scholarly journals Affinity purification of the novel cysteine proteinase papaya proteinase IV, and papain from papaya latex

1989 ◽  
Vol 261 (2) ◽  
pp. 469-476 ◽  
Author(s):  
D J Buttle ◽  
A A Kembhavi ◽  
S L Sharp ◽  
R E Shute ◽  
D H Rich ◽  
...  
Keyword(s):  
Cysteine Proteinase ◽  
Affinity Purification ◽  
Molecular Size ◽  
Exchange Chromatography ◽  
The Novel ◽  
Protein Substrates ◽  
Papaya Latex ◽  
Sepharose 4B ◽  
Spray Dried

A procedure is described for the purification of a previously undetected cysteine proteinase, which we have called papaya proteinase IV, from spray-dried latex of the papaya (Carica papaya) plant. The purification involves affinity chromatography on Gly-Phe-aminoacetonitrile linked to CH-Sepharose 4B, with elution by 2-hydroxyethyl disulphide at pH 4.5. The product thus obtained is a mixture of almost fully active papain and papay proteinase IV, which are then separated by cation-exchange chromatography. A preliminary characterization of papaya proteinase IV showed it to be very similar to chymopapain in both molecular size and charge. However, the new enzyme is immunologically distinct from the previously characterized cysteine proteinases of papaya latex. It also differs in its lack of activity against the synthetic substrates of the other papaya proteinases, in its narrow specificity against protein substrates and its lack of inhibition by chicken cystatin. Papaya proteinase IV is abundant, contributing almost 30% of the protein in spray-dried papaya latex, and contamination of chymopapain preparations with this enzyme may account for some of the previously reported heterogeneity of chymopapain.

Characterization of proteinases in extracts of adultHaemonchus contortus, the ovine abomasal nematode

Parasitology ◽  
1993 ◽  
Vol 106 (4) ◽  
pp. 395-404 ◽  
Author(s):  
D. P. Knox ◽  
D. L. Redmond ◽  
D. G. Jones
Keyword(s):  
Immune Responses ◽  
Haemonchus Contortus ◽  
Cysteine Proteinase ◽  
Molecular Size ◽  
Acidic Ph ◽  
Blood Proteins ◽  
Protein Substrates ◽  
Host Immune Responses ◽  
Ph Range

SUMMARYThe degradation of several protein substrates, including the blood proteins haemoglobin, albumin and fibrinogen, by proteinases present in extracts of adultHaemonchus contortuswas examined over a broad pH range. These proteinases were further characterized on the basis of substrate specificity, inhibitor sensitivity and molecular size by spectrophotometric and substrate gel analysis. The majority of the proteinases capable of degrading the blood proteins tested were active at acidic pH and could be ascribed to the cysteine proteinase class. In addition, evidence is presented that these proteinases are differentially recognized and inhibited by immune sera and that parasites capable of withstanding protective host immune responses exhibit modified expression of proteinases.

scholarly journals Roles of the novel coiled-coil protein Rng10 in septum formation during fission yeast cytokinesis

2016 ◽  
Vol 27 (16) ◽  
pp. 2528-2541 ◽  
Author(s):  
Yajun Liu ◽  
I-Ju Lee ◽  
Mingzhai Sun ◽  
Casey A. Lower ◽  
Kurt W. Runge ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Rho Gtpases ◽  
Cellular Localization ◽  
Affinity Purification ◽  
Coiled Coil ◽  
The Novel ◽  
Septum Formation ◽  
Division Site ◽  
And Function

Rho GAPs are important regulators of Rho GTPases, which are involved in various steps of cytokinesis and other processes. However, regulation of Rho-GAP cellular localization and function is not fully understood. Here we report the characterization of a novel coiled-coil protein Rng10 and its relationship with the Rho-GAP Rga7 in fission yeast. Both rng10Δ and rga7Δ result in defective septum and cell lysis during cytokinesis. Rng10 and Rga7 colocalize on the plasma membrane at the cell tips during interphase and at the division site during cell division. Rng10 physically interacts with Rga7 in affinity purification and coimmunoprecipitation. Of interest, Rga7 localization is nearly abolished without Rng10. Moreover, Rng10 and Rga7 work together to regulate the accumulation and dynamics of glucan synthases for successful septum formation in cytokinesis. Our results show that cellular localization and function of the Rho-GAP Rga7 are regulated by a novel protein, Rng10, during cytokinesis in fission yeast.

