Behavior of L-threonine-degrading enzymes during liver regeneration

We examined the effects of a two-thirds hepatectomy in the adult rat on the activities of the three L-threonine-degrading enzymes, L-threonine dehydratase, L-threonine aldolase and L-threonine dehydrogenase. Noticeable variations were observed which did not occur in either sham-operated or turpentine-treated rats and were not linked to food intake. They were considered specific to the regenerating liver. When the reactions were followed in vitro, L-threonine deaminase and L-threonine aldolase were significantly lower for the first 12-24 h: L-threonine dehydrogenase decreased only after 48 h. These results are linked to a decrease in the enzyme concentration in the tissue. L-Serine and L-threonine liver concentrations increased 2-3-fold during the same periods. When the activities were evaluated in vivo, the levels of the first two enzymes remained constant for 24 h, but increased after 48 h; L-threonine dehydrogenase increased between 12 and 48 h. The in vivo activity of the enzymes was reflected by total L-threonine degradation, which had a single sharp peak at 48 h. The asynchronous variations in enzyme activity are related to the differences in protein metabolism which occur in the regenerating liver, and are the consequence of a new transient differential control. The changes observed are significant in liver regeneration; they regulate the consumption and the serum and liver levels of L-serine and L-threonine, setting them aside for protein synthesis. They minutely control the flux of amino acids toward gluconeogenesis, since, during the first 48 h after partial hepatectomy, the production of glucose is ensured principally by lactate; the contribution of L-threonine seems to be more significant only at 48 h. These findings are useful in the study of the regulation of the enzymes involved in amino acid metabolism during liver regeneration.     

L-serine deaminating enzymes in Escherichia coli crude extracts

(a) The measured L-serine deaminating activity of a crude bacterial extract may originate from L-serine deaminase, from biosynthetic L-threonine deaminase, or from degradative L-serine deaminase. Nevertheless, the contribution of the individual enzymes can be determined.(b) About a half of the L-serine deaminating activity of wild type E. coli bacteria, grown in synthetic minimal medium, originates from L-serine deaminase and about half from biosynthetic L-threonine deaminase.(c) Ninety percent of L-serine deaminating activity of wild type E. coli bacteria, grown in yeast extract-tryptone medium, originates from L-serine deaminase, and the remainging ten percent from the degradative L-threonine deaminase.(d) Conditions have been established in which threonine deaminases are eliminated and the activity of L-serine deaminase alone could be measured, even in crude extracts.     

Purification and characterization of zeta-crystallin/quinone reductase from guinea pig liver.

zeta-Crystallin, a major lens protein of certain mammalian species, has recently been characterized as a novel and active NADPH:quinone oxidoreductase. Here we report the purification of this protein from guinea pig liver by utilizing sequentially: ammonium sulphate precipitation, Blue Sepharose affinity, cation exchange and hydrophobic chromatography steps. This four-step isolation procedure yielded 118-fold purification and a specific activity of 6 U/mg protein when assayed in the presence of 9,10-phenanthrenequinone. Kinetic, immunological and physical properties of this protein have been found to be identical with those of guinea pig lens zeta-crystallin. Western blot analysis using antibodies raised against zeta-crystallin peptides demonstrated the presence of substantial amounts of this protein in human liver homogenates.     

An improved method for purification of L-threonine deaminase from rat liver

L-threonine deaminase was obtained at a high degree of purity from rat liver. Two main steps were added to the previously reported procedure: cryoprecipitation and chromatofocusing (in the presence of a specific KCl concentration). The purification factor was 3,090 and the specific activity 989. The method is very reproducible and convenient. It gives the highest specific activity and the highest degree of purity of the enzyme recorded to date.     

