scholarly journals THE ENZYMATIC HYDROLYSIS OF AMINO ACID β-NAPHTHYLAMIDES II. PARTIAL PURIFICATION AND PROPERTIES OF A PARTICLE-BOUND COBALT-ACTIVATED RAT KIDNEY AMINOPEPTIDASE

1966 ◽  
Vol 14 (5) ◽  
pp. 401-413 ◽  
Author(s):  
K. FELGENHAUER ◽  
G. G. GLENNER
Keyword(s):  
Amino Acid ◽  
Rat Kidney ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Disc Electrophoresis ◽  
Partial Purification ◽  
Soluble Enzyme ◽  
Solvent Fractionation ◽  
Biochemical Systems ◽  
Hydrolysis Of

In order to determine the relationship and characteristics of the soluble and tissue-bound (so-called "lyo" and "desmo") components of enzymes in histochemical and biochemical systems, a biochemical investigation of the hydrolysis of l-leucyl β-naphthylamide in rat kidney was undertaken. By ultracentrifugation, autolysis, salt and solvent fractionation procedures and DEAE-cellulose column chromatography primary soluble and tissuue-bound enzymes were separated and partially purified. The soluble enzyme was found to be inhibited by p-chloromercuribenzoate and reactivated by cysteine. The tissue-bound enzyme was found to be inhibited by o-phenanthroline and reactivated by Co++, and hydrolyzed peptides and amino acid β-naphthylamides at rates differing markedly from those of the soluble enzyme and commercial leucine aminopeptidase. Disc electrophoresis of the tissue-bound enzyme preparation demonstrated two protein bands, both revealing enzymic activity with almost identical hydrolytic characteristics, i.e., isozymes. This evidence established the identity of the tissue-bound Co++-activated enzyme with that found histochemically in the brush border of the proximal tubuli, and indicated its functional capacity as that of an aminopolypeptidase. It was also concluded that in the system investigated the "lyo" and "desmo" components represented, in reality, distinct enzymes with unique characteristics.

scholarly journals Partial purification of a 6-methyladenine mRNA methyltransferase which modifies internal adenine residues

1992 ◽  
Vol 288 (1) ◽  
pp. 233-240 ◽  
Author(s):  
M T Tuck
Keyword(s):  
Rat Liver ◽  
Rat Kidney ◽  
Cell Activity ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Partial Purification ◽  
Methyltransferase Activity ◽  
Nuclear Extracts ◽  
Normal Rat ◽  
Minor Form

Two forms of a 6-methyladenine mRNA methyltransferase have been partially purified using a T7 transcript coding for mouse dihydrofolate reductase as an RNA substrate. Both enzyme forms modify internal adenine residues within the RNA substrate. The enzymes were purified 357- and 37-fold respectively from nuclear salt extracts prepared from HeLa cells using DEAE-cellulose and phosphocellulose chromatography. The activity of the first form of the enzyme eluted from DEAE-cellulose (major form) was at least 3-fold greater than that of the second (minor form). H.p.l.c. analysis of the hydrolysed, methylated mRNA substrates demonstrated that both forms of the enzyme produced only 6-methyladenine. The two forms of the enzyme differed in their RNA substrate specificity as well as in the dependence for a 5′ cap structure. The 6-methyladenine mRNA methyltransferase activity was found to be elevated in HeLa nuclei as compared with nuclear extracts from rat kidney and brain. Enzymic activity could not be detected in nuclei from either normal rat liver or regenerating rat liver. In the case of the HeLa cell, activity could only be detected in nuclear extracts, with a small amount in the ribosomal fraction. Other HeLa subcellular fractions were void of activity.

Partial purification and characterization of an extracellular proteinase from Aeromonas hydrophila strain A4

1988 ◽  
Vol 55 (1) ◽  
pp. 97-107 ◽  
Author(s):  
Efstathios Alichanidis
Keyword(s):  
Aeromonas Hydrophila ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Partial Purification ◽  
Significant Activity ◽  
Metal Chelators ◽  
Purification And Characterization ◽  
Tris Maleate ◽  
Ph Range

