Production of an alkaline proteinase fromConidiobolus coronatus and its use to resolvedl-phenylalanine anddl-phenylglycine

An isolate ofConidiobolus coronatus (NCIM 1238) showed high proteinase activity (20.1 U/ml) and productivity (600 U/l.h) when 1% (w/v) glucose or sucrose was used as the carbon source in shake flasks. Addition of organic nitrogen sources, casein (2%), soybean flour (4%), liver extract (2%) or Edamin-S (2%), enhanced growth and proteinase production up to three-fold and seven-fold, respectively. The system was successfully run up to 6 l in a laboratory fermenter with a productivity of 600 U/l.h. The proteinase was successfully used to resolve the recemic mixtures ofdl-phenylalanine anddl-phenylglycine and thus could replace the currently used subtilisin.     

Purification and characterization of a bovine colostrum low molecular weight cysteine proteinase inhibitor

Large amounts of cysteine proteinase inhibitors were found in bovine colostrum. One had a molecular weight of 90,000, and the other a molecular weight of 10,500. The concentrations of both these inhibitors were highest the day after parturition, and were about one-tenth as much on day 7. The lower molecular weight inhibitor was purified by acid treatment, ammonium sulfate fractionation, gel filtration on Sephadex G-50, CM-Sephadex chromatography and rechromatography on Sephadex G-50. The purified preparation gave a single band on SDS-polyacrylamide gel electrophoresis. This inhibitor contained one tryptophanyl residue and one cystinyl residue, and did not contain a free thiol group. Values obtained for its isoelectric point (pI) were 10.0 and 10.3. This material strongly inhibited cathepsin B, cathepsin H, and papain. the higher molecular weight inhibitor was partially purified. It had a pI of 4.2 and inhibited papain, cathepsin H, and cathepsin B.     

Applications of alkaline protease fromConidiobolus in animal cell culture

Summary Alkaline protease fromConidiobolus has been tested as a substitute for trypsin in animal cell culture. Applications in the dissociation of cells for primary cell cultures, maintenance of cell lines and the production of G-bands on metaphase chromosomes are described. The advantages of a microbial enzyme for such applications are discussed.     

Purification and characterization of a low molecular weight cysteine proteinase inhibitor from bovine muscle

A low-Mr tight binding proteinase inhibitor was purified from bovine muscle by alkaline denaturation of cysteine proteinases, gel filtration on Sephadex G-75 and affinity chromatography on carboxymethyl-papain-Sepharose. Chromatofocusing separated three isoforms which are similar in their Mr of about 14 000, their stability with heating at 80 degrees C and their inhibitory activity towards cathepsin H, cathepsin B and papain. The equilibrium constants (Ki) were determined for these three cysteine proteinases but for cathepsin H, association (kass) and dissociation (kdiss) rate constants were also evaluated. Ki values of 56 nM and 8.4 nM were found for cathepsin B and cathepsin H, respectively. For papain, Ki was in the range of 0.1-1 nM. The kinetic features of enzyme-inhibitor binding suggest a possible role for this low-Mr protein inhibitor in controlling 'in vivo' cathepsin H proteolytic activity. With regard to cathepsin B, such a physiological role was less evident.     

Isolation of a low molecular weight active fragment of potato proteinase inhibitor IIb.

A low molecular weight active fragment of potato proteinase inhibitor IIPB was obtained by incubating the inhibitor with an equimolar amount of trypsin [EC 3.4.21.4] at pH 8 and 30 degrees for 16 hr, followed by gel filtration through Sephadex G-50, treatment with trichloroacetic acid, and CM-cellulose chromatography. The purified active fragment consisted of a single peptide chain with a molecular weight of 4,300, comprising 39 amino acid residues. It retained very strong inhibitory activity against chymotrypsin [EC 3.4.21.1] and subtilisin [EC 3.4.21.14]. However, the yield of this active fragment was rather low and was variable. On further incubation with trypsin, it was converted into smaller inactive peptides.     

