Alpha-2-macroglobulin-like protease inhibitor from the egg white of cuban crocodile (Crocodylus rhombifer)

A high molecular weight protease inhibitor was purified from the egg white of Cuban crocodile (Crocodylus rhombifer). It inhibited the casein hydrolyzing activity of trypsin, subtilisin and papain. Its native molecular weight was 730,000 and it consisted of four subunits of equal molecular weight, each pair of which were disulfide bonded. The amino acid composition, circular dichroic spectrum and electron micrographs of this protein are also presented. Upon incubation with trypsin this protein yielded a fragment of Mr = 80,000, similar in size to the one known to originate from alpha 2-macroglobulin under the same conditions. The molecular parameters of this protein and the broad inhibitory activity towards thiol and serine proteases with different substrate specificities suggest that it is a protein closely related to alpha 2-macroglobulin in mammalian serum. From its native molecular weight and amino acid composition we believe that this protein is also a reptilian counterpart of the avian ovomacroglobulin described by Miller and Feeney (3).     

Human senile cataractous lens protease. Isolation and some chemical characteristics

A proteolytic enzyme was isolated from human senile cataractous lens by anion-exchange and gel-filtration chromatography. Sedimentation and zone-electrophoretic experiments indicated a high degree of homogeneity for the enzyme. A molecular weight of 27000 was calculated from measurements of sedimentation velocity and diffusion coefficient. Chelating agents decreased activity which could be restored by addition of certain bivalent metal ions. Di-isopropyl phosphorofluoridate and phenylmethanesulphonyl fluoride inhibit the proteolytic activities. Optimum rates of hydrolysis were observed at pH5.2.     

Isolation and characterization of an intracellular aminopeptidase from the extreme thermophilic archaebacterium Sulfolobus solfataricus

An intracellular aminopeptidase (EC 3.4.11.-) was purified from the extreme thermophilic archaebacterium, Sulfolobus solfataricus. The molecular weight of the native enzyme was about 320,000, as calculated by gel-filtration studies, and a subunit Mr of 80,000 was estimated by SDS-polyacrylamide gel electrophoresis. The temperature optimum of the enzyme was at 75 degrees C and the pH optimum was found to be 6.5. The aminopeptidase was highly active against the chromogenic substrates L-Leu-p-NA and L-Ala-p-NA. The enzyme was inhibited by EDTA, but the activity could be partially restored by removal of the EDTA and incubation with Co2+ or Mn2+. Bestatin, a typical inhibitor of aminopeptidase, fully inhibited the enzyme activity, but inhibitors of serine proteinases had no effect. Beside a high thermostability, the enzyme showed a remarkable stability against 6 M urea, organic solvents and acetonitrile.     

Purification and characterization of methyl-accepting chemotaxis protein methyltransferase I in Bacillus subtilis.

A methyltransferase that methylates one of the proteins involved in chemotactic adaptation to sensory stimuli in Bacillus subtilis was purified to homogeneity. The enzyme utilizes S-adenosylmethionine as donor for a methyl group that is transferred to a glutamate residue in a 69 000-mol.wt. membrane protein and also to a protein of 19 000 mol.wt. The molecular weights of the denatured enzyme by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and of the native enzyme by gel-filtration chromatography both show the protein to be a 44 000-mol.wt. monomer. Isoelectric focusing of the purified methyltransferase showed the protein to be a single species with isoelectric point pI 5.4. On the basis of a molecular weight of 44 000, the molar absorption coefficient at 262 nm of the enzyme is 10.9 x 10(4) M-1 . cm-1. The Km of the enzyme for S-adenosylmethionine is about 2 microM. The Ki for S-adenosylhomocysteine is about 0.2 microM. Ca2+ is a competitive inhibitor of methylation, with a Ki of 0.065 microM. The enzyme methylates membranes from the wild-type more efficiently than membranes isolated from a mutant strain defective in chemotaxis. The enzyme is unable to methylate Escherichia coli membranes.     

