scholarly journals A novel type of putrescine (diamine)-acetylating enzyme from the nematode Ascaris suum

1989 ◽  
Vol 260 (1) ◽  
pp. 265-269 ◽  
Author(s):  
R M Wittich ◽  
R D Walter
Keyword(s):  
Parasitic Nematode ◽  
Ascaris Suum ◽  
Deae Cellulose ◽  
Reproductive Tissue ◽  
Freezing And Thawing ◽  
Acetyl Coa ◽  
Michaelis Constants ◽  
The Common ◽  
Derivatives Of ◽  
Thawing Procedure

A cytosolic enzyme catalysing the acetylation of the diamines putrescine, cadaverine, 1,3-diaminopropane and 1,6-diaminohexane has been partially purified from reproductive tissue of the intestinal parasitic nematode Ascaris suum. The enzyme formed N-acetylated derivatives of the above diamines when incubated in the presence of acetyl-CoA. The Michaelis constants (Km) for the above diamines were 0.25 nM, 0.1 mM, 1.25 mM and 0.4 mM respectively, and the apparent Km for acetyl-CoA was 7.7 microM. sym-Norspermidine was also acetylated by this enzyme preparation, and, at a much lower rate, the enzyme acted on sym-norspermine. The common polyamines, spermidine and spermine, and histones were not substrates. Purification steps involved a freezing-and-thawing procedure to release enzyme activity from unknown inhibitors, DEAE-cellulose chromatography and affinity chromatography on cadaverine-Sepharose, from which the enzyme was eluted by increasing ionic strength. The enzyme exhibited an apparent Mr of about 38,000-40,000, and it consisted of at least two subunits, of which the catalytic one had an Mr of about 13,000. The partially purified enzyme showed no deacetylase activity, and its activity was competitively inhibited by the product N-acetylputrescine, but not by CoA. The name putrescine N-acetyltransferase is suggested for this enzyme, which may have an important function in the degradation of diamines of lower eukaryotes.

Autoprothrombin C in Irregular Blood Clotting

1962 ◽  
Vol 08 (01) ◽  
pp. 001-020
Author(s):  
Walter H. Seegers ◽  
Eva Marciniak
Keyword(s):  
Blood Clotting ◽  
Hemophilia A ◽  
Deae Cellulose ◽  
Hemophilia B ◽  
Factor Vii ◽  
Factor X ◽  
The Common ◽  
Autoprothrombin C ◽  
Derivatives Of ◽  
Autocatalytic Activation

SummaryIn this study autoprothrombin C was considered in relationship to the hemorrhagic diseases. Concentrates of autoprothrombin C were made from purified prothrombin known to be homogeneous by several criteria. These autoprothrombin C preparations were free of thrombin, and were almost a single component when analyzed by centrifugation. Autoprothrombin C corrected the partial thromboplastin time and produced prothrombin consumption in all the plasmas obtained from patients with hemorrhagic diseases except parahemophilia. In the case of hemophilia B prothrombin consumption was obtained with the addition of purified prothrombin or purified autoprothrombin II. In the case of Stuart plasma prothrombin consumption was rapid after the addition of purified prothrombin or purified prothrombin chromatographed on Amberlite ICR-50, but not after the addition of purified prothrombin that was chromatographed on DEAE cellulose. The latter prothrombin is an abnormal prothrombin molecule and does not readily yield autoprothrombin C. It is concluded that Stuart prothrombin is abnormal and that this is a molecular disease. There is no need to postulate the existence of factor X to account for the irregular prothrombin activation in Stuart plasma. The most important question considered was how can autoprothrombin C be generated from prothrombin to promote the autocatalytic activation of prothrombin. Certain prothrombin molecules do not yield this enzyme, but normal prothrombin does. However, it does so only under certain conditions of activation. By applying the new knowledge of prothrombin chemistry to blood clotting irregularities in hemorrhagic diseases the following main considerations highlight the common deviations: 1. Abnormal prothrombin molecule, 2. Changes related to the derivatives of prothrombin, 3. Accessories needed for the generation of autoprothrombin C so it can function in auto-catalysis are irregular, and 4. Accessories needed for the function of autoprothrombin C after it is out of the prothrombin molecule are irregular. In the first group is Stuart Plasma. In the second group falls hemophilia B (autoprothrombin II) and the so-called factor VII deficiency (autoprothrombin I). In the third group is hemophilia A. In the fourth group is parahemophilia, and the platelet abnormalities.

