Purification and properties of subunits of sterol ester hydrolase from rat pancreas

Calame, K.B.; Gallo, Linda L.; Cheriathundam, Elizabeth; Vahouny, George V.; Treadwell, C. R.

doi:10.1016/0003-9861(75)90227-1

Cited by 51 publications

(8 citation statements)

References 20 publications

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“…In method A (see Materials and Methods), cytosol was prepared from frozen tissue in the presence of digitonin and was dialyzed at pH 5.1. This method is similar to conditions previously reported (Calame et al" 1975;Rudd et al" 1987), and it is now known that this does not preserve the more labile 72-kDa enzyme. Hence, method A facilitates the isolation and purification of the 67-kDa form.…”

Section: Quantitation Of Cholesterol Esterase Bysupporting

confidence: 56%

“…The existence of the 67and 72-kDa enzyme forms and their different lability to proteolysis bear importantly on previous attempts to isolate and characterize cholesterol esterase. These methods have usually provided a 67-kDa protein (Calame et al, 1975;Van Den Bosch et al, 1973). Most purification schemes (Calame et al, 1975;Rudd et al, 1987;Albro et al, 1976) use digitonin and low concentrations of benzamidine during the disruption of pancreatic tissue, conditions that favor release of the 67-kDa form (unpublished observations) and proteolytic degradation of the 72-kDa enzyme.…”

Section: Discussionmentioning

confidence: 99%

“…These methods have usually provided a 67-kDa protein (Calame et al, 1975;Van Den Bosch et al, 1973). Most purification schemes (Calame et al, 1975;Rudd et al, 1987;Albro et al, 1976) use digitonin and low concentrations of benzamidine during the disruption of pancreatic tissue, conditions that favor release of the 67-kDa form (unpublished observations) and proteolytic degradation of the 72-kDa enzyme. Under these conditions, as the purification proceeds, not only does the yield diminish, but the final homogeneous enzyme may simply be a minor pancreatic component that is less susceptible to proteolytic degradation and that contributes in our experience less than 10% of the total cholesterol esterase activity.…”

Section: Discussionmentioning

confidence: 99%

“…One of the critical steps in cholesterol absorption is the pancreatic cholesterol esterase catalyzed cleavage of cholesteryl esters, one of the dietary forms of ingested cholesterol (Vahouny & Treadwell, 1964). This lipolytic enzyme has been extensively investigated from a variety of adult species, including man, pig, rat, and cow (Lombardo et al, 1978;Rudd et al, 1987;Calame et al, 1975; Van Den Bosch et al, 1973), but there have been no studies from the corresponding juvenile animals or children. Thus, biochemical details of cholesterol absorption have remained largely speculative in children, without comparative knowledge of enzyme structure, function, and regulation.…”

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confidence: 99%

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Pancreatic cholesterol esterases. 1. Pancreatic cholesterol esterase induction during maturation

Cox¹,

Leung²,

Kyger³

et al. 1990

Biochemistry

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The activities of pancreatic cholesterol esterase from calf and cow pancreas were examined in detail. A 1300-fold enhancement of enzymatic activity was found after maturation, even though cholesterol esterase activity levels in other organs did not change from the juvenile to the adult species. Radioimmunoassays also showed that the calf pancreas contained at least 100-fold less cholesterol esterase protein. Decreased amounts of protein were not due to enhanced proteolysis, since cytosol from cow pancreas degrades exogenously added cholesterol esterase faster than that from calf pancreas. Rather, enhancement of pancreatic cholesterol esterase activity associated with bovine maturation was the result of specific, increased synthesis of a 72-kDa enzyme. This labile 72-kDa cholesterol esterase species was purified to homogeneity by a two-step process in 75% yield and is the major form of bovine pancreatic cholesterol esterase (99%). A much less abundant 67-kDa species, accounting for less than 1% of total pancreatic cholesterol esterase activity, was also purified to homogeneity in a similar two-step process. These results demonstrate that a specific form of pancreatic cholesterol esterase is induced during maturation, and they bear importantly on understanding juvenile cholesterol metabolism as related to dietary absorption of this sterol.