Structure and function correlations between the rat liver threonine deaminase and aminotransferases

The rat liver threonine deaminase is a cytoplasmic enzyme that catalyses the pyridoxal-phosphate-dependent dehydrative deamination of L-threonine and L-serine to ammonia and alpha-ketobutyrate and pyruvate, respectively, in vivo. During deamination, a molecule of the cofactor is converted to pyridoxamine phosphate. Recently, the ability of this enzyme to accomplish an inverse half-reaction, restoring pyridoxal-phosphate and L-alanine or L-aminobutyrate, respectively, from pyruvate or 2-oxobutyrate, was reported. In order to investigate the molecular mechanisms of this transaminating activity, a molecular model of rat liver threonine deaminase was constructed on the basis of sequence homology with the biosynthetic threonine deaminase of Escherichia coli, the crystal structure of which is known. The model has structural features shared by aminotransferases, suggesting that tertiary structural elements may be responsible for the transaminating activity observed for rat liver threonine deaminase.     

Swine liver l-leucine aminopeptidase: improved purification procedure

An l-leucine aminopeptidase, having a specificity toward the substrate l-leucine amide, was purified 1084-fold from swine liver with a yield of 50.7 per cent. Purification procedure was carried out using successively centrifugation at 105 000 × g fractionation by ammonium sulfate, DEAE Sephacel chromatography and zonal ultracentrifugation.Enzyme homogeneity and purity studies were carried out by analytical ultracentrifugation and polyacrylamide gel electrophoresis.In SDS gel polyacrylamide a single band was observed. It corresponded to a 55 000 molecular weight protein.     

Purification and characterization of the larvicidal toxin of Bacillus sphaericus 1593M

We present here a procedure for purifying the larvicidal toxin from sporulating cells of Bacillus sphaericus 1593M and describe some of the biochemical and biophysical properties of this toxin. The procedure involves solubilization of the cell-wall/membrane bound toxin by sonication of cells followed by repeated rounds of freezing and thawing at 50 degrees C. Further purification involved Sephadex G-100 and DEAE Sephacel chromatography. We show by Sephadex G-100 chromatography that at pH 7.5 the smallest active form of the toxin has an Mr of 38,000 and that this toxin can reversibly aggregate to molecular forms of a size higher than 2 X 10(5) Mr. By shifting the pH from 7.5 to 8.5 only the aggregated forms can be observed.     

Detection and purification of a liver microsomal enzyme inducing the release of a glycopeptide inhibitor of hepatocyte proliferation from human alpha 2 macroglobulin

The interaction rat liver microsomes/human alpha 2 macroglobulin releases in vivo an inhibitory peptide of the hepatocyte proliferation. Treatment of Triton X 100 on adult rat liver microsomes enables the solubilisation of a proteolytic enzyme. Its partial purification was obtained by Ultrogel AcA 44 filtration followed by a DEAE Sephacel chromatography. This enzyme shows a proteolytic activity in presence of calcium on synthetic substrates including Phe, Tyr and Trp. A whole enzyme inhibition is got after treatment by DFP or benzamidine. In presence of highly purified human alpha 2 macroglobulin this enzyme releases a glycopeptide of low molecular weight, which inhibits the hepatocyte proliferation during the G1-S transition in baby rat.     

Purification of a syngeneic cross-reactive autotumorolytic factor

An autotumorolytic fraction (ATF) derived from sera of mice bearing spontaneous mammary adenocarcinoma has been purified by sequential chromatography on Sephacryl 200 SF, DEAE Sephacel, hydroxylapatite, and Sephacryl 200 SF. At each stage of the purification, tumorolytic activity was assessed by the ability of the active material to induce lysis of a 2-week-old mammary tumor, and verified by histopathological analysis. Active fractions obtained during the stages of purification were capable of inducing lysis of tumors in both the original donor mice and the syngeneic tumor-bearing mice. Lytic activity was specific only for tumor tissue. This syngeneic crossreactivity of partially purified materials permitted pooling of serum samples for purification purposes. The resultant final preparation of ATF is an homogenous protein fraction, as verified by gradient SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography. The molecular weight of active ATF is approximately 50,000 Da, as determined by gradient SDS-PAGE.     

Purification and characterization of 2-oxoaldehyde dehydrogenase from rat liver.

