Evidence that HetR protein is an unusual serine-type protease.

The hetR gene plays a very important role in cell differentiation of heterocystous cyanobacteria. To understand the mechanism of the hetR gene product in regulation of heterocyst differentiation, the recombinant HetR protein (rHetR) was overproduced in Escherichia coli. Purified rHetR was unstable and degraded easily in solution. Phenylmethanesulfonyl fluoride, a serine-type protease inhibitor, prevented the degradation and was shown to modify covalently rHetR. Dansyl fluoride (DnsF), another serine-type protease inhibitor, also covalently modifies rHetR as shown by electrophoresis and electroblotting of the labeled rHetR and by MS. The labeling of rHetR with phenylmethanesulfonyl fluoride and DnsF was at the same site of rHetR and required Ca2+. S179N-rHetR, a mutant protein from strain 216 of Anabaena PCC 7120, which cannot differentiate heterocysts because of the mutation, was also overproduced and characterized. Although S170N-rHetR still can be labeled with DnsF, no proteolysis was observed, suggesting that Ser179 is involved in proteolytic activity. DnsF-labeled rHetR was digested with trypsin, and the labeled peptide was isolated and sequenced. The labeled peptide matches a sequence from HetR. These results show that HetR is a protease.

Visualization of proteins by modification of lysines, cysteines, and phosphorylated serines facilitates sample preparation for microsequencing.

A procedure for visualization and sensitive detection of protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent sample preparation for sequence analysis is described. This procedure utilizes either fluorescent or visible tags for certain amino acids in protein molecules, e.g., lysines modified with dansyl/dabsyl chloride and cystines/cysteines or phosphorylated serines modified with iodoacetamidofluorescein (I-15) after proper sample pretreatments. Modifications are performed prior to SDS-PAGE, eliminating the need for fixing, staining, and destaining as required for the conventional procedures. After electrophoresis, the fluorescent or visible bands are excised from the gel, homogenized in microcentrifuge tubes, and soaked in an appropriate buffer to release the separated proteins into solution. Enzymatic digestion can then be carried out in solution for better efficiency of digestion and recovery. The subsequent HPLC mapping and collection of protein digests are performed on PE Applied Biosystems Model 173A MicroBlotter. The separated peptides containing tagged amino acids are visible on the PVDF membrane and can be excised for direct sequence analysis. This approach has been employed for selectively isolating the lysine, cysteine, or phosphorylated serine containing peptides using model proteins. The sequencing results of the peptides generated from premodified proteins demonstrate that this approach facilitates sample preparation for microsequence analysis at low picomole level. Overall recoveries of 20-30% by sequencing initial yields have been achieved using our model proteins.

Structural determination of the essential serine and glycosylation sites of carboxypeptidase P.

Through a series of kinetic studies involving the inactivation effects of diisopropylfluorophosphate, an affinity label that modifies the active site serine residue involved in the mechanism of action, it has been firmly established that carboxypeptidase P (CPP) requires a serine residue for catalytic activity. The essential kinetic parameters were determined to be 1.33 mM for the apparent dissociation constant with a limiting half-life of inactivation of 20.1 min. Structural elucidation of the primary amino acid sequence surrounding the essential serine, and comparing that with the reactive site of carboxypeptidase Y (CPY), revealed a significant degree of homology at the active site between these two enzymes. These regions, however, were quite divergent from other known serine proteases, leading to the speculation that these serine exopeptidases may comprise a unique family in the overall classification of serine proteases. It was established that CPY could be inactivated with either of the classic histidine affinity labels tosylphenylalanylchloromethyl ketone (TPCK) or carbobenzoxyphenylalanylchloromethyl ketone (ZPCK) with Ki's of 1.2 and 12.8 microM, respectively. This is in marked contrast to CPP, which was unaffected by saturating levels of the known histidine affinity labels, TPCK, tosyllysylchloromethyl ketone, or ZPCK. This point may be a significant element in differentiating specificity among these two serine proteases. Further investigation into the structural nature of CPP revealed that it is a glycoprotein with a single site of carbohydrate attachment. In addition, the carbohydrate moiety itself appears to contribute 1217 Da to the overall molecular weight and it is characterized as an asparagine linked high mannose type. This is significantly different from CPY with its four sites of carbohydrate attachment contributing approximately 17% to its molecular weight.

