Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 15 de 15
Filter
Add more filters










Publication year range
1.
J Biol Chem ; 275(4): 2349-58, 2000 Jan 28.
Article in English | MEDLINE | ID: mdl-10644685

ABSTRACT

Biochemical and enzymatic characterization of the novel human subtilase hSKI-1 was carried out in various cell lines. Within the endoplasmic reticulum of LoVo cells, proSKI-1 is converted to SKI-1 by processing of its prosegment into 26-, 24-, 14-, 10-, and 8-kDa products, some of which remain tightly associated with the enzyme. N-terminal sequencing and mass spectrometric analysis were used to map the cleavage sites of the most abundant fragments, which were confirmed by synthetic peptide processing. To characterize its in vitro enzymatic properties, we generated a secreted form of SKI-1. Our data demonstrate that SKI-1 is a Ca(2+)-dependent proteinase exhibiting optimal cleavage at pH 6.5. We present evidence that SKI-1 processes peptides mimicking the cleavage sites of the SKI-1 prosegment, pro-brain-derived neurotrophic factor, and the sterol regulatory element-binding protein SREBP-2. Among the candidate peptides encompassing sections of the SKI-1 prosegment, the RSLK(137)- and RRLL(186)-containing peptides were best cleaved by this enzyme. Mutagenesis of the latter peptide allowed us to develop an efficiently processed SKI-1 substrate and to assess the importance of several P and P' residues. Finally, we demonstrate that, in vitro, recombinant prosegments of SKI-1 inhibit its activity with apparent inhibitor constants of 100-200 nM.


Subject(s)
Proprotein Convertases , Protein Processing, Post-Translational , Serine Endopeptidases/biosynthesis , Amino Acid Sequence , Base Sequence , Cell Line , DNA Primers , Humans , Molecular Sequence Data , Peptide Mapping , Serine Endopeptidases/chemistry , Serine Endopeptidases/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
2.
DNA Cell Biol ; 9(10): 737-48, 1990 Dec.
Article in English | MEDLINE | ID: mdl-2264928

ABSTRACT

There is differential regulation of liver mRNA levels of rat (r) and mouse (m) plasma kallikrein (PK), as observed on Northern blots. Affinity purification of mPK and rPK, microsequencing, and radioimmunoassay in either rat or mouse showed that the difference in mRNA levels does not appreciably affect the circulating PK concentration. Nuclear run-off assays demonstrated that the regulation of the mRNA level of PK is post-transcriptionally controlled. Complete cDNA sequence determination of mPK was achieved using a combination of polymerase chain reaction and lambda gt11 library screening procedures. Within the coding region, the overall sequence homology between mPK and rPK is about 91-92% in amino acid and nucleotide sequence. Although the 3' noncoding segment of mPK is shorter than that of rPK, we calculate a 53% homology with a 5% higher A/T content for mPK. The largest difference is found at the 5' end of the mRNAs: whereas rPK is predicted from its gene structure to have a 167-nucleotide leader sequence, mPK is expected to have more than 605 nucleotides, of which the last 291 are very similar to those found in the rPK gene. The regulation of the mRNA stability and/or turnover rate of PK may possibly be affected by its 5' end in a species-dependent manner.


Subject(s)
DNA/genetics , Kallikreins/genetics , Sequence Homology, Nucleic Acid , Amino Acid Sequence , Animals , Blotting, Northern , Chromatography, High Pressure Liquid , Cricetinae , Electrophoresis, Polyacrylamide Gel , Female , Humans , Infant, Newborn , Kallikreins/blood , Kallikreins/isolation & purification , Mice , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Messenger/analysis , Radioimmunoassay , Rats , Species Specificity , Swine , Transcription, Genetic
3.
Biochem J ; 243(1): 195-203, 1987 Apr 01.
Article in English | MEDLINE | ID: mdl-3606570

ABSTRACT

Bovine seminal plasma was shown to contain three similar proteins, called BSP-A1, BSP-A2 and BSP-A3. Both BSP-A1 and BSP-A2 were shown to be molecular variants of a recently characterized peptide called PDC-109. They seem to differ only in their degree of glycosylation and otherwise seem to possess an identical amino acid composition. The work in the present paper deals with the complete characterization of the third member of this series, namely BSP-A3. The complete amino acid sequence revealed that it is composed of 115 amino acids and predicts a Mr of 13,403. An analysis of the primary structure of BSP-A3 revealed a high degree of internal homology, with two homologous domains composed of 39 (residues 28-66) and 43 (residues 73-115) amino acids. An exhaustive computer-bank search for the similarity of this sequence to any known protein, or segment thereof, revealed two significant homologies. The first is between PDC-109 and BSP-A3, which is so high that we can confidently predict that both proteins evolved from a single ancestral gene. The collagen-binding domain of bovine fibronectin (type II sequence) was also found to be highly homologous to both BSP-A3 and PDC-109.


