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C L Hew

Publications and source records attributed to C L Hew.

At least 91 records · Page 5Linked to original sources
PubMed1986

Structure of an antifreeze polypeptide precursor from the sea raven, Hemitripterus americanus.

The cystine-rich antifreeze polypeptides (AFP) from sea raven were fractionated by reverse-phase high performance liquid chromatography into several components, with SR2 (Mr 17,000) as the major AFP. Sea raven AFP cDNA clones were isolated from a liver cDNA library using a synthetic oligonucleotide, and the identity of one of the clones, C2-1, was confirmed by hybridization selection and cell-free translation. C2-1 encodes a pre-AFP of 195 amino acids with no evidence of any profragments. Comparison of the deduced amino acid sequence with partial peptide sequences from SR2 showed substitutions in at least four amino acid positions, suggesting that C2-1 cDNA codes for a minor component. Both the primary and the predicted secondary structures of sea raven AFP are completely different from those of other fish AFP. This further confirms that sea raven AFP belongs to a different class of antifreezes. The high frequency of reverse turns and the presence of paired hydrophilic amino acids in these structures are striking features of the protein and may contribute to their antifreeze action.

Amino Acid Sequence↗
PubMed1986

Biosynthesis of antifreeze polypeptides in the winter flounder. Characterization and seasonal occurrence of precursor polypeptides.

The precursor proteins for winter flounder antifreeze polypeptide (AFP) were isolated from liver using gel filtration chromatography and reverse-phase high-performance liquid chromatography. Two major pro-antifreezes (Mr 5000), corresponding to the precursors for AFP-6 and AFP-8, were characterized by amino acid analyses and automated Edman degradation. These precursors showed significant antifreeze activity. The pro-antifreezes were synthesized in the liver seasonally as demonstrated by immunoblotting and in vitro liver incorporation studies. No mature AFP were detected in liver, thus indicating that the processing of pro-antifreezes, including amidation of the C-termini, occurred mainly in the serum. The function(s) of the prosequences, if any, remain unclear.

Amino Acids↗
PubMed1986

Molecular cloning and sequencing of salmon gonadotropin beta subunit.

Gonadotropin (GTH) was purified from the pituitaries of the Pacific chinook salmon using a combination of stepwise ethanol precipitation and concanavalin-A affinity chromatography. The alpha and beta subunits were dissociated and fractionated by C-18 reverse-phase high-performance liquid chromatography with a 0.1% trifluoroacetic acid/acetonitrile gradient. An enriched cDNA library was screened for the beta-GTH gene(s) using two synthetic oligonucleotides based on partial protein data. A positive, full-size clone (E3) was identified and sequenced. It contains 657 base pairs and codes for a 142-amino-acid precursor protein. The mature salmon beta-GTH (119 amino acids) is structurally homologous to human luteinizing hormone and chorionic gonadotropin. The effect of testosterone implantation on pituitary GTH and beta-GTH mRNA was examined with radioimmunoassay and Northern blot analysis. There was a corresponding increase in both the pituitary GTH and mRNA levels.

Amino Acid Sequence↗
PubMed1986

Single crystals of a winter flounder antifreeze polypeptide.

Component 6 of the winter flounder's antifreeze polypeptides has been crystallized. The space group is P21, with cell parameters of a = 38.14 A, b = 37.19 A, c = 21.82 A, beta = 101.5 degrees. There are two molecules of 3300 Mr per asymmetric unit.

Animals↗
PubMed1985

Structure of an antifreeze polypeptide and its precursor from the ocean pout, Macrozoarces americanus.

Serum antifreeze polypeptides (AFP) from Newfoundland ocean pout have been resolved by ion exchange chromatography and reverse phase high performance liquid chromatography into at least 12 components. The protein sequences of three of the AFP were determined using a combination of protein Edman degradation and cDNA sequencing. The AFP precursor protein encodes for a preprotein of 87 amino acids with no obvious prosequences. Two of the AFP (SP1-A and SP1-C) were separate gene products with minor amino acid sequence differences. The protein structure of SP1-C precursor is MKSVILTGLLFVLLCVDHMTASQSVVAT QLIPINTALTPAMMEGKVTNPIGIPFAEMSQIVGKQVNTPVAKGQTLMPNMVKTYVAGK. The third AFP (SP1-B) is a post-translation modification product of SP1-C. These experiments indicate that the ocean pout AFP are a multigene family with protein structure different from any other known polypeptide antifreezes.

