Purification and characterization of protease So, a cytoplasmic serine protease in Escherichia coli

A new cytoplasmic endoprotease, named protease So, was purified to homogeneity from Escherichia coli by conventional procedures with casein as the substrate. Its molecular weight was 140,000 when determined by gel filtration on Sephadex G-200 and 77,000 when estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Thus, it appears to be composed of two identical subunits. Protease So had an isoelectric point of 6.4 and a K(m) of 1.4 muM for casein. In addition to casein, it hydrolyzed globin, glucagon, and denatured bovine serum albumin to acid-soluble peptides but did not degrade insulin, native bovine serum albumin, or the "auto alpha" fragment of beta-galactosidase. A variety of commonly used peptide substrates for endoproteases were not hydrolyzed by protease So. It had a broad pH optimum of 6.5 to 8.0. This enzyme is a serine protease, since it was inhibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride. Although it was not inhibited by chelating agents, divalent cations (e.g., Mg(2+)) stabilized its activity. Protease So was sensitive to inhibition by N-tosyl-l-phenylalanine chloromethyl ketone but not by N-tosyl-l-lysine chloromethyl ketone. Neither ATP nor 5'-diphosphate-guanosine-3'-diphosphate affected the rate of casein hydrolysis. Protease So was distinct from the other soluble endoproteases in E. coli (including proteases Do, Re, Mi, Fa, La, Ci, and Pi) in its physical and chemical properties and also differed from the membrane-associated proteases, protease IV and V, and from two amino acid esterases, originally named protease I and II. The physiological function of protease So is presently unknown.     

Protease So from Escherichia coli preferentially degrades oxidatively damaged glutamine synthetase

After oxidative damage (e.g. induced with iron, ascorbate, and oxygen), the inactivated glutamine synthetase is selectively hydrolyzed in extracts of Escherichia coli. We therefore tested if glutamine synthetase treated with this system is hydrolyzed preferentially by any of the known E. coli proteases. Protease So, a cytoplasmic serine protease, was found to degrade the oxidized form of glutamine synthetase to acid-soluble peptides 5-10 times faster than the native glutamine synthetase. Degradation of the oxidized glutamine synthetase was inhibited by EDTA and stimulated 5-10-fold by Mg2+, Ca2+, or Mn2+, even though casein hydrolysis by protease So is not affected by divalent cations. Apparently, these cations affect the conformation of this substrate, making it more susceptible to proteolytic attack. Protease Re, another cytoplasmic protease, also degrades preferentially the oxidized form of glutamine synthetase and seems to correspond to the glutamine synthetase-degrading activity recently described by Roseman and Levine [1987) J. Biol. Chem. 262, 2101-2110). However, it is much less active in this reaction than protease So. No other soluble E. coli protease, including Do, Ci, Mi, Fa, Pi, or the ATP-dependent proteases Ti and La (the lon product), appears to degrade this oxidized protein. These results suggest that protease So participates in the hydrolysis of oxidatively damaged proteins and that E. coli has multiple systems for degrading different types of aberrant proteins.     

Design, synthesis, and evaluation of [188Re]organorhenium-labeled antibody fragments with renal enzyme-cleavable linkage for low renal radioactivity levels