Purification and characterization of a low molecular weight cysteine proteinase inhibitor from bovine muscle

A low-M_r tight binding proteinase inhibitor was purified from bovine muscle by alkaline denaturation of cysteine proteinases, gel filtration on Sexphadex G-75 and affinity chromatography on carboxymethyl-papain-Sepharose. Chromatofocusing separated three isoforms which are similar in their M_r of about 14 000, their stability with heating at 80°C and their inhibitory activity towards cathepsin H, cathepsin B and papain. The equilibrium constants (K_i) were determined for these three cysteine proteinases but for cathepsin H, association (k_ass) and dissociation (k_diss) rate constants were also evaluated. K_i values of 56 nM and 8.4 nM were found for cathepsin B and cathepsin H, respectively. For papain, K_i was in the range of 0.1–1 nM. The kinetic features of enzyme-inhibitor binding suggest a possible role for this low-M_r protein inhibitor in controlling ‘in vivo’ cathepsin H proteolytic activity. With regard to cathepsin B, such a physiological role was less evident.     

Rapid alkaline transfer of low molecular weight DNA from NuSieve GTG agarose gels

The rapid alkaline transfer of high molecular weight DNA from agarose gels to nylon membranes has greatly decreased the time required for setup of Southern transfers. This technique has been used to resolve genomic DNA greater than 1000 base pairs by conventional electrophoresis on 1% agarose gels followed by alkaline transfer to nylon membrane. Now we report that this rapid alkaline method can be used for the transfer of low molecular weight DNA fragments (10 to 1000 base pairs) from NuSieve GTG agarose gels to nylon membrane.     

Semiquantitative determination of low molecular weight proteinase inhibitors in the presence of high molecular weight inhibitors by a modified radial diffusion assay

The radial diffusion assay is very suitable for the determination of proteinase inhibitors in biological fluids. By combining radial diffusion and ultrafiltration, it has become possible to directly determine low molecular weight proteinase inhibitors in mixtures with inhibitors of higher molecular weight. By this modification the inhibitor solutions to be investigated are not pipetted into wells as usually, but are applied on small pieces of dialysis membranes lying on the gel. The exclusion limit of the membrane must be of a magnitude that the inhibitors of higher molecular weight are retained, whereas the inhibitors of lower molecular weight can diffuse into the gel. The modified method can be used for the direct determination of e.g. aprotinin (Mr 6500) in the presence of alpha 1-proteinase inhibitor (Mr 54,000), ovoinhibitor (Mr 50,000) and ovomucoid (Mr 27,000), respectively. The modified method is suitable for the direct determination of low molecular weight inhibitors of trypsin and papain in serum, synovial fluid and saliva. Tissue extracts containing 4 M guanidine hydrochloride or 6 M urea can be investigated directly, too.     

Phosphatidylserine modulates calf intestine alkaline phosphatase activity towards low and high molecular weight substrate

Pretreatment of calf intestine alkaline phosphatase with phosphatidylserine resulted in an inhibition of the phosphatase activity towards low - (p-nitrophenylphosphate) and high (phosphohistone) molecular weight substrate. Phosphatidylcholine, irrespectively of the substrate used did not cause enzyme modulation. 12-O-tetradecanoylphorbol-13-acetate, 1,2-diolein as well certain retinoids, known to effect phosphatidylserine-sensitive enzyme systems (Castagna, M. et al. 1982, J. Biol. Chem. 257, 7847-7851; Gmeiner, B. 1986, Biochim. Biophys. Acta 856, 392-394) had no influence on the modulated phosphatase. The lipid interacting drug trifluoperazine inhibited the enzyme activity towards phosphohistone, but not towards p-nitrophenylphosphate as a substrate. The results indicate that acidic phospholipid may play a role in activity modulation of calf intestine membranous alkaline phosphatase activity.     

Isolation and immunological studies of high and low molecular weight cysteine proteinase inhibitors in bovine serum

Two kinds of cysteine proteinase inhibitor (M_r 145 000 and M_r 15 500) were purified from bovine serum. These purified inhibitors showed a single band on SDS-polyacrylamide gel electrophoresis, respectively. The isoelectric point of the high molecular weight inhibitor was found to be 4.4 and that of the low molecular weight inhibitor was 8.6. The high molecular weight inhibitor inhibited papain and cathepsin H, but had little activity against cathepsin B. While the low molecular weight inhibitor was a strong inhibitor of papain and cathepsin H and showed a weak inhibition of cathepsin B. These two inhibitors showed different immunological reactivities.     

