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Partial purification of cathepsins from salmon muscleTing, Chao-Yun 15 March 1965 (has links)
Cathepsins are intracellular proteinases that hydrolyze the
peptide bonds of proteins. These enzyme have been implicated in
the tenderization of aging beef, with the deterioration of radiation-stabilized
meats on storage, and in the spoilage of fish prior to
processing. Hence, the cathepsins of edible muscles are of concern
to the food scientist.
The purpose of the research reported herein was to develop
procedures for the purification of the cathepsin from salmon muscle.
The availability of a purified preparation of salmon muscle cathepsins
should stimulate interest and research in the characterization of
these enzymes and lead to better means for the control of catheptic
activity in fish muscle.
Results from these investigations indicate that salmon muscle
cathepsins exhibit pH optima at 3.7, 6.9, and 8.5 when Folin's reagent was used to determine the products of protein hydrolysis;
whereas, pH optima at 3.7 and 7.3 were obtained when the products
of protein hydrolysis were determined by absorption at 280 mμ.
Possible reasons for the differences in pH optima are discussed.
It was decided to attempt the purification of the cathepsin optimally
active at pH 3.7. The stability of this enzyme was found to be maximal
at pH 6.5.
The purification was accomplished by extracting the salmon
muscle cathepsin with two parts 0.2 N KC1. The pH of this crude
extract was adjusted to 5.5 and the precipitated proteins were removed
by centrifugation before the pH of the supernatant was readjusted
to the original pH of the extract. Upon dialysis of this fraction
against 0.005 M phosphate (pH 6.5) a precipitate formed and
was removed by centrifugation. The catheptic activity was precipitated
from the surpernatant at 0.50 saturation with (NH₄)₂SO₄. The
precipitate was recovered by centrifugation, dissolved in 0.005 M
phosphate (pH 6.5), and dialyzed against the same buffer. A precipitate
formed and was removed by centrifugation. This fraction was
placed on a 2.5 x 35 cm column of DEAE-cellulose equilibrated
with the starting buffer (0.005 M phosphate at pH 6.5). A concave
concentration gradient was used to elute the proteins from the ion-exchange
resin. Final buffer was 0.005 M phosphate (pH 6.5) containing
0.5 N NaCl. The absorbance (280 mμ) of the column effluent was continuously recorded and 10 ml fractions of the effluent were
collected. Fractions comprising the various protein peaks were
combined and concentrated by lyophilization. Two catheptic enzymes
appeared to be separated by this procedure with maximum purification
of 117 fold with 6.8 per cent recovery.
During the development of the procedure, it was found that the
fractions obtained from column chromatography could not be successfully
concentrated by ultrafiltration, pervaporation, or "Aquacide
#2" and that the presence of cysteine in the eluting buffer was
not beneficial. / Graduation date: 1965
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Comparative activity and partial characterization of cathepsins in three species of Pacific soleGeist, Gary Michael 27 April 1973 (has links)
Graduation date: 1973
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Characterization of oxyanion hole mutants of the cysteine proteases papain and cathepsin B ** check pdfCarrier, Julie, 1959- January 1992 (has links)
Note:
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Synthesis and evaluation of CA clan cysteine inhibitors : a thesis submitted in partial fulfilment of the requirements for the degree of Master of Science in Chemistry at the University of Canterbury /Millar, Tarek Lawson. January 2008 (has links)
Thesis (M. Sc.)--University of Canterbury, 2008. / Typescript (photocopy). Includes bibliographical references (leaves 124-131). Also available via the World Wide Web.
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Transcriptional regulation of cathepsin L during mouse mammary gland involution a test of STAT3 involvement /Patel, Amita. January 2006 (has links)
Thesis (M.S.)--Villanova University, 2006. / Biology Dept. Includes bibliographical references.
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The role of cathepsin L in involution and the termination of lactation in the mouse mammary glandStairiker, Patricia A. January 2006 (has links)
Thesis (M.S.)--Villanova University, 2006. / Biology Dept. Includes bibliographical references.
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A study of the role of redox potential in lysosomal function.Meinesz, Richard Edward. 11 October 2013 (has links)
No abstract available. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1996.
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Genetische Analyse des Cathepsin L bei chronischer PankreatitisHerms , Max 13 July 2012 (has links) (PDF)
Die chronische Pankreatitis (CP) ist eine wiederkehrende, entzündliche Erkrankung des Pankreas. In den letzten Jahren wurden mehrere Kandidatengene, die zur Entstehung einer CP prädisponieren, identifiziert. Zu diesen Genen gehören PRSS1, PRSS2, SPINK1, CFTR und CTRC. Der Pathogenese der genetisch bedingten CP scheint dabei eine frühzeitige, intrapankreatische Aktivierung von Trypsin zugrunde zu liegen.