Renal localization of radiolabeled antibody fragments constitutes a problem in targeted imaging and radiotherapy. We have reported that Fab fragments labeled with 3'-[131I]iodohippuryl Nepsilon-maleoyl-lysine (HML) showed markedly low renal radioactivity levels even shortly after injection, due to a rapid and selective release of m-[131I]iodohippuric acid by the action of brush border enzymes. To estimate the applicability of the molecular design to metallic radionuclides, [188Re]tricarbonyl(cyclopentadienylcarbonate)rhenium ([188Re]CpTR-COOH) was conjugated with Nepsilon-tert-butoxycarbonyl-glycyl-lysine or Nepsilon-maleoyl-glycyl-lysine to prepare [188Re]CpTR-GK-Boc or [188Re]CpTR-GK. The cleavage of the glycyl-lysine linkage of the two compounds generates a glycine conjugate of [188Re]CpTR-COOH ([188Re]CpTR-Gly), which possesses in vivo behaviors similar to those of m-iodohippuric acid. The hydrolysis rate of the peptide bond in [188Re]CpTR-GK-Boc was compared with that in 3'-[125I]iodohippuryl Nepsilon-Boc-lysine ([125I]HL-Boc) using brush border membrane vesicles (BBMVs) prepared from rat kidneys. [188Re]CpTR-GK was conjugated to thiolated Fab fragments to prepare [188Re]CpTR-GK-Fab. The biodistribution of radioactivity after injection of [188Re]CpTR-GK-Fab was compared with that of [125I]HML-Fab and [188Re]CpTR-Fab prepared by conjugating N-hydroxysuccinimidyl ester of [188Re]CpTR-COOH with antibody fragments. While [188Re]CpTR-GK-Boc liberated [188Re]CpTR-Gly in BBMVs, [125I]HL-Boc liberated m-[125I]iodohippuric acid at a much faster rate. In addition, although [125I]HL-Boc was hydrolyzed by both metalloenzymes and nonmetalloenzymes, metalloenzymes were responsible for the cleavage of the peptide linkage in [188Re]CpTR-GK-Boc. In biodistribution studies, [188Re]CpTR-GK-Fab exhibited significantly lower renal radioactivity levels than did [188Re]CpTR-Fab. However, the renal radioactivity levels of [188Re]CpTR-GK-Fab were slightly higher than those of [125I]HML-Fab. The analysis of urine samples collected for 6 h postinjection of [188Re]CpTR-GK-Fab showed that [188Re]CpTR-Gly was the major radiometabolite. In tumor-bearing mice, [188Re]CpTR-GK-Fab significantly reduced renal radioactivity levels without impairing the radioactivity levels in tumor. These findings indicate that the molecular design of HML can be applied to metallic radionuclides by using a radiometal chelate of high inertness and by designing a radiometabolite of high urinary excretion when released from antibody fragments following cleavage of a glycyl-lysine linkage. This study also indicates that a change in chemical structure of a radiolabel attached to a glycyl-lysine linkage significantly affected enzymes involved in the hydrolysis reaction. Since there are many kinds of enzymes that cleave a variety of peptide linkages on the renal brush border membrane, selection of a peptide linkage optimal to a radiometal chelate of interest may provide radiolabeled antibody fragments that exhibit renal radioactivity levels similar to those of [131I]HML-labeled ones. The in vitro system using BBMVs might be useful for selecting an appropriate peptide linkage.     

Identification of macrophage cell-surface binding sites for cationized bovine serum albumin.

Autoimmune diseases are characterized by the presence of autoantibodies often restricted to host proteins exhibiting charge rich domains. Charged polypeptides elicit strong immune responses, and cationized bovine serum albumin and other cationic proteins are significantly more immunogenic than their less charged counterparts. These phenomena may involve enhanced protein uptake by macrophages, resulting in greater processing and presentation of antigenic peptide-MHC complexes to T-cells. We compared macrophage cell-surface binding and uptake of native and cationized bovine serum albumin. Specific binding of [125I]cationized bovine serum albumin to THP-1 macrophages in vitro was 11-16 fold greater than for native albumin. Half-maximal inhibition of [125I]cationized albumin binding was observed at 10-7M ligand. The specificity of [125I]cationized bovine serum albumin binding and uptake was further studied in terms of competitive inhibition of proteolysis by proteins of varying charge content. Cationized bovine serum albumin, but not native albumin, inhibited proteolysis of [125I]cBSA. Calf thymus histones also inhibited cBSA degradation. High concentration of myelin basic protein was moderately effective at blocking cBSA degradation, while myoglobin and beta lactalbumin showed no inhibition. These results indicate that specific cell-surface binding sites which occur on macrophages may mediate selective uptake of certain proteins with highly charged domains including some autoantigens.     

Regulation of glutamine synthetase, aspartokinase, and total protein turnover in Klebsiella aerogenes

