Purification and characterization of a ribonuclease from human spleen. Immunological and enzymological comparison with nonsecretory ribonuclease from human urine

A ribonuclease has been isolated from human spleen (RNase HS) by means of acid extraction, ammonium sulphate fractionation, successive column chromatographies on CM-cellulose, heparin-actigel, and poly(G)-agarose, and double gel-filtration on Sephadex G-75. The purified preparation was homogeneous as judged by SDS/PAGE. RNase HS was found to be a glycoprotein, containing three fucose, one mannose and five glucosamine residues/molecule, with a molecular mass of 17 kDa as determined by both SDS/PAGE and gel filtration. The catalytic properties and structural features, including its amino acid composition and the amino acid sequence of the N-terminal 35 residues, indicated that the enzyme was strictly related to nonsecretory RNase isolated from human urine and liver. In particular, the amino acid sequence of the N-terminal was identical with that of urine nonsecretory RNase and eosinophil-derived neurotoxin. Furthermore, analyses using three different antibodies specific to RNase HS, urine nonsecretory RNase and urine secretory RNase, indicated that RNase HS was not immunologically distinguishable from urine nonsecretory RNase, but clearly so from urine secretory RNase. However, the carbohydrate compositions of RNase HS and urine nonsecretory RNase were found to differ. It therefore remains to be resolved whether or not the tissue of origin of nonsecretory RNase in urine is the spleen.     

Characterization of a unique nonsecretory ribonuclease from urine of pregnant women.

We have reported previously [Sakakibara, et al. (1991) Chem. Pharm. Bull. 39, 146-149] that a protein purified from a partially purified pharmaceutical preparation of human chorionic gonadotropin (a urinary protein preparation from pregnant women) is a unique nonsecretory ribonuclease (RNase)-like protein on the basis of its amino terminal sequence homology. We purified the protein further from the same materials by gel filtration and reversed-phase column chromatographies with RNase activity as an index. The purified protein was designated RNase UpI-2. The catalytic activity and its sensitivity to inhibition by divalent cations suggest that the protein is related to nonsecretory RNase. The estimated molecular weight of RNase UpI-2 (38 kDa) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was significantly higher than that of urinary nonsecretory RNases (13 to 19 kDa) reported so far. After trifluoromethanesulfonic acid treatment, the molecular weight of RNase UpI-2 was reduced and approached that of nonsecretory RNase, which indicated that the protein contains a significant amount of carbohydrate (approximately 50%). RNase UpI-2 was immunoreactive with antibodies to a nonsecretory RNase, RNAase 1 [Yasuda et al. (1988) Biochim. Biophys. Acta 965, 185-194]. By immunoblot analysis of the protein freshly prepared from various urine samples, it was shown that a considerable amount of RNase UpI-2 is present in urine of pregnant women, but only a trace of RNase UpI-2, if any, was detected in urine of nonpregnant women and men. These results suggest the possibility that RNase UpI-2 may have been formed via a specific protein modification in pregnant women.     

Amino acid sequence of ribonuclease Ap1 from Aspergillus pallidus

The complete amino acid sequence of an extracellular guanyl-specific RNase from Aspergillus pallidus fungi has been established. The RNase contains 104 amino acid residues (Mr 11,029). Its primary structure was analyzed basing on the automated Edman degradation of the carboxymethylated RNase followed by tryptic digestion and sequencing of the resultant hydrolysate. An additional structural information was obtained by means of the automatic sequencing of the cyanogen bromide peptide mixture and by studying the kinetics of the RNase's digestion with carboxypeptidase Y.     

Amino acid sequence determination of guanyl-specific ribonuclease Sa from Streptomyces aureofaciens

Using automated Edman degradation of two nonfractionated peptide mixtures of tryptic and staphylococcal protease digests of the protein, the complete amino acid sequence of the guanyl-specific ribonuclease Sa from Streptomyces aureofaciens was established. Ribonuclease Sa contains 96 amino acid residues (Mr 10,566). A 50% sequence homology of ribonuclease Sa to the guanyl-specific ribonuclease St from S. erythreus was found.     

Amino acid sequence and S-S bonds of Penicillium brevicompactum guanyl-specific ribonuclease

The primary structure of Penicillium brevicompactum guanyl-specific RNase was determined. The enzyme consists of 102 amino acid residues, Mr 10801. The 4 cysteine residues of the RNase are linked in pairs by disulfide bonds: Cys2-Cys10, Cys6-Cys101. P. brevicompactum RNase structure is similar to RNase T1; the degree of homology is 66%.     

