Hormone-sensitive lipase, a key enzyme in fatty acid mobilization, overall energy homeostasis, and possibly steroidogenesis, is acutely controlled through reversible phosphorylation by catecholamines and insulin. The 757-amino acid sequence predicted from a cloned rat adipocyte complementary DNA showed no homology with any other known lipase or protein. The activity-controlling phosphorylation site was localized to Ser563 in a markedly hydrophilic domain, and a lipid-binding consensus site was tentatively identified. One or several messenger RNA species (3.3, 3.5, or 3.9 kilobases) were expressed in adipose and steroidogenic tissues and heart and skeletal muscle. The human hormone-sensitive lipase gene mapped to chromosome 19 cent-q13.3.

Apolipoprotein(a) [apo(a)] is a glycoprotein with Mr %280,000 that is disulfide linked to apolipoprotein B in lipoprotein(a) particles. Elevated plasma levels of lipoprotein(a) are correlated with atherosclerosis. Partial amino acid sequence of apo(a) shows that it has striking homology to plasminogen. Plasminogen is a plasma serine protease zymogen that consists of five homologous and tandemly repeated do-

2-Azidoadenine [32P]nucleotide was bound specifically at catalytic or noncatalytic nucleotide binding sites on beef heart mitochondrial F. ATPase. In both cases, photolysis resulted in nearly exclusive labeling of the I8 subunit. The modified enzyme was digested with trypsin, and labeled peptides were purified by reversed-phase high-pressure liquid chromatography. Amino acid sequence analysis of the major 32P-labeled tryptic fragments showed 13-subunit Tyr-368 to be present at noncatalytic sites and /8 Tyr-345 to be present at catalytic sites. From the relationship between the degree of inhibition and extent of modification, it is estimated that one-third of the catalytic sites or two-thirds of the noncatalytic sites must be modified to give near-complete inhibition of catalytic activity.The ATP synthase FtFl, found in mitochondria, chloroplasts, and bacteria, couples proton translocation to ATP synthesis during oxidative phosphorylation and photophosphorylation. The F1 component can be readily solubilized and has a a3f33y&e subunit structure. F1 contains the catalytic sites for ATP synthesis, but when detached from the membrane it is only capable of net ATP hydrolysis (1, 2).Of the six nucleotide binding sites present on the beef heart mitochondrial enzyme, MF1, only three sites exchange bound nucleotide rapidly during hydrolysis of MgATP at pH 8.0 (3). Evidence has been presented that at least two (4-6) and probably all three (7-12) of the exchangeable sites participate sequentially in catalysis at high substrate concentration. The three nonexchangeable sites, referred to here as noncatalytic sites, have been suggested to play a structural or regulatory role (for example, see refs. 13 and 14).A valuable approach to the characterization of nucleotide binding sites on F1 has been the use of affinity and photoaffinity analogs (15). Perhaps the most promising probes are the 2-azidoadenine nucleotides (11,16,17). Their anti conformation resembles that of F1-bound ADP and ATP; thus, they bind very tightly and serve as good substrates (18). Procedures described by Kironde and Cross (19) Garin et al. (22). We also report here the effects of derivatization on catalytic activity. MATERIALS AND METHODSPreparation of MF1. MF1 was isolated from beef heart mitochondria as described (23) Photoaffinity Labeling. To label a noncatalytic nucleotide site, in a typical experiment 60 ,ul of 14.5 mM 2-azido-[,B,y-32P]ATP was added with rapid mixing to 450 ul of 75 ,uM MF1[2,1] and incubated for 15 min. Unbound ligand was removed on a 3-ml centrifuge column, and MgATP was added to a final concentration of 15 mM to displace 2-azido-ANP from catalytic sites. After 1 min, the enzyme was passed through a second 3-ml centrifuge column and diluted to 2 ml. Four 500-,ul aliquots were placed on a shallow plastic tray 8 mm from a Minerallight lamp and photolyzed at the short wavelength setting for 30 min.To 5715The publication costs of this article were defrayed in part by page charge payment. This article must therefore be here...

