Discontinuities in the topography of alcohol dehydrogenase-sodium dodecyl sulphate complexes revealed by mutagenesis and proteolysis.

Experiments utilizing proteolytic mapping of maize Alcohol dehydrogenase-1 protein (EC 1.1.1.1; ADH) showed that, in the presence of sodium dodecyl sulphate (SDS), two discrete areas of the protein molecule were hypersensitive to digestion with Staphylococcus aureus V8 proteinase. These areas were mapped to points 11 and 27 kDa along the 41 kDa polypeptide. Furthermore, ADH1 proteins isolated from the ethyl methanesulphonate-induced mutants U725 and W586 showed both a change in electrophoretic mobility in SDS gels, and an altered V8 proteinase digestion pattern. Protein from U725 migrated in SDS gels as though it was 2 kDa smaller than wild-type ADH protein and lacked the 11 kDa cleavage site. Protein from W586 lacked the 30 kDa cleavage site and migrated as if it was 3 kDa smaller than wild type. The possible relationships between proteinase cleavage sites, mutations and SDS gel mobilities are discussed.     

Activation by hemoglobin of the Ca2+-requiring neutral proteinase of human erythrocytes: structural requirements

The proenzyme form of the Ca2+-requiring neutral proteinase of human erythrocytes (procalpain) is converted to the active proteinase (calpain) by low concentrations of Ca2+ in the presence of appropriate substrates such as beta-hemoglobin or heme-free beta-globin chains. Modification of these substrates by limited proteolysis with calpain abolishes their ability to promote the conversion of procalpain. A similar requirement for the presence of unmodified beta-hemoglobin or heme-free beta-globin chains is observed for the autocatalytic inactivation of calpain. The conversion of procalpain to calpain is accompanied by a small decrease in the molecular mass of the catalytic subunit, from 80 kDa to 75 kDa; however, the activation is not accelerated by the addition of a small quantity of calpain. The autocatalytic inactivation of active CANP is related to the disappearance of the 75 kDa subunit and the formation of smaller peptide fragments.     

Exogenous adenosine 3': 5'-monophosphate can release yeast from catabolite repression.

A new simple fast and reproducible purification procedure for the proteinase from rat liver mitochondria has been worked out. The specificity of cleavage of peptide bonds in glucagon, oxidized A and B chains of insulin and yeast proteinase B inhibitor by the proteinase of the inner mitochondrial membrane has been studied. The proteinase hydrolyzed three peptide bonds in glucagon, Tyr (13) - Leu (14), Trp (25) - Leu (26) and Phe (22) - Val (23) (minor cleavage site); none in the insulin A chain; one in the B chain of insulin, Tyr (16) - Leu (17); and three in the yeast proteinase B inhibitor, Phe (4) - Ile (5), Phe (20) - Leu (21) and Tyr (41) - Thr (42) (minor cleavage site).Thus, the mitochondrial proteinase cleaves peptide bonds at the carboxyl site of an aromatic amino acid and the amino site of a leucine, isoleucine, threonine or valine. The comparison with chymotrypsin A shows that the mitochondrial proteinase cleaves peptide bonds in a more restricted manner.     

The amino acid sequence and reactive site of a single-headed trypsin inhibitor from wheat endosperm

The sequence of a trypsin inhibitor, isolated from wheat endosperm, is reported. The primary structure was obtained by automatic sequence analysis of the S-alkylated protein and of purified peptides derived from chemical cleavage by cyanogen bromide and digestion withStaphylococcus aureus V8 protease. This protein, named wheat trypsin inhibitor (WTI), which is comprised of a total of 71 amino acid residues, has 12 cysteines, all involved in disulfide bridges. The primary site of interaction (reactive site) with bovine trypsin has been identified as the dipeptide arginyl-methionyl at positions 19 and 20. WTI has a high degree of sequence identity with a number of serine proteinase inhibitors isolated from both cereal and leguminous plants. On the basis of the findings presented, this protein has been classified as a single-headed trypsin inhibitor of Bowman-Birk type.     

Properties and specificity of a second metal chelator-sensitive proteinase in the keratinolytic larvae of the webbing clothes moth.

