Purification and properties of a proteolytic enzyme from French beans.

1. A proteolytic enzyme with some features of a carboxypeptidase has been purified some 1180-fold from the sap of French beans (Phaseolus vulgaris var. Prince). A bright blue protein, plastocyanin, was separated from the enzyme by DEAE-cellulose chromatography. 2. Unlike carboxypeptidase A or B of animal origin, there is no evidence that the enzyme is a metalloprotein. There was no stimulation of activity by a number of metal ions, reducing agents or 2-mercapto-ethanol. Neither EDTA nor 1,10-o-phenanthroline inhibited the enzyme. 3. The proteolytic enzyme from beans, readily soluble at neutral or slightly acidic pH values, has a pH optimum of pH5.6 for the hydrolysis of leucine from benzyloxy-carbonylglycyl-l-leucine. Solutions of the enzyme in 0.1m-sodium acetate, pH5.5, lose about 2% of their activity/week at 4 degrees . Virtually no loss of activity results after prolonged storage at -15 degrees . 4. Incubation of the bean enzyme with peptides indicates that the enzyme will release acidic, neutral and basic amino acid residues as well as proline, although adjacent acidic residues in a peptide appear to inhibit the enzyme. The possibility of endopeptidase activity in the purified preparation requires further examination.     

The structure,regulation, and function of human matrix metalloproteinase-13

Matrix metalloproteinase-13 (MMP-13) is a proteolytic enzyme that belongs to a large family of extracellular matrix-degrading endopeptidases that are characterized by a zinc-binding motif at their catalytic sites. MMP-13 has a key role in the MMP activation cascade and appears to be critical in bone metabolism and homeostasis. It also has an important role in tumor invasion and metastasis. This commentary provides a detailed overview of the regulatory mechanisms, structure, and function of human MMP-13 and highlights the key factors involved in the biology of this important molecule.     

The Structure,Regulation, and Function of Human Matrix Metalloproteinase-13

Matrix metalloproteinase-13 (MMP-13) is a proteolytic enzyme that belongs to a large family of extracellular matrix-degrading endopeptidases that are characterized by a zinc-binding motif at their catalytic sites. MMP-13 has a key role in the MMP activation cascade and appears to be critical in bone metabolism and homeostasis. It also has an important role in tumor invasion and metastasis. This commentary provides a detailed overview of the regulatory mechanisms, structure, and function of human MMP-13 and highlights the key factors involved in the biology of this important molecule.     

Marked increase in ascorbate oxidase protein in pumpkin callus by adding copper

Ascorbate oxidase from pumpkin (Cucurbita sp.) was purified from a commercially available preparation. A single polypeptide band with M_r 64,000 was detected after sodium dodecylsulfate-polyacrylamide gel electrophoresis of the purified enzyme. In double immunodiffusion tests, antiserum against the purified preparation formed a single precipitin line with the crude extract from pumpkin fruit tissue or the callus as well as with the purified preparation. Immunological blotting method showed that mol wt of ascorbate oxidase subunit in pumpkin callus was the same as that of the purified preparation. Analysis with the single radial immunodiffusion method showed that the increase in ascorbate oxidase activity during the growth of pumpkin callus correlated with an increase in the enzyme protein. Furthermore, enzyme protein in the callus grown in the presence of 10 micromolar CuSO₄ for 2 weeks was about eight times that grown in the presence of 0.1 micromolar CuSO₄. The synthesis of ascorbate oxidase in pumpkin callus may be induced by copper, a prosthetic metal of the enzyme, or copper may help stabilize the enzyme against proteolytic breakdown.     

Characterization of an exosite binding inhibitor of matrix metalloproteinase 13

Matrix metalloproteinase 13 (MMP13) is a key enzyme implicated in the degradation of the extracellular matrix in osteoarthritis. Clinical administration of broad spectrum MMP inhibitors such as marimastat has been implicated in severe musculo-skeletal side effects. Consequently, research has been focused on designing inhibitors that selectively inhibit MMP13, thereby circumventing musculo-skeletal toxicities. A series of pyrimidine dicarboxamides were recently shown to be highly selective inhibitors of MMP13 with a novel binding mode. We have applied a molecular ruler to this exosite by dual inhibition studies involving a potent dicarboxamide in the presence of two metal chelators of different sizes. A larger hydroxamate mimic overlaps and antagonizes binding of the dicarboxamide to the exosite whereas the much smaller acetohydroxamate synergizes with the dicarboxamide. These studies elucidate the steric requirement for compounds that fit exclusively into the active site, a mandate for generating highly selective MMP13 inhibitors.     