When suspensions of Klebsiella aerogenes are incubated in a nitrogen-free medium there is a gradual decrease in the levels of acid-precipitable protein and of aspartokinase III (lysine-sensitive) and aspartokinase I (threonine-sensitive) activities. In contrast, the level of glutamine synthetase increases slightly and then remains constant. Under these conditions, the glutamine synthetase and other proteins continue to be synthesized as judged by the incorporation of [14C]leucine into the acid-precipitable protein fraction and into protein precipitated by anti-glutamine synthetase antibodies, by the fact that growth-inhibiting concentrations of chloramphenicol also inhibit the incorporation of [14C]leucine into protein and into protein precipitated by anti-glutamine synthetase antibody, and by the fact that chloramphenicol leads to acceleration in the loss of aspartokinases I and III and promotes a net decrease in the level of glutamine synthetase and its cross-reactive protein. The loss of aspartokinases I and III in cell suspensions is stimulated by glucose and is inhibited by 2,4-dinitrophenol. Glucose also stimulates the loss of aspartokinases and glutamine synthetase in the presence of chloramphenicol. Cell-free extracts of K. aerogenes catalyze rapid inactivation of endogenous glutamine synthetase as well as exogenously added pure glutamine synthetase. This loss of glutamine synthetase is not associated with a loss of protein that cross-reacts with anti-glutamine synthetase antibodies. The inactivation of glutamine synthetase in extracts is not due to adenylylation. It is partially prevented by sulfhydryl reagents, Mn2+, antimycin A, 2,4-dinitrophenol, EDTA, anaerobiosis and by dialysis. Following 18 h dialysis, the capacity of extracts to catalyze inactivation of glutamine synthetase is lost but can be restored by the addition of Fe2+ (or Ni2+) together with ATP (or other nucleoside di- and triphosphates. After 40-60 h dialysis Fe3+ together with NADH (but not ATP) are required for glutamine synthetase inactivation. The results suggest that accelerated protein degradation in cells exposed to nitrogen-limited conditions reflects the differential destruction of some proteins, including aspartokinases I and III, in order to sustain the biosynthesis of others such as glutamine synthetase. The loss of glutamine synthetase activity in cell-free extracts is likely mediated in part by mixed-function oxidation systems and could represent a 'marking' step in protein turnover.     

Purification of a protease from Escherichia coli with specificity for oxidized glutamine synthetase

A soluble Escherichia coli protease has been identified and purified to homogeneity. The protease cleaves glutamine synthetase which has been modified by mixed function oxidation; native glutamine synthetase is not a substrate. Using [14C]glutamine synthetase as a substrate (prepared by growing E. coli on 14C-labeled amino acids), protease activity was assayed by determining the release of trichloroacetic acid-soluble material. The pure protease cleaves glutamine synthetase near the carboxyl terminus yielding 4,500 and 47,000 Mr products. The characteristics of this enzyme distinguish it from proteases previously purified from E. coli. These characteristics include a molecular weight of 75,000, alkaline pH optimum, lack of inhibition by serine protease inhibitors, and the ability to degrade insulin and casein. Oxidation of glutamine synthetase and other enzymes can be catalyzed by a variety of mixed function oxidase systems from bacterial and mammalian sources. Mixed function oxidation may be a "signal" or "marker" which consigns a protein for proteolytic degradation. Susceptibility to oxidation is subject to metabolic regulation, thereby providing control of proteolytic turnover. Isolation of a protease specific for modified glutamine synthetase provides the enzymatic basis for the specificity of this scheme.     

Comparison of biotin binding protein of pregnant rat serum with rat serum albumin

The purified biotin binding protein of pregnant rat serum was shown to be immunologically similar to rat serum albumin as assessed by a sensitive radioimmunoassay. In radioimmunoassay for rat biotin binding protein, the binding of [¹²⁵I] rat biotin binding protein to anti-chicken egg yolk biotin binding protein antibodies was displaced by both rat serum (10–100 nl) and purified rat serum albumin (0.1–10 ng). Similarly, in radioimmunoassay for rat serum albumin the binding of [¹²⁵I] rat serum albumin to either anti-rat serum albumin antibodies or anti-chicken egg yolk biotin binding protein antibodies was displaced by unlabelled rat biotin binding protein at comparable concentration range (0·5–10 ng). Significant fractions of radioiodinated rat biotin binding protein and rat serum albumin bound to antibodies to chicken egg yolk biotin binding protein. In immature rats, the circulating half-lives of rat biotin binding protein and rat serum albumin were determined to be 12 and 17 h respectively. The rat biotin binding protein and rat serum albumin were analysed by techniques that exploit their physicochemical properties. They displayed similar electrophoretic mobilities in alkaline as well as denaturing sodium dodecyl sulphate-polyacrylamide gels. However, in nonequilibrium pH gradient polyacrylamide gel electrophoresis, they resolved clearly. In two-dimensional tryptic peptide map analysis, the two proteins showed similarities as well as significant differences in the relative distribution patterns of their iodopeptides. These results showed that the primary structure of rat biotin binding protein and rat serum albumin were different in finer details despite the fact that they shared significant immunological cross-reactivity.     