The partial purification (123-fold) of 2-oxoaldehyde dehydrogenase (2-oxoaldehyde:NAD(P)+ oxidoreductase, 1.2.1.23) from rat liver was carried out using a purification procedure which involved (NH4)2SO4 fractionation, DEAE-Sephadex chromatography, Blue-Dextran affinity chromatography and CM-Sephadex chromatography. A single form of the enzyme was observed, mol. wt. approx. 50000 by gel chromatography. 2-Oxoaldehyde dehydrogenase appears to be highly specific for NADP+ and methylglyoxal. No activity is observed in the absence of certain amines which have vicinal amino and hydroxyl groups. The only known amine which activates the enzyme at physiological pH is L-serine methyl ester, suggesting that the regulation of this enzyme in vivo may require a derivative of serine.     

Detection and properties of L-threonine-L-serine dehydratase in human liver]

It has been shown that in liver extract of men deceased by different causes, L-threonine and L-serine dehydratase activities probably, belonging to only one enzyme--L-threonine-L-serine dehydratase--are found. Both activities and their ratios depend on K+ concentration both in the buffer used for enzyme extraction and in the reaction medium. Before extraction of active and stable forms of enzyme the liver is to homogenized in a buffer containing 0.15 M KCl. Both enzymatic activities have a pH-optimum at pH 9.6--10.0. It was shown that D-isomers of threonine and serine are not dehydratated and do not inhibit dehydratation of L-isomers. Studies of dependence of L-threonine and L-serine dehydratase reaction rates on temperature showed that at any temperature ranges the energy activation values are higher for the L-threonine dehydratase reaction than for the L-serine dehydratase reaction and that the ratio reaction rates for both reactions depends on temperature.     

Purification, stabilization, and characterization of rat hepatic triglyceride lipase

Hepatic triglyceride lipase (H-TGL) was purified to near homogeneity from heparin-containing rat liver perfusates with the following column chromatography steps: heparin-Sepharose affinity chromatography, anion-exchange chromatography on DEAE-Sephacel, and gel filtration on Ultrogel AcA 34. A final specific activity of 45,000 μmol fatty acid/mg/h was obtained with an overall 31% recovery of catalytic activity. The heparin-Sepharose step resulted in a 20-fold purification, while the DEAE and gel filtration steps led to further purification with complete recovery of activity. An extensive survey of various detergents as potential stabilizers of H-TGL activity led to the selection of Triton N-101 for use in the column buffers of the DEAE and gel filtration steps. Relative to initial H-TGL activity upon dilution in buffer without detergent, recoveries between 90 and 100% were consistently obtained with Triton N-101-containing buffers following a 24-h incubation at 20°C. In contrast after a 24-h incubation at 20°C those control samples lacking detergent were at least 95% inactivated. The highly purified H-TGL exhibited a single major band by sodium dodecyl sulfate-electrophoresis. The use of DEAE chromatography and stabilization of H-TGL with Triton N-101 are the improvements in purification that resulted in an 8-fold enhancement in specific activity relative to the highest previous report of purification from rat liver perfusates.     

Serine utilization by Klebsiella aerogenes.

Klebsiella aerogenes was found to contain a specific L-serine dehydrase that was induced by threonine, glycine or leucine, but not by its substrate. Cellular concentrations were sensitive to carbon rather than nitrogen sources in the growth medium. A nonspecific isoleucine-sensitive L-threonine dehydrase supplemented the specific L-serine dehydrase activity. K. aerogenes also contains a leucine-inducible L-threonine dehydrogenase which probably initiated a threonine-utilization pathway in which the serine-specific dehydrate participated. Strains that were altered in their ability to metabolize serine differed in either L-serine dehydrase or L-threonine dehydrase activity. Thus, K. aerogenes growing on L-serine as a sole nitrogen source relies upon two enzymes that metabolize the amino acid as subsidiary functions.     

