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cell biology Cell biology (also cellular biology or cytology) is a branch of biology that studies the structure, function, and behavior of cells. All living organisms are made of cells. A cell is the basic unit of life that is responsible for the living and ...
, microsomes are heterogeneous vesicle-like artifacts (~20-200 nm diameter) re-formed from pieces of the
endoplasmic reticulum The endoplasmic reticulum (ER) is, in essence, the transportation system of the eukaryotic cell, and has many other important functions such as protein folding. It is a type of organelle made up of two subunits – rough endoplasmic reticulum ( ...
(ER) when
eukaryotic Eukaryotes () are organisms whose cells have a nucleus. All animals, plants, fungi, and many unicellular organisms, are Eukaryotes. They belong to the group of organisms Eukaryota or Eukarya, which is one of the three domains of life. Bacte ...
cells are broken-up in the
laboratory A laboratory (; ; colloquially lab) is a facility that provides controlled conditions in which scientific or technological research, experiments, and measurement may be performed. Laboratory services are provided in a variety of settings: physicia ...
; microsomes are not present in healthy, living cells. Rough (containing
ribosomes Ribosomes ( ) are macromolecular machines, found within all cells, that perform biological protein synthesis (mRNA translation). Ribosomes link amino acids together in the order specified by the codons of messenger RNA (mRNA) molecules to f ...
) and smooth (without ribosomes) microsomes are made from the endoplasmic reticulum through
cell disruption Cell disruption is a method or process for releasing biological molecules from inside a cell. Methods The production of biologically interesting molecules using cloning and culturing methods allows the study and manufacture of relevant molecule ...
. These microsomes have an inside that is exactly the same as the endoplasmic reticulum lumen. Both forms of microsomes can be purified by a process known as equilibrium density
centrifugation Centrifugation is a mechanical process which involves the use of the centrifugal force to separate particles from a solution according to their size, shape, density, medium viscosity and rotor speed. The denser components of the mixture migrate ...
. Rough and smooth microsomes do differ in their proteins and rough microsomes have shown occurrence of translation and translocation at the same time besides certain exceptions from proteins in yeast.


Signal Hypothesis

Microsomes play a role in the signal hypothesis. This hypothesis explores in vitro protein translation for a
mRNA In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of Protein biosynthesis, synthesizing a protein. mRNA is ...
encoding secretory protein. When microsomes are present, the proteins showed to be the correct size. Without the microsomes, the proteins turned out to be larger than normal.


Cell-free Protein Synthesis

This relates to
cell-free protein synthesis Cell-free protein synthesis, also known as '' in vitro'' protein synthesis or CFPS, is the production of protein using biological machinery in a cell-free system, that is, without the use of living cells. The ''in vitro'' protein synthesis environ ...
. Cell-free protein synthesis that is without microsomes has no way for incorporation into the microsomes to happen. This means that when microsomal membranes are presented later there isn’t the removal of the signal sequence. With microsomes there, cell-free protein synthesis demonstrates cotranslational transport of the protein into the microsome and therefore the removal of the signal sequence. This process produces a mature protein chain. Studies have looked into the cell-free protein synthesis process when microsomes have their bound ribosomes stripped away from them. This explained certain details about endoplasmic reticulum signal sequences. Normally, a
secretory protein A secretory protein is any protein, whether it be endocrine or exocrine, which is secreted by a cell. Secretory proteins include many hormones, enzymes, toxins, and antimicrobial peptides. Secretory proteins are synthesized in the endoplasmic reti ...
only has its signal sequence removed if the microsomes are there for
protein synthesis Protein biosynthesis (or protein synthesis) is a core biological process, occurring inside Cell (biology), cells, homeostasis, balancing the loss of cellular proteins (via Proteolysis, degradation or Protein targeting, export) through the product ...
due to the secretory protein being incorporated into the microsomes. Protein transport doesn’t happen if there is a late addition of microsomes after the completion of the protein synthesis process. Protein extrusion into a microsome can be described by multiple factors. A protein has been extruded if it is resistant to
proteases A protease (also called a peptidase, proteinase, or proteolytic enzyme) is an enzyme that catalyzes (increases reaction rate or "speeds up") proteolysis, breaking down proteins into smaller polypeptides or single amino acids, and spurring the for ...
, is not resistant to proteases when detergents are present, or is glycosylated by enzymes residing in the microsomes. Additionally, another sign that a protein has been extruded is
signal peptidase Signal peptidases are enzymes that convert secretory and some membrane proteins to their mature or pro forms by cleaving their signal peptides from their N-termini. Signal peptidases were initially observed in endoplasmic reticulum (ER)-der ...
cleaving off the N-terminal signal peptide inside the microsome that may cause the protein to be smaller in size.