Experimental study of a new biological preparation for treating E. coli infection and Proteus dysbacteriosis in young infants

The action of a new biological preparation intended for the treatment of E. coli infection and Proteus dysbacteriosis in children has been studied in experiments on mice and suckling rabbits. The protective activity of anti-E. coli and anti-Proteus lactoglobulin has been found to exceed that of normal lactoglobulin 6- to 13-fold. When subjected to the action of proteolytic enzymes of the gastrointestinal tract, the preparation retains its preventive properties on the level of the native preparation, which confirms the possibility of the oral administration of the preparation in clinical practice without protecting it from the proteolytic action of the enzymes of the gastrointestinal tract.     

Mechanical forces-induced human osteoblasts differentiation involves MMP-2/MMP-13/MT1-MMP proteolytic cascade

Matrix metalloproteinase (MMP) family proteins play diverse roles in many aspects of cellular processes such as osteoblastic differentiation. Besides, mechanical forces that occur in 3D collagen gel promote the osteoblastic phenotype and accelerate matrix mineralization. Although MMPs have been involved in bone differentiation, the proteolytic cascades triggered by mechanical forces are still not well characterized. In this study, we have investigated the contribution of both proteolytic cascades, MMP-3/MMP-1 and MMP-2/MMP-13/MT1-MMP in the differentiation of human osteoblasts cultured in a floating type I collagen lattice (FL) versus an attached collagen lattice (AL). Compared to AL, contraction of human osteoblasts-populated FL led to a fast (1 day) induction of alkaline phosphatase (ALP), bone sialoprotein (BSP), osteoprotegerin (OPG), and Runx-2 expression. At day 4, osteocalcin (OC) overexpression preceded the formation of calcium-containing nodule formation as assessed by X-ray analyses. MMP-1 and MMP-3 were produced to similar extent by cells cultured in FL and AL, whereas contraction of collagen lattices triggered both mRNA overexpression of MMP-2, MMP-13, and MT1-MMP (i.e., MMP-14), and their activation as evidenced by Western blotting or zymographic analyses. Down-regulating MT1-MMP expression or activity either by siRNA transfection or supplementation of culture medium with TIMP-1 or TIMP-2 highlighted the contribution of that enzyme in OC, ALP, and OPG expression. MMP-2 and MMP-13 were more directly involved in BSP expression. So, these results suggest that the main proteolytic cascade, MMP-2/MMP-13/MT1-MMP, and more particularly, its initial regulator MT1-MMP is involved in osteoblast differentiation through mechanical forces.     

Proteolytic Enzyme Preparation from Pseudomonas fragi: Its Action on Pig Muscle

An extracellular preparation from Pseudomonas fragi with proteolytic enzyme activity was isolated, and its action on meat proteins and meat protein ultrastructure was studied. First, a suitable growth medium for proteolytic enzyme production was determined, and a method for partial purification of the proteolytically active fraction was developed. The enzyme preparation displayed optimal proteolytic activity at neutral pH and 35 C. Proteolytic activity was irreversibly lost by mild heat treatment. The enzyme preparation was tested for its ability to hydrolyze isolated pig muscle proteins. Myofibrillar protein was rapidly degraded, G-actin and myosin were broken down at a slower rate, and the sarcoplasmic proteins were least susceptible to hydrolysis. Electron micrographs of pork muscle showed that the proteolytic enzyme preparation caused a complete loss of dense material from the Z line. Similarities are discussed between the action of P. fragi extracellular proteolytic enzyme(s) on meat and normal bacterial spoilage of meat.     