Three-Dimensional Structure of Cloned T3 Connector Protein at 1.6nm Resolution

1990 ◽  
Vol 48 (1) ◽  
pp. 282-283
Author(s):  
José L. Carrascosa ◽  
José M. Valpuesta ◽  
Hisao Fujisawa
Keyword(s):  
Dna Sequences ◽  
Expression Vector ◽  
Three Dimensional ◽  
Deae Cellulose ◽  
Dna Packaging ◽  
Dimensional Structure ◽  
Two Dimensional ◽  
Freezing And Thawing ◽  
Three Dimensional Structure ◽  
Dimensional Reconstruction

The head to tail connector of bacteriophages plays a fundamental role in the assembly of viral heads and DNA packaging. In spite of the absence of sequence homology, the structure of connectors from different viruses (T4, Ø29, T3, P22, etc) share common morphological features, that are most clearly revealed in their three-dimensional structure. We have studied the three-dimensional reconstruction of the connector protein from phage T3 (gp 8) from tilted view of two dimensional crystals obtained from this protein after cloning and purification.DNA sequences including gene 8 from phage T3 were cloned, into Bam Hl-Eco Rl sites down stream of lambda promotor PL, in the expression vector pNT45 under the control of cI857. E R204 (pNT89) cells were incubated at 42°C for 2h, harvested and resuspended in 20 mM Tris HC1 (pH 7.4), 7mM 2 mercaptoethanol, ImM EDTA. The cells were lysed by freezing and thawing in the presence of lysozyme (lmg/ml) and ligthly sonicated. The low speed supernatant was precipitated by ammonium sulfate (60% saturated) and dissolved in the original buffer to be subjected to gel nitration through Sepharose 6B, followed by phosphocellulose colum (Pll) and DEAE cellulose colum (DE52). Purified gp8 appeared at 0.3M NaCl and formed crystals when its concentration increased above 1.5 mg/ml.

scholarly journals Anthroponyms of Jewish Women in the 16th Century Grand Duchy of Lithuania

2012 ◽  
Vol 21 (26) ◽  
pp. 200-207
Author(s):  
Jūratė Čirūnaitė
Keyword(s):  
Average Length ◽  
Family Members ◽  
16Th Century ◽  
Canonical Forms ◽  
Family Status ◽  
Social Origin ◽  
Jewish Women ◽  
The Common ◽  
Grand Duchy Of Lithuania ◽  
Derivatives Of

The most popular names among Jewish women in 16th century Lithuania were Simcha, Marjam, Anna, Debora. The names were most frequently recorded as diminutives (63.3%), with only 36.4% appearing in canonical forms. The smallest group comprises names formed using only anthroponyms that were derived from those of (male) family members (29.6%). 35.2% of the namings are recorded as mixed type. The same number of women are recorded using only names in the documents.Personal names are included in 70.4% of recorded women’s namings. Andronyms (anthroponyms formed from the spouse’s name) were found in 64.8% of all the records. 9.3% of women’s namings include anthroponyms formed using the spouse’s patronymic. Only 1.9% of namings had a female patronymic (the derivative of the suffix -owna/-ewna).One-member female namings prevail (59.3%). Two-member namings comprise 33.3%. Three members are found in 5.6% of the namings, while four-membered ones comprise 1.9%. The average length of the namings is 1.5 times that of the anthroponyms.Common words explaining anthroponyms were found in 68.5% of the namings. Common words related to religion prevail (51.4%). 29.7% of the common words characterize relationships or family status, and only 10.8% describe occupation, post or trade (vocation). Common words describing descent (social origin) comprise only 8.1% of all the women’s namings.Namings consisting only of anthroponyms of family members can be subdivided into the following subgroups: 1) derivatives of the suffix -owaja/-ewaja; 2) derivatives of the suffix ‑owaja/-ewaja; 3) derivatives of the suffix -owaja/-ewaja + the genitive of a male patronymic; 4) derivatives of the suffix -owaja/-ewaja + a male patronymic + the genitive of a male patronymic. Namings without anthroponyms consisting of family members included names and names with common words. Mixed namings consisted of: 1) a name + a derivative of the suffix -owaja/-ewaja; 2) a derivative of the suffix -owaja/-ewaja + the genitive of a male patronymic + a name; 3) a derivative of the suffix -owaja/-ewaja + the genitive of a male patronymic + a name + a female patronymic.The most popular type of naming is a recorded name.