SummaryAn extracellular metalloproteinase from Aeromonas hydrophila strain A4, isolated from milk, was purified by a factor of 300 by chromatogrpahy on DEAE-cellulose and Sephadex G-150. The enzyme had a mol. wt of 43000 and contained 2 g atom Ca/mol. It was active over a pH range 4·8–9·5 and had optimum activity on casein at pH 7·0 with Km = 0·17 mM. It was strongly inactivated by metal chelators and the apoenzyme was fully reactivated with Ca2+, Mn2+ or Co2+. Heavy metal ions such as Ag+, Hg2+, Fe2+, Zn2+, Cd2+, Ni2+ and Cu2+ totally or partly inactivated the enzymic activity at 5 mM concentration. The enzyme was not inactivated by diisopropylfluorophosphate, soyabean trypsin inhibitor or sulphydryl group reagents. It was optimally active at 45 °C; above 50 °C activity declined rapidly, but significant activity persisted at 4 °C. It was heat labile in phosphate or Tris-maleate buffer but exogenous Ca2+ afforded protection.

scholarly journals Peptidase Activity of Amidinated Thrombin

1977 ◽  
Author(s):  
E.P. Kang
Keyword(s):  
Amino Acid ◽  
Catalytic Efficiency ◽  
Enzymic Activity ◽  
Peptidase Activity ◽  
Amino Acid Residues ◽  
Small Peptides ◽  
Human Thrombin ◽  
Small Change ◽  
Hydrolysis Of ◽  
Hydrolysis Activity

Human thrombin, free of plasminogen and plasmin, was treated with ethyl acetimidate hydrochloride in order to modify the lysyl residues of the protein. By monitoring the enzymic activity in the modification mixture, it was found that the reaction was completed in about one hour and the loss of activity of thrombin was proportional to the amount of modification. After the removal of the excess ethyl acetimidate, approximately 25% of the clotting activity and of the hydrolysis activity for small peptides remained. Amino acid analysis of this modified thrombin indicated about 80% of the lysyl residues had been modified with no apparent change of other amino acid residues. By studying the thrombolytic hydrolysis of Bz-phe-val-arg-pNA, the kcat of the amidinated thrombin was about 8% of the control while the KM Secreased to 0.056 μM from 0.098 μM. The modification of the lysyl residues of thrombin, therefore, has lowered the catalytic efficiency of the enzyme with a rather small change in binding affinity. This suggests that modification of lysyl residues in the neighborhood of the active site hinders the catalytic hydrolysis of the small peptides.

scholarly journals Purification of carboxypeptidase B from human pancreas

1977 ◽  
Vol 163 (2) ◽  
pp. 253-260 ◽  
Author(s):  
D V Marinkovic ◽  
J N Marinkovic ◽  
E G Erdös ◽  
C J G Robinson
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Amino Acid Content ◽  
Disc Electrophoresis ◽  
Human Pancreas ◽  
Peptidase Activity ◽  
Ph Optimum ◽  
Carboxypeptidase B

Carboxypeptidase B of the human pancreas was purified by chromatography on DEAE-cellulose and CM-cellulose columns. Two forms of the enzyme, named carboxypeptidase B1 and B2, were separated. They have similar mol.wts. (34250 +/- 590) as established by polyacrylamide-gel disc electrophoresis and by gel filtration. Carboxypeptidase B2 migrates further towards the anode in disc electrophoresis. When the amino acid content of the enzymes was analysed, carboxypeptidase B2 had four more glycine and three more aspartic acid residues than had form B1. The amino acid sequence of the human carboxypeptidase B1 differs from that of the bovine enzyme only in two places in the N-terminal 20-amino-acid sequence. The N-terminal amino acid in carboxypeptidase B1 and B2 is alanine. The peptide ‘map’ of the tryptic digest of carboxypeptidase B1 contained more peptides than did that of form B2. The Km, the Vmax. and the pH optimum of the cleavage of the peptide substrate hippurylarginine and the ester substrate hippurylargininic acid were similar for both enzymes. CoCl2 accelerated the peptidase activity, and cadmium acetate enhanced the esterase activity, of human carboxypeptidases B1 and B2. Urea and sodium dodecyl sulphate inhibited the enzymes.

scholarly journals THE ENZYMATIC HYDROLYSIS OF AMINO ACID β-NAPHTHYLAMIDES: I. PREPARATION OF AMINO ACID AND DIPEPTIDE β-NAPHTHYLAMIDES

1965 ◽  
Vol 13 (1) ◽  
pp. 57-64 ◽  
Author(s):  
G. G. GLENNER ◽  
L. A. COHEN ◽  
J. E. FOLK
Keyword(s):  
Amino Acid ◽  
Enzymatic Hydrolysis ◽  
Biochemical Systems ◽  
Hydrolysis Of ◽  
Acid Salts