Induction of creatine kinase in immature rat uteri by zearalenone, an estrogenic mycotoxin

Intraperitoneal administration of alpha-zearalenone (ZEN) to female immature rats induced synthesis of a uterine protein which has been identified as creatine kinase. The induced enzyme was purified to homogeneity by chromatography on DEAE Sephacel and Hydroxyapatite Ultrogel. Guinea pig antiserum against estrogen-induced uterine, rat uterine creatine kinase crossreacted with the ZEN-induced enzyme, indicating that ZEN exhibits an early estrogenic response in a manner analogous to natural estrogens.     

Purification and some physicochemical properties of slow-reacting substance of anaphylaxis (SRS-A) from sensitized guinea pig lung.

The slow-reacting substance of anaphylaxis (SRS-A) generated by antigen challenge of sensitized guinea pig lung fragments was partially purified and the physicochemical properties of this activity were studied. The SRS-A recovered from antigen challenged lung preparations of 600 animals was used for the purification procedure. Treatment with organic solvents, extraction with 80% ethanol, Sephadex LH-20 chromatography with 80% ethanol, and DEAE-Sephadex A-25 chromatography in 60% methanol eluted with 0.0 to 0.1 M NaCl in 60% methanol was the purification sequence finally adopted. Overall recovery of SRS-A bioactivity was 60% with a specific activity of 2.52 units/ng of dry weight. This represented a 1.67 million-fold purification over the starting material. The DEAE-Sephadex A-25 step alone provided a 7600-fold purification. This highly purified SRS-A had an apparent molecular weight of 380 to 400 daltons. The bioactivity was acid labile and alkaline stable and was blocked by low concentrations of the SRS-A antagonist FPL 55712. The SRS-A was thermostable in aqueous media and displayed enhanced bioactivity after heating at 60 C for 60 min. These results indicate that we have developed a highly efficient new approach to the isolation of guinea pig SRS-A, which also may be useful in the study of SRS-A from other tissues or species. The physicochemical properties of guinea pig SRS-A appear to be very similar to those of SRS-A from other species.     

Identification, purification, and characterization of a thermophilic imidase from pig liver

This study investigates thermophilic imidase activity of the liver. We demonstrate that imidase catalyzes the hydrolysis of imides at a temperature substantially higher than that of its native environment. Then, a thermophilic imidase is purified to homogeneity from pig liver, and its thermoproperties are studied. About 2500-fold of purification and 15% yield of imidase activity are obtained after ammonium sulfate precipitation, octyl, DEAE, chelation, and gel filtration chromatography. While avoiding heat treatment for the protein purification, this study also indicates that only one enzyme is responsible for the imidase activity. This homogenous enzyme prefers to catalyze hydrolysis of imides at above 60 degrees C rather than at the body temperature of a pig. Although stable at below 50 degrees C, imidase quickly loses its activity at above 65 degrees C. Thus, the temperature effect on imidase activity is limited mainly by its thermostability. Substrate specificity of imidase is also temperature dependent. Our results demonstrate that the hydrolysis of physiological substrates is the most temperature dependent and that of hydantoins is the least temperature dependent. When increasing the reaction temperature from 25 to 60 degrees C, specific activities increase 50- and 60-fold for dihydrouracil and dihydrothymine, respectively. The temperature effect on the K(m) and V(max) of imidase is substrate dependent.     

Chromatofocusing of mammalian estrone sulfate sulfohydrolase activity

Estrone sulfate sulfohydrolase (estrogen sulfatase) activity was solubilized by treatment with Triton X-100 from 105,000 g pellets of guinea pig uterus, testis and brain, as well as from rat liver and human placenta. The solubilized forms were subjected to chromatofocusing in the fast protein liquid chromatography (FPLC) system and on conventional columns packed in our laboratory. The guinea pig tissue pattern was complex. Uterus showed peaks of activity with apparent pI's of 9.11 and 7.6; testis contained 3 peaks with pI's of 9.18, 8.7 and 7.5; brain possessed peaks with pI's of 9.28 and 8.6. In each case the major activity peak was that with pI greater than 9. Rat liver activity chromatofocused as a single peak of apparent pI = 6.87 and the human placental enzyme also showed a single, though broad, peak, of pI = 6.57. This suggests not only that the guinea pig enzyme(s) differs markedly from those of rat liver and human placenta, but that there may be qualitative differences between the forms in the three guinea pig tissues. Chromatofocusing behaviour was not independent of the specific exchange resins and ampholytes utilized. The recovered enzyme activity was fairly stable and it seems that chromatofocusing could be a useful step in purification of the guinea pig enzyme(s), particularly the main form possessing a pI greater than 9.     