Characterization of 5-HT receptors in the parasitic nematode, Ascaris suum

Parasitology ◽  
2001 ◽  
Vol 122 (02) ◽  
Author(s):  
J.E. TRIM ◽  
L. HOLDEN-DYE ◽  
J. WILLSON ◽  
M. LOCKYER ◽  
R.J. WALKER
Keyword(s):  
Parasitic Nematode ◽  
Ascaris Suum

The ABA-1 Allergen of the Parasitic Nematode Ascaris suum: Fatty Acid and Retinoid Binding Function and Structural Characterization

Biochemistry ◽  
1995 ◽  
Vol 34 (20) ◽  
pp. 6700-6710 ◽  
Author(s):  
Malcolm W. Kennedy ◽  
Andrew Brass ◽  
Alan B. McCruden ◽  
Nicholas C. Price ◽  
Sharon M. Kelly ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Structural Characterization ◽  
Parasitic Nematode ◽  
Ascaris Suum ◽  
Binding Function

Phosphoenolpyruvate Carboxylase of Thiobacillus thiooxidans. Kinetic and Metabolic Control Properties

1972 ◽  
Vol 50 (2) ◽  
pp. 158-165 ◽  
Author(s):  
R. L. Howden ◽  
H. Lees ◽  
Isamu Suzuki
Keyword(s):  
Enzyme Activity ◽  
Competitive Inhibitor ◽  
Acetyl Coa ◽  
Ph Optimum ◽  
Pep Carboxylase ◽  
Thiobacillus Thiooxidans ◽  
Powerful Activator ◽  
Michaelis Constants ◽  
Noncompetitive Inhibitor ◽  
Decreasing Ph

Phosphoenolpyruvate (PEP) carboxylase (orthophosphate:oxalacetate carboxy-lyase (phosphorylating), EC 4.1.1.31) was purified 19-fold from Thiobacillus thiooxidans. The level of enzyme activity was dependent on culture age. No enzyme activity could be obtained from frozen cells.The pH optimum of the enzyme was determined to be around 8.0. Apparent Michaelis constants were determined for the substrates:phosphoenolpyruvate (1.4, 1.5 mM), bicarbonate (0.4, 1.1 mM), and magnesium (1.1, 0.8 mM) at pH 7.0 and 8.0, respectively. Acetyl-CoA was found to be a powerful activator of this enzyme, with the degree of activation increasing with decreasing pH. The concentration of acetyl-CoA to obtain half-maximal activation, however, remained fairly constant and low, namely 1.2 and 1.0 μM at pH 7.0 and 8.0, respectively. L-Aspartate and L-malate were strong inhibitors of enzyme activity. In the presence of aspartate at pH 7.0 the double reciprocal activity plots for PEP became nonlinear, a characteristic of negative cooperativity. These plots became linear with the addition of acetyl-CoA with aspartate now acting as a noncompetitive inhibitor with respect to PEP. At pH 8.0, the same plots were linear with aspartate acting as a competitive inhibitor of PEP. All the other effectors of PEP carboxylase from Salmonella typhimurium and Escherichia coli were found to be ineffective towards the enzyme from T. thiooxidans.

scholarly journals Ligand binding on to maize (Zea mays) malate synthase: a structural study

1994 ◽  
Vol 303 (2) ◽  
pp. 413-421 ◽  
Author(s):  
S Beeckmans ◽  
A S Khan ◽  
L Kanarek ◽  
E Van Driessche
Keyword(s):  
Zea Mays ◽  
Ligand Binding ◽  
Conformational Change ◽  
Limited Proteolysis ◽  
Enzymic Activity ◽  
Malate Synthase ◽  
Acetyl Coa ◽  
Original Activity ◽  
Kinetic Measurements ◽  
Michaelis Constants