The properties of a second metal chelator-sensitive proteinase (metalloproteinase 2) from the larvae of the webbing clothes moth, Tineola bisselliella, have been studied. The pH optimum for casein digestion was 9-4 and the enzyme showed high stability between pH 8 and 11, but very poor stability at acid pH. The proteinase was inhibited by EDTA, but not by an EDTA-calcium complex. EDTA inhibition could be reversed by addition of a slight excess of calcium or zinc ions. The cleavage specificity of metalloproteinase 2 against the A and B chains of S-carboxymethyl insulin was almost identical to that found previously for metalloproteinase 1.     

The coordinate synthesis and cotranslational assembly of type I procollagen

Nascent polysome-associated type I procollagen pro-alpha-chains isolated from chick embryo tendon fibroblasts were examined for their proteinase resistance. The distribution of chain sizes and their proteinase resistance were also determined following chain elongation in an in vitro readout system in the absence of chain initiation factors. Chains were labeled with [14C]proline in the cells and with [3H]proline in the readout system. Differences in the ratios of 14C to 3H in the double-labeled nascent chains before and after chymotryptic digestion, determined by slicing and counting polyacrylamide gels after electrophoresis, permitted analysis of the relative stabilities of in vivo and in vitro elongated portions of the chains. In confirmation of earlier work, the polysome-bound nascent procollagen contained chymotrypsin, chymotrypsin plus trypsin, and pepsin-resistant alpha-chain size components. The readout system data showed that the full length chains produced in the cell were more resistant to digestion than the fully elongated readout-completed chains. The protease resistance of the chains was taken to indicate the registration of the chains prior to the induction of helix formation during the isolation procedure. These data support the model in which chain selection and folding are facilitated by the organization of the attachment of the ribosomes to the endoplasmic reticulum surface.     

Isolation and properties of an elastase-like proteinase from horse blood leucocytes

The rapid, two steps method of purification of an elastase-like proteinase from cytoplasmic granules of horse leucocytes is described. This enzyme called the proteinase 1 is released easily from isolated granules in the low ionic strength solutions in opposite to the other two molecular forms of which one differs slightly in isoelectric point from the other. The enzyme is a typical neutral proteinase of a broad substrate specificity and wide pH optimum. In a physiological conditions the enzyme is built of two polypeptide chains of molecular weight about 30000 and 20000, respectively.     

A comparative study of the structure of egg-white riboflavin binding protein from the domestic fowl and Japanese quail.

1. The riboflavin binding proteins from domestic fowl and Japanese quail have been isolated and their structures compared by circular dichroism, fluorescence and peptide mapping. 2. The two proteins have similar secondary structures, but differ in their tertiary structures as reflected in the environments of aromatic amino acid side chains. 3. Differences in amino acid sequence between the proteins are indicated by the digestion patterns obtained with thermolysin, chymotrypsin and V8 proteinase from Staphylococcus aureus. Both proteins are resistant to digestion by trypsin.     

Multiple sequence-specific DNA binding activities are eluted from chicken nuclei at low ionic strengths.

DNA sequence-specific binding proteins eluted from chicken erythrocyte and thymus nuclei, and fractionated as described by Emerson and Felsenfeld (19), have been investigated by filter binding and footprint analyses. The erythrocyte nuclear protein fraction specifically binds to at least two sites within the 5' flanking chromatin hypersensitive site of the chicken beta A-globin gene, and to a site 5' to the human beta-globin gene. The major chicken beta A globin gene binding site [G)18CGGGTGG) and the human beta-globin gene binding site [TA)6(T)8C(T)4) occur at or near sequences which are hypersensitive to S1 nuclease cleavage in supercoiled plasmids. Downstream, the second chicken beta A-globin gene binding site includes the beta-globin gene CACCC consensus sequence. Filter binding studies also show other sequence specific binding activities to human N-ras and human (but not chicken) c-myc gene sequences.     

Oxidized lipoproteins inhibit surfactant phosphatidylcholine synthesis via calpain-mediated cleavage of CTP:phosphocholine cytidylyltransferase