Subject(s)
Prostatic Secretory Proteins , Proteins , Semen/analysis , Amino Acid Sequence , Animals , Cattle , Chromatography, High Pressure Liquid , Collagen , Computers , Fibronectins , Male , Oxidation-Reduction , Peptide Fragments/analysis , Seminal Plasma Proteins
4.
Anal Biochem ; 160(2): 251-66, 1987 Feb 01.
Article in English | MEDLINE | ID: mdl-3578753

ABSTRACT

Analytical single-column chromatographic methods have been developed for determining all methylated basic amino acids, isodesmosine, desmosine, the amino sugars glucosamine and galactosamine, the diastereoisomers of 5-hydroxylysine, and related compounds at picomole levels in protein and tissue hydrolysates. Complete resolution of all these unique basic amino acids as discrete peaks was achieved in 5.4 on a 50 X 0.28-cm microcolumn of Dionex type DC-4A spherical resin (9.0 +/- 0.5 micron) using updated instrumentation commonly available for amino acid analysis. The column was operated at 5.65 ml/h with two 0.35 M sodium citrate buffers (pH 5.700 and 4.501), at two temperatures (31.5 and 73 degrees C). Excellent resolution of all omega-N-methylarginines and related compounds was also achieved in 3 h using a 17.5 X 0.28-cm microcolumn of Dionex DC-5A resin (sized to 6.0 +/- 0.5 microns), two citrate buffers (0.21 M Na+, pH 5.125; 0.35 M Na+, pH 5.700), a buffer flow rate of 5.75 ml/h, and a temperature of 52 degrees C. Complete separation of all other amino acids found in protein or tissue hydrolysates including S-carboxymethyl cysteine, 4-hydroxyproline, methionine S,S-dioxide, and the amino sugars was also carried out in 95 min using a 23.5 X 0.28-cm microcolumn of Dionex DC-5A resin. The use of purified microcolumn buffers gave smooth baselines without interference from artifacts or minor hydrolysate components. The major advantages of these methods are: first, their high resolving power; second, their high sensitivity which is comparable and in some aspects superior to the newer instruments; and third, their high reproducibility (100 +/- 2.5%) and low operating costs. These methods should be especially valuable for determining myosin, actin, and elastin in tissue hydrolysates from the amounts of N tau-methylhistidine, desmosine, or isodesmosine present, respectively, and for studying protein methylation, hydroxylation, cross-linking formation, and the turnover rates of contractile and connective tissue proteins in biological systems.


Subject(s)
Amino Acids/analysis , Amino Sugars/analysis , Chromatography, Ion Exchange , Elastin/analysis , Hydroxylysine/analysis , Methylation , Proteins/analysis
5.
FEBS Lett ; 181(1): 57-63, 1985 Feb 11.
Article in English | MEDLINE | ID: mdl-3918887

ABSTRACT

The complete synthesis of the C-terminal 28 residues segment 67-94 of human seminal plasma beta-Inhibin, called beta 2-Inhibin, is reported. The Inhibin-like activity of the native 94 amino acids beta-Inhibin is compared to that of the synthetic replica of beta 2-Inhibin. In all assays used both peptides effectively suppress the FSH release induced by LHRH but have little effect on the LH release. In the mouse both peptides are equipotent on a mole basis. In the rat the synthetic beta 2-Inhibin is 3-10 times more potent than beta-Inhibin. Both peptides are active in rat anterior pituitary primary culture assays where maximum suppression of FSH release induced by LHRH occurs around 300 pmol/ml of beta 2-Inhibin. In contrast, maximum suppression of FSH release in the mouse pituitary assay occurs at 10-15 pmol/ml of either Inhibin.


Subject(s)
Inhibins , Peptide Fragments , Peptides/chemical synthesis , Prostatic Secretory Proteins , Semen/analysis , Amino Acid Sequence , Chromatography, High Pressure Liquid , Follicle Stimulating Hormone/metabolism , Gonadotropin-Releasing Hormone/pharmacology , Humans , Luteinizing Hormone/metabolism , Peptides/pharmacology , Pituitary Gland/drug effects
6.
FEBS Lett ; 175(2): 349-55, 1984 Oct 01.
Article in English | MEDLINE | ID: mdl-6434350

ABSTRACT

The complete sequence of a 94 amino acid human seminal plasma polypeptide exhibiting inhibin-like activity is presented. This molecule, called beta-inhibin, selectively and specifically suppresses the release of pituitary FSH in vivo as well as in vitro. It does not affect the secretion of LH. Such a novel acidic protein contains a very basic C-terminal segment which is easily cleaved by mild tryptic digestion. It is predicted that the FSH inhibiting activity may reside within this region of the molecule. This would imply a post Gln-Arg cleavage to release the basic C-terminal active moiety.