A kinetic and ligand binding study on maize (Zea mays) malate synthase is presented. It is concluded from kinetic measurements that the enzyme proceeds through a ternary-complex mechanism. Michaelis constants (Km,glyoxylate and Km,acetyl-CoA) were determined to be 104 microM and 20 microM respectively. C.d. measurements in the near u.v.-region indicate that a conformational change is induced in the enzyme by its substrate, glyoxylate. From these studies we are able to calculate the affinity for the substrate (Kd,glyoxylate) as 100 microM. A number of inhibitors apparently trigger the same conformational change in the enzyme, i.e. pyruvate, glycollate and fluoroacetate. Another series of inhibitors bearing more bulky groups and/or an extra carboxylic acid also induce a conformational change, which is, however, clearly different from the former one. Limited proteolysis with trypsin results in cleavage of malate synthase into two fragments of respectively 45 and 19 kDa. Even when no more intact malate synthase chains are present, the final enzymic activity still amounts to 30% of the original activity. If trypsinolysis is performed in the presence of acetyl-CoA, the cleavage reaction is appreciably slowed down. The dissociation constant for acetyl-CoA (Kd,acetyl-CoA) was calculated to be 14.8 microM when the glyoxylate subsite is fully occupied by pyruvate and 950 microM (= 50 x Km) when the second subsite is empty. It is concluded that malate synthase follows a compulsory-order mechanism, glyoxylate being the first-binding substrate. Glyoxylate triggers a conformational change in the enzyme and, as a consequence, the correctly shaped binding site for acetyl-CoA is created. Demetallization of malate synthase has no effect on the c.d. spectrum in the near u.v.-region. Moreover, glyoxylate induces the same spectral change in the absence of Mg2+ as in its presence. Nevertheless, malate synthase shows no activity in the absence of the cation. We conclude that Mg2+ is essential for catalysis, rather than for the structure of the enzyme's catalytic site.

scholarly journals Neuronal N-acetyl-β-D-glucosaminidase. Evidence for its biosynthesis in vitro

1976 ◽  
Vol 158 (3) ◽  
pp. 513-527 ◽  
Author(s):  
J A Khawaja ◽  
O Z Sellinger
Keyword(s):  
Concanavalin A ◽  
Microsomal Fraction ◽  
Deae Cellulose ◽  
Neuronal Cell ◽  
Amino Acid Mixture ◽  
Acid Mixture ◽  
Freezing And Thawing ◽  
Cellulose Acetate Electrophoresis ◽  
Fluorescent Band

Neuronal cell bodies, isolated in bulk from 8-day-old rat cerebral cortices, were incubated in the presence of a 3H-labelled amino acid mixture, and subcellular fractions isolated by differential centrifugation. The particulate fractions were frozen/thawed in 0.20 M-sucrose/0.1 M-KCl [Selling et al. (1973) Biochim. Biophys. Acta 315, 128-146] and the profiles of acid-insoluble radioactivity and N-acetyl-beta-D-glucosaminidase (glucosaminidase) activity compared in the resulting non-sedimentable fractions by DEAE-cellulose chromatography and cellulose acetate electrophoresis. Radioactivity and glucosaminidase activity co-migrated to a significant extent. Electrophoresis revealed that after 1 min of incubation 42% of the radioactivity of the non-sedimentable microsomal fraction after freezing and thawing co-migrated with an intensely fluorescent band of glucosaminidase activity. Since the pellet fraction obtained on freezing/thawing the microsomal fraction contained up to 75% of the RNA, 95% of the radioactivity and 45% of the glucosaminidase, a detailed study of the association between its radioactivity and nascent glucosaminidase activity was undertaken. After 1 and 2 min of incubation, followed by centrifugation of the microsomal pellet on 35-60% (w/v) sucrose density gradients, radioactivity and glucosaminidase activity exhibited parallel profiles in the region of heavy polyribosomes and at the top of the gradient which contains spontaneously released nascent polypeptide chains. DEAE-cellulose chromatography of these chains revealed glucosaminidase A to be the principal nascent glucosaminidase component, with glucosaminidases B and C as minor peaks. After 2 min of incubation, all of the glucosaminidase components appeared labelled, and glucosaminidase A exhibited two distinct sub-components. The pattern of glucosaminidase labelling in the soluble and microsomal fractions suggested that newly formed glucosaminidase molecules traverse both the cellular sap and the lumen of the endoplasmic reticulum. Only glucosaminidase A reacted specifically with concanavalin A and radioactive glucosaminidase A could be successfully regenerated by treatment with alpha-methyl-D-glucoside. Glucosaminidase A and a substantial portion of the radioactivity associating with it could be readily converted into glucosaminidase B by re-chromatography on DEAE-cellulose and by reaction of the concanavalin A-glucosaminidase A complex with methyl glucosides.

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