Isolation and characterization of protease do from Escherichia coli, a large serine protease containing multiple subunits

A new cytoplasmic proteolytic enzyme in Escherichia coli, named protease Do, has been purified to near homogeneity. The enzyme is an endoprotease that degrades casein, denatured bovine serum albumin, and globin but shows little or no hydrolytic activity against insulin, growth hormone, native bovine serum albumin, or a variety of commonly used peptide substrates. The molecular size of the enzyme was large, and it could be isolated in different preparations in either of two forms. One showed a molecular weight of about 500,000 on gel filtration and a sedimentation coefficient of 15.9 S on sucrose gradient centrifugation. The other appeared to be about 300,000 and sedimented at 12.7 S. No interconversion between the two forms and no other difference in the properties was found. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) shows that both forms contain a major 54,000-dalton band and three additional minor polypeptides with molecular weights of 45,000, 44,000, and 42,000. These minor polypeptides appear to result from autolytic degradation of the major protein as demonstrated by peptide mapping with Staphylococcus aureus V8 protease. Thus, protease Do appears to contain a single subunit of 54,000, and can exist either as a decamer or as a hexamer or pentamer. The enzyme is a serine protease. It is sensitive to diisopropyl fluorophosphate (DFP) but not to metal chelating agents, sulfhydryl blocking groups, certain chloromethyl ketones, or various peptide aldehyde inhibitors. The enzyme covalently binds [3H]DFP, and the labeled subunit was visualized on SDS-polyacrylamide gels by fluorography. When cells growing in rich broth enter stationary phase, the relative concentration of protease Do increases more than twofold.     

Purification and properties of Azotobacter vinelandii glutamine synthetase.

A procedure is described for the purification of glutamine synthetase from the nitrogen-fixing organism Azotobacter vinelandii. Electron micrographs of the enzyme reveal a dodecameric arrangement of its subunits in two superimposed hexagonal rings similar to the glutamine synthetase of Escherichia coli. Disc eleetrophoresis in the presence of sodium dodecyl sulfate and sedimentation studies show a subunit molecular weight of 56,500 and a sedimentation coefficient (s_20,w) of the native enzyme of 20.0 S. Like the E. coli enzyme, the glutamine synthetase of A. vinelandii is regulated by adenylylation/deadenylylation. This finding was derived from (a) studies on the effect of snake venom phosphodiesterase treatment on the catalytic and spectral properties of enzyme isolated from cells grown on a nitrogen-rich medium, (b) the identification of the AMP released by the phosphodiesterase by thin-layer chromatography, (c) the selective precipitation of adenylylated enzyme with antibodies directed against adenylylated bovine serum albumin, and (d) the in vitro incorporation of radioactivity from [¹⁴C]ATP into deadenylylated enzyme in the presence of either crude extract from A. vinelandii or partially purified adenylyl transferase from E. coli. The state of adenylylation appears to have a similar influence on the catalytic properties of A. vinelandii glutamine synthetase as on those of the E. coli enzyme, with the exception that the deadenylylated form of the A. vinelandii glutamine synthetase is almost inactive in the Mn-dependent transferase reaction.     

Regulation of glutamine synthetase, aspartokinase, and total protein turnover in Klebsiella aerogenes