Endogenous inhibitor of cysteine proteases from the human kidney

A thermo- and acid stable inhibitor of cysteine proteinases was isolated from the human kidney by successive procedures--acid fractionation, gel-filtration on Sephadex G-75, affinity chromatography on papain-sepharose. The final purification factor was 650 fold. The inhibitor molecular weight was equal to 12 kDa. The values of Ki measured by different methods are (7.9-9.4) X 10(-4) M for papain and (7.1-8.0) X 10(-10) M for purified human kidney cathepsin B. In experiments with papain, inhibitor kass and kd were 1.1 X 10(6) M-1 s-1 and 9.0 X 10(-4) s-1, respectively. The inhibitor did not influence the trypsin activity, its properties being similar to those of related thermo- and acid-stable inhibitors from other human and animal tissues.     

Purification, characterization, and complete amino acid sequence of a trypsin inhibitor from amaranth (Amaranthus hypochondriacus) seeds.

A protein proteinase inhibitor was purified from a seed extract of amaranth (Amaranthus hypochondriacus) by precipitation with (NH4)2SO4, gel-filtration chromatography, ion-exchange chromatography, and reverse-phase high-performance liquid chromatography. It is a 69-amino acid protein with a high content of valine, arginine, and glutamic acid, but lacking in methionine. The inhibitor has a relative molecular weight of 7400 and an isoelectric point of 7.5. It is a serine proteinase inhibitor that recognizes chymotrypsin, trypsin, and trypsin-like proteinase activities extracted from larvae of the insect Prostephanus truncatus. This inhibitor belongs to the potato-I inhibitor family, showing the closest homology (59.5%) with the Lycopersicum peruvianum trypsin inhibitor, and (51%) with the proteinase inhibitor 5 extracted from the seeds of Cucurbita maxima. The position of the lysine-aspartic acid residues present in the active site of the amaranth inhibitor are found in almost the same relative position as in the inhibitor from C. maxima.     

Presence of angiotensin-converting enzyme in follicular fluids of porcine ovaries and its possible involvement in the intrafollicular breakdown of bradykinin

The follicular fluid of porcine ovaries contains a metalloenzyme capable of hydrolyzing the synthetic substrate, benzyloxycarbonyl-Val-Lys-Met-MCA. This enzyme was purified by ammonium sulfate fractionation followed by column chromatography on DEAE-cellulose, CM-cellulose, Zn(2+)-chelating Cellulofine, and Diol-300 gel-filtration columns. The molecular weight of the purified enzyme was estimated to be 170,000 by SDS-PAGE and 400,000 by gel-filtration analysis, suggesting that the native enzyme is a dimer of the 170-kDa subunit polypeptide. The enzyme activity was drastically enhanced by the presence of chloride ion, and strongly inhibited by captopril and bradykinin potentiator B. A 9-residue peptide containing a processing site of human amyloid precursor protein was degraded by its dipeptidyl carboxypeptidase activity. Furthermore, the purified protein was recognized by specific antibody raised against human angiotensin-converting enzyme. The enzyme rapidly degraded bradykinin in vitro. These results indicate that benzyloxycarbonyl-Val-Lys-Met-MCA-hydrolyzing enzyme is a porcine angiotensin-converting enzyme, and that the enzyme may play a role in bradykinin turnover within the follicles of porcine ovaries.     

A re-evaluation of the molecular size of duck ε-crystallin and its comparison with avian lactate dehydrogenases

A biochemical comparison of ε-crystallin isolated from the duck lens and lactate dehydrogenase of chicken heart has been made in order to establish the structural and functional identities of these two proteins. The native molecular weight of ε-crystallin was re-examined by combining sedimentation and gel-filtration data. It was found that ε-crystallin is 150 kDa in contrast to the 120 kDa reported previously for this crystallin. Subunit cross-linking experiments corroborated that lactate dehydrogenase and ε-crystallin both exist as tetramers of four identical subunits in their native quaternary structures. Amino acid compositions plus N-terminal analyses revealed no differences between the two proteins. Duck ε-crystallin exhibited high enzymatic activity of lactate dehydrogenases even after a long period of storage, and showed characteristic thermostability at 50°C for several hours. Comparison of the enzyme activity of duck lens homogenate with those of heart, liver and muscle tissues revealed that duck lens is a much richer source than other tissues for the isolation and characterization of this important enzyme which appears also as a structural protein in the lens.     