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Section: Quantitation Of Cholesterol Esterase Bysupporting

confidence: 56%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Pancreatic cholesterol esterases. 1. Pancreatic cholesterol esterase induction during maturation

Cox¹,

Leung²,

Kyger³

et al. 1990

Biochemistry

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“…In the deaggregated form, the purified cocaine esterase showed a decrease in thermal stability at 470C, implying that some destabilizing physical change was induced by the binding of cholate or the deaggregation of the enzyme. This is in contrast to the mammalian lipases, whose thermal stabilities have been reported to increase in the presence of bile acids; however, these enzymes do not undergo a bile acid-induced deaggregation (8,41).…”

Section: Discussionmentioning

confidence: 95%

Identification of a cocaine esterase in a strain of Pseudomonas maltophilia

1992

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A strain of Pseudomonas maltophilia (termed MB11L) which was capable of using cocaine as its sole carbon and energy source was isolated by selective enrichment. An inducible esterase catalyzing the hydrolysis of cocaine to ecgonine methyl ester and benzoic acid was identified and purified 22-fold. In the presence of the solubilizing agent cholate, cocaine esterase had a native Mr of 110,000 and was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be a monomer. In the absence of cholate, cocaine esterase had a native Mr of 410,000 and probably existed as a tetramer. The pH optimum of the enzyme was 8.0, and the Km values for cocaine, ethyl benzoate, and ethyl 2-hydroxybenzoate were 0.36, 1.89, and 1.75 mM, respectively.Inhibition studies indicated that the enzyme was a serine esterase, possibly possessing a cation-binding site similar to those of mammalian acetylcholinesterase and the atropine esterase of Pseudomonas putida PMBL-1.The cocaine esterase ofP. maltophilia MBl1L showed no activity with atropine, despite the structural similarity of cocaine and atropine.Microbial enzyme activities against alkaloids have been investigated as models of mammalian metabolism and as potential sources of new therapeutic compounds (16,17,38). Of the tropane alkaloids, the microbial metabolism of atropine has received the most significant attention. Corynebacterium belladonae catabolizes atropine via esterolytic hydrolysis of the tropic acid moiety followed by dehydrogenation, ring opening, and deamination of the tropane ring (27,28). A wide range of pseudomonads were also found to utilize atropine as their sole source of carbon and nitrogen (34). Further studies with nine of these strains identified two distinct classes of atropine esterase which possessed different physical and chemical properties (31). The atropine esterase from Pseudomonas putida PMBL-1 has been purified and extensively characterized (15,40,(43)(44)(45). Probing of the active site of the enzyme indicated an active serine residue and a classical charge relay system (43,44), although sequence analysis (15) and structure prediction (42) implied little homology to the serine protease families. Atropine esterase showed stereospecificity toward the (-)-isomer of atropine, (-)-hyoscyamine, and appeared to favor esters of tropic acid over those of acetic acid (34). Interestingly, the enzyme displayed no activity with the structurally related tropane alkaloid cocaine (34). In this paper, we describe the isolation of a strain of Pseudomonas maltophilia capable of using cocaine as its sole carbon and energy source and the partial purification and characterization of a cocaine esterase from this organism. Solutions and buffers were prepared by using water purified by a Milli-RO-60 system (Millipore Waters UK Ltd., Watford, United Kingdom). Ecgonine hydrochloride was prepared from cocaine by using the method of Findlay (11). The purity of the product was established by thin-layer chromatography (TLC), gas chromatography (GC), and HPLC analyses. Ecg...

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Luminal Events in Gastrointestinal Lipid Digestion

Borgström

Patton

1991

Comprehensive Physiology

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Purification and properties of subunits of sterol ester hydrolase from rat pancreas

Cited by 51 publications

References 20 publications

Pancreatic cholesterol esterases. 1. Pancreatic cholesterol esterase induction during maturation

Pancreatic cholesterol esterases. 1. Pancreatic cholesterol esterase induction during maturation

Identification of a cocaine esterase in a strain of Pseudomonas maltophilia

Luminal Events in Gastrointestinal Lipid Digestion

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