When suspensions of Klebsiella aerogenes are incubated in a nitrogen-free medium there is a gradual decrease in the levels of acid-precipitable protein and of aspartokinase III (lysine-sensitive) and aspartokinase I (threonine-sensitive) activities. In contrast, the level of glutamine synthetase increases slightly and then remains constant. Under these conditions, the glutamine synthetase and other proteins continue to be synthesized as judged (a) by the incorporation of [¹⁴C]leucine into the acid-precipitable protein fraction and into protein precipitated by anti-glutamine synthetase antibodies, (b) by the fact that growth-inhibiting concentrations of chloramphenicol also inhibit the incroporation of [¹⁴C]leucine into protein and into protein precipitated by anti-glutamine synthetase antibody, and (c) by the fact that chloramphenicol leads to acceleration in the loss of aspartokinases I and III and promotes a net decrease in the level of glutamine synthetase and its cross-reactive protein. The loss of aspartokinases I and III in cell suspensions is stimulated by glucose and is inhibited by 2,4-dinitrophenol. Glucose also stimulates the loss of aspartokinases and glutamine synthetase in the presence of chloramphenicol. Cell-free extracts of K. aerogenes catalyze rapid inactivation of endogenous glutamine synthetase as well as exogeneously added pure glutamine synthetase. This loss of glutamine synthetase is not associated with a loss of protein that cross-reacts with anti-glutamine synthetase antibodies. The inactivation of glutamine synthetase in extracts is not due to adenylylation. It is partially prevented by sulfhydryl reagents, Mn²⁺, antimycin A, 2,4-dinitrophenol, EDTA, anaerobiosis and by dialysis. Following 18 h dialysis, the capacity of extracts to catalyze inactivation of glutamine synthetase is lost but can be restored by the addition of Fe²⁺ (or Ni²⁺ together with ATP (or other nucleoside di- and triphosphates. After 40–60 h dialysis Fe³⁺ together with NADH (but not ATP) are required for glutamine synthetase inactivation. The results suggest that accelerated protein degradation in cells exposed to nitrogen-limited conditions reflects the differential destruction of some proteins, including aspartokinases I and III, in order to sustain the biosynthesis of others such as glutamine synthetase. The loss of glutamine synthetase activity in cell-free extracts is likely mediated in part by mixed-function oxidation systems and could represent a ‘marking’ step in protein turnover.     

LOCALIZATION OF POLYSOME-BOUND ALBUMIN AND SERINE DEHYDRATASE IN RAT LIVER CELL FRACTIONS

The polysomes involved in albumin and serine dehydratase synthesis were identified and localized by the binding to rat liver polysomes of anti-rat serum albumin and anti-serine dehydratase [¹²⁵I]Fab dimer and monomer. Techniques were developed for the isolation of undegraded free and membrane-bound polysomes and for the preparation of [¹²⁵I]Fab monomers and dimers from the IgG obtained from the antisera to the two proteins, rat serum albumin and serine dehydratase. The distribution of anti-rat serum albumin [¹²⁵I]Fab dimer in the polysome profile is in accordance with the size of polysomes that are expected to be synthesizing albumin. By direct precipitation, it has been demonstrated that nascent chains isolated from the membrane-bound polysomes by puromycin were precipitated by anti-rat serum albumin-IgG at a level of 5–6 times those released from free polysomes. Anti-rat serum albumin-[¹²⁵I]Fab dimer reacted with membrane-bound polysomes almost exclusively compared to the binding of nonimmune, control [¹²⁵I]Fab dimer; a significant degree of binding of anti-rat serum albumin-[¹²⁵I]Fab to free polysomes was also obtained. The [¹²⁵I]Fab dimer made from normal control rabbit serum does not react with polysomes from liver at all and this preparation will not interact with polysomes extracted from tissues that do not synthesize rat serum albumin. Both anti-serine dehydratase-[¹²⁵I]Fab monomer and dimer react with free and bound polysomes from livers of animals fed a chow diet or those fed a high 90% protein diet and given glucagon. In the latter instance, however, it is clear that the majority of the binding occurs to the bound polysomes. Furthermore, the specificity of this reaction may be further shown by the use of kidney polysomes that do not normally synthesize serine dehydratase. When these latter polysomes are isolated, even after the addition of crude and purified serine dehydratase, no reaction with anti-serine dehydratase-Fab fragments could be demonstrated. These results indicate that the reaction of the Fab fragments are specific for polysomes that synthesize rat serum albumin or rat liver serine dehydratase. Furthermore, they demonstrate that even with this high degree of specificity, some polysomes in the fraction labeled "free" are in the process of synthesizing rat serum albumin while bound polysomes to a significant, if not major, degree are the site of the synthesis of rat liver serine dehydratase.     

A putative protein-sequestration site involving intermediate filaments for protein degradation by autophagy. Studies with microinjected purified glycolytic enzymes in 3T3-L1 cells.