A re-evaluation of the molecular size of duck epsilon-crystallin and its comparison with avian lactate dehydrogenases

A biochemical comparison of epsilon-crystallin isolated from the duck lens and lactate dehydrogenases of chicken heart has been made in order to establish the structural and functional identities of these two proteins. The native molecular weight of epsilon-crystallin was re-examined by combining sedimentation and gel-filtration data. It was found that epsilon-crystallin is 150 kDa in contrast to the 120 kDa reported previously for this crystallin. Subunit cross-linking experiments corroborated that lactate dehydrogenase and epsilon-crystallin both exist as tetramers of four identical subunits in their native quaternary structures. Amino acid compositions plus N-terminal analyses revealed no differences between the two proteins. Duck epsilon-crystallin exhibited high enzymatic activity of lactate dehydrogenases even after a long period of storage, and showed characteristic thermostability at 50 degrees C for several hours. Comparison of the enzyme activity of duck lens homogenate with those of heart, liver and muscle tissues revealed that duck lens is a much richer source than other tissues for the isolation and characterization of this important enzyme which appears also as a structural protein in the lens.     

Purification and characterization of glyoxalase I from Hansenula mrakii

Glyoxalase I was purified from Hansenula mrakii IFO 0895 which was resistant to 25 mM methylglyoxal. The molecular weight of the purified enzyme was calculated to be 38,000 by both gel-filtration of Sephadex G-150 and SDS-PAGE. The enzyme was almost specific to methylglyoxal (K_m = 0.91 mM). The activity of the enzyme was not inhibited by metal ion chelators such as EDTA, which is a potent inhibitor for glyoxalase Is from other sources.     

Metal binding activity of pectin isolated from seagrass Zostera marina and its derivatives

Low esterified pectin was isolated from the seagrass Zostera marina. Dynamics of the isolation process for the pectin from this seagrass was investigated. Two pectin derivatives: galacturonide (A) with molecular weight 30.55 kDa and galacturonide (B) with molecular weight 3.94 kDa were obtained using acidic hydrolysis of the native pectin from Zostera marina. Molecular weight parameters (Mw, Mn, Mw/Mn) of this pectin and its derivatives as well as of commercial apple pectin were determined using gel-filtration chromatography method. Comparative assessment of Cd²⁺, Pb²⁺, Y³⁺-binding activity of the native Zostera pectin, galacturonides A and B, and commercial apple pectin was performed. The results showed that galacturonide A with molecular weight 30.55 kDa possesses highest metal-binding capacity and may be considered as a candidate for development of medicines with metal-binding activity.     

Study of structural heterogeneity of polymerized hemoglobin using biochemical and immunochemical methods

The structural heterogeneity of crosslinked pyridoxalated hemoglobin (polyHb-PLP) have been investigated by the methods of gel-filtration, isoelectric focusing and immunoelectrophoresis. It was established that the heterogeneity of poly-Hb-PLP is mainly depicted by polydispersion of molecular weight and less by the changes in the isoelectric properties. The qualitative difference has been shown between the immunochemical properties of the native hemoglobin and poly-Hb-PLP, which gives the suggestion of the formation of new antigenic determinants as a result of chemical modification and of more expressed antigenicity of modified hemoglobin compared with native protein. Poly-Hb-PLP has wide antigenic spectrum that corresponds to its structural heterogeneity. The data obtained suggest that the decrease in the antigenic activity of poly-Hb-PLP can be achieved by the lowering of the part of high molecular weight components.     

Physicochemical and kinetic properties of acid phosphatase from Saccharomyces cerevisiae

Acid phosphatase from yeast Saccharomyces cerevisiae was purified, and its physicochemical and kinetic properties were investigated. The sedimentation coefficient has been determined to be s0(20,w) = 13.6 S. The diffusion constant has been found to be 3.9 X 10(-7) cm2s-1, and the calculated partial specific volume was v = 0.663 cm3/g. From these data, a molecular weight of 252,000 was calculated. Electrophoresis on gel slabs, with a linear concentration gradient of polyacrylamide (4-30%), showed size heterogeneity of the native enzyme preparation and indicated an apparent molecular weight in the range of 170,000 to 360,000. In the presence of sodium dodecyl sulfate, the molecular weight was in the range of 82,000 to 165,000, indicating dimeric structure of the native enzyme, which was confirmed by cross-linking experiments. Isoelectric focusing demonstrated charge heterogeneity of enzyme preparation. From CD spectrum it was calculated that the enzyme contains about 29% of alpha-helical structure. Excitation at 278 nm gave an emission fluorescence spectrum with a maximum at 340 nm. Amino acid analysis revealed a high content of aspartic acid, serine, and threonine. Glycine is found as the NH2-terminal amino acid. Initial velocity dependence on substrate concentration, as well as on pH, and thermostability studies indicated the presence of at least two enzyme forms in the preparation.     

