Pressure homogenization of mammalian tissues

Hunter, Margaret J.; Commerford, S. L.

doi:10.1016/0006-3002(61)90553-4

Cited by 176 publications

(50 citation statements)

References 14 publications

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“…Microsome preparation for P450 activity measurement was performed in the absence of protease inhibitors. For protein quantification, cells were additionally subjected to nitrogen cavitation (450 psi for 15 min at 4°C) in buffer A (Hunter and Commerford, 1961;Kamiie et al, 2008). The obtained homogenates were centrifuged at 10,800g for 20 min at 4°C, and the supernatants were collected and ultracentrifuged at 100,000g for 60 min at 4°C.…”

Section: Methodsmentioning

confidence: 99%

Simultaneous Absolute Protein Quantification of Transporters, Cytochromes P450, and UDP-Glucuronosyltransferases as a Novel Approach for the Characterization of Individual Human Liver: Comparison with mRNA Levels and Activities

et al. 2011

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ABSTRACT:The purpose of the present study was to determine the absolute protein expression levels of multiple drug-metabolizing enzymes and transporters in 17 human liver biopsies, and to compare them with the mRNA expression levels and functional activities to evaluate the suitability of the three measures as parameters of hepatic metabolism. Absolute protein expression levels of 13 cytochrome P450 (P450) enzymes, NADPH-P450 reductase (P450R) and 6 UDPglucuronosyltransferase (UGT) enzymes in microsomal fraction, and 22 transporters in plasma membrane fraction were determined using liquid chromatography/tandem mass spectrometry. CYP2C9, CYP2E1, CYP3A4, CYP2A6, UGT1A6, UGT2B7, UGT2B15, and P450R were abundantly expressed (more than 50 pmol/mg protein) in human liver microsomes. The protein expression levels of CYP3A4, CYP2B6, and CYP2C8 were each highly correlated with the corresponding enzyme activity and mRNA expression levels, whereas for other P450s, the protein expression levels were better correlated with the enzyme activities than the mRNA expression levels were. Among transporters, the protein expression level of organic anion-transporting polypeptide 1B1 was relatively highly correlated with the mRNA expression level. However, other transporters showed almost no correlation. These findings indicate that protein expression levels determined by the present simultaneous quantification method are a useful parameter to assess differences of hepatic function between individuals.

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Section: Methodsmentioning

confidence: 99%

Simultaneous Absolute Protein Quantification of Transporters, Cytochromes P450, and UDP-Glucuronosyltransferases as a Novel Approach for the Characterization of Individual Human Liver: Comparison with mRNA Levels and Activities

et al. 2011

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“…A buffer containing 250 mM-Tris-HC1 pH 7.5, 2 mM-MgC12, 3 mM-dithiothreitol, 0.5 mM-CaClz, 2 mM-ATP and 0.1 mM-phenylmethylsulphonyl fluoride was added to each dish, and the cells were scraped offwith a rubber policeman. The material from three dishes was collected in a total volume of 1.2 ml and subjected to nitrogen cavitation (Hunter & Commerford, 1961;Svardal & Pryme, 1978). Nitrogen was let through the apparatus for 5 min at low pressure to evacuate the air.…”

Section: Methodsmentioning

confidence: 99%

Evidence that Deletion of Coding Sequences in the 5' End of the Thymidine Kinase Gene of Herpes Simplex Virus Type 1 Affects the Stability of the Gene Products

Haarr

Flatmark²

1987

Journal of General Virology

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SUMMARYThe experiments described in the present work were designed to study the function of the N-terminal end of thymidine kinase (TK) encoded by herpes simplex virus type 1. Specifically we were interested to know whether this end was involved in binding of the enzyme to other molecules, had any influence on its subcellular localization or affected one or more of the activities associated with the enzyme. A parental enzyme and a deletion mutant, lacking the 45 N-terminal amino acids, derived from this strain, were used. Thymidine kinase from the parental virus bound to DNA-Sepharose, but the truncated enzyme did not. This was apparently not due to a specific ability to bind to DNA, since immunofluorescence studies indicated that both the normal and the deleted TK were mainly located in the cytoplasm, preferentially in the perinuclear region. Phosphorylation of thymidine as well as the amounts of TK potypeptides were markedly reduced at late times after infection with the mutant, but not to the same extent after infection with the wild-type. The deleted TK gene was efficiently transcribed as shown by hybridization of RNA to a probe specific for the gene, and this RNA directed the synthesis in vitro of TK polypeptides. Deletion of the 5' end of the gene seems to affect the stability of either the enzyme or TK-specific mRNA, or both. The TMP phosphorylating activity seems to be particularly destabilized relative to the thymidine phosphorylating activity.

