Hepatic lipase: a member of a family of structurally related lipases.

Partial amino acid sequence of rat hepatic lipase was obtained by gas-phase microsequence analysis of proteolytic fragments. Sequence comparison to bovine lipoprotein lipase and porcine pancreatic lipase reveals a highly conserved region existing among these three physiologically distinct lipolytic enzymes. In a stretch of 36 amino acid residues previously reported for pancreatic lipase (De Caro, J., Boudouard, M., Bonicel, J., Guidoni, A., Desnuelle, P. and Rovery, M. (1981) Biochim. Biophys. Acta 671, 129-138), nineteen residues are identical for all three enzymes, whereas 27 of 36 are identical in rat hepatic lipase and bovine lipoprotein lipase. The fact that this primary structural conservation extends to three different animal species emphasizes the conclusion that these lipolytic enzymes comprise a protein family originating from a common ancestral gene.

Isolation and sequence analysis of human bombesin-like peptides.

The decapeptide form of human gastrin releasing peptide was isolated from acid extracts of liver tissue containing a metastatic human bronchial carcinoid tumor. A larger form also was isolated and partially characterized. During gel permeation chromatography the major immunoreactive peak eluted in the same region as synthetic gastrin releasing decapeptide while a second minor immunoreactive peak eluted near gastrin releasing peptide. Bombesin-like immunoreactivity (BLI) was purified by successive applications to reverse phase high pressure liquid chromatography (HPLC) columns. After four successive HPLC purifications a single peak of bombesin-like immunoreactivity was detected. Amino acid analysis, microsequence analysis and coelution with synthetic peptide indicated that the predominant form present in metastatic tumor tissue was identical to the decapeptide form of canine gastrin-releasing peptide. The less abundant form was purified by cation exchange chromatography followed by reverse phase high pressure liquid chromatography. Partial microsequence analysis of this peptide, through the first 11 residues, was Val-Pro-Leu-Pro-Ala-Gly-Gly-Gly-Thr-Val-Leu. This sequence differed from that of hog heptacosapeptide gastrin releasing peptide at positions 1,3,4 and 5 and from the canine peptide as positions 1,3,5, and 7.

Isolation and characterization of a neurotensin-like decapeptide from a canine upper small intestinal extract.

Neurotensin-like immunoreactivity can be detected in extracts of canine upper gastrointestinal mucosa when measured by carboxyl terminal but not by amino terminal antibodies to neurotensin. The nature of this immunoreactive material was characterized by complete purification on gel filtration and HPLC followed by peptide microsequence analysis. The structure obtained was Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-(Leu), identical in structure to the carboxyl terminal decapeptide of neurotensin. It cannot, however, be excluded that this neurotensin decapeptide was generated from a larger neurotensin-like peptide during the extraction procedure by a physiological or artificial enzymatic process. Since carboxyl terminal neurotensin fragments containing eight or more residues have full biological activity, this peptide may be responsible for neurotensin-like biological activities within the mucosa of, or after release from, the upper gut.

Primary structure of a glycosylated DNA-binding domain in human plasma fibronectin.

The complete amino acid sequence of a DNA-binding domain isolated from human plasma fibronectin after limited trypsin digestion has been obtained. It contains 132 amino acids and one biantennary glycosyl unit at residue 104, for an estimated Mr of 16,931. The fragment can be purified by a two-step procedure consisting of DNA-affinity chromatography and reverse-phase high performance liquid chromatography. It can also be purified by heparin-affinity chromatography. The domain is unusual in its susceptibility to tryptic-like cleavages even by neutral or aromatic residue-specific proteases. It has no cysteine residues and is predicted to favor a beta-sheet structure by Chou and Fasman analysis. Based on this analysis we have proposed a model which exhibits a clustering of aromatic and basic residues, consistent with similar involvement of basic and aromatic residues in other DNA-binding proteins. The net charge of the domain at neutral pH (+1, without sialic acid) argues against a nonspecific charge interaction with polyanionic macromolecules such as DNA and heparin. Internal sequence repeats occur at intervals of 30, 60, and 90 residues, thus suggesting a maximum size for a repetitive building block which gave rise to this domain.

Amino-terminal amino acid sequence of murine colony-stimulating factor 1.

We report the amino acid composition, the NH2-terminal sequence, and the tryptic map by HPLC for murine L-cell colony-stimulating factor 1 (CSF-1). CSF-1 purified by affinity chromatography was S-carboxymethylated under denaturing conditions and subjected to sequence microanalysis. CSF-1 contains four or five cysteine residues per subunit and approximately 30-40% carbohydrate by weight. Three contiguous cysteine residues were located in the NH2-terminal sequence. An additional two cysteine residues were located on the tryptic map. Previous studies as well as immunoblotting studies reported here suggest that sulfhydryl bridging plays a major role in maintaining the conformation of the protein. Carbohydrate was located on two tryptic fragments. The findings of a single NH2-terminal sequence in high yield and a relatively simple tryptic map (15 major peptides) suggest that CSF-1 contains two identical subunits. The NH2-terminal sequence of CSF-1 has only limited homology to insulin or insulin-like hormones but no homology with granulocyte/macrophage-CSF or interleukin 3.