A low molecular weight antioxidant decreases weight and lowers tumor incidence

Stable free radical nitroxides are potent antioxidants possessing superoxide dismutase- and catalase-mimetic activity that protect cells and animals against a variety of oxidative insults. Tempol, as a representative nitroxide, was evaluated for its influence on weight maintenance and spontaneous tumor incidence in C3H mice. Tempol administered in either the drinking water or food did not show any untoward effects and prevented animals from becoming obese. Tempol-treated animals' leptin levels were reduced. Long-term treatment with Tempol significantly decreased tumorigenesis when compared to controls (10 vs. 40%, respectively). Selected tissues from Tempol-treated animals exhibited elevated levels of mitochrondrial uncoupling protein-2 (UCP-2) and HSP70. The present data suggest that nitroxides upregulate UCP-2, obviate weight gain, and decrease age-related spontaneous tumor incidence. As a class, nitroxides may provide overall health benefits by contributing to decreased obesity and tumor incidence.     

Three low molecular weight cysteine proteinase inhibitors of human seminal fluid: Purification and enzyme kinetic properties

The cystatins form a superfamily of structurally related proteins with highly conserved structural folds. They are all potent, reversible, competitive inhibitors of cysteine proteinases (CPs). Proteins from this group present differences in proteinase inhibition despite their high level of structural similarities. In this study, three cysteine proteinase inhibitors (CPIs) of low molecular weight were isolated from human seminal fluid (HSF) by affinity chromatography on carboxymethyl (CM)-papain–Sepharose column, purified using various chromatographic procedures and checked for purity on sodium-dodecyl PAGE (SDS-PAGE). Matrix-assisted laser desorption-ionization-time-of flight-mass spectrometry (MALDI-TOF-MS) identified these proteins as cystatin 9, cystatin SN, and SAP-1 (an N-terminal truncated form of cystatin S). All three CPIs suppressed the activity of papain potentially and showed remarkable heat stability. Interestingly SAP-1 also inhibits the activity of trypsin, chymotrypsin, pepsin, and PSA (prostate specific antigen) and acts as a cross-class protease inhibitor in in vitro studies. Using Surface Plasmon Resonance, we have also observed that SAP-1 shows a significant binding with all these proteases. These studies suggest that SAP-1 is a cross-class inhibitor that may regulate activity of various classes of proteases within the reproductive systems. To our knowledge, this is the first report about purification of CPIs from HSF; the identification of such proteins could provide better insights into the physiological processes and offer intimation for further research.     

A new retinoid-binding protein with a low molecular weight

Cellular retinoid-binding proteins and nuclear receptors may mediate the intracellular transport and the action of retinoids in the control of differentiation and tumorigenesis. We report a new retinoid-binding protein (Ret BP) with a molecular size of 4,000 that binds retinol, retinoic acid, and some of their derivatives. Purification of Ret BP from chick skin cytosol involved DEAE-Sephadex, Sephadex G-100, and Mono Q column chromatography. The Ret BP-retinoid complex eluted at 195 mM NaCl during Mono Q column chromatography using a 0-300 nM NaCl gradient. Superose-12 column chromatography indicated a molecular size of 4,000 for Ret BP. The binding protein showed a pI of 6.8 on electrofocusing in ampholines of pH 3-10. Ret BP may act as an affinant for retinoids in the cell, and may serve to dispense the ligands to their respective functionally active sites.     

A low molecular weight DNA polymerase from wheat embryos

The study of plant DNA polymerases lags far behind that concerning their animal or yeast counterpart. In this work we describe the first extensive purification to apparent homogeneity, as well as a detailed biochemical and immunological characterization, of a low molecular weight DNA polymerase (DNA polymerase C_I) purified from wheat embryos. The monomeric enzyme is a basic protein having a molecular weight of 52 kDa. Polyclonal antibodies raised in rabbits against DNA polymerase C_I did not inhibit animal DNA polymerases and or wheat DNA polymerase A, whereas wheat DNA polymerases C_II and B were much less affected than the C_I enzyme. Several properties of enzyme C_I were studied. Some known inhibitors of DNA polymerase activity including aphidicolin, phosphonoacetic acid and heparin, did not affect DNA polymerase C_I while the activity of this enzyme was strongly inhibited by ddTTP and N-ethylmaleimide. The polyamine spermine decreased markedly the enzyme activity, while spermidine produced a strong stimulation at the same concentrations that spermine inhibited the enzyme. The best template for this enzyme is poly dA-oligo dT, although polymerase C_I can recognize significantly some synthetic polyribonucleotide templates (poly rC-oligo dG, poly rA-oligo dT) but only at a given protein/template primer ratio. The enzyme is blocked at the amino terminus, thus preventing the automatic sequencing of the protein. The amino acid analysis showed a striking similarity with the animal low molecular weight DNA polymerase . The latter observation, as well as the effect of inhibitors (except N-ethylmaleimide which does not inhibit the animal polymerase) indicate that the DNA polymerase described in this work is a plant DNA polymerase very similar to the low molecular weight animal DNA polymerase , an enzyme believed to be involved in nuclear DNA repair.     