A general approach for characterizing glycosylation sites of glycoproteins.

Recent advances in glycobiology have greatly stimulated carbohydrate research; however, improving techniques for identification and isolation of specific glycosylation sites in protein structure analysis remains a challenge. We report here a practical approach utilizing a membrane staining technique on Problott, a PVDF-type membrane, to screen glycoproteins and glycopeptides derived from enzymatic digests of glycoproteins. To improve the detection sensitivity, an amplified staining technique using biotinylated lectins, avidin, and biotinylated peroxidase was employed. In addition, we describe a micro-batch affinity binding procedure to isolate glycopeptides from complex glycoprotein enzymatic digests. These protocols allow us to start with a subnanomole quantity of glycoprotein and locate the glycosylation sites; isolate glycopeptides in a homogeneous form; and perform amino acid composition, amino acid sequence, and mass analyses on the isolated glycopeptides. The characterization of glycosylation site of a model glycoprotein, carboxypeptidase P, of which the structure is still largely unknown, has been investigated.

Human DNA polymerase alpha catalytic polypeptide binds ConA and RCA and contains a specific labile site in the N-terminus.

The catalytic polypeptide of DNA polymerase alpha is often observed in vitro as a family of phosphopolypeptides predominantly of 180 and 165 kDa derived from a single primary structure. The estimated Mr of this polypeptide deduced from the full-length cDNA is 165 kDa. Immunoblot analysis with polyclonal antibodies against peptides of the N- and C-termini of the deduced primary sequence indicates that the observed family of polypeptides from 180 kDa to lower molecular weight results from proteolytic cleavage from the N-terminus. Antibodies against the N-terminal peptide detect only the 180 kDa species suggesting that this higher molecular weight polypeptide may be the result of posttranslational modification of the 165 kDa primary translation product. The catalytic polypeptide is not only phosphorylated but is also found to react with lectins ConA and RCA. N-terminal sequencing of the isolated catalytic polypeptide from human cells and of the recombinant fusion proteins indicates that the often observed 165 kDa polypeptide is the in vitro proteolytic cleavage product of the modified 180 kDa protein at the specific site between lys123 and lys124 within the sequence -RNVKKLAVTKPNN-.

Neo-kyotorphin, an analgesic peptide isolated from human lung carcinoma.

A pentapeptide with analgesic activity has been isolated from human lung squamous cell carcinoma and from three other types of propagated tumors of human lung small-cell carcinoma (SCC), adenoma (AD) and large-cell carcinoma (LCC) in nude mice. The amino acid sequence of the peptide has been revealed to be H-Thr-Ser-Lys-Tyr-Arg-OH, which is exactly the same as that of neo-kyotorphin, an analgesic peptide originally isolated from bovine brain [(1982) Life Sci. 31, 1733]. No neo-kyotorphin could be isolated from normal lung tissue using the same procedures as those used for carcinomas. The results suggest that the presence of neo-kyotorphin in the lung carcinoma may represent the ectopic expression of peptide hormone. Our findings constitute the first example of a human lung carcinoma producing analgesic peptide.

Isolation and sequencing of a new biologically active peptide from human lung carcinoma.

A biologically active peptide designated hLCP has been isolated and purified to homogeneity from human lung carcinoma by means of acidic extraction and successive chromatography on Sephadex G-50, Toyopearl HW-40 F and reverse-phase high performance liquid chromatography columns. Analysis showed that peptide consists of thirteen amino acids. Primary structure of hLCP has been deduced by double-coupling Edman degradation combined with enzyme digestion as H-Ser-Pro-Pro-Asp-Gly-Lys-Lys-Glx-Ser-Ala-Asp-Val-Lys-OH. hLCP possessed significant excitatory activity on an electrical stimulation induced contraction. No hLCP could be detected in normal lung tissue. The possibility of using hLCP as a biochemical marker in the clinic for the early detection of lung carcinoma is being investigated.

Isolation, amino acid sequence, synthesis, and biological activity of some oligopeptides from porcine spinal cord.