Amino Acid Sequence↗
PubMed1985

Structures of shorthorn sculpin antifreeze polypeptides.

The amino acid sequences of the two major antifreeze polypeptides (AFP) from the shorthorn sculpin have been determined using an automatic protein sequencer and enzymic digestion. These two polypeptides, SS-3 and SS-8, consist of 33 and 45 amino acid residues respectively. The N-terminal methionyl residue is blocked in both the polypeptides. When aligned for maximum structural similarity these two AFP are 80% homologous, and there appears a deletion of 12 amino acid residues at the N-terminal portion of SS-3. Like the winter flounder AFP, both the sculpin AFP also contain the 11-amino-acid repeat sequences. The secondary structure of the sculpin AFP is mainly alpha-helical as deduced from circular dichroic spectral data. The helical content of SS-8 is high (73%), while that of SS-3 is moderate (about 45%). The latter exhibits a relatively weak antifreeze activity. Removal of the blocked N-terminal residue in SS-8 did not alter the helical content significantly but did reduce the antifreeze activity. Helical contents of proteolytically generated fragments of AFP are much lower, and they are devoid of activity. The alpha-helix in the SS-8 component is seen to be amphiphilic in character. The relevance of this feature to the mechanism of the antifreeze action is briefly discussed.

Amino Acid Sequence↗
PubMed1985

Antifreeze protein genes are tandemly linked and clustered in the genome of the winter flounder.

We have used genomic Southern blots and restriction maps of genomic clones to examine the organization of the antifreeze protein multigene family in the winter flounder. The majority of the approximately equal to 40 antifreeze protein (AFP) genes in this fish are present in 7- to 8-kilobase-pair (kbp) elements of DNA, which are iterated as tandem direct repeats. Each repeat contains a single antifreeze protein gene that is 1 kbp long, and all of these genes have the same transcriptional orientation. Although the repeated elements are highly homologous, they do show some restriction site and restriction length polymorphisms. When flounder genomic DNA is digested with restriction endonucleases that do not cut within the repeats, most of the antifreeze protein genes reside in fragments that are at least 40 kbp long, representing clusters of five or more repeats in tandem. After genomic DNA is digested with Xba I or Xho I, these genes are present in fragments of exceptionally high molecular weight, suggesting that the clusters themselves are grouped together in the genome. The AFP gene locus may have evolved by gene amplification as recently as 10(6) years ago in response to the onset of the Cenozoic ice age.

Animals↗
PubMed1984

Antifreeze protein genes of the winter flounder.

A genomic library of the winter flounder in lambda phage Charon 4 was screened with antifreeze protein cDNA. Nine genomic clones were isolated from three haploid genome equivalents of phage and were grouped into three classes on the basis of restriction mapping and heteroduplex analysis. The three chromosomal regions are non-overlapping and each contain two antifreeze protein genes spaced from 3 to 7 kilobase pairs apart. Genomic Southern blots indicate that these six non-allelic genes represent only 10-20% of the complement of this large multigene family. One gene has been identified by sequencing as a variant of the gene encoding the most abundant antifreeze protein. This gene is 1.0 kilobase pair long and contains an intervening sequence of 0.6 kilobase pairs between the coding region for the bulk of the signal sequence and that coding for the proprotein.

Amino Acid Sequence↗
PubMed1984

Accumulation of winter flounder antifreeze messenger RNA after hypophysectomy.

The effect of hypophysectomy (hypex) on winter flounder antifreeze mRNA accumulation in the liver was examined. Hypophysectomy resulted in a significant decrease in serum freezing temperature, and increases in liver weights, total liver poly(A)+ RNA and antifreeze polypeptides (AFP) mRNA accumulation. The identity of the AFP mRNA in hypex animals was confirmed by gel electrophoresis, cell-free translation, and Northern blot hybridization techniques. Cytoplasmic dot hybridization analysis indicated that the AFP mRNA level in hypex fish approximated that observed in winter animals actively synthesizing AFP. An increase in AFP mRNA was detectable as early as the first day after hypophysectomy and by Day 7 reached 25% of the level found in fish actively synthesizing AFP mRNA during the winter months. Since AFP mRNA is found at very low levels in the control flounder, we suggest that its accumulation after hypophysectomy depends on accelerated transcription. The pituitary gland appears to regulate the liver AFP mRNA level by a negative transcriptional control mechanism.