A novel metal-free low molecular weight superoxide dismutase mimic

2-Ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine (OXANOH), the one-electron reduction product of the stable nitroxide radical, 2-ethyl-2,5,5-trimethyl-3-oxazolidinoxyl (OXANO), is reportedly oxidized by superoxide, and its oxidation has been proposed as a method for assaying superoxide. We find that superoxide can both reduce OXANO and oxidize OXANOH. The respective rate constants, k1 and k2, were determined using two superoxide-generating systems (xanthine oxidase/xanthine as well as ionizing radiation). OXANOH oxidation and OXANO reduction are both inhibitable by superoxide dismutase, pH-dependent (4.5-9.3), and result in a steady state distribution of [OXANO] and [OXANOH], independent of their initial concentrations, i.e. the OXANO/OXANOH couple exhibits a metal-independent superoxide dismutase-like function. Thus it provides a prototype for future development of improved low molecular weight superoxide dismutase mimics which will also function in cellular hydrophobic (aprotic) compartments such as membranes.     

Purification of a liver alkaline protease which degrades oxidatively modified glutamine synthetase. Characterization as a high molecular weight cysteine proteinase

A nonlysosomal alkaline protease which degrades the oxidatively modified form of Escherichia coli glutamine synthetase has been purified to apparent homogeneity from rat and mouse liver acetone powders. Its molecular weight was determined to be 300,000 by Sephacryl S-300 gel filtration but results of further studies using high pressure liquid chromatography gel filtration suggest a value of 650,000. Examination of the subunit structure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed multiple bands of molecular weights between 22,000 and 34,000. The alkaline protease was inhibited by thiol reagents. Phenylmethylsulfonyl fluoride, aprotinin, leupeptin, antipain, and chymostatin partially inhibited the protease. The inhibition by phenylmethylsulfonyl fluoride was prevented by dithiothreitol, and alpha 1-antitrypsin and soybean trypsin inhibitor did not inhibit. No inhibition was observed with metalloprotease inhibitors. The alkaline protease is active over a broad range of pH with optimum activity for the degradation of oxidized glutamine synthetase around pH 9.0. Its activity is not stimulated by MgATP. A study of the products of insulin B chain degradation demonstrated major cleavage sites at Gln13-Ala14, Leu15-Tyr16, Cys(SO3H)19-Gly20, Gln4-His5, and Leu17-Val18. Based on its endopeptidase activity and its inhibitor specificity, the alkaline protease should be classified as a cysteine proteinase. It appears to be distinct from previously described proteinases and is likely involved in nonlysosomal mechanisms of intracellular protein turnover.     

A low molecular weight PSMA-based fluorescent imaging agent for cancer

We synthesized YC-27 3 to provide a fluorescent imaging agent for the prostate-specific membrane antigen (PSMA), a marker for hormone-independent prostate cancer and tumor neovasculature, with suitable pharmacokinetics for use in vivo. Immediate precursor trifluoroacetate salt of 2-(3-{5-[7-(5-amino-1-carboxy-pentylcarbamoyl)-heptanoylamino]-1-carboxy-pentyl}-ureido)-pentanedioic acid 2 was conjugated with a commercially available near-infrared light-emitting dye (IRDye 800CW) to provide 3 in 72% yield. YC-27 3 demonstrated a PSMA inhibitory activity of 0.37 nM and was capable of generating target-to-nontarget ratios of at least 10 in PSMA-expressing PC3-PIP vs. PSMA-negative PC3-flu tumors in vivo. YC-27 3 may be useful for study of PSMA-expressing tissue in preclinical models or for intraoperative guidance.