Four oligopeptides, designated SCP-3, SCP-4, SCP-5, and SCP-6, have been isolated and purified to homogeneity from porcine spinal cord. The amino acid sequences have been determined as pyroGlu-Gly, pyroGlu-Gly-Gly, Met-Met-Gly, and Asp-Ala-Gly-Ala-Gly, respectively. All of these peptides have been synthesized by conventional liquid-phase or solid-phase methods. The synthetic and extracted peptides showed identical behavior in a reverse-phase high-performance liquid chromatography system. SCP-3 and SCP-4 exhibited some significant inhibitory activity on the electrical stimulation-induced contractions of longitudinal muscle strip of guinea pig ileum, and SCP-5 showed some stimulating effect on the same preparation. The physiological significance of these purified peptides is being investigated.

Isolation and NH2-terminal sequence of a highly conserved human and porcine pituitary protein belonging to a new superfamily. Immunocytochemical localization in pars distalis and pars nervosa of the pituitary and in the supraoptic nucleus of the hypothalamus.

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.

Characterization of multiple forms of porcine anterior pituitary proopiomelanocortin amino-terminal glycopeptide.

Chromatography on a molecular sieve column of a preparation of porcine proopiomelanocortin N-terminal glycopeptide purified from anterior pituitary resulted in the isolation of three forms of the peptide with respective apparent Mr 21 000, 17 500, and 13 500 on polyacrylamide/sodium dodecyl sulfate gel. Determination of the amino acid composition of each peptide revealed that the form with a molecular weight of 17 500 corresponds to the 80 amino acid residue porcine N-terminal glycopeptide (PNT 1-80) previously characterized [Larivière, N., Seidah, N.G., & Chrétien, M. (1981) Int. J. Pept. Protein Res. 18, 487-491]. The forms with molecular weight of 21 000 and 13 500 correspond respectively to longer and shorter forms of the N-terminal glycopeptide. The high molecular weight form contains 107 amino acid residues. Sequencing of the fragments obtained after cleavage of the molecule with cyanogen bromide and Myxobacter Lys-C protease indicated that an extension of 27 amino acid residues is linked to PNT 1-80 through a -Lys-Arg-sequence. The sequence of the extension is reported. The low molecular weight form corresponds to the first 61 residues of PNT 1-80. Pronase digestion of the peptide and dansylation of the digest revealed the presence of a residue of phenylalanine amide at position 61. A general model for the maturation of the N-terminal glycopeptide of proopiomelanocortin in porcine anterior pituitary is presented.

The primary structure of human beta-lipotropin. Further peptide sequencing resolves the controversy and suggests the existence of only one human beta-lipotropin.

Human beta-lipotropin isolated in Hungary from frozen pituitary glands was purified by high-performance liquid chromatography in Canada. The amino acid sequence of the first 30 residues was determined. Trypsin, trypsin/papain, and trypsin/thermolysin fragments were obtained for the disputed region containing residues 9-25 of beta-lipotropin. Their amino acid composition and sequence established beyond doubt that only one human beta-lipotropin sequence is present. These results suggest the presence of only one gene coding for human pro-opiomelanocortin, the precursor of adrenocorticotropin and beta-endorphin and resolve the controversy over the sequence of human beta-lipotropin.

Isolation and NH2-terminal sequence of a novel porcine anterior pituitary polypeptide. Homology to proinsulin, secretin and Rous sarcoma virus transforming protein TVFV60.

An Mr 21 000 polypeptide, designated APPG, has been purified by reverse-phase, high-performance liquid chromatography (RP-HPLC), from acid extracts of porcine anterior pituitary glands. This acidic protein possesses an isoelectric point of 4.9. Amino acid analysis shows that it is not a glycoprotein and estimates it to contain about 173 amino acids. NH2-terminal sequence analysis allowed the determination of the first 50 residues unambiguously. A computer data bank search using a mutation data matrix and comparison with 269 012 protein segments indicated that this is a novel polypeptide sequence. However, this search revealed suggestive sequence homologies to a number of peptides of known sequence, including duck proinsulin (30%), Rous sarcoma virus transforming protein TVFV60 (24%) and pig secretin (26%).