Digestive processes of haematophagous insects. XI. Partial purification and some properties of six proteolytic enzymes from the tsetse fly Glossina morsitans morsitans Westwood (Diptera: Glossinidae)

1976 ◽  
Vol 54 (11) ◽  
pp. 1950-1959 ◽  
Author(s):  
R. H. Gooding ◽  
B. M. Rolseth
Keyword(s):  
Proteolytic Enzymes ◽  
Substrate Inhibition ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Glossina Morsitans Morsitans ◽  
Tsetse Fly ◽  
Partial Purification ◽  
Glossina Morsitans ◽  
Molecular Weights ◽  
Diethylaminoethyl Cellulose

By O-(diethylaminoethyl)cellulose (DEAE-cellulose) chromatography, affinity chromatography, and Sephadex gel filtration, six proteolytic enzymes active in alkaline medium have been found in the digestive portion of the midgut (tissue and lumen contents) of adult Glossina morsitans morsitans. By use of synthetic substrates the enzymes have been characterized as aminopeptidase (AP; EC. 3.4.11.1), carboxypeptidase A (CPA; EC 3.4.12.2), carboxypeptidase B (CPB; EC 3.4.12.3), trypsin (EC 3.4.21.4), a trypsinlike enzyme designated proteinase VI, and a chymotrypsinlike enzyme designated proteinase VII. By Sephadex G-100 gel filtration the molecular weights were estimated to be 20 000 for trypsin, 19 000 for proteinase VI, 35 500 for proteinase VII, 30 000 for CPA, 22 000 for CPB, and [Formula: see text] for AP. The Km values (mg/ml) for haemoglobin were 3.43 for trypsin, 2.45 for proteinase VI, 3.68 for proteinase VII, and 2.42 for CPA. The Km values for casein were 1.22 for trypsin and 1.38 for proteinase VII. Casein showed substrate inhibition when hydrolyzed by proteinase VI and VII. Neither haemoglobin nor casein was hydrolyzed by AP and CPB. The pH optima were determined for hydrolysis of casein and the synthetic substrates.

scholarly journals Purification and characterization of two human erythrocyte arylamidases preferentially hydrolysing N-terminal arginine or lysine residues

1978 ◽  
Vol 175 (3) ◽  
pp. 1051-1067 ◽  
Author(s):  
K K Mäkinen ◽  
P L Mäkinen
Keyword(s):  
Substrate Inhibition ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Gel Permeation Chromatography ◽  
Ph Optimum ◽  
Preparative Scale ◽  
Molecular Weights ◽  
Gel Permeation ◽  
Lysine Residues ◽  
Enzyme I

Two arylamidases (I and II) were purified from human erythrocytes by a procedure that comprised removal of haemoglobin from disrupted cells with CM-Sephadex D-50, followed by treatment of the haemoglobin-free preparation subsequently with DEAE-cellulose, gel-permeation chromatography on Sephadex G-200, gradient solubilization on Celite, isoelectric focusing in a pH gradient from 4 to 6, gel-permeation chromatography on Sephadex G-100 (superfine), and finally affinity chromatography on Sepharose 4B covalently coupled to L-arginine. In preparative-scale purifications, enzymes I and II were separated at the second gel-permeation chromatography. Enzyme II was obtained as a homogeneous protein, as shown by several criteria. Enzyme I hydrolysed, with decreasing rates, the L-amino acid 2-naphtylamides of lysine, arginine, alanine, methionine, phenylalanine and leucine, and the reactions were slightly inhibited by 0.2 M-NaCl. Enzyme II hydrolysed most rapidly the corresponding derivatives of arginine, leucine, valine, methionine, proline and alanine, in that order, and the hydrolyses were strongly dependent on Cl-. The hydrolysis of these substrates proceeded rapidly at physiological Cl- concentration (0.15 M). The molecular weights (by gel filtration) of enzymes I and II were 85 000 and 52 500 respectively. The pH optimum was approx. 7.2 for both enzymes. The isoelectric point of enzyme II was approx. 4.8. Enzyme I was activated by Co2+, which did not affect enzyme II to any noticeable extent. The kinetics of reactions catalysed by enzyme I were characterized by strong substrate inhibition, but enzyme II was not inhibited by high substrate concentrations. The Cl- activated enzyme II also showed endopeptidase activity in hydrolysing bradykinin.