Exposure of chloroplast F1 ATPase to 2-azido-ATP results in the noncovalent tight binding of 2-azido-ATP or 2-azido-ADP to noncatalytic or to catalytic sites. Subsequent photolysis results in covalent labeling of adjacent tryptic peptides of the &subunit. Binding at noncatalytic sites results in labeling of tyrosine 385 by an ATP or an ADP moiety. Binding at catalytic sites results in labeling of tyrosine 362 by only an ADP moiety. Similar labeling patterns are observed for the heat-activated or the membrane-bound enzymes.

A successful approach has been developed for the sequencing of apolipoprotein B based upon the procedure of Cleveland et al. [(1977) J. Biol. Chem. 252, 1102–1106] involving limited proteolysis in the presence of sodium dodecyl sulfate. Staphylococcus aureus protease was employed to produce large peptides which were isolated in relatively pure form by preparative gel electrophoresis. Two peptides were partially sequenced using spinning‐cup microsequencing techniques. The sequences are: Peptide R2‐5, ‐Ala ‐ Leu ‐ Val ‐ Gly ‐ Ile ‐ Asn ‐ Gly ‐ Glu ‐ Ala ‐ Asn ‐ Leu ‐ Asp ‐ Phe ‐ Leu ‐ Asn ‐ Ile ‐ Pro ‐ Leu ‐ Arg‐ Ile ‐ Pro‐ Pro‐Met‐Arg‐(Arg)‐; and Peptide R3‐1, ‐Leu‐Val‐Ala‐Lys‐Pro‐Ser‐Val‐Ser‐Val‐Glu‐Phe‐Val‐Thr ‐Asn‐Met‐Gly‐Ile‐Ile‐Ile‐Pro‐Lys‐Phe‐Ala‐Arg‐. Several stretches of residues suitable for the construction of oligonucleotide probes have been identified.

Pancreatic polypeptide (PP) has been isolated from rat pancreas by gel filtration, ion exchange chromatography, and high performance liquid chromatography. The isolation was monitored with a RIA, using antibody to the carboxyl-terminal hexapeptide of bovine PP. Rat PP contains 36 amino acids and is similar in composition to PP from other mammalian sources. The single methionine residue in the peptide appears to oxidize easily to the sulfoxide, thereby giving rise to two immunoactive peaks on high performance liquid chromatography. Reduction to the native peptide can be accomplished with mercaptoethanol. The PP content of rat pancreas is about 2 mg/kg. The amino acid sequence of rat PP is Ala-Pro-Leu-Glu-Pro-Met-Tyr-Pro-Gly-Asp- Tyr-Ala-Thr-His-Glu-Gln-Arg-Ala-Gln-Tyr-Glu-Thr-Gln-Leu-Arg-Arg-Tyr-Ile- Asn-Thr-Leu-Thr-Arg-Pro-Arg-Tyr-NH2. This sequence preserves characteristics necessary for stabilization of the compact globular conformation found in avian PP.

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Cholecystokinin (CCK)-like immunoreactivity (CCK-LI) in a pool of 12 dog brains was extracted sequentially into boiling water and cold 2% trifluoroacetic acid. Gel filtration on Sephadex G-50 revealed three main molecular forms detected by a carboxyl-terminal antibody; one was eluted in the position of CCK-58 (58 amino acid residues long); a second, in the position of CCK-8; and a third, near the radioactive iodide marker. When the CCK-LI was purified by affinity chromatography using carboxyl-terminal CCK antibody followed by three steps of reversed-phase high-pressure liquid chromatography, three components were isolated and characterized by sequence microanalysis. The smallest component was the pentapeptide common to gastrin and CCK. The second peak was eluted in the same region as synthetic CCK octapeptide, and sequence analysis showed that the chemical structure of this biologically active region of canine CCK is identical to that found in sheep and pig brains. The 22-residue aminoterminal sequence of brain CCK-58 was: Ala-Val-Gln-LysVal-Asp-Gly-Glu-Pro-Arg-Ala-His-Leu-Gly-Ala-LeuLeu-Ala-Arg-Tyr-Ile-Gln-, the same as the sequence found for canine intestinal CCK-58 from this pool of dogs. This is the same sequence others have reported for porcine brain CCK-58 lacking nine amino acid residues (CCK-58 desnonapeptide) except that the porcine peptide had a serine in position 9. The canine CCK amino-terminal sequence differed from the se-