scholarly journals Partial Chemical Characterization of S-Carboxymethylated Chains of Rabbit Fibrin(OGEN): Evidence for Separate Chain Biosynthesis

1977 ◽  
Author(s):  
B. Alving ◽  
G. Murano ◽  
D. Walz
Keyword(s):  
Amino Acid ◽  
Specific Activity ◽  
Chemical Characterization ◽  
Molecular Size ◽  
Exchange Chromatography ◽  
12 Steps ◽  
Polypeptide Chains ◽  
Sds Electrophoresis ◽  
Derivatives Of

The purpose of this study was twofold: 1) chemically characterize the isolated polypeptide chains of rabbit fibrin(ogen), and 2) explore their mode of biosynthesis. The three S-carboxy-methyl polypeptide chain derivatives of rabbit fibrin (α, β and γ) were isolated by cation exchange chromatography. Their amino acid composition was similar to the human with a methionine distribution (mole/mole) as follows: γ = 9; β = 14, α = 14. Their molecular size, (SDS electrophoresis) was estimated as follows: γ = 46,000; β = 54,000; α = 63,500. The N-terminal amino acid sequence (12 steps) of the β derivative was:Gly-His-Arg-Pro-Ile-Asp-Arg-Arg-Arg-Glu-Glu-Leu-. To determine whether the three chains are synthesized sequentially (one continuous chain, later split into three) or in parallel, turpentine-stimulated male New Zealand rabbits were given ~40 μCi of [75Se] selenomethionine (SeM) and its incorporation into fibrinogen (F) was followed. F was clotted from plasma samples, washed, reduced, and constituent chains separated by gel electrophoresis in the presence of SDS-urea. The radioactivity of each chain (expressed as percent of total F radioactivity) was determined, and the specific methionine radioactivity calculated for each chain isolated at 20, 25, and 30 min after SeM injection. During this interval the specific activity of the α and the γ chains was essentially the same (within 3%) while that of the β chain was 42 to 97% greater than that of the α chain. The similar activity of the α and γ chains during the early phase of SeM incorporation suggests that these two chains are not synthesized sequentially, rather they are synthesized in parallel.

scholarly journals Purification and characterization of a digestive cysteine proteinase from the American lobster (Homarus americanus)

1989 ◽  
Vol 263 (2) ◽  
pp. 439-444 ◽  
Author(s):  
M V Laycock ◽  
T Hirama ◽  
S Hasnain ◽  
D Watson ◽  
A C Storer
Keyword(s):  
Proteinase Inhibitors ◽  
Cysteine Proteinase ◽  
Gel Filtration ◽  
Homarus Americanus ◽  
Exchange Chromatography ◽  
American Lobster ◽  
Digestive Juice ◽  
Effects Of Ph ◽  
Km Value

A new cysteine proteinase was isolated from the digestive juice of the American lobster (Homarus americanus). The enzyme was purified by a combination of affinity and ion-exchange chromatography and gel filtration. The cysteine proteinase accounted for 80% of the proteolytic activity in the lumen of the hepatopancreas. The most potent heavy-metal inhibitors were Hg, Cu, and Ag ions. Inhibition by organic proteinase inhibitors, including E-64 [L-trans-epoxysuccinyl-leucylamido-(4-guanidino)butane] and activation of the enzyme by 2-mercaptoethanol and dithiothreitol are characteristic of cysteine proteinases. Several similarities to papain are noted and include the N-terminal sequence, of which 22 of the first 28 amino acids are identical. Some notable differences are the higher Mr of 28,000 compared with 23,350 for papain, and the low isoelectric point (pI 4.5) of the lobster enzyme. The effects of pH and temperature on catalytic activity of the lobster proteinase were studied with benzyloxycarbonylalanine p-nitrophenyl ester as the substrate. The kcat./Km value was effectively temperature-independent between 10 and 60 degrees C. The pH-activity profile for the lobster enzyme revealed four apparent protonation states, of which only two are active.