Rapid high-yield purification of canine intestinal motilin and its complete sequence determination.

Canine motilin has been purified from small amounts of canine intestine in a form suitable for microsequence analysis. The sequence determined is: Phe-Val-Pro-Ile-Phe-Thr-His-Ser-Glu-Leu-Gln-Lys-Ile-Arg-Glu-Lys-Glu-Arg- Asn-Lys - Ile-Arg-Asn-Lys-Gly-Gln. Canine motilin differs from porcine motilin at five positions. The rapid, high-yield (24% overall yield) microisolation techniques used for canine motilin should be suitable for the isolation of other basic peptides found in low levels in tissue that is available only in limited amounts. These methods should make the isolation and sequence determination of human brain and gut peptides more readily achievable.

New molecular forms of cholecystokinin. Microsequence analysis of forms previously characterized by chromatographic methods.

Cholecystokinin previously has been characterized by chromatographic and immunological techniques. Analysis of cDNA has revealed the structure of preprocholecystokinin. The predicted processing sites of preprocholecystokinin cannot account for the multiple forms of cholecystokinin detected. This report details the isolation and characterization of cholecystokinin peptides containing 58, 39, 33, 25, 18, 8, 7, and 5 amino acids. None of the cleavages that occurred to form these peptides was at the carboxyl side of double basic residues. Cholecystokinin 25, 18, and 7 have not previously been isolated and identified by sequence analysis. The processing that forms these peptides includes cleavage after single basic residues for the 58, 39, 33, and 8 amino acid peptides. The 25, 18, 7, and 5 amino acid peptides must be formed by other endopeptidases or combinations of endo- and exopeptidases. The analysis of this series of peptides provides the chemical basis for structural differences in cholecystokinin molecules previously demonstrated by chromatographic and immunological methods. These structures provide insight into tissue-specific processing that occurs for this important regulatory peptide in intestine and brain.

Cloning and tissue-specific expression of mouse macrophage colony-stimulating factor mRNA.

Macrophage colony-stimulating factor (CSF-1) stimulates the production of macrophages from bone marrow progenitor cells. We have identified a cDNA clone for murine CSF-1 by antibody screening of a mouse L-cell cDNA library in the expression vector lambda gt11. A screen of about 150,000 recombinant plaques yielded 6 clones that reacted well with an antibody raised against denatured and reduced mouse L-cell CSF-1. These clones were further screened with synthetic oligonucleotides based on the amino-terminal amino acid sequence of CSF-1. One clone, which hybridized to the oligonucleotides, was sequenced and found to contain a single open reading frame. This encompassed 68 amino acids of the mature protein, including the entire amino-terminal sequence we previously reported. This is preceded by what appears to be a 31 amino acid signal peptide. Blot analysis showed that this cDNA hybridizes to a major mRNA species of about 4.5 kilobases (kb) as well as several smaller, less abundant mRNA species (3.8, 2.3, and 1.4 kb) present in mouse L cells. A similar pattern of hybridization was observed with mRNA from a human pancreatic carcinoma cell line that produces CSF-1. Striking differences in the qualitative and quantitative expression of mRNA species for CSF-1 were observed in various mouse tissues. Liver expressed primarily a 1.4-kb species, heart and lung expressed primarily a 4.5-kb species, brain expressed high levels of both the 4.5-kb and 1.4-kb species, and intestine lacked detectable CSF-1 transcripts. Southern blot analysis suggests that the CSF-1 gene is present as a single copy in the mouse haploid genome and that it is not rearranged or amplified in L cells.

Homology of lipoprotein lipase to pancreatic lipase.

Bovine milk lipoprotein lipase was subjected to amino acid sequence analysis. The first 19 amino-terminal residues were Asp-Arg-Ile-Thr-Gly-Gly-Lys-Asp-Phe-Arg-Asp-Ile-Glu-Ser-Lys-Phe-Ala-Leu- Arg. In addition, reversed-phase high-performance liquid chromatography of a tryptic digest of reduced and alkylated lipase resolved a number of peptides, five of which contained cysteine. Sequence analysis of the tryptic peptides revealed in most instances a close homology to porcine pancreatic lipase. Based on this homology, the relative alignment of the sequenced lipoprotein lipase peptides can be made. In addition, a potential binding site for the triacylglycerol substrate and a carbohydrate-binding domain for lipoprotein lipase are postulated.