Several glycolytic enzymes (lactate dehydrogenase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase) were radiolabelled by [125I]iodination, conjugation with 125I-labelled Bolton & Hunter reagent and reductive [3H]methylation, and their degradative rates after microinjection into 3T3-L1 cells compared with that of the extracellular protein bovine serum albumin. Although the albumin remains largely cytosolic in recipient cells, the glycolytic enzymes rapidly (less than 30 min) become insoluble, as measured by detergent and salt extractions. The microinjected glycolytic enzymes appear to form disulphide-linked aggregates, are found in a cell fraction rich in vimentin-containing intermediate filaments and histones (nuclear-intermediate-filament fraction), and are degraded slowly by a lysosomal mechanism, as judged by the effects of inhibitors (NH4Cl, leupeptin, 3-methyladenine). 125I-labelled bovine serum albumin appears to be degraded rapidly and non-lysosomally. Prolonged treatment (96 h) of cultured cells with leupeptin results in the accumulation of pulse-labelled ([35S]methionine for 24 h) endogenous cell proteins in the detergent-and salt-non-extractable residue, but NH4Cl and 3-methyladenine do not have this effect. The findings are in terms of the interpretation of experiments involving microinjection of proteins to study intracellular protein protein degradation by autophagy.     

Alteration of immunological properties of bovine serum albumin by covalent attachment of polyethylene glycol.

Methoxypolyethylene glycols of 1900 and 5000 daltons have been attached covalently to bovine serum albumin using cyanuric chloride as the coupling agent. When sufficient polymer is attached, the modified bovine serum albumin appears to lose its immunogenicity in the rabbit and, on intramuscular or intravenous injection, elicits antibodies neither to itself nor to native bovine serum albumin. It does not react with antibodies raised against native bovine serum albumin. Bovine serum albumin to which methoxypolyethylene glycol has been attached exhibits a blood circulating life in the rabbit rather similar to native bovine serum albumin, except that it is not removed from circulation by the eventual development of antibodies. Modified bovine serum albumins which had been iodinated with 125I, or prepared with [14C]cyanuric chloride, were injected intravenously in rabbits. Both labels appeared almost quantitatively in the urine after 30 days. The modified bovine serum albumins showed substantial changes in properties, such as solubility, electrophoretic mobility in acrylamide gel, ion exchange chromatography, and sedimentation, as compared with the unmodified protein.     

Development of a new thiol-reactive prosthetic group for site-specific labeling of biomolecules with radioactive iodine

In this report, we describe the radiosynthesis of a new thiol-targeting prosthetic group for efficient radioactive iodine labeling of biomolecules. Radioiodination using the precursor 3 was performed to obtain ¹²⁵I-labeled tetrazole 4b with high radiochemical yield (73%) and radiochemical purity. Using the radiolabeled 4b, a single free cysteine containing peptide and human serum albumin were labeled with ¹²⁵I in modest-to-good radiochemical yields (65–99%) under mildly reactive conditions. A biodistribution study of [¹²⁵I]7 in normal ICR mice exhibited lower thyroid uptake values than those of ¹²⁵I-labeled human serum albumin prepared via a traditional radiolabeling method. Thus, [¹²⁵I]7 could be employed as an effective radiotracer for molecular imaging and biodistribution studies. The results clearly demonstrate that 4b has the potential to be effectively implemented as a prosthetic group in the preparation of radiolabeled biomolecules.     

Subcellular distribution of various proteases in Escherichia coli.

It has been reported recently that Escherichia coli cells contain eight distinct soluble enzymes capable of degrading proteins to acid-soluble material. Two are metalloproteases that degrade [125I]insulin but not larger proteins: protease Pi, which is identical to protease III, is restricted to the periplasm, and protease Ci is restriction to the cytoplasm. The six others (named Do, Re, Mi, Fa, So, and La, which is the ATP-dependent protease) are serine proteases that degrade [14C]globin and [3H]casein, but not insulin. One of these (Mi) is localized to the periplasm, and one (Re) is distributed equally between the two cellular fractions. The others are present only in the cytoplasm.     

Protein deamidase from germinating wheat grains.

A new enzyme catalyzing the deamidation of seed storage proteins was found in germinating wheat grains and was partially purified. It also acts on egg lysozyme, horse hemoglobin and reduced RNAse, glutamine and Gly-L-Gln-L-Tyr. No activity was observed when using ovalbumin, serum albumin, RNAse, insulin, asparagine and an asparagine-containing peptide. Only glutaminyl residues appear to be deamidated by this enzyme. It differs from transglutaminase and proved to be a true protein deamidase.     