Purification and molecular weight determination of glucose-6-phosphate dehydrogenase and malic enzyme from mouse and Drosophila.

A facile two-step procedure was employed for simultaneous purification of glucose-6-phosphate dehydrogenase and malic enzyme from mouse (strain $\text{DBA}\text{2J}$) and Drosophila melanogaster. This involved the use of an 8-(6-aminohexyl)-amino-2′,5′-ADP-Sepharsoe affinity column chromatography followed by DEAE-Sephadex chromatography. The native and subunit molecular weights of these two homogeneous enzymes were determined by gel-filtration chromatography and SDS-polyacrylamide gel electrophoresis. From this study, it was concluded that the two enzymes are tetrameric and have native molecular weights between 200,000 and 280,000 in both species.     

Molecular weights of nitrogenase components from Azotobacter vinelandii.

Meniscus depletion sedimentation equilibrium ultracentrifuge experiments were performed on purified MoFe and Fe proteins of $\text{Azotobacter vinelandii}$. The MoFe protein was found to have a molecular weight of 245,000, using an experimentally confirmed partial specific volume of 0.73. The MoFe protein formed one band on sodium dodecyl sulfate gel electrophoresis and had a subunit molecular weight of 56,000. The subunit molecular weight from ultracentrifuge experiments in 8 M urea was 61,000. The molecular weight of the Fe protein was calculated to be 60,500 in meniscus depletion experiments. Similar experiments in 8 M urea solvent indicated a subunit molecular weight of 30,000. A subunit molecular weight of 33,000 was obtained from sodium dodecyl sulfate gel electrophoresis experiments.     

Purification and characterization of an acetyl-CoA hydrolase from Saccharomyces cerevisiae

Acetyl-CoA hydrolase, which hydrolyzes acetyl-CoA to acetate and CoASH, was isolated from Saccharomyces cerevisiae and demonstrated by protein sequence analysis to be NH2-terminally blocked. The enzyme was purified 1080-fold to apparent homogeneity by successive purification steps using DEAE-Sepharose, gel filtration and hydroxylapatite. The molecular mass of the native yeast acetyl-CoA hydrolase was estimated to be 64 +/- 5 kDa by gel-filtration chromatography. SDS/PAGE analysis revealed that the denatured molecular mass was 65 +/- 2 kDa and together with that for the native enzyme indicates that yeast acetyl-CoA hydrolase was monomeric. The enzyme had a pH optimum near 8.0 and its pI was approximately 5.8. Several acyl-CoA derivatives of varying chain length were tested as substrates for yeast acetyl-CoA hydrolase. Although acetyl-CoA hydrolase was relatively specific for acetyl-CoA, longer acyl-chain CoAs were also hydrolyzed and were capable of functioning as inhibitors during the hydrolysis of acetyl-CoA. Among a series of divalent cations, Zn2+ was demonstrated to be the most potent inhibitor. The enzyme was inactivated by chemical modification with diethyl pyrocarbonate, a histidine-modifying reagent.     

N-acetyl-D-galactosamine/N-acetyl-D-glucosamine--recognizing lectin from the snail Cepaea hortensis: purification, chemical characterization, cloning and expression in E. coli