We investigated effects of pro-atherogenic oxidized lipoproteins on phosphatidylcholine (PtdCho) biosynthesis in murine lung epithelial cells (MLE-12). Cells surface-bound, internalized, and degraded oxidized low density lipoproteins (Ox-LDL). Ox-LDL significantly reduced [3H]choline incorporation into PtdCho in cells by selectively inhibiting the activity of the rate-regulatory enzyme, CTP:phosphocholine cytdylyltransferase (CCT). Ox-LDL coordinately increased the cellular turnover of CCTalpha protein as determined by [35S]methionine pulse-chase studies by inducing the calcium-activated proteinase, calpain. Forced expression of calpain or exposure of cells to the calcium ionophore, A23187, increased CCTalpha degradation, whereas overexpression of the endogenous calpain inhibitor, calpastatin, attenuated Ox-LDL-induced CCTalpha degradation. The effects of Ox-LDL on CCTalpha breakdown were attenuated in calpain-deficient cells. In vitro calpain digestion of CCTalpha isolated from cells transfected with truncated or internal deletion mutants indicated multiple cleavage sites within the CCTalpha primary structure, leading to the generation of a 26-kDa (p26) fragment. Calpain hydrolysis of purified CCTalpha generated p26, which upon NH2-terminal sequencing localized a calpain attack site within the CCTalpha amino terminus. Expression of a CCTalpha mutant where the amino-terminal cleavage site and a putative carboxyl-terminal hydrolysis region were modified resulted in an enzyme that was significantly less sensitive to proteolytic cleavage and restored the ability of cells to synthesize surfactant PtdCho after Ox-LDL treatment. Thus, these results provide a critical link between proatherogenic lipoproteins and their metabolic target, CCTalpha, resulting in impaired surfactant metabolism.     

Protein chemical characterization of Mucor pusillus aspartic proteinase. Amino acid sequence homology with the other aspartic proteinases, disulfide bond arrangement and site of carbohydrate attachment

The amino acid sequence of Mucor pusillus aspartic proteinase was determined by analysis of fragments obtained from cleavage of the enzyme by CNBr and limited tryptic digestion. The proteinase is a single polypeptide chain protein containing 361 amino acid residues, cross-linked by two disulfide bonds. A sugar moiety composed of two GlcNAc residues and four neutral sugar residues is asparagine-linked to the chain. The sequence of M. pusillus proteinase is highly homologous with the M. miehei proteinase (83% identity). The homology with other aspartic proteinases is low (22-24%) and indicates that the Mucor proteinases diverged at an early evolutionary phase. The most conservative regions of the molecule are those involved in catalysis and forming the binding cleft and the core region of the molecule.     

Location of the abi, col and imm genes on pHU011, a colicin Ib plasmid derivative

A cleavage site map of pHU011, a derivative of the colicin Ib plasmid containing the complete SalI-B fragment ligated to pBR322, has been determined. Sites of cleavage by PstI were determined using the Smith and Birnstiel [Nucl. Acids Res. 3 (1976) 2387-2398] method of mapping, whereas those for XbaI, XhoI, and HindIII were determined by double digestions or digestion of isolated fragments. In addition, the sites of the abi gene, which causes the abortive infection by T5 bacteriophage, and of the colicin (col) gene have been determined. The results indicate that these genes are not contiguous.     

A novel proteinase from human pancreas

A cholesterol-binding protein was previously isolated from human pancreas [Sziegoleit (1982) Biochem. J. 207, 573-582] and shown to consist of a single polypeptide chain with an apparent Mr of 28 000 and an isoelectric point of pH 4.9. In further investigations, a proteolytic activity was observed to be present in preparations of this protein. The enzyme activity was not dissociable from the cholesterol-binding protein. It decreased in the presence of sodium dodecyl sulphate or urea parallel to degradation of the protein, indicating autodegradation in the presence of these denaturants. Glucagon digestion studies indicated the carbonyl bond of alanine to be a favoured site of the enzymic cleavage. The proteinase was inactive against chromogenic substrates relatively specific for elastase, trypsin and chymotrypsin, but was found to cleave benzyloxycarbonylalanine p-nitrophenyl ester efficiently. The enzyme was inactivated by phenylmethanesulphonyl fluoride and was thus classified as a serine proteinase. Autoradiographic studies demonstrated binding to serum alpha 1-antitrypsin and alpha 2-macroglobulin in a similar manner to that observed with other pancreatic endo-proteinases. The collective results indicate that the isolated protein, provisionally named 'cholesterol-binding pancreatic proteinase', is a novel proteinase of the human pancreas. Quantitative measurements indicate that it comprises 4-6% of total protein in pancreatic secretions.     

Cleavage of Type II and III collagens with mammalian collagenase: site of cleavage and primary structure at the NH2-terminal portion of the smaller fragment released from both collagens.

