Editing Microsome (section)

== Pulse-Chase experiments ==

Microsomes also play a part in the [[Pulse-chase experiments|Pulse-Chase experiments]]. The Pulse-Chase experiments showed that secreted proteins move across the endoplasmic reticulum membrane when the membranes are purified. It was important to take the endoplasmic reticulum away from the rest of the cell to look into translocation but this isn’t possible due to how delicate and interconnected it is. This allowed microsomes to come into play as they have the majority of the biochemical properties of the endoplasmic reticulum. The microsomes are formed through homogenizing the cells and small closed [[Vesicle (biology and chemistry)|vesicles]] with ribosomes outside being formed from rough endoplasmic reticulum breakdown. When microsomes were treated with protease, it was found that the [[polypeptide]] made by ribosomes ended in the microsomal lumen. This takes place even though the proteins are made on the cytosolic face of the endoplasmic reticulum membrane.

Other experiments have shown that microsomes have to be introduced before about the first 70 [[amino acid]]s are translated for the secretory protein to go into the microsomal lumen. At this point, 40 amino acids are sticking out from the ribosome and the 30 amino acids after that are in the ribosomal channel. Cotranslational translocation explains that transport into the endoplasmic reticulum lumen of secretory proteins starts with the protein still bound to the ribosomes and not completely synthesized.<ref>Lodish, H. F. et al. (2008). Molecular Cell Biology. W.H. Freeman.</ref>
Microsomes can be concentrated and separated from other cellular debris by [[differential centrifugation]]. Unbroken cells, [[Cell nucleus|nuclei]], and [[mitochondria]] sediment out at 10,000 g (where g is the Earth's gravitational acceleration), whereas soluble enzymes and fragmented ER, which contains [[cytochrome P450]] (CYP), remain in solution. At 100,000 g, achieved by faster centrifuge rotation, ER sediments out of solution as a pellet but the soluble enzymes remain in the [[Precipitation (chemistry)|supernatant]]. In this way, cytochrome P450 in microsomes is concentrated and isolated. Microsomes have a reddish-brown color, due to the presence of the [[heme]]. Because of the need for a multi-part protein-system, microsomes are necessary to analyze the [[Metabolism|metabolic activity]] of CYPs. These CYPs are highly abundant in livers of rats, mice and humans, but present in all other organs and organisms as well.

To get microsomes containing a specific CYP or for high amounts of active enzyme, microsomes are prepared from [[Sf9 (cells)|Sf9 insect cells]] or in yeast via [[heterologous expression]]. Alternatively expression in ''[[Escherichia coli]]'' of whole or truncated proteins can also be performed.<ref name="Pan_2011">{{cite journal | vauthors = Pan Y, Abd-Rashid BA, Ismail Z, Ismail R, Mak JW, Ong CE | title = Heterologous expression of human cytochromes P450 2D6 and CYP3A4 in Escherichia coli and their functional characterization | journal = The Protein Journal | volume = 30 | issue = 8 | pages = 581–91 | year = 2011 | pmid = 22001938 | doi = 10.1007/s10930-011-9365-6 | s2cid = 26020065 }}</ref><ref name="Schwarz_2010">{{cite journal | vauthors = Schwarz D, Kisselev P, Honeck H, Cascorbi I, Schunck WH, Roots I | title = Co-expression of human cytochrome P4501A1 (CYP1A1) variants and human NADPH-cytochrome P450 reductase in the baculovirus/insect cell system | journal = Xenobiotica; the Fate of Foreign Compounds in Biological Systems | volume = 31 | issue = 6 | pages = 345–56 | year = 2001 | pmid = 11513247 | doi = 10.1080/00498250110055947 | s2cid = 43124584 }}</ref> Therefore, microsomes are a valuable tool for investigating the metabolism of compounds (enzyme inhibition, clearance and [[metabolite]] identification) and for examining drug-drug interactions by ''[[in vitro]]''-research. Researchers often select microsome lots based on the enzyme activity level of specific CYPs. Some lots are available to study specific populations (for example, lung microsomes from smokers or non-smokers) or divided into classifications to meet target CYP activity levels for inhibition and [[Metabolomics|metabolism studies]].

Microsomes are used to mimic the activity of the endoplasmic reticulum in a test tube and conduct experiments that require [[Protein biosynthesis|protein synthesis]] on a membrane. They provide a way for scientists to figure out how proteins are being made on the ER in a cell by reconstituting the process in a test tube.

Keefer et al. looked into how human liver microsomes and human [[hepatocyte]]s are used to study metabolic stability and inhibition for in vitro systems. Going into their similarities and differences can shine light on the mechanisms of [[metabolism]], passive permeability, and transporters. It was shown that passive permeability is important in metabolism and enzyme inhibition in human hepatocytes. Also, P-gp efflux has a smaller role in this same area. Also, liver microsomes are more predictive than hepatocytes of in vivo clearance when they give higher intrinsic clearance than the hepatocytes.<ref>Keefer, C. et al. (2020). Mechanistic insights on clearance and inhibition discordance between liver microsomes and hepatocytes when clearance in liver microsomes is higher than in hepatocytes. European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences. Retrieved November 29, 2022, from {{PMID|32927071}}</ref>