Amino acid sequence of the human fibronectin receptor

The amino acid sequence deduced from cDNA of the human placental fibronectin receptor is reported. The receptor is composed of two subunits: an alpha subunit of 1,008 amino acids which is processed into two polypeptides disulfide bonded to one another, and a beta subunit of 778 amino acids. Each subunit has near its COOH terminus a hydrophobic segment. This and other sequence features suggest a structure for the receptor in which the hydrophobic segments serve as transmembrane domains anchoring each subunit to the membrane and dividing each into a large ectodomain and a short cytoplasmic domain. The alpha subunit ectodomain has five sequence elements homologous to consensus Ca2+-binding sites of several calcium-binding proteins, and the beta subunit contains a fourfold repeat strikingly rich in cysteine. The alpha subunit sequence is 46% homologous to the alpha subunit of the vitronectin receptor. The beta subunit is 44% homologous to the human platelet adhesion receptor subunit IIIa and 47% homologous to a leukocyte adhesion receptor beta subunit. The high degree of homology (85%) of the beta subunit with one of the polypeptides of a chicken adhesion receptor complex referred to as integrin complex strongly suggests that the latter polypeptide is the chicken homologue of the fibronectin receptor beta subunit. These receptor subunit homologies define a superfamily of adhesion receptors. The availability of the entire protein sequence for the fibronectin receptor will facilitate studies on the functions of these receptors.     

Amino acid sequence of human tumor derived angiogenin

The amino acid sequence and disulfide bond pairing of human tumor derived angiogenin, the first tumor angiogenesis factor to be isolated in pure form from human sources, have been determined by conventional sequencing techniques adapted and applied to nanomole and subnanomole levels of material. Angiogenin, obtained from conditioned media of a human colonic adenocarcinoma cell line, is a single-chain protein consisting of 123 amino acids with the following sequences: less than Glu1-Asp-Asn-Ser-Arg-Tyr-Thr-His- Phe-Leu-Thr-Gln-His-Tyr-Asp15-Ala-Lys-Pro-Gln-Gly-Arg-Asp-Asp- Arg-Tyr-Cys-Glu-Ser-Ile-Met30- Arg-Arg-Arg-Gly-Leu-Thr-Ser-Pro-Cys-Lys-Asp-Ile-Asn-Thr- Phe45-Ile-His-Gly-Asn-Lys-Arg-Ser -Ile-Lys-Ala-Ile-Cys-Glu-Asn-Lys60-Asn-Gly-Asn-Pro-His-Arg-Glu-Asn -Leu-Arg-Ile -Ser-Lys-Ser-Ser75 -Phe-Gln-Val-Thr-Thr-Cys-Lys-Leu-His-Gly-Gly-Ser-Pro-Trp-Pro90-Pro -Cys-Gln-Tyr -Arg-Ala-Thr-Ala -Gly-Phe-Arg-Asn-Val-Val-Val105-Ala-Cys-Glu-Asn-Gly-Leu-Pro-Val- His-Leu-Asp-Gln-Ser-Ile-Phe120-Arg-Arg-Pro123-OH. Three disulfide bonds link the half-cystinyl residues 26-81, 39-92, and 57-107. The sequence is homologous to that of the pancreatic ribonucleases with 35% identity and many of the remaining residues conservatively replaced. Similarities are especially apparent around the major active-site residues His-12, Lys-41, and His-119 of ribonuclease which are conserved as are three of the four disulfide bonds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid sequence of human liver cathepsin B

The complete amino acid sequence of cathepsin B (EC 3.4.22.1) from human liver was determined. The 252-residue sequence was obtained by automated solid-phase Edman degradation of the light and heavy chain resulting from limited proteolysis of the single-chain enzyme and of fragments produced by cyanogen bromide and enzymatic cleavage of the heavy chain. Human liver cathepsin B has 83.7% identical residues with the corresponding enzyme from rat liver. Comparison of both mammalian cathepsin B sequences with the sequence of papain provides further evidence that lysosomal and plant cysteine proteinases have evolved from a common ancestor and share a similar catalytic mechanism.     

Amino acid sequence of human von Willebrand factor

The complete amino acid sequence of human von Willebrand factor (vWF) is presented. Most of the sequence was determined by analysis of the S-carboxymethylated protein. Some overlaps not provided by the protein sequence analysis were obtained from the sequence predicted by the nucleotide sequence of a cDNA clone [Sadler, J.E., Shelton-Inloes, B.B., Sorace, J., Harlan, M., Titani, K., & Davie, E.W. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6391-6398]. The protein is composed of 2050 amino acid residues containing 12 Asn-linked and 10 Thr/Ser-linked oligosaccharide chains. One of the carbohydrate chains is linked to an Asn residue in the sequence Asn-Ser-Cys rather than the usual Asn-X-Ser/Thr sequence. The sequence of von Willebrand factor includes several regions bearing evidence of internal gene duplication of ancestral sequences. The protein also contains the tetrapeptide sequence Arg-Gly-Asp-Ser (at residues 1744-1747), which may be a cell attachment site, as in fibronectin. The amino- and carboxyl-terminal regions of the molecule contain clusters of half-cystinyl residues. The sequence is unique except for some homology to human complement factor B.     