Collagenase cleavage of human Type II and III collagens has been studied using a highly purified preparation of rabbit tumor collagenase. Progress of the reactions in solution was followed by viscometry and the results indicated that under the conditions employed Type III collagen molecules were cleaved at approximately five times the rate of Type II molecules. Cleavage products of the reactions were isolated in denatured form by agarose molecular sieve chromatography. The molecular weights and amino acid compositions of the products demonstrated that Type II and III molecules had been cleaved at the characteristic three-quarter, one-quarter locus, giving rise to a large fragment derived from the NH2-terminal portion of the molecule and a smaller fragment representing the COOH-terminal region. The amino acid sequence at the NH2-terminal portion of the smaller fragment derived from Type II collagen was determined to be Ile-Ala-Gly-Gln-Arg, and the corresponding region from Type III collagen was found to have the sequence Leu-Ala Gly-Leu-Arg. These sequences for alpha1(II) and alpha1(III) chains adjacent to the site of collagenase cleavage along with previous data for alpha1(I) and alpha2 chains indicate that the minimum specific sequence required for collagenase cleavage is Gly-Ile-Ala or Gly-Leu-Ala. Inspection of the available sequence data for collagen alpha chains indicates that the latter sequences are found in at least three additional locations at which collagenase cleavage does not occur. Each of the sequences which are apparently not substrates for collagenase, however, are followed by a Gly-X-Hyp sequence. We suggest, then, that a minimum of five residues in collagen alpha chains COOH-terminal to the cleavage site comprise the substrate recognition site.     

Supercoil-dependent recognition of specific DNA sites by chromosomal protein HMG 2

The ability of the chromosomal high mobility group protein HMG 2 to recognize supercoil-dependent structures within the chicken adult beta-globin gene was investigated by examining its ability to protect such sites from digestion by S1 nuclease. Low molar ratios of HMG 2 were found to be sufficient for complete inhibition of S1 cleavage of a supercoiled plasmid containing the globin gene. Furthermore, HMG 2 protected an S1 cleavage site within the 5'-flanking region of the globin gene, in preference to a palindromic S1 site within the plasmid vector.     

The amino-acid sequence of multiple lectins of the acorn barnacle Megabalanus rosa and its homology with animal lectins

The amino-acid sequence of a lectin isolated from the coelomic fluid of the acorn barnacle Megabalanus rosa has been determined. The lectin (Mr 140,000) is a multimeric protein whose subunit consists of 173 amino acids and one carbohydrate chain attached to Asn-39. The amino-acid sequence was determined by the manual sequencing of peptides derived from the protein by digestion with Staphylococcus aureus V8 proteinase, lysine endopeptidase and chymotrypsin, as well as fragments produced by cleavage with cyanogen bromide. The amino-acid sequence of the lectin was compared with the sequence of one (Mr 64,000) of the multiple lectins of M. rosa. They are distinct molecules in spite of a significant homology in their amino-acid sequences. The amino-acid sequence includes some regions homologous to those in other invertebrate lectins, such as sea urchin and flesh fly lectins, and vertebrate lectins. This is the first report to show the amino-acid sequence of multiple lectins isolated from an invertebrate.     

Proteinases in human polymorphonuclear leukocytes. Purification and characterization of an enzyme which cleaves denatured collagen and a synthetic peptide with a Gly-Ile sequence

Polymorphonuclear leukocytes have been shown to contain proteolytic enzymes which are capable of degrading connective tissue proteins such as native collagen. In this study, proteolytic enzymes were extracted from human polymorphonuclear leukocytes and a neutral proteinase was extensively purified and characterized. The activity of this enzyme was monitored by degradation of denatured [ 3H ]proline-labeled type I collagen or by cleavage of a synthetic dinitrophenylated peptide with a Gly-Ile sequence. The enzyme was readily separated from leukocyte collagenase by concanavalin-A--Sepharose affinity chromatography and further purified by QAE-Sephadex ion-exchange chromatography and gel filtration on Sephacryl S-200. The purified enzyme had a molecular weight of approximately 105000, its pH optimum was about 7.8, and it was inhibited by Na2EDTA and dithiothreitol, but not by fetal calf serum. The enzyme degraded genetically distinct type I, II, III, IV and V collagens, when in a non-helical form, but not when in native triple-helical conformation. Dansyl-monitored end-group analyses, combined with digestion by carboxypeptidase A, indicated that the enzyme cleaved denaturated type I collagen at Gly-Xaa sequences, in which Xaa can be leucine, isoleucine, valine, phenylalanine, lysine, or methionine. Thus, the purified enzyme referred to here as Gly-Xaa proteinase, is a neutral proteinase, which may be of importance in inflammatory disease processes by degrading further collagen peptides which have been rendered non-helical as a result of collagenase cleavage.     