Isolation and characterization of the lytic enzyme from Bacillus subtilis 797

The isolation procedure and some properties of the lytic enzyme produced by Bacillus subtilis 797 and capable of hydrolyzing the E. coli K-12 cells are described. Using hydrophobic chromatography on DNP-hexamethylene diamine Sepharose 4B and ion-exchange chromatography on DEAE-cellulose, a highly purified enzyme preparation with mol. weight of 28000, pI 8.2 has been obtained. The amino acid composition of the enzyme has been determined: Asp--37, Thr--17, Ser--34, Glu--15, Pro--14, Gly--17, Ala--36, Val--28, Met--4, Ile--11, Leu--17, Tyr--9, Phe--4, Lys--18, His--5, Arg--4. The enzyme is inhibited by a specific inhibitor of serine proteinases--benzylsulfonylfluoride, and is insensitive to EDTA and iodoacetic acid. The lytic enzyme has a proteolytic activity and splits the peptide substrate of bacterial serine proteinases--p-nitroanilide benzyloxycarbonyl-L-analyl-L-alanyl-L-leucine; the maxima for both activities lie within the pH range of 7.8-8.5. The lytic protease has the highest stability at pH 6-10. In some of its properties the enzyme is similar to serine proteinase from Bac. subtilis, i. e. subtilisins.     

Dienelactone hydrolase from Pseudomonas sp. strain B13.

Dienelactone hydrolase (EC 3.1.1.45) catalyzes the conversion of cis- or trans-4-carboxymethylenebut-2-en-4-olide (dienelactone) to maleylacetate. An approximately 24-fold purification from extracts of 3-chlorobenzoate-grown Pseudomonas sp. strain B13 yielded a homogeneous preparation of the enzyme. The purified enzyme crystallized readily and proved to be a monomer with a molecular weight of about 30,000. Each dienelactone hydrolase molecule contains two cysteinyl side chains. One of these was readily titrated by stoichiometric amounts of p-chloromercuribenzoate, resulting in inactivation of the enzyme; the inactivation could be reversed by the addition of dithiothreitol. The other cysteinyl side chain appeared to be protected in the native protein against chemical reaction with p-chloromercuribenzoate. The properties of sulfhydryl side chains in dienelactone hydrolase resembled those that have been characterized for bacterial 4-carboxymethylbut-3-en-4-olide (enol-lactone) hydrolases (EC 3.1.1.24), which also are monomers with molecular weights of about 30,000. The amino acid composition of the dienelactone hydrolase resembled the amino acid composition of enol-lactone hydrolase from Pseudomonas putida, and alignment of the NH2-terminal amino acid sequence of the dienelactone hydrolase with the corresponding sequence of an Acinetobacter calcoaceticus enol-lactone hydrolase revealed sequence identity at 8 of the 28 positions. These observations foster the hypothesis that the lactone hydrolases share a common ancestor. The lactone hydrolases differed in one significant property: the kcat of dienelactone hydrolase was 1,800 min-1, an order of magnitude below the kcat observed with enol-lactone hydrolases. The relatively low catalytic activity of dienelactone hydrolase may demand its production at the high levels observed for induced cultures of Pseudomonas sp. strain B13.     

The isolation and characterization of phosphofructokinase from the epithelial cells of rat small intestine.

1. Only a single phosphofructokinase isoenzyme is present in the mucosa of rat small intestine. 2. Mucosal phosphofructokinase was purified to yield a homogeneous preparation of specific activity 175 units/mg of protein. 3. The native enzyme is a tetramer, with monomer Mr 84 500 +/- 5000. 4. The native enzyme may be degraded by the action of endogenous proteinases to give two products with the same specific activity as the native enzyme: degradation occurs in the order native enzyme leads to proteolytic product 1 leads to proteolytic product 2. 5. Proteolytic product 1 has a greater mobility in cellulose acetate electrophoresis at pH8 and binds more strongly to DEAE-cellulose than does native enzyme; the converse is true for proteolytic product 2. 6. Proteolytic product 1 is a tetramer with a monomer Mr about 74 300; proteolytic product 2 is also a tetramer. 7. Native enzyme can only be prepared in the presence of proteinase inhibitors; partial purifications based on simple fractionation of crude mucosal extracts in the absence of proteinases inhibitors contain proteolytic product 2 as the main component and proteolytic product 1 together with little native enzyme. 8. Purified native mucosal phosphofructokinase displayed little co-operativity with respect to fructose 6-phosphate at pH 7.0 and was only weakly inhibited by ATP.     