Pulse-Chase experiments

Microsomes also play a part in the 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
vesicles Vesicle may refer to: ; In cellular biology or chemistry * Vesicle (biology and chemistry), a supramolecular assembly of lipid molecules, like a cell membrane * Synaptic vesicle ; In human embryology * Vesicle (embryology), bulge-like features o ...
with ribosomes outside being formed from rough endoplasmic reticulum breakdown. When microsomes were treated with protease, it was found that the
polypeptide Peptides (, ) are short chains of amino acids linked by peptide bonds. Long chains of amino acids are called proteins. Chains of fewer than twenty amino acids are called oligopeptides, and include dipeptides, tripeptides, and tetrapeptides. A p ...
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 acids Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha am ...
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. Microsomes can be concentrated and separated from other cellular debris by
differential centrifugation In biochemistry and cell biology, differential centrifugation (also known as differential velocity centrifugation) is a common procedure used to separate organelles and other sub-cellular particles based on their sedimentation rate. Although o ...
. Unbroken cells, nuclei, and
mitochondria A mitochondrion (; ) is an organelle found in the Cell (biology), cells of most Eukaryotes, such as animals, plants and Fungus, fungi. Mitochondria have a double lipid bilayer, membrane structure and use aerobic respiration to generate adenosi ...
sediment out at 10,000 g (where g is the Earth's gravitational acceleration), whereas soluble enzymes and fragmented ER, which contains
cytochrome P450 Cytochromes P450 (CYPs) are a Protein superfamily, superfamily of enzymes containing heme as a cofactor (biochemistry), cofactor that functions as monooxygenases. In mammals, these proteins oxidize steroids, fatty acids, and xenobiotics, and are ...
(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
supernatant In an aqueous solution, precipitation is the process of transforming a dissolved substance into an insoluble solid from a super-saturated solution. The solid formed is called the precipitate. In case of an inorganic chemical reaction leading ...
. In this way, cytochrome P450 in microsomes is concentrated and isolated. Microsomes have a reddish-brown color, due to the presence of the
heme Heme, or haem (pronounced / hi:m/ ), is a precursor to hemoglobin, which is necessary to bind oxygen in the bloodstream. Heme is biosynthesized in both the bone marrow and the liver. In biochemical terms, heme is a coordination complex "consisti ...
. Because of the need for a multi-part protein-system, microsomes are necessary to analyze the
metabolic activity Metabolism (, from el, μεταβολή ''metabolē'', "change") is the set of life-sustaining chemical reactions in organisms. The three main functions of metabolism are: the conversion of the energy in food to energy available to run cell ...
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 insect cells or in yeast via
heterologous expression Heterologous expression refers to the expression of a gene or part of a gene in a host organism that does not naturally have the gene or gene fragment in question. Insertion of the gene in the heterologous host is performed by recombinant DNA techno ...
. Alternatively expression in ''
Escherichia coli ''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Escher ...
'' of whole or truncated proteins can also be performed. Therefore, microsomes are a valuable tool for investigating the metabolism of compounds (enzyme inhibition, clearance and
metabolite In biochemistry, a metabolite is an intermediate or end product of metabolism. The term is usually used for small molecules. Metabolites have various functions, including fuel, structure, signaling, stimulatory and inhibitory effects on enzymes, c ...
identification) and for examining drug-drug interactions by ''
in vitro ''In vitro'' (meaning in glass, or ''in the glass'') studies are performed with microorganisms, cells, or biological molecules outside their normal biological context. Colloquially called "test-tube experiments", these studies in biology an ...
''-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 metabolism studies. Microsomes are used to mimic the activity of the endoplasmic reticulum in a test tube and conduct experiments that require
protein synthesis Protein biosynthesis (or protein synthesis) is a core biological process, occurring inside Cell (biology), cells, homeostasis, balancing the loss of cellular proteins (via Proteolysis, degradation or Protein targeting, export) through the product ...
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
hepatocytes A hepatocyte is a cell of the main parenchymal tissue of the liver. Hepatocytes make up 80% of the liver's mass. These cells are involved in: * Protein synthesis * Protein storage * Transformation of carbohydrates * Synthesis of cholesterol, b ...
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 Metabolism (, from el, μεταβολή ''metabolē'', "change") is the set of life-sustaining chemical reactions in organisms. The three main functions of metabolism are: the conversion of the energy in food to energy available to run cell ...
, 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.