scholarly journals Characterization of human liver α-D-mannosidase purified by affinity chromatography

1976 ◽  
Vol 153 (3) ◽  
pp. 579-587 ◽  
Author(s):  
N C Phillips ◽  
D Robinson ◽  
B G Winchester
Keyword(s):  
Affinity Chromatography ◽  
Concanavalin A ◽  
Human Liver ◽  
Early Stage ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Exchange Chromatography ◽  
Lysosomal Storage ◽  
Sepharose 4B

Human liver acidic α-D-mannosidase was purified 1400-fold by a relatively short procedure incorporating chromatography on concanavalin A-Sepharose and affinity chromatography on Sepharose 4B-epsilon-aminohexanoylmannosylamine. In contrast with the acidic enzymic activity the neutral α-mannosidase did not bind to the concanavalin A-Sepharose so the two types of α-mannosidase could be separated at an early stage in the purification. The only significant glycosidase contaminant after affinity chromatography on the mannosylamine ligand was α-L-fucosidase, which was selectively removed by affinity chromatography on the corresponding fucosylamine ligand. The final preparation was free of other glycosidase activities. The pI of the purified enzyme was increased from 6.0 to 6.45 on treatment with neuraminidase. Although the pI and the mol.wt. (220 000) suggested that α-mannosidase A had been purified selectively, ion-exchange chromatography on DEAE-cellulose indicated that the preparation consisted predominantly of α-mannosidase B. This discrepancy is discussed in relation to the basis of the multiple forms of human α-mannosidase. The purified enzyme completely removed the α-linked non-reducing terminal mannose from a trisaccharide isolated from the urine of a patient with mannosidosis. A comparison of the activity of the pure enzyme towards the natural substrate and synthetic substrates suggests that the same enzymic activity is responsible for hydrolysing all the substrates. These results validate the use of synthetic substrates for determining the mannosidosis genotype. They are also further evidence that mannosidosis is a lysosomal storage disease resulting from a deficiency of acidic α-mannosidase.

scholarly journals Structure of chymopapain M the late-eluted chymopapain deduced by comparative modelling techniques and active-centre characteristics determined by pH-dependent kinetics of catalysis and reactions with time-dependent inhibitors: the Cys-25/His-159 ion-pair is insufficient for catalytic competence in both chymopapain M and papain

1994 ◽  
Vol 300 (3) ◽  
pp. 805-820 ◽  
Author(s):  
M P Thomas ◽  
C M Topham ◽  
D Kowlessur ◽  
G W Mellor ◽  
E W Thomas ◽  
...  
Keyword(s):  
Cation Exchange ◽  
Cysteine Proteinase ◽  
Ion Pair ◽  
Exchange Chromatography ◽  
Catalytic Site ◽  
Time Dependent ◽  
Cation Exchange Chromatography ◽  
Pka Values ◽  
Full Expression ◽  
Papaya Latex