Cathepsin B (CTSB), eine in Lysosomen vorkommenden Protease, ist in der Lage Trypsinogen zu aktivieren. Genetisch zeigte sich eine Assoziation der p.L26V Variante bei tropisch-kalzifizierender CP, welche bei idiopathischer CP nicht bestätigt wurde. Neben CTSB ist CTSL die am zweithäufigsten vorkommende lysosomale Protease. Funktionelle Untersuchungen zeigten, dass CTSL ein inaktives Trypsin freisetzt. Im Mausmodell zeigten sich bei Ctsl-/- Tieren bei experimentell induzierter Pankreatitis zwei Effekte. Zum einen war die Trypsinaktivität erhöht, zum anderen verlief die Pankreatitis milder, da vermehrt Apoptose anstelle von Nekrose der Azinuszellen auftrat.
In dieser Studie wurde mittels uni-direktionaler DNA-Sequenzierung das gesamte CTSL1 untersucht. Dabei fanden wir insgesamt drei seltene nicht-synonyme Varianten. Die Variante c.5A>C (p.N2T, rs112682750) fanden wir bei einem Patienten, wobei diese Variante bereits bei Kontrollen beschrieben wurde. Die Varianten c.126+1G>A und c.915A>C (p.E305D) lagen bei jeweils einer Kontrolle vor. Sowohl seltene als auch häufige Varianten und die berechneten Haplotypen zeigten keinen signifikanten Verteilungsunterschied zwischen Patienten und Kontrollen. Demnach besteht keine Assoziation von Varianten des CTSL1 und CP.
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Identification of potential exosite in cathepsin V necessary for elastin degradationChen, Li Hsuen 11 1900 (has links)
Besides collagen, elastin is the most common connective tissue structural protein in
vertebrates and similar to collagen relatively resistant to non-specific degradation.
Typical elastolytic proteases are the serine-dependent pancreatic and leukocyte elastases,
the Zn-dependent matrix metalloproteinase 12, and several lysosomal cysteine proteases.
Among the cysteine cathepsins, cathepsins S, K and V are highly potent elastases with
cathepsin V displaying the highest activity among all known mammalian elastases.
Despite a shared amino acid sequence identity of over 80% between cathepsins V and L
and very similar subsite specificities, only cathepsin V has a potent elastase activity
whereas cathepsin L lacks it. A series of chimera mutants containing various proportions
of cathepsin V and cathepsin L were constructed in an attempt to define a specific region
needed for elastin degradation. It was found that retaining the peptide sequence region
from amino acids 89 to 119 of cathepsin V preserves the mutant’s elastolytic activity
against elastin-Rhodamine conjugates whereas the region FTVVAPGK (amino acids
112-119) contributes approximately 60% of activity retention. Several additional mutant
proteins involving mutual swapping of residues VDIPK (amino acids 113-117) of
cathepsin L with residues TVVAPGK (amino acids 113-119) of cathepsin V, deletion of
Glyl 18 from cathepsin V, and insertion of Gly between Prol 16 and Lysi 17 in cathepsin
L were constructed and evaluated for their elastolytic activities. The results obtained with
those mutant cathepsin proteins support the importance of the amino acid region spanning
the residues from 112 to 119 in cathepsin V. Based on the 3-D structure of cathepsin V,
this peptide region is located below subsite binding pocket S2 and forms a wall-like
barrier which may act as an exosite for the productive binding of cross-linked elastin.
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Identification of potential exosite in cathepsin V necessary for elastin degradationChen, Li Hsuen 11 1900 (has links)
Besides collagen, elastin is the most common connective tissue structural protein in
vertebrates and similar to collagen relatively resistant to non-specific degradation.
Typical elastolytic proteases are the serine-dependent pancreatic and leukocyte elastases,
the Zn-dependent matrix metalloproteinase 12, and several lysosomal cysteine proteases.
Among the cysteine cathepsins, cathepsins S, K and V are highly potent elastases with
cathepsin V displaying the highest activity among all known mammalian elastases.
Despite a shared amino acid sequence identity of over 80% between cathepsins V and L
and very similar subsite specificities, only cathepsin V has a potent elastase activity
whereas cathepsin L lacks it. A series of chimera mutants containing various proportions
of cathepsin V and cathepsin L were constructed in an attempt to define a specific region
needed for elastin degradation. It was found that retaining the peptide sequence region
from amino acids 89 to 119 of cathepsin V preserves the mutant’s elastolytic activity
against elastin-Rhodamine conjugates whereas the region FTVVAPGK (amino acids
112-119) contributes approximately 60% of activity retention. Several additional mutant
proteins involving mutual swapping of residues VDIPK (amino acids 113-117) of
cathepsin L with residues TVVAPGK (amino acids 113-119) of cathepsin V, deletion of
Glyl 18 from cathepsin V, and insertion of Gly between Prol 16 and Lysi 17 in cathepsin
L were constructed and evaluated for their elastolytic activities. The results obtained with
those mutant cathepsin proteins support the importance of the amino acid region spanning
the residues from 112 to 119 in cathepsin V. Based on the 3-D structure of cathepsin V,
this peptide region is located below subsite binding pocket S2 and forms a wall-like
barrier which may act as an exosite for the productive binding of cross-linked elastin.
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