Animals↗
PubMed1983

Seasonal variation in the level of antifreeze protein mRNA from the winter flounder.

Livers from winter flounder collected at monthly intervals throughout the year were analyzed for their content of antifreeze protein mRNA. Putative mRNA was detected in liver RNAs from summer fish by liquid hybridization to kinetically purified antifreeze protein cDNA. These mRNA sequences were shown by Northern blot analysis to be of the same length as mature antifreeze protein mRNA isolated from winter fish, and were able to direct the incorporation of alanine into a translation product which comigrated with antifreeze preproprotein. The build-up of antifreeze protein mRNA levels in the autumn and their decline in the spring to summer levels were measured by liquid hybridization. These seasonal fluctuations match closely, but slightly precede, the rise and fall in plasma osmolality due to the presence of antifreeze protein. In mid-winter 0.5% of the total liver RNA is antifreeze protein mRNA, and although by late August the level of this mRNA has declined to 0.0007% of the total RNA, at no time during the summer is the mRNA undetectable. These results suggest that antifreeze protein production is more likely to be regulated by changing the rate of transcription of their genes than by switching them between active and inactive states.

Acclimatization↗
PubMed1982

DNA sequence coding for an antifreeze protein precursor from winter flounder.

A cDNA made to antifreeze protein mRNA of the winter flounder was cloned in the plasmid pBR322 and its sequence was determined by the method of Maxam and Gilbert. Its sequence codes for a precursor protein that is 82 amino acids in length. This precursor has both a signal polypeptide and a prosequence before the mature protein of 38 amino acid residues. The mature protein matches in composition one of the alanine-rich serum antifreeze proteins that was purified by ion-exchange and reverse-phase chromatography. The composition of the pro- sequence is similar to that of the native protein except that it contains five prolines. The mature protein, but not the pro- sequence, contains three of the 11-residue repeats previously observed [Lin, Y. & Gross, J. K. (1981) Proc. Natl. Acad. Sci. USA 78, 2825-2829] in two other antifreeze protein components.

Amino Acid Sequence↗
PubMed1982

Radioimmunoassay for the antifreeze polypeptides of the winter flounder: seasonal profile and immunological cross-reactivity with other fish antifreezes.

The polymerization of winter flounder antifreeze polypeptide (AFP) with glutaraldehyde to increase its antigenicity and the conjugation of the AFP to bovine serum albumin so that the AFP could carry a radioiodine label have allowed the development of a convenient and reliable radioimmunoassay (RIA) for the antifreeze. A seasonal concentration profile of serum AFP as determined by the RIA corresponded closely with its activity profile. Winter serum concentrations of AFP measured by the RIA (7-11 mg/mL) agreed well with antifreeze recovery from Sephadex G-75 chromatography of serum (6-10 mg/mL), but less so with concentration estimates reported from activity measurements (12-25 mg/mL) (Petzel, D. H., Reisman, H. M. & DeVries, A. L. (1980) J. Exp. Zool. 211, 63-69). A small but measurable immunological cross-reactivity was detected between the antiflounder AFP and other fish antifreeze polypeptides. Such cross-reactivity was quite unexpected considering the large compositional and structural differences among the various AFP and its significance remains unclear.

Animals↗
PubMed1981

Antifreeze proteins from the sea raven, Hemitripterus americanus. Further evidence for diversity among fish polypeptide antifreezes.