The heterogeneity of brewer's yeast old yellow enzyme

Heterogeneity of brewer's yeast old yellow enzyme (OYE) was found by anion-exchange high-performance liquid chromatography (HPLC) as well as by 13C-NMR spectroscopy of [4a-13C]FMN reconstituted into apo OYE. Though the OYE sample prepared according to the conventional procedure gave a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the OYE sample was found to consist of five species on anion-exchange HPLC. The 13C-NMR spectrum of the [4a-13C]FMN-reconstituted OYE gave multiple peaks corresponding to 4a-13C. This multiplicity indicates that this OYE preparation possesses heterogeneity in the environment surrounding FMN, i.e., the active site of OYE. The different species of OYE were separately obtained by preparative HPLC on an anion-exchange column. These species as well as the unresolved sample showed identical mobility on SDS-PAGE and similar but slightly different NADPH oxidase activities. This heterogeneity was shown not to have resulted from proteolytic modification during the conventional purification procedure, which includes autolysis of the yeast cells, since the enzyme extracted by mechanical destruction of the yeast cells in the presence of various protease inhibitors exhibited identical heterogeneity. The pure OYE forms obtained by preparative anion-exchange HPLC are homogeneous in the flavin environment as revealed by a single 13C-NMR signal for the [4a-13C]FMN-reconstituted species.     

Cytochromec oxidase metal centers: Location and function

Cytochromec oxidase ofParacoccus denitrificans is spectroscopically and functionally very similar to the mammalian enzyme. However, it has a very much simpler quaternary structure, consisting of only three subunits instead of the 13 of the bovine enzyme. The known primary structure of theParacoccus denitrificans subunits, the knowledge of a large number of sequences from other species, and data on the controlled proteolytic digestion of the enzyme allow structural restrictions to be placed on the models describing the binding of the active metal centers to the polypeptide structure.     

Acetylation of spermidine and methylglyoxal bis(guanylhydrazone) in baby-hamster kidney cells (BHK-21/C13).

Treatment of BHK-21/C13 cells with methylglyoxal bis(guanylhydrazone) (MGBG) induced the cytosolic form of spermidine N1-acetyltransferase. It stabilized the enzyme against proteolytic degradation, but the drug did not affect the enzyme activity in vitro. MGBG was itself acetylated by BHK-21/C13 cells, but at only one-tenth the rate at which spermidine was acetylated. Acetylation occurred almost exclusively in the nuclear fraction. The product was identified as N-acetyl-MGBG by h.p.l.c., by using [3H]acetyl-CoA and [14C]MGBG as co-substrates. The results suggest that the acetylation of MGBG by BHK-21/C13 cells occurs by a different acetyltransferase enzyme from that which acetylates spermidine.     

Activation of the vaccinia virus nicking-joining enzyme by trypsinization

The vaccinia virus nicking-joining (NJ) enzyme has been purified to homogeneity from a preparation of virus cores. The virus-specific DNA-dependent enzyme, which does not require ATP, is a single polypeptide of Mr 50,000 and possesses both endonuclease and ligase activities. The principal end product of the enzyme activity, following incubation with closed circular DNA of sufficient linking deficiency, is a linear DNA in which one of the termini has become cross-linked by the in vitro formation of a hairpin. The ability of the NJ enzyme to cross-link DNA is significantly enhanced by in vitro proteolysis. The enzymatic properties of the proteolytic digestion product, a 44-kDa polypeptide, differ in several other ways from the intact NJ enzyme. In particular, the specific activity is enhanced and the ionic strength optimum is shifted toward higher salt concentrations. It is suggested that the purified 50-kDa species is a pronuclease that is activated by proteolytic processing.     

13C NMR studies of D- and L-phenylalanine binding to cobalt(II) carboxypeptidase A

13C NMR T1 and T2 measurements have been performed on cobalt(II) substituted carboxypeptidase A in the presence of carboxylate-13C-enriched L- and D-phenylalanine. Upon binding to the cobalt enzyme, the longitudinal and transverse relaxation rates T1p-1 and T2p-1 of these inhibitors are enhanced significantly compared to the zinc enzyme, allowing both determination of an affinity constant for inhibitor binding, K, and calculation of the metal-13C carboxylate distances. The L-and D- Phe concentration dependence of T2p-1 yields affinity constants of 290 +/- 60M-1 and 670 +/- 90M-1. The distance measurements calculated for Co-13C from T1p-1 are 0.39 +/- 0.04 and 0.42 +/- 0.04 nm for L-Phe and D-Phe. Both values are too great for direct coordination of their carboxylate groups to the metal atom. Upon formation of their respective ternary enzyme.Phe.N3- complexes, the distances are essentially unaltered. In conjunction with electronic absorption studies on these complexes it can be concluded that N3-, but not the amino acid carboxylate, is bound to the metal.     