Chymopapain M, the monothiol cysteine proteinase component of the chymopapain band eluted after chymopapains A and B in cation-exchange chromatography, was isolated from the dried latex of Carica papaya and characterized by kinetic and chromatographic analysis. This late-eluted chymopapain is probably a component of the cysteine proteinase fraction of papaya latex discovered by Schack [(1967) Compt. Rend. Trav. Lab. Carlsberg 36, 67-83], named papaya peptidase B by Lynn [(1979) Biochim. Biophys. Acta 569, 193-201] and partially characterized by Polgár [(1981) Biochim. Biophys. Acta 658, 262-269] and is the enzyme with unusual specificity characteristics (papaya proteinase IV) that Buttle, Kembhavi, Sharp, Shute, Rich and Barrett [Biochem. J. (1989) 261, 469-476] claimed to be a previously undetected cysteine proteinase eluted from a cation-exchange column near to the early-eluted chymopapains. A study of the time-dependent chromatographic consequences of thiol-dependent proteolysis of the components of papaya latex is reported. Chymopapain M was isolated by (i) affinity chromatography followed by separation from papain using cation-exchange f.p.l.c. on a Mono S HR5/5 column and (ii) cation-exchange chromatography followed by an unusual variant of covalent chromatography by thiol-disulphide interchange. The existence in chymopapain M of a nucleophilic interactive Cys/His catalytic-site system analogous to those in papain (EC 3.4.22.2) and other cysteine proteinases was deduced from the characteristics shape of the pH-second-order rate constant (k) profiles for its reactions with 2,2′-dipyridyl disulphide and ethyl 2-pyridyl disulphide. Analysis of the pH-k data for the reactions of chymopapain M with the 2-pyridyl disulphides and with 4,4′-dipyridyl disulphide permits the assignment of molecular pKa values of 3.4 and 8.7 to the formation and subsequent dehydronation of the Cys-S-/His-Im+H state of the catalytic site and reveals three other kinetically influential ionizations with pKa values 3.4, 4.3 and 5.6. The pH-dependences of kcat./Km for the hydrolysis of N-acetyl-L-Phe-Gly-4-nitroanilide at 25.0 degrees C and I0.1 M catalysed by chymopapain M and papain were determined. For both enzymes, little catalytic activity (5-7% of the maximal) develops consequent on formation of the catalytic site Cys-S-/His-Im+H ion-pair state (across pKa 3.4 for both enzymes). For papain, full expression of Kcat./Km for the uncharged substrate requires only the additional hydronic dissociation with pKa 4.2. By contrast, full expression of kcat./Km for chymopapain M requires additional hydronic dissociation with pKa values of 4.3 and 5.6.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals The purification and characterization of glucokinase from the thermophile Bacillus stearothermophilus

1986 ◽  
Vol 237 (2) ◽  
pp. 415-420 ◽  
Author(s):  
C R Goward ◽  
R Hartwell ◽  
T Atkinson ◽  
M D Scawen
Keyword(s):  
Large Scale ◽  
Bacillus Stearothermophilus ◽  
Specific Activity ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Purification And Characterization ◽  
Km Value ◽  
Extraneous Material ◽  
Sepharose 4B

Homogeneous glucokinase (EC 2.7.1.2) from the thermophile Bacillus stearothermophilus was isolated on the large scale by using four major steps: precipitation of extraneous material at pH 5.5, ion-exchange chromatography on DEAE-Sepharose, pseudo-affinity chromatography on Procion Brown H-3R-Sepharose 4B and gel filtration on Ultrogel AcA 34. The purified enzyme had a specific activity of about 330 units/mg of protein and was shown to exist as a dimer of subunit Mr 33,000. Kinetic parameters for the enzyme were determined with a variety of substrates. The glucokinase was highly specific for alpha-D-glucose, and the only other sugar substrate utilized was N-acetyl-alpha-D-glucosamine. The enzyme shows Michaelis-Menten kinetics, with a Km value of 150 microM for alpha-D-glucose. The glucokinase was maximally active at pH 9.0.

Isolation of Noncollagenous Proteins from Gingival Connective Tissue

1988 ◽  
Vol 67 (8) ◽  
pp. 1109-1113 ◽  
Author(s):  
U. Schlagenhauf ◽  
A.S. Narayanan ◽  
R.C. Page
Keyword(s):  
Connective Tissue ◽  
Type I Collagen ◽  
Deae Cellulose ◽  
Molecular Size ◽  
Type I ◽  
Connective Tissues ◽  
Noncollagenous Proteins ◽  
And Function ◽  
Sepharose 4B

Noncollagenous proteins form an integral part of gingiva and other connective tissues. We have performed studies aimed at purification and partial characterization of the gingival noncollagenous proteins. Healthy gingival tissues from mongrel dogs were extracted in neutral buffers, acetic acid, and 6 mol/L urea. Immunoblots using anti-keratin antibodies and CNBr peptide patterns revealed that the majority of the proteins present in these extracts were keratins. To exclude keratins, gingival connective tissue was separated from the epithelium and then extracted. Acid extracts of the connective tissue contained very little protein, whereas urea extracts contained collagen and other noncollagenous proteins. The noncollagenous proteins present in the urea extract were partially purified by DEAE-cellulose chromatography and separated by affinity chromatography through a Sepharose 4B-type I collagen column. At least eight proteins, which ranged in molecular size from 15 to 75 kilodaltons, were obtained by this procedure. We conclude that keratins are major components of whole gingiva extracts and that epithelium must first be removed in order for connective tissue proteins to be obtained. The gingival connective tissue appears to contain several collagen-binding proteins, and these proteins may play an important role in the structure and function of the gingival matrix.