Digestive processes of haematophagous insects. XIII. Evidence for the digestive function of midgut proteinases of Glossina morsitans morsitans Westwood (Diptera: Glossinidae)

1977 ◽  
Vol 55 (9) ◽  
pp. 1557-1562 ◽  
Author(s):  
R. H. Gooding
Keyword(s):  
Amino Acids ◽  
Free Amino Acids ◽  
Free Amino ◽  
Proteolytic Enzymes ◽  
Gel Filtration ◽  
Glossina Morsitans Morsitans ◽  
Glossina Morsitans ◽  
Anterior Section ◽  
Digestive Function ◽  
Posterior Section

Trypsin (EC 3.4.21.4), proteinase VI, proteinase VII, aminopeptidase (EC 3.4.11.1), carboxypeptidase A (EC 3.4.12.2), and carboxypeptidase B (EC 3.4.11.3) occur in the posterior section of the midgut of unfed male and female Glossina morsitans morsitans Westwood, and their activities rise after a blood meal. Only traces of these enzymes occur in the anterior section of the midgut. Elution profiles of proteins during Sephadex gel filtration of the anterior midgut at various times after feeding, and the low ratio of free amino acids to protein in the lumen of the anterior section of the midgut indicate that no significant hydrolysis of protein takes place there. Results of Sephadex® gel filtration of material from the posterior section of the midgut indicate that proteins are rapidly converted to peptides and free amino acids, which occur in high concentration there. The results are interpreted as indicating that digestion of proteins takes place only in the posterior section of the midgut, and that all six proteolytic enzymes have a digestive function.

Serological responses in rabbits used to maintain uninfected, laboratory-reared tsetses (Glossina morsitans morsitans Westwood) (Diptera: Glossinidae)

1979 ◽  
Vol 57 (4) ◽  
pp. 705-710 ◽  
Author(s):  
K. R. Parker
Keyword(s):  
Salivary Gland ◽  
Salivary Glands ◽  
Gel Filtration ◽  
Glossina Morsitans Morsitans ◽  
Immunological Reaction ◽  
Glossina Morsitans ◽  
Anamnestic Response ◽  
Molecular Weights ◽  
Antigen Antibody

Antibodies, reacting with homogenatesof salivary glands, were produced in rabbits exposed to Glossina morsitans morsitans Westwood. Precipitating antibodies and high titres of haemagglutinating antibodies occurred in all exposed rabbits. Precipitating antibodies, identified using immunoelectrophoresis, immunodiffusion, and precipitin ring tests, developed within 11 days of exposure. As many as seven antigen–antibody precipitin arcs were identified using immunoelectrophoresis. All precipitating antigens in the salivary glands had molecular weights greater than 25 000 (determined by Sephadex gel filtration); the salivary gland anticoagulant was not shown to be antigenic. No precipitating immunological reaction occurred between rabbit sera and tsetse hindguts or midguts. Titres of sera from rabbits receiving a second exposure to tsetses, following a period of no exposure, followed an anamnestic response.

scholarly journals α-Crystallin. The isolation and characterization of distinct macromolecular fractions

1971 ◽  
Vol 124 (2) ◽  
pp. 337-343 ◽  
Author(s):  
Abraham Spector ◽  
Lu-Ku Li ◽  
Robert C. Augusteyn ◽  
Arthur Schneider ◽  
Thomas Freund
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Chemical Difference ◽  
Molecular Weights ◽  
Isolation And Characterization ◽  
Different Populations ◽  
Molecular Weight Fractions

α-Crystallin was isolated from calf lens periphery by chromatography on DEAE-cellulose and gel filtration. Three distinct populations of macromolecules have been isolated with molecular weights in the ranges approx. 6×105−9×105, 0.9×106−4×106and greater than 10×106. The concentration of macromolecules at the molecular-weight limits of a population are very low. The members of the different populations do not appear to be in equilibrium with each other. Further, in those molecular-weight fractions investigated, no equilibrium between members of the same population was observed. The population of lowest molecular weight comprises 65–75% of the total material. The amino acid and subunit composition of the different-sized fractions appear very similar, if not identical. The only chemical difference observed between the fractions is the presence of significant amounts of sugar in the higher-molecular-weight fractions. Subunit molecular weights of approx. 19.5×103and 22.5×103were observed for all α-crystallin fractions.