The antifreeze proteins of the sea raven, Hemitripterus americanus, were isolated and compared with other fish antifreeze proteins. The sea raven contains one major protein of molecular weight 14,000-16,000 with little or no carbohydrate. Except for its similar seasonal appearance, the sea raven antifreeze protein differs from other polypeptide antifreeze in its amino acid composition, secondary structure, and immunological specificity. Amino acid analysis of sea raven antifreeze showed that it contains a high amount of half-cystine, hydrophilic amino acids, and only an average amount of alanine. In contrast, all other fish antifreeze proteins contain approximately 60% alanine and no half-cystine residues. Furthermore, the sea raven antifreeze protein is sensitive to sulfhydryl reagents. The antifreeze activity was decreased by 67% in the presence of 0.01 M dithiothreitol. Circular dichroism studies indicated the absence of significant amounts of alpha-helix and the possible presence of beta-structure. Antibodies raised against the antifreeze protein did not cross-react with the known polypeptide antifreeze from the winter flounder and shorthorn sculpin (Hew, C. L., Fletcher, G. L., and Ananthanarayanan, V. S. (1980) Can. J. Biochem. 58, 377-383). A specific radioimmunoassay was developed for the sea raven antifreeze protein and was used to quantitate the protein concentration in the fish. The seasonal profile obtained by radioimmunoassay was compatible with the antifreeze activity determined with a freezing point osmometer.

Amino Acids↗
PubMed1980

Isolation and characterization of the antifreeze protein messenger RNA from the winter flounder.

The messenger RNA coding for the serum antifreeze protein of the winter flounder (Pseudopleuronectes americanus) was extracted from the liver polysomes of fish caught in November and was purified by oligo(dT)-cellulose chromatography and sucrose density gradient centrifugation. The length of the mRNA was 520 nucleotides measured by agarose gel electrophoresis in the pesence of methyl mercury. It sedimented between trout protamine mRNA (6 S) and rabbit globin globin mRNA (9-10 S) on sucrose density gradients. The purified antifreeze protein mRNA assayed in the mRNA-dependent reticulocyte lysate translation system incorporated 10 times as much alanine as arginine into a single translation product. the molecular weight of the primary translation product was 11,700 estimated by sodium dodecyl sulfate-gel electrophoresis. Antifreeze protein mRNA was the only major mRNA species present in liver polysomes of fish caught in November but was absent in the liver polysomes of fish caught in April. This finding emphasizes the seasonal nature of antifreeze protein production in the winter flounder. A 32P-labeled cDNA prepared from the antifreeze protein mRNA was hybridized against total cellular RNAs from flounder liver. These RNA excess hybridizations showed that antifreeze protein mRNA is several orders of magnitude more abundant in liver in mid-November than in late August. This suggests that transcriptional control is involved in the seasonal expression of the antifreeze protein gene.

Alanine↗
PubMed1980

Antifreeze proteins from the shorthorn sculpin, Myoxocephalus scorpius: isolation and characterization.

The antifreeze proteins (AFP) of the shorthorn sculpin, Myoxocephalus scorpius, were isolated and compared with the AFP of the winter flounder. The shorthorn sculpin was found to contain one major and one minor antifreeze protein with an approximate molecular weight of 10 000--11 000 in the winter. The major AFP was isolated and characterized. It had many characteristics in common with the flounder AFP studied in our laboratories. These characteristics included its seasonal appearance, size, amino acid composition, the abundance of alanine in the composition, the extent of antifreeze activity, and the nature of the secondary structure. It is suggested that both the sculpin and flounder AFP are structurally homologous and belong to the same type of polypeptide antifreeze.

Alanine↗
PubMed1979

Cell-free synthesis of rat liver zinc-thioneins.

The induction of rat liver zinc-thioneins mRNA was studied in a wheat germ cell-free translation system. Liver poly A rich polysomal RNA was isolated from rats which had been injected with zinc sulfate 5 h previously. These RNA preparations stimulated the incorporation of [35S]cystine into trichloroacetic acid insoluble proteins when assayed in the cell-free synthetic system. The translation products were characterized by Sephadex G-75 chromatography in 8 M urea--50 mM beta-mercaptoethanol, by disc gel electrophoresis in 4 M area--Tris-glycine buffer (pH 9.2), and by peptide fingerprinting with pepsin. These results were identical with authentic rat liver zinc-thioneins. The zinc-thioneins mRNA activity in the control rats, however, was minimal. The stimulation in zinc-thioneins synthesis observed in the cell-free synthesis was similar to the increased synthesis of these polypeptides in vivo.

Amino Acids↗