A double-antibody radioimmunoassay for serum progesterone using progesterone-3-(O-carboxymethyl) oximino-[125I]-iodo-histamine as radioligand.

A reliable, convenient and economical radioimmunoassay (RIA) for serum progesterone has been established and tested. This procedure employs diethyl ether extraction followed by RIA utilizing rabbit anti-11 alpha-hydroxyprogesterone 11-hemisuccinyl-bovine serum albumin (progesterone-11 alpha-BSA) serum, progresterone-3-(O-carboxymethyl) oximino-[125I]-iodohistamine (progesterone-3-[125I]) as radioligand and goat anti-rabbit gamma globulin as second antibody. In conjunction with antiprogesterone-11 alpha-BSA serum, the overall assay specificity of the progesterone-3-[125I] RIA is similar to that of the [3H]-progesterone method using dextran-coated charcoal. The results of serum progesterone measurements during the menstrual cycle obtained by the progesterone-3-[125I] RIA appear comparable to those of [3H]-progesterone assays which employ similar anti-progesterone-11 alpha-BSA sera. The progesterone-3-[125I] double-antibody RIA, however, is more convenient and less expensive than the [3H]-progesterone RIA method.     

Highly efficient method for 125I-radiolabeling of biomolecules using inverse-electron-demand Diels–Alder reaction

In this report, we present a rapid and highly efficient method for radioactive iodine labeling of trans-cyclooctene group conjugated biomolecules using inverse-electron-demand Diels–Alder reaction. Radioiodination reaction of the tetrazine structure was carried out using the stannylated precursor 2 to give ¹²⁵I-labeled product ([¹²⁵I]1) with high radiochemical yield (65 ± 8%) and radiochemical purity (>99%). For radiolabeling application of [¹²⁵I]1, trans-cyclooctene derived cRGD peptide and human serum albumin were prepared. These substrates were reacted with [¹²⁵I]1 under mild condition to provide the radiolabeled products [¹²⁵I]6 and [¹²⁵I]8, respectively, with excellent radiochemical yields. The biodistribution study of [¹²⁵I]8 in normal ICR mice showed significantly lower thyroid uptake values than that of ¹²⁵I-labeled human serum albumin prepared by a traditional radiolabeling method. Therefore [¹²⁵I]8 will be a useful radiolabeled tracer in various molecular imaging and biological studies. Those results clearly demonstrate that [¹²⁵I]1 will be used as a valuable prosthetic group for radiolabeling of biomolecules.     

Glutamate Synthetase in Developing Cotyledons of Pisum sativum

Glutamate synthetase (glutamine[amide]:α ketoglutarate amino transferase oxidoreductase) activity has been demonstrated in the developing cotyledons of Pisum sativum L. cv. Burpeeana. The enzyme appears to be soluble and is specific for glutamine as amide donor. The enzyme activity is greater with NADH than with NADPH as electron donor.     

一种米曲霉蛋白酶的酶学性质及其在酪蛋白磷酸肽制备中的应用

【目的】分离纯化米曲霉蛋白酶的主要组分,分析其酶学特性,并应用于酪蛋白磷酸肽(Casein phosphopeptides,CPPs)的制备。【方法】采用硫酸铵盐析、DEAE-Sepharose FF阴离子交换层析和Butyl-sepharose HP疏水层析对米曲霉蛋白酶进行分离纯化,SDS-PAGE检测分子量与纯度,MALDI-TOF-MS检测酶切位点。【结果】得到一种蛋白酶组分(命名为PE),分子量大小为58 kD左右。该酶最适反应条件为55 °C,pH 8.0,酶活被Fe3+抑制,被Mn2+激活。以酪蛋白为底物时,Km=0.36 g/L,最大反应速率Vm=18.18 mg/(L?min)。蛋白酶PE对牛胰岛素B链上-Leu-Cys-、-Val-Glu-、-Tyr-Leu-和-Arg-Gly-组成的肽键有较高的切割能力,酶切位点较多。利用其水解酪蛋白,通过钡-乙醇沉淀法得到CPPs,产率为15.87%,摩尔氮磷比r (N/P)为6.17,得到的CPPs可以使钙沉淀推迟35 min。【结论】利用米曲霉蛋白酶水解酪蛋白产生CPPs,为其在功能性食品加工方面的应用提供有利的参考。