The acid salts of amino acid β-naphthylamides have been found to have greater stability and solubility than their corresponding free bases. For the purpose of applying these substrates in subsequent studies in histochemical and biochemical systems, the syntheses and characteristics of a large variety of previously unreported intermediates, acid salts and free bases of amino acid β-naphthylamides and their analogues are described.

scholarly journals Ca2+-independent cyclic GMP phosphodiesterases from rat liver and HTC hepatoma cells

1983 ◽  
Vol 213 (2) ◽  
pp. 379-386 ◽  
Author(s):  
G J Strewler ◽  
M A Danello ◽  
V C Manganiello ◽  
M Vaughan
Keyword(s):  
Cyclic Amp ◽  
Rat Liver ◽  
Cyclic Gmp ◽  
Rat Kidney ◽  
Deae Cellulose ◽  
Hepatoma Cells ◽  
Partial Hydrolysis ◽  
Kinetic Behaviour ◽  
Lysosomal Proteinase ◽  
Hydrolysis Of

We have separated and characterized a Ca2+- and calmodulin-insensitive cyclic nucleotide phosphodiesterase from rat liver supernatant as well as an analogous enzyme from HTC hepatoma cells. Chromatography of rat liver supernatant on DEAE-cellulose in the presence and subsequently in the absence of 0.1 mM-CaCl2 resulted in the separation of two distinct phosphodiesterase activities, both of which preferentially hydrolysed cyclic GMP rather than cyclic AMP. One enzyme, E-Ib, was activated in the presence of Ca2+ and calmodulin, and the other, E-Ia, was not. The E-Ia enzyme, which did not bind to calmodulin-Sepharose, had Mr 325 000 and displayed anomalous kinetic behaviour [Km (cyclic GMP) 1.2 microM; Km (cyclic AMP) 15.4 microM]. The E-Ib enzyme, which bound to calmodulin-Sepharose in the presence of Ca2+, had Mr 150 000 and exhibited Michaelis-Menten kinetics for hydrolysis of cyclic GMP [Km (basal) 6.5 microM; Km (activated) 12.0 microM]. E-Ia activity was diminished by incubation with alpha-chymotrypsin and was unaffected by the action of a rat kidney lysosomal proteinase. Partial hydrolysis of E-Ib enzyme by alpha-chymotrypsin or the kidney proteinase resulted in irreversible activation of the enzyme. The E-I enzyme isolated from HTC hepatoma cells was similar to the rat liver E-Ia enzyme in many respects. Its apparent Mr was 325 000. Its activity was unaffected by calmodulin in the presence of Ca2+ or by incubation with the kidney proteinase, and was decreased by digestion with alpha-chymotrypsin. Unlike the liver E-Ia enzyme, however, the hepatoma enzyme exhibited normal kinetic behaviour, with Km (cyclic GMP) 3.2 microM. Although HTC cells contain two other phosphodiesterases analogous to those in rat liver and a calmodulin-like activator of phosphodiesterase, no calmodulin-sensitive phosphodiesterase was detected.

Partial purification of phosphodiesterase I from microsomes of rat intestinal mucosa

1970 ◽  
Vol 48 (10) ◽  
pp. 1141-1150 ◽  
Author(s):  
I. Hynie ◽  
S. H. Zbarsky
Keyword(s):  
Aqueous Phase ◽  
Intestinal Mucosa ◽  
Phosphate Buffer ◽  
High Speed ◽  
Deae Cellulose ◽  
Amyl Alcohol ◽  
Partial Purification ◽  
Soluble Enzyme ◽  
Enzyme Solution ◽  
Alkaline Phosphomonoesterase

Intestinal mucosa was homogenized in Krebs–Ringer phosphate buffer, pH 7.4, containing 6% Dextran. After centrifugation, the sediment was rehomogenized in 0.24 M sucrose and the homogenate centrifuged. The supernatant material from both steps was combined and centrifuged at high speed through a layer of 25% sucrose to yield a pellet of microsomes. This pellet was suspended in Tris–HCl buffer, pH 8.4, made isotonic in KCl, and the suspension was sonicated and centrifuged. A suspension of the sediment in Tris–HCl buffer was shaken with t-amyl alcohol to yield the soluble enzyme in the aqueous phase. The enzyme was purified further by chromatography on DEAE-cellulose using elution with a KCl gradient in Tris–HCl buffer, pH 8, made 6 M in urea. Phosphodiesterase I and alkaline phosphomonoesterase were eluted in peaks which overlapped only partially enabling the collection of phosphodiesterase I free of Phosphomonoesterase. The enzyme solution was concentrated and freed of urea by pressure dialysis.

