Studies on proteolytic activity in commercial myoglobin preparations.
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ABSTRACT:
SUBMITTER: Goldspink DF
PROVIDER: S-EPMC1178192 | biostudies-other | 1971 Dec
REPOSITORIES: biostudies-other
Similar Datasets
Project description:1. The proteolytic activities of several gamma-globulin preparations were tested. These included sulphate-precipitated human and bovine preparations and human and bovine Cohn fraction II preparations as well as purified gamma-globulin preparations. Up to 14mg. of diffusible peptides and glycopeptides/g. of gamma-globulin was liberated after dialysis and up to 10mg. of peptides/g. after incubation and trichloroacetic acid precipitation, as products of the degradation process in incubated gamma-globulin. 2. in-Aminohexanoic acid and p-chloromercuribenzoic acid, as well as heating at 60 degrees for 40min., were shown to inhibit strongly these proteolytic activities. Streptokinase was shown to activate strongly the proteolytic activity of all the human preparations (sulphate-precipitated, Cohn fraction II, and purified gamma-globulin). 3. Two distinct pH optima were shown for human and bovine gamma-globulin preparations: one at pH8, the other at pH3.8 (the latter activity could be demonstrated only in the presence of cysteine). 4. Both (131)I-labelled human Cohn fraction II and bovine fibrinogen were attacked by a sulphate-precipitated preparation of gamma-globulin. Of the synthetic substrates tested toluene-p-sulphonyl-l-arginine methyl ester was hydrolysed by both the sulphate-precipitated and Cohn fraction II preparations, as was benzoyl-l-arginine amide at pH5, but only in the presence of cysteine. 5. These data are interpreted to indicate that at least two enzymes are present in gamma-globulin preparations, one being similar to the plasmin system, the other similar to cathepsin B.
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Project description:The present study reports the perplexing results that came about because of seriously impure commercially available reagents. Commercial reagents and chemicals are routinely ordered by scientists and expected to have been rigorously assessed for their purity. Unfortunately, we found this assumption to be risky. Extensive work was carried out within our laboratory using commercially sourced preparations of the small leucine-rich proteoglycans (SLRPs), decorin and biglycan, to investigate their influence on nerve cell growth. Unusual results compelled us to analyse the composition and purity of both preparations of these proteoglycans (PGs) using both mass spectrometry (MS) and Western blotting, with and without various enzymatic deglycosylations. Commercial 'decorin' and 'biglycan' were found to contain a mixture of PGs including not only both decorin and biglycan but also fibromodulin and aggrecan. The unexpected effects of 'decorin' and 'biglycan' on nerve cell growth could be explained by these impurities. Decorin and biglycan contain either chondroitin or dermatan sulfate glycosaminoglycan (GAG) chains whereas fibromodulin only contains keratan sulfate and the large (>2500 kDa), highly glycosylated aggrecan contains both keratan and chondroitin sulfate. The different structure, molecular weight and composition of these impurities significantly affected our work and any conclusions that could be made. These findings beg the question as to whether scientists need to verify the purity of each commercially obtained reagent used in their experiments. The implications of these findings are vast, since the effects of these impurities may already have led to inaccurate conclusions and reports in the literature with concomitant loss of researchers' funds and time.
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Project description:Hops (Humulus lupulus L. Cannabaceae) is an economically important crop, that has drawn more attention in recent years due to its potential pharmaceutical applications. Bitter acids (prenylated polyketides) and prenylflavonoids are the primary phytochemical components that account for hops resins medicinal value. We have previously reported on utilizing untargeted NMR and MS metabolomics for analysis of 13 hops cultivars, revealing for differences in α- versus β-bitter acids composition in derived resins. In this study, effect of ratios of bitter α- to β-acids in hop resins to cytotoxicity of hop resins was investigated. In vitro cell culture assays revealed that β-acids were more effective than α-acids in growth inhibition of PC3 and HT29 cancer cell lines. Nevertheless, hop resins enriched in β-acids showed comparable growth inhibition patterns to α-enriched resins and suggesting that bioactivity may not be easily predicted by metabolomics and/or gross metabolic profiling in hops.
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Project description:1. The physical, chemical and enzymic properties of subfragment 1 prepared from myosin of rabbit skeletal muscle by using two different concentrations of insoluble papain were compared. 2. Subfragment 1 prepared by using a myosin/papain ratio of 2000: 1 (by wt.) migrated on electrophoresis in non-dissociating conditions as a single enzymically active band. When prepared with a myosin/papain ratio of 200: 1 the preparation consisted of two enzymically active components of slightly different electrophoretic mobility. 3. The two types of preparation were obtained in similar yield and possessed similar specific adenosine triphosphatase activities when determined in the presence of Ca(2+). 4. Gel electrophoresis in the presence of 8m-urea showed that both preparations contained three light components. The component of molecular weight 15500 was apparently identical with one of the light-chain components of myosin (Ml(1)). The other two light-chain components of subfragment 1 were not identical with any of the light-chain components of myosin. 5. The heavy-chain fraction of subfragment 1 prepared by using low concentrations of papain dissociated into components with molecular weights of 87000, 69000 and 26000 on electrophoresis in sodium dodecyl sulphate. The heavy-chain fraction of subfragment 1 prepared by using higher concentrations of papain contained components with molecular weights of 69000 and 53000 and relatively increased amounts of the component of molecular weight 26000. 6. The isolated 26000 dalton component had an amino acid composition similar to that of the heavy-chain fraction of subfragment 1 and contained 3-methylhistidine and mono-and tri-N(epsilon)-methyl-lysine. It was homogeneous on electrophoresis in the presence of sodium dodecyl sulphate but gave two bands on electrophoresis in 8m-urea.
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