Purification and characterization of serine racemase from a hyperthermophilic archaeon, Pyrobaculum islandicum

Pyrobaculum islandicum is an anaerobic hyperthermophilic archaeon that is most active at 100 degrees C. A pyridoxal 5'-phosphate-dependent serine racemase called Srr was purified from the organism. The corresponding srr gene was cloned, and recombinant Srr was purified from Escherichia coli. It showed the highest racemase activity toward L-serine, followed by L-threonine, D-serine, and D-threonine. Like rodent and plant serine racemases, Srr is bifunctional, showing high L-serine/L-threonine dehydratase activity. The sequence of Srr is 87% similar to that of Pyrobaculum aerophilum IlvA (a putative threonine dehydratase) but less than 32% similar to any other serine racemases and threonine dehydratases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration analyses revealed that Srr is a homotrimer of a 44,000-molecular-weight subunit. Both racemase and dehydratase activities were highest at 95 degrees C, while racemization and dehydration were maximum at pH 8.2 and 7.8, respectively. Unlike other, related Ilv enzymes, Srr showed no allosteric properties: neither of these enzymatic activities was affected by either L-amino acids (isoleucine and valine) or most of the metal ions. Only Fe2+ and Cu2+ caused 20 to 30% inhibition and 30 to 40% stimulation of both enzyme activities, respectively. ATP inhibited racemase activity by 10 to 20%. The Km and Vmax values of the racemase activity of Srr for L-serine were 185 mM and 20.1 micromol/min/mg, respectively, while the corresponding values of the dehydratase activity of L-serine were 2.2 mM and 80.4 micromol/min/mg, respectively.     

Effects of starvation, a hyperprotein diet and treatment with nicotinamide on L-threonine aldolase and allothreonine aldolase in the rat liver

In order to evaluate if the aminoacid L-threonine and its isomer allothreonine are metabolized by a single protein or by two specific enzymatic proteins, in the rat liver, we have studied the behaviour of L-threonine aldolase activity and allothreonine aldolase activity in rats which are injected with nicotinamide (1 g/ Kg) or have received a special diet containing 91% of casein or that have been starved for several days. The L-threonine aldolase enhances its activity in all three cases, while the allothreonine aldolase does not show significative changes of activity. These different behaviours of the two enzymatic activities, support the hypothesis that in the rat liver two proteins are necessary for the metabolism of the L-threonine and its isomer allothreonine.     

Purification from goat antiserum of immunoglobulins against G6PD from rabbits]

DEAE Affi-Gel Blue (Bio-Rad) provides an efficient and rapid fractionation of human serum proteins by a single chromatographic step. When goat serum is applied to the matrix and chromatography is performed following the procedure utilized for the human serum proteins, the elution pattern changes and the Ig purification is not satisfactory. We achieved a better Ig purification from goat serum by the following improved procedure. We performed first an AS-40 fractionation followed by extensive dialysis in 50 mM Na-citrate pH 5.7. The sample was then loaded onto a P11 column equilibrated in the same buffer. The fraction eluted at Vo contained total IgG and the other serum proteins, except beta-globulins which were eluted with 0.24 M phosphate. Peak 1 concentrated and dialyzed in 20 mM phosphate buffer pH 8 was then applied to a DEAE Affi-Gel Blue column, equilibrated in the same buffer. Two protein peaks were eluted from this column and electrophoretically characterized as: peak 1, containing a pure Ig fraction (70% yield), peak 2 with albumin and other contaminating serum proteins. When goat antiserum is obtained against a specific protein, our technique may be suitably employed to purify polyclonal antibodies for immunoprecipitation studies.