Autolytic enzyme system of Streptococcus faecalis. V. Nature of the autolysin-cell wall complex and its relationship to properties of the autolytic enzyme of Streptococcus faecalis

Cell walls from exponential-phase cultures of Streptococcus faecalis ATCC 9790 contain an autolysin (a beta-N-acetylmuramide glycanhydrolase, E.C. 3.2.1.17) which has been isolated from trypsin-speeded wall autolysates. The autolysin, which was excluded from Bio-Gel P-60, was further fractionated by diethylaminoethyl (DEAE)-cellulose chromatography or filtration on Bio-Gel P-200. After DEAE-cellulose chromatography, which removed most of the wall polysaccharide, autolysin activity was extremely labile and was rapidly lost at -20 C, even in the presence of albumin. The P-60-excluded enzyme was rapidly bound by walls at both 37 C (50% bound in about 1 min) and 0 C (50% bound in less than 4 min). Wall-bound autolysin could not be removed by 1.0 m ammonium acetate (pH 6.9). Autolysin was also bound by walls that had been extracted with 10% trichloroacetic acid or treated with 0.01 n periodate, suggesting that the nonpeptidoglycan wall polymers are not important for binding. Wall-bound autolysin was more stable than the soluble enzyme to proteinase digestion, acetone (40%), 8 m urea (at 0 C), or to inactivation at 56 C. Two bacterial neutral proteinases (which do not hydrolyze ester bonds) activated latent wall-bound autolysin, suggesting that activation results from the cleavage of one or more peptide bonds. The group A streptococcal proteinase activated latent autolysin but differed from the other proteinases in that it did not inactivate soluble autolysin. The results suggest that the autolysin is not covalently linked to the wall. The high affinity of the walls for the autolysin appears to be responsible for the firm, not easily reversed binding.     

Analysis of bacteriophage phi X174 gene A protein-mediated termination and reinitiation of phi X DNA synthesis. II. Structural characterization of the covalent phi X A protein-DNA complex

In the preceeding paper (Brown, D. R., Roth, M. J., Reinberg, D., and Hurwitz, J. (1984) J. Biol. Chem. 259, 10545-10555), it was shown that following bacteriophage phi X174 (phi X) DNA synthesis in vitro using purified proteins, the phi X A protein could be detected covalently linked to nascent 32P-labeled DNA. This phi X A protein-[32P]DNA complex was the product of the reinitiation reaction. The phi X A protein-[32P]DNA complex could be trapped as a protein-32P-oligonucleotide complex by the inclusion of ddGTP in reaction mixtures. In this report, the structure of the phi X A protein-32P-oligonucleotide complex has been analyzed. The DNA sequence of the oligonucleotide bound to the phi X A protein has been determined and shown to be homologous to the phi X (+) strand sequence immediately adjacent (3') to the replication origin. The phi X A protein was directly linked to the 5' position of a dAMP residue of the oligonucleotide; this residue corresponded to position 4306 of the phi X DNA sequence. The phi X A protein-32P-oligonucleotide complex was exhaustively digested with either trypsin or proteinase K and the 32P-labeled proteolytic fragments were analyzed. Each protease yielded two different 32P-labeled peptides in approximately equimolar ratios. The two 32P-labeled peptides formed after digestion with trypsin (designated T1 and T2) and with proteinase K (designated PK1 and PK2) were isolated and characterized. Digestion of peptide T1 with proteinase K yielded a product which co-migrated with peptide PK2. In contrast, peptide T2 was unaffected by digestion with proteinase K. These results suggest that the phi X A protein contains two active sites that are each capable of binding covalently to DNA. The peptide-mononucleotide complexes T1-[32P]pdA and T2-[32P]pdA were isolated and subjected to acid hydrolysis in 6.0 N HCl. In each case, the major 32P-labeled products were identified as [32P] phosphotyrosine and [32P]Pi. This indicates that each active site of the phi X A protein participates in a phosphodiester linkage between a tyrosyl moiety of the protein and the 5' position of dAMP.