Cleavage of the ADAMTS13 propeptide is not required for protease activity

ADAMTS13 belongs to the "a disintegrin and metalloprotease with thrombospondin repeats" family, and cleaves von Willebrand factor multimers into smaller forms. For several related proteases, normal folding and enzymatic latency depend on an NH2-terminal propeptide that is removed by proteolytic processing during biosynthesis. However, the ADAMTS13 propeptide is unusually short and poorly conserved, suggesting it may not perform these functions. ADAMTS13 was secreted from transfected HeLa cells with a half-time of 7 h and the rate-limiting step was exported from the endoplasmic reticulum. Deletion of the propeptide did not impair the secretion of active ADAMTS13, indicating that the propeptide is dispensable for folding. Furin was shown to be sufficient for ADAMTS13 propeptide processing in two ways. First, mutation of the furin consensus recognition site prevented propeptide cleavage in HeLa cells and resulted in secretion of pro-ADAMTS13. Second, furin-deficient LoVo cells secreted ADAMTS13 with the propeptide intact, and cotransfection with furin restored propeptide cleavage. In both cell lines, secreted pro-ADAMTS13 had normal proteolytic activity toward von Willebrand factor. In cells coexpressing both ADAMTS13 and von Willebrand factor, pro-ADAMTS13 cleaved pro-von Willebrand factor intracellularly. Therefore, the ADAMTS13 propeptide is not required for folding or secretion, and does not perform the common function of maintaining enzyme latency.     

Enzyme preparations for research on protein hydrolysates and amino acid mixtures not containing peptides

Some properties and the hydrolysing ability of two novel enzyme preparations, a proteolytic preparation "C" from Acremonium chrysogenum (Cephalosporium acremonium) and a peptidase preparation (the producer from the family Pseudomanadaceae), are described. The preparations can be used for obtaining protein hydrolysates with different ratios of free amino acids and peptides. The protein hydrolysis with the preparation "C" enables one to obtain hydrolysates containing 13-18% of free amino acids. The further treatment of the hydrolysates with the peptidase preparation results either in complete hydrolysis of the remained peptide fractions or in obtainment of solutions containing from 60 to 85% of free amino acids and low-molecular weight peptides.     

Purification and properties of a ribosomal protein methylase from Eschericha coli Q13.

Ribosomal protein methylase has been purified from Escherichia coli strain Q13 using methyl-deficient 50S subunits as substrates. The purified enzyme (or enzyme complex) which is devoid of rRNA methylating activity is quite stable and has a pH optimum around 8.0. The Km for S-adenosyl-L-methionine is 3.2 muM. The molecular weight of the enzyme is 3.1 X 10(4); minor methylating activity was also detected for protein peaks with molecular weights of 1.7 X 10(4) and 5.6 X 10(4). Protein L11 is the major protein methylated by the purified enzyme. Product analysis revealed the presence of N epislon-trimethyllysine, a methylated neutral amino acid(s) previously observed in protein L11 and N epislon-monomethyllysine. Free ribosomal proteins were much better substrates for the methylation, indicating that methylation of 50S ribosomal proteins can occur before the complete assembly of the 50S ribosomal subunit.     

4-N-trimethylaminobutyraldehyde dehydrogenase: purification and characterization of an enzyme from Pseudomonas sp. 13CM

4-N-trimethylaminobutyraldehyde dehydrogenase from Pseudomonas sp. 13CM was purified 14-fold to apparent homogeneity by hydrophobic chromatography on a Phenyl-Toyopearl, and affinity chromatography was done on a 5'-AMP Sepharose4B in the presence of dithiothreitol. The enzyme was found to be a trimer with identical 55 kDa subunits. The isoeletric point was found to be 5.5. The optimum temperature and pH were 40 degrees C and pH 10.0. The purified enzyme was further characterized with respect to substrate specificity, kinetic parameters, and analog inhibition. The K(m) values for 4-N-trimethylaminobutyraldehyde, 4-dimethylaminobutyraldehyde, and NAD(+) were 7.4, 51, and 125 microM respectively. The enzyme was inhibited by SH reagents, and by heavy metal ions.