Amino acid sequence of the ribonuclease inhibitor from porcine liver reveals the presence of leucine-rich repeats

The primary structure of the ribonuclease inhibitor from pig liver has been determined by amino acid sequence analysis. The N alpha-acetylated polypeptide chain of 456 amino acids consists of 15 homologous leucine-rich repeats, characterized by leucyl residues at constant positions. Two types of alternating repeats occur, 29 (A) and 28 (B) residues long. The degree of identity between repeats of a given type ranged from 25 to 60%. Only one deletion in the B-repeat was necessary to perfectly align the leucyl residues between the two repeats. Leucine-rich repeats have previously been found in four membrane-bound proteins and one extracellular protein, and their amphiphilic character suggested that they could be involved in membrane binding. Ribonuclease inhibitor is the first example of a cytoplasmic protein containing this type of repeat. It seems likely, therefore, that leucine-rich repeats can have functions other than forming membrane binding structures.     

Sequence of human eosinophil-derived neurotoxin cDNA: identity of deduced amino acid sequence with human nonsecretory ribonucleases

Several clones of human eosinophil-derived neurotoxin (EDN) cDNA have been isolated from a lambda gt10 cDNA library prepared from mRNA derived from noninduced HL-60 cells. The amino acid (aa) sequence deduced from the coding sequence of the EDN cDNA is identical to the aa sequence of urinary nonsecretory RNase. Comparison of the aa and/or nucleotide (nt) sequences of EDN and other proteins possessing ribonucleolytic activity, namely bovine seminal RNase, human and rat pancreatic RNases, eosinophil cationic protein (ECP), and human angiogenin, shows extensive identity at half-cystine residues and at aa of active sites. Differences in aa sequences at the active sites are often the result of single nt changes in the codons. The data presented here support the concept of a RNase gene superfamily containing secretory and nonsecretory RNases, angiogenin, EDN and ECP.     

Amino acid sequence of the active site of human pseudocholinesterase

The usualE ₁ ^u and atypicalE ₁ ^a human pseudocholinesterases (acylocholine acylhydrolase, EC 3.1.1.8) were purified to homogeneity. The active-site serine residue was conjugated with diisopropyl fluorophosphate and digested with trypsin. The tryptic peptide containing the active site was isolated by gel filtration followed by two-dimensional paper chromatography and electrophoresis. The amino acid sequence of the active site peptide obtained from the usualE ₁ ^u enzyme was found to be Gly-Glu-Ser-Ala-Gly-Ala-Ser-Ala-Val-Ser-Leu. A remarkable structural homology exists between the human and the horse enzymes in their active sites. From the difference in electrophoretic mobility of the active-site peptides obtained from the usual and atypical enzymes, the probable structure of the atypical human enzyme was deduced as Gly-His-Ser-Ala-Gly-Ala-Ser-Ala-Val-Ser-Leu.     

Amino acid sequence of human acidic fibroblast growth factor

The complete amino acid sequence of human brain acidic fibroblast growth factor (aFGF) has been established. Human aFGF consists of 140 amino acids and is highly homologous to bovine aFGF (11 amino acid replacements). Results from experiments involving alkylation of cysteine residues are compatible with the possibilities that in aFGF all three cysteines exist as free sulfhydryls, or alternatively, that a disulfide bridge is present but cannot be identified due to disulfide scrambling caused by the SH group of the remaining cysteine. A potential glycosylation site Asn114-Gly115-Ser116 is present in aFGF but the mitogen does not bind to lectins suggesting that it may not be glycosylated.     

Amino acid sequence of human erythrocyte carbonic anhydrase C

Human carbonic anhydrase C is the high-specific-activity form of the enzyme found in human red cells. A proposal for the primary structure of this enzyme is presented. Trypsin-catalyzed hydrolysis was restricted to the arginyl bonds of the protein by blocking the lysyl side chains by amidination. The arginine fragments were isolated and their amino acid sequences determined. The sequence contains 259 amino acid residues in a single polypeptide chain devoid of disulfide bonds. The structure obtained should be adequate to permit a detailed interpretation of the 2.0 Å X-ray crystallographic model of the enzyme previously determined. The primary structures of the human B and C enzymes have about 60% identities.