Characterization of immunoreactive motilin from the rat small intestine

1990 ◽  
Vol 68 (8) ◽  
pp. 1124-1130 ◽  
Author(s):  
L. B. Vogel ◽  
J. C. Brown
Keyword(s):  
Biological Activity ◽  
Small Intestine ◽  
Molecular Size ◽  
Reversed Phase ◽  
Exchange Chromatography ◽  
Muscle Strip ◽  
Rat Small Intestine ◽  
Fixation Techniques ◽  
Staining Methods

Immunocytochemistry, radioimmunoassay, chromatography, and biological assay using a rabbit isolated duodenal muscle strip preparation were used in attempting to characterize motilin from the rat small intestine. Several different antisera and monoclonal antibodies directed against natural porcine motilin were used. A variety of fixation techniques using Bouin's, paraformaldehyde, and benzoquinone with different staining methods including, fluorescein-conjugated second antibody, peroxidase–antiperoxidase or peroxidase-conjugated second antibody techniques were used. All methods failed to detect immunoreactive motilin cells in the rat small intestine. The same antisera were used in radioimmunoassays for motilin to evaluate extracts of rat intestinal tissue. Two of these detected immunoreactive motilin in gut extracts, and these antisera showed a different distribution for the peptide. Samples containing immunoreactive motilin obtained from cation exchange chromatography on SP-Sephadex-G25 were concentrated and assayed for biological activity in a rabbit duodenal muscle strip preparation. Desensitization of duodenal tissue to porcine motilin could be demonstrated by pretreatment with this peptide. The biological activity of partially purified rat intestinal immunoreactive motilin was not prevented by pretreatment of the tissue with motilin. Further purification of this preparation on Bio-Gel P-10 yielded an immunoreactive motilin peak that co-eluted with natural porcine motilin. Rat intestinal immunoreactive motilin did not co-elute with natural porcine motilin following high pressure liquid chromatography on a Waters μBondapak C18 reversed-phase column using a linear gradient of water–acetonitrile (10–45%) over 30 min. Although of similar molecular size, rat motilin is probably structurally dissimilar to other mammalian motilins.Key words: motilin, radioimmunoassay, immunocytochemistry.

scholarly journals Characterization of phospholipid methylation in rat brain myelin

1995 ◽  
Vol 307 (1) ◽  
pp. 239-244 ◽  
Author(s):  
V Tsvetnitsky ◽  
L Auchi ◽  
A Nicolaou ◽  
W A Gibbons
Keyword(s):  
Rat Brain ◽  
Affinity Purification ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Methyl Transferase ◽  
Sds Page ◽  
Triton X ◽  
Predominant Product

Highly purified rat brain myelin was solubilized in Triton X-100 and myelin phospholipid N-methyltransferase was characterized. The enzyme activities were separated by isoelectric focusing and ion-exchange chromatography. The phospholipid methyl-transferase has shown at least four peaks of activity with pIapp. values of 4.5, 5.2, 6.2 and 8.4. After affinity purification each of these activities revealed a close set of bands of approx. 65 kDa on SDS/PAGE. These data together with those from preparative SDS/PAGE separations suggested that rat brain myelin contains three acidic and at least one basic phospholipid-methylating isoenzymes and that the major isoenzyme in each case is approx. 65 kDa in size. While the predominant product of the reaction catalysed by all detected isoforms was monomethylated phosphatidylethanolamine, the least acidic isoform (pIapp. 6.2) also formed about 20% phosphatidylcholine, suggesting that these isoenzymes may play different roles in vivo.

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