Subject(s)
Arginine , Glutamine , Inhibins/isolation & purification , Peptides/isolation & purification , Prostatic Secretory Proteins , Semen/analysis , Amino Acid Sequence , Animals , Follicle Stimulating Hormone/metabolism , Humans , Indicators and Reagents , Inhibins/pharmacology , Kinetics , Male , Mice , Peptide Fragments/analysis , Pituitary Gland/drug effects , Pituitary Gland/metabolism , Rats , Trypsin/metabolism
7.
Arch Biochem Biophys ; 225(2): 525-34, 1983 Sep.
Article in English | MEDLINE | ID: mdl-6625600

ABSTRACT

The isolation and purification of a 21,000-Da (pI 4.9) novel protein from porcine anterior pituitary and whole human pituitary is described. Comparison of the NH2-terminal sequence of the first 77 and 81 residues of the human and porcine homologs shows only one conservative substitution at residue 12, namely an Ala for a Thr between these two species. Such high sequence homology is also reflected in their amino acid composition. A computer data-bank search using a mutation data matrix and comparison with 338,327 segments of proteins revealed that this substance should be classified as belonging to a new protein superfamily. Immunocytochemical staining, using an antibody produced against a synthetic fragment, revealed the presence of immunostainable material in the anterior and posterior lobe of the pituitary and in the supraoptic nucleus of the hypothalamus. No staining was observed in the intermediate lobe of the pituitary. Furthermore, purified neurointermediate lobe secretory granule preparations were also shown to contain this novel polypeptide.


Subject(s)
Nerve Tissue Proteins/isolation & purification , Pituitary Gland, Anterior/analysis , Pituitary Gland, Posterior/analysis , Proteins/isolation & purification , Supraoptic Nucleus/analysis , Animals , Chromatography, High Pressure Liquid , Computers , Humans , Molecular Weight , Organ Specificity , Species Specificity , Swine
8.
J Chromatogr ; 266: 213-24, 1983 Aug 26.
Article in English | MEDLINE | ID: mdl-6630350

ABSTRACT

The coupling of high-performance liquid chromatography, gel-permeation, and reversed-phase chromatography with microsequencing proved to be advantageous for the unambiguous determination of specific sites in pro-opiomelanocortin, cleaved by secretory granule lysates of pig anterior pituitary. This system allows the unambiguous identification of a major chymotrypsin-like enzyme activity, optimal at pH 8.0, in the granule preparation, with a specificity directed towards some Phe decreases X and Tyr decreases X cleavage sites. The approach used emphasizes the necessity to use methodologies leading to the unambiguous determination of conversion activities.


Subject(s)
Cytoplasmic Granules/metabolism , Pituitary Gland, Anterior/metabolism , Pituitary Hormones, Anterior/metabolism , Protein Precursors/metabolism , Amino Acid Sequence , Animals , Binding Sites , Cell Fractionation , Chromatography, High Pressure Liquid , Hydrogen-Ion Concentration , Peptides/analysis , Pro-Opiomelanocortin , Rats , Swine
11.
J Biol Chem ; 256(15): 7977-84, 1981 Aug 10.
Article in English | MEDLINE | ID: mdl-6267033

ABSTRACT

The isolation and complete purification of a human glycopeptide representing the major immunoreactive form of the pituitary NH2-terminal segment of pro-opiomelanocortin is presented. The complete sequence of this peptide was determined following CNBr fragmentation and it is shown to be 76 amino acids long. It bears an O-glycosylation site at Thr 45 and an N-glycosidic linkage at Asn 65. Compared to the reported genomic DNA sequence (Chang, A. C. Y., Cochet, M., and Cohen, S. N. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 4890-4894), one variation exists, namely Arg 22 replacing Gly 22. Two disulfide bridges linking Cys 2 to 8 and Cys 20 to 24 have been determined. Based on the sequence and disulfide bridge localization, a large degree of homology exists between the NH2-terminal sequence of the human peptide and all calcitonins, especially porcine calcitonin. The human NH2-terminal peptide is shown to stimulate the release of aldosterone from isolated cells of a human adrenal tumor, in at least equipotency to adrenocorticotropic hormone and the porcine NH2-terminal analogue, which is 100 times more potent than angiotensin II. Finally, this reported sequence completes that of the human DNA which was lacking the first 19 amino acids due to the presence of a 2-kilobase intron.


Subject(s)
Adrenocorticotropic Hormone , Aldosterone/metabolism , Glycopeptides , Peptide Fragments , Pituitary Gland/analysis , Pituitary Hormones, Anterior , Pro-Opiomelanocortin , Adrenal Gland Neoplasms/metabolism , Adrenocorticotropic Hormone/pharmacology , Amino Acid Sequence , Animals , Base Sequence , Cyanogen Bromide , DNA , Genes , Glycopeptides/pharmacology , Humans , Iodoacetamide , Pituitary Hormones, Anterior/pharmacology , Species Specificity , Swine
15.
Anal Biochem ; 91(2): 403-9, 1978 Dec.
Article in English | MEDLINE | ID: mdl-9762125

ABSTRACT

A single-column amino acid analysis method is presented for use in structural studies of glycoproteins. The system gives excellent resolution of glucosamine, galactosamine, cysteic acid, CM-cysteine, AE-cysteine, the internal standard norleucine, and all amino acids normally present in protein hydrolysates.


Subject(s)
Amino Acids/analysis , Chemistry Techniques, Analytical/methods , Buffers , Chromatography, Ion Exchange/methods , Cysteine/analysis , Glycoproteins/chemistry , Hexosamines/analysis , Indicators and Reagents
SELECTION OF CITATIONS
SEARCH DETAIL
...