scholarly journals Host-seeking efficiency can explain population dynamics of the tsetse fly Glossina morsitans morsitans in response to host density decline

2017 ◽  
Vol 11 (7) ◽  
pp. e0005730 ◽  
Author(s):  
Jennifer S. Lord ◽  
Zinhle Mthombothi ◽  
Vitalis K. Lagat ◽  
Fatumah Atuhaire ◽  
John W. Hargrove
Keyword(s):  
Population Dynamics ◽  
Host Density ◽  
Glossina Morsitans Morsitans ◽  
Tsetse Fly ◽  
Glossina Morsitans ◽  
Host Seeking

scholarly journals Two Tsetse Fly Species, Glossina palpalis gambiensis and Glossina morsitans morsitans, Carry Genetically Distinct Populations of the Secondary Symbiont Sodalis glossinidius

2005 ◽  
Vol 71 (12) ◽  
pp. 8941-8943 ◽  
Author(s):  
Anne Geiger ◽  
Gérard Cuny ◽  
Roger Frutos
Keyword(s):  
Genetic Diversity ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Glossina Morsitans Morsitans ◽  
Tsetse Fly ◽  
Glossina Morsitans ◽  
Glossina Palpalis Gambiensis ◽  
Sodalis Glossinidius ◽  
Glossina Palpalis

ABSTRACT Genetic diversity among Sodalis glossinidius populations was investigated using amplified fragment length polymorphism markers. Strains collected from Glossina palpalis gambiensis and Glossina morsitans morsitans flies group into separate clusters, being differentially structured. This differential structuring may reflect different host-related selection pressures and may be related to the different vector competences of Glossina spp.

scholarly journals Isolation of palmitoyl-CoA hydrolases from human blood platelets

1981 ◽  
Vol 199 (3) ◽  
pp. 639-647 ◽  
Author(s):  
R K Berge ◽  
L E Hagen ◽  
M Farstad
Keyword(s):  
Human Blood ◽  
Blood Platelets ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Apparent Molecular Weight ◽  
Cytosol Fraction ◽  
Molecular Weights ◽  
Apparent Homogeneity ◽  
Coa Hydrolase ◽  
Human Blood Platelets

The palmitoyl-CoA hydrolase activity, which in human blood platelets is mainly localized in the cytosol fraction [Berge, Vollset & Farstad (1980) Scand. J. Clin. Lab. Invest. 40, 271--279], was found to be extremely labile. Inclusion of glycerol or palmitoyl-CoA stabilized the activity during preparation. Gel-filtration studies revealed multiple forms of the enzyme with molecular weights corresponding to about 70 000, 40 000 and 24 000. The relative recovery of the mol.wt.-70 000 form was increased by the presence of 20% (v/v) glycerol or 10 microM-palmitoyl-CoA. The three enzyme forms are probably unrelated, since they were not interconvertible. The three different species of palmitoyl-CoA hydrolase were purified by DEAE-cellulose and hydroxyapatite chromatography, isoelectric focusing and high-pressure liquid chromatography (h.p.l.c.) to apparent homogeneity. The three enzymes had isoelectric points (pI) of 7.0, 6.1 and 4.9. The corresponding molecular weights were 27 000--33 000, 66 000--72 000 and 45 000--49 000, calculated from h.p.l.c. and Ultrogel AcA-44 chromatography. The apparently purified enzymes were unstable, as most of the activity was lost during purification. The enzyme with an apparent molecular weight of 45 000--49 000 was split into fractions with molecular weights of less than 10 000 by re-chromatography on h.p.l.c. concomitantly with a loss of activity. The stimulation of the activity by the presence of serum albumin seems to depend on the availability of palmitoyl-CoA, as has been reported for other palmitoyl-CoA hydrolases. [Berge & Farstad (1979) Eur. J. Biochem. 96, 393--401].

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