino Acid Sequence↗
PubMed1975

Early estrogen action. Stimulation of the synthesis of methylated ribosomal and transfer RNAs.

The effects of estrogen on the rates of incorporation in vivo of radioactive uridine and Me-methionine, administered together, into RNA in the uterus of the ovariectomized adult rat have been measured. The ratio of incorporation of methionine to uridine during a 45-min labeling period was increased several-fold by hormone treatment. The increased rate of methylation was apparent in the uterus taken from the rat administered estrogen for 1 h, and the effect was more striking following 2 and 3 h of hormone treatment. This stimulation of methylation of RNA occurred in association with an increase in the whole-organ concentration of RNA. Analysis of the doubly-labeled uterine RNA on sucrose gradients revealed that the methylated species were mainly ribosomal and transfer RNA. These results show that very little methylation of RNA occurs in the atrophied uterus of the ovariectomized rat. During the first 3 h following estrogen administration to the ovariectomized animal, an increasing percentage of the newly synthesized RNA formed by the uterus is methylated ribosomal and transfer RNA. This result is discussed in light of recent studies of the efficiency of processing of ribosomal precursor RNA, as well as the synthesis of high-molecular-weight heterogeneous RNA in the early action of estrogen.

Animals↗
PubMed1972

Early estrogen action: stimulation of the metabolism of high molecular weight and ribosomal RNAs (estradiol-17 -RNA isolation-uterus-ribosomal RNA precursors-actinomycin D).

Samples of RNA, isolated from uteri of ovariectomized adult rats treated with estrogen, have been analyzed on sucrose gradients. Treatment with estrogen either for 20 min or 2 hr increased the specific activity of all classes of uterine RNA, but produced no significant alteration in the distribution of radioactivity in the gradients, when animals received [(3)H]uridine intraperitoneally 15 min before they were killed. After labeling periods of 30 min, 1 hr, or 2 hr, however, the RNAs isolated from animals treated with estrogen had a smaller percentage of rapidly sedimenting (faster than 28S) species of RNA than did RNA from animals not treated with the hormone. The decreased percentage of high molecular weight RNA correlated with increases in both the specific activity of 28S and 18S RNA and the concentration of RNA in the whole organ. The labeled RNA of high molecular weight was also demonstrated, by the use of actinomycin D in vivo, to have a more rapid turnover rate in the estrogen-stimulated uterus. Our results indicate that estrogen increases not only the rate of synthesis of ribosomal RNA in the uterus of the ovariectomized adult rat, but also the rate or efficiency of processing of precursor RNA species of high molecular weight.

Animals↗