scholarly journals Purification of endopeptidase-24.11 (‘enkephalinase’) from pig brain by immunoadsorbent chromatography

1983 ◽  
Vol 215 (3) ◽  
pp. 519-523 ◽  
Author(s):  
J M Relton ◽  
N S Gee ◽  
R Matsas ◽  
A J Turner ◽  
A J Kenny
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Deae Cellulose ◽  
Single Step ◽  
Endopeptidase 24.11 ◽  
Pig Brain ◽  
Endopeptidase Activity ◽  
Hydrolysis Of ◽  
The Brain ◽  
Subunit Size

Membrane preparations from striatum of pig brain contain endopeptidase activity towards iodoinsulin B-chain. Only 50% of the hydrolysis of insulin B-chain is inhibitable by phosphoramidon, and DEAE-cellulose chromatography can resolve the phosphoramidon-sensitive and -insensitive activities. The former activity (now designated ‘endopeptidase-24.11’) is responsible for hydrolysis of [D-Ala2,Leu5]enkephalin and is identical with an enzyme in brain that has previously been referred to as ‘enkephalinase’. Pig striatal endopeptidase-24.11 has now been purified to homogeneity in a single step by immunoadsorbent chromatography using a monoclonal antibody. The overall purification was 23 000-fold, with a yield of 30%. The brain enzyme appears to be identical with kidney endopeptidase-24.11 in amino acid composition as well as by immunological and kinetic criteria. However, it differs slightly in apparent subunit size (Mr = 87 000), attributable to differences in glycosylation.

scholarly journals Biosynthesis of proline in Pseudomonas aeruginosa. Partial purification and characterization of γ-glutamyl kinase

1979 ◽  
Vol 181 (1) ◽  
pp. 215-222 ◽  
Author(s):  
R V Krishna ◽  
T Leisinger
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Kinase Activity ◽  
Wild Type Strain ◽  
Deae Cellulose ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Gamma Glutamyl ◽  
Hydrolysis Of ◽  
Molecular Sieving

A gamma-glutamyl kinase (ATP-L-glutamate 5-phosphotransferase) was purified about 85-fold from crude extracts of Pseudomonas aeruginosa strain PAO 1 by (NH4)2SO4 precipitation, molecular-sieving by Sephadex G-150 and DEAE-cellulose chromatography. The molecular weight of this enzyme was 84,000. The preparation catalysed formation of gamma-glutamyl hydroxamate from L-glutamate, ATP and Mg2+ or Mn2+ with concomitant hydrolysis of ATP to ADP + Pi. L-Proline inhibited the gamma-glutamyl kinase activity by 50% at 5 mM and almost completely at 30 mM. The inhibition of L-proline was non-competitive, wherease L-methionine-DL-sulphoximine inhibited the enzyme competitively. Proline was found to inhibit the gamma-glutamyl kinase activity of the wild-type strain and of representatives of two of the three transductional classes of proline-auxotrophic mutants. Strain PAO 879, a mutant representing the third transductional class of proline auxotrophs, lacked proline-inhibitible gamma-glutamyl kinase. Thiol-blocking reagents inhibited the gamma-glutamyl kinase and this effect was prevented by dithiothreitol.

Anticoagulants Produced by Thrombin from Fibrin, the Effect on Blood Coagulation, Some Physical Characteristics

1971 ◽  
Vol 26 (02) ◽  
pp. 211-223 ◽  
Author(s):  
Ch R. Muirhead ◽  
D. C Triantaphyllopoulos
Keyword(s):  
Blood Coagulation ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Fibrin Clot ◽  
Disc Electrophoresis ◽  
Physical Characteristics ◽  
Advanced Stage ◽  
Kallikrein Inhibitor ◽  
Shed Blood ◽  
Complete Lysis

SummaryChromatographed thrombin in the presence of both 50 Kallikrein inhibitor units of Trasylol per ml and 0.1 M E-ACA solubilized fibrin and the products of lysis possessed anticoagulant properties. The peak of the antithrombic activity coincided with the time of complete lysis of the fibrin clot, plasmin lysed fibrin exhibited the peak of its antithrombic activity much earlier. The effect of thrombin lysed fibrin on the prothrombin consumption of shed blood was found to be inhibitory.The products of the digestion of fibrin by thrombin and by plasmin, isolated at an advanced stage of proteolysis were compared by gel filtration, disc electrophoresis and DEAE cellulose chromatography. Differences in physical characteristics of these fibrin breakdown products offer evidence that they were produced by two different enzymes.

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