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“…Whole cells (including the nuclei) could be broken by placing the cell suspension in a nitrogen cavitation bomb (7) at a pressure of 500 lbs./inch2 at 4°. The cells were ruptured by rapid transfer to ambient pressure.…”

Section: Methodsmentioning

confidence: 99%

Enhancement of DNA Synthesis in a Mammalian Cell-Free System by Trypsin Treatment

Brown¹,

Stubblefield²

1974

Proc. Natl. Acad. Sci. U.S.A.

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DNA synthesis in a broken cellular preparation of Chinese hamster cells was enhanced approximately 10-fold by a brief trypsin treatment. Alphachywotrypsin also enhanced the, synthesis, whereas Pronase did not. The trypsin appears to be acting on a compopent of the nucleus. Evidence suggests that the trypsin is not removing protein from the DNA, but may be activating the system some other way.Much remains to be learned about DNA replication, especially in eukaryotic systems. The presence of histones, other chromosomal proteins, and the nuclear envelope provide extra complications for such studies 'in eukaryotes. Using cell culture systems, Taylor has shown that the nascent DNA is replicated in 6S units and then joined by a ligase (1). The same mechanism exists in prokaryotes (2). Recently it has been shown that a short chain of RNA is associated with the nascent DNA, and it has been suggested that this RNA must be synthesized before the DNA can be replicated (3). Silverman and Mirsky (4) have shown that exogenous DNA polymerase and RNA polymerase both have much less access to the DNA in isolated nuclei or chromatin than in isolated DNA.Using isolated nuclei, chromatin, and DNA, we have studied DNA synthesis using the endogenous DNA polymerase. In the course of such experiments we found a striking effect of trypsin on the rate of DNA synthesis. Suspecting at first that the effect might be due to the removal of histones from the DNA, we were surprised to find that this was not the explanation. The key experiments will be reported in this paper; a preliminary abstract was published earlier (5). MATERIALS AND METHODSCulture&. Chinese hamster fibroblast cultures (Don and Don-C) were grown as monolayers in McCoy's 5a medium supplemented with 10% fetal-calf serum (v/v).I8olation of Nuclei and(Cytosol&. Nuclei were isolated by a modification of the method of Wray and Stubblefield (6). The isolation buffer contained 1 mM CaCl2, 0.5 mM piperazine-NN'-bis(2-ethanesulfonic acid) monosodium monohydrate "PIPES" (Calbiochem) at pH 6.7, and 0.5 M hexylene glycol. Cells were either removed by a brief trypsin treatment or else scraped off in isolation buffer with a rubber policeman. In either case, the cells were washed once in 4 ml of isolation buffer (5 X 106 cells per ml) at 40 and then pelleted by centrifugation at 800 X g for 5 min at 4°. The pellet was then resuspended in isolation buffer at approximately 2 X 107 cells per ml. The nuclei were isolated by rapid 2432 passage of the suspension through a 22-gauge needle with the bevel placed against the side of the centrifuge tube. This was repeated until clean nuclei were obtained, as determined by phase microscopy. The resulting preparation is called the cellular lysate. The nuclei can be removed from the suspension by centrifugation at 800 X g for 5 min at 4°. In some cases the supernatant from this was' then centrifuged again at 100,000 X g at 4°. The supernatant from the high-speed centrifugation is termed the cytoplasmic cyto8ol. The pellet of nuclei was resuspende...

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Pressure homogenization of mammalian tissues

Cited by 176 publications

References 14 publications

Simultaneous Absolute Protein Quantification of Transporters, Cytochromes P450, and UDP-Glucuronosyltransferases as a Novel Approach for the Characterization of Individual Human Liver: Comparison with mRNA Levels and Activities

Simultaneous Absolute Protein Quantification of Transporters, Cytochromes P450, and UDP-Glucuronosyltransferases as a Novel Approach for the Characterization of Individual Human Liver: Comparison with mRNA Levels and Activities

Evidence that Deletion of Coding Sequences in the 5' End of the Thymidine Kinase Gene of Herpes Simplex Virus Type 1 Affects the Stability of the Gene Products

Enhancement of DNA Synthesis in a Mammalian Cell-Free System by Trypsin Treatment

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