From the albumin gland of the snail Cepaea hortensis we isolated and characterized a new N-acetyl-D-galactosamine/N-acetyl-D-glucosamine (GalNAc/GlcNAc) specific lectin (CHA-II) which was purified by a combination of affinity chromatography on GalNAc-agarose and gel filtration. The purified native lectin was found to be a multimeric protein, as revealed by SDS-PAGE and MALDI-TOF analysis. In SDS-PAGE the denatured and reduced lectin showed two bands of molecular masses with 17 and 15.5 kDa which reacted equally with anti-CHA-II rabbit antiserum. The lectin was O- and N-glycosylated with [(Gal)2-Man]2-Man-GlcNAc-GlcNAc-Asn as a probable structure for the oligosaccharide. Isoelectric focusing revealed a heterogeneous protein of at least four bands around pH 8.7. Tryptic peptides of CHA-II were N-terminally sequenced and highly degenerated gene specific oligonucleotide primers (GSPs) had been constructed. Using total RNA isolated from albumin glands, cDNAs were produced by the running race technique. Specific PCR fragments were obtained by PCR using GSPs, the universal primer and 5'- or 3'-RACE-cDNAs. The amplified fragments were cloned into the vector pDrive and were sequenced. The resulting total cDNA sequence consisted of 496 base pairs including an open reading frame of 360 base pairs which encoded a protein of 120 amino acids. The protein carried a putative signal peptide. The mature protein was predicted to comprise 99 amino acid residues with a calculated molecular weight of 11,239 Da. The PCR fragment encoding the mature protein was cloned into the vector pQE30 and expressed in E. coli. Recombinant CHA-II lectin was produced as inclusion bodies and extracted by 6 M guanidine hydrochloride. After refolding, the recombinant CHA-II agglutinated specifically human red blood cells of groups A and AB. In immunodiffusion experiments using rabbit antiserum raised against the native lectin, the protein showed a precipitation line of identity with the native lectin.     

Protein chemical and kinetic characterization of recombinant porcine ribonuclease inhibitor expressed in Saccharomyces cerevisiae

A cDNA encoding porcine ribonuclease inhibitor was used to express this protein in yeast under control of the PHO5 promoter. The recombinant protein was purified to homogeneity with a yield of 0.2 mg/g of yeast cells (wet weight) and was found to be indistinguishable from the inhibitor isolated from porcine liver on the basis of the following criteria: the amino acid composition, the number of free sulfhydryl groups, the molecular weight of the native and the denatured protein, peptide mapping, and amino acid sequence analysis of the N- and C-terminal regions of the protein. A simple method was developed for measuring accurately the slow, tight-biding kinetics of the inhibition of ribonuclease by ribonuclease inhibitor. From the dependence of the observed inhibition constant on the substrate concentration, it could be concluded that RI was competitive with the substrate UpA. The dependence of the observed association rate constant on the substrate concentration was consistent with a two-step mechanism in which the substrate only competed in the second (isomerization) step. The values for the inhibition constant for the inhibition of RNase by the recombinant inhibitor, 67 fM, the association rate constant, 1.5 x 10(8) M-1.s-1, and the dissociation rate constant, 8.3 x 10(-6) s-1, were in good agreement with those obtained for the porcine liver RNase inhibitor.     

类产碱假单胞菌杀虫物质的分离纯化和鉴定

类产碱假单胞菌是一株昆虫病原菌,该菌对草地蝗虫、竹蝗等具有良好的致死作用,经鉴定,该菌对蝗虫具有毒杀作用的物质为其代谢分泌到胞外的一种蛋白质。类产碱假单胞菌培养物经硫酸按沉淀,SephadexG-100凝胶过滤及DEAE-SephadexA-50阴离子交换柱层析,纯化获得的杀虫蛋白只含一种亚基,分子量25100,等电点5．16,含17种氨基酸,其中谷氨酸含量最高,胱氨酸含量最低,最大吸收峰为278．3nm。     

Purification and characterization of arginase from Neurospora crassa

We have purified an enzymatically active form of arginase from a wild-type strain of Neurospora crassa to homogeneity. The enzyme has a subunit molecular weight of 38,300 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native protein migrated as a hexamer during gel-filtration chromatography with an apparent molecular weight of 266,000. The enzyme exhibited hyperbolic kinetics at pH 9.5 with an apparent Km for arginine of 131 mM. Antiserum was prepared against the purified enzyme and used to demonstrate the existence of three cross-reactive proteins in crude extracts of wild-type N. crassa. One of these proteins corresponded to the purified protein, whereas the other two were of molecular weights 41,700 and 26,800, respectively. Using the same antiserum, we found that rat liver, but not rat kidney, contains immunoreactive material. We also detected two proteins in extracts of Saccharomyces cerevisiae that were weakly cross-reactive with the antiserum. These data provide evidence for the existence of multiple forms of arginase in fungi as well as in mammals.