MTP

Iqbal, Jahangir, and Al-Qarni studied the
microsomal triglyceride transfer protein Microsomal triglyceride transfer protein large subunit is a protein that in humans is encoded by the ''MTTP'' gene. MTP encodes the large subunit of the heterodimeric microsomal triglyceride transfer protein. Protein disulfide isomerase (PDI) comp ...
(MTP). MTP is an endoplasmic reticulum resident protein and assists in transferring neutral lipids to nascent
apolipoprotein B Apolipoprotein B (ApoB) is a protein that in humans is encoded by the gene. Function Apolipoprotein B is the primary apolipoprotein of chylomicrons, VLDL, Lp(a), IDL, and LDL particles (LDL—commonly known as "bad cholesterol" when in refer ...
. MTP has a large use for abetalipoproteinemia patients with MTP mutations because of how it affects the assembly and secretion of apoB-containing
lipoproteins A lipoprotein is a biochemical assembly whose primary function is to transport hydrophobic lipid (also known as fat) molecules in water, as in blood plasma or other extracellular fluids. They consist of a triglyceride and cholesterol center, sur ...
. These MTP mutations are linked with not having circulation of the apoB-containing lipoproteins. MTP is also involved with cholesterol ester and cluster of differentiation 1d biosynthesis. Transferring
sphingolipids Sphingolipids are a class of lipids containing a backbone of sphingoid bases, a set of aliphatic amino alcohols that includes sphingosine. They were discovered in brain extracts in the 1870s and were named after the mythological sphinx because o ...
to apoB-containing lipoproteins also falls under the ability of MTP. MTP works with the
homeostasis In biology, homeostasis (British English, British also homoeostasis) Help:IPA/English, (/hɒmɪə(ʊ)ˈsteɪsɪs/) is the state of steady internal, physics, physical, and chemistry, chemical conditions maintained by organism, living systems. Thi ...
of
lipids Lipids are a broad group of naturally-occurring molecules which includes fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides, phospholipids, and others. The functions of lipids include ...
and lipoproteins and is related to certain pathophysiological conditions and
metabolic diseases Inborn errors of metabolism form a large class of genetic diseases involving congenital disorders of enzyme activities. The majority are due to defects of single genes that code for enzymes that facilitate conversion of various substances (substrat ...
. Wang et al. explored
drug metabolism Drug metabolism is the metabolic breakdown of drugs by living organisms, usually through specialized enzymatic systems. More generally, xenobiotic metabolism (from the Greek xenos "stranger" and biotic "related to living beings") is the set of ...
in vitro using human liver microsomes and human liver S9 fractions. The study found significant differences between human liver microsomes and human liver S9 fractions in drug-metabolizing enzyme and transporter protein concentrations.  The protein-protein correlations of these drug-metabolizing enzymes and transporters was determined relating to the two hepatic preparations.Wang, X. et al. (2020, January). Comparative proteomics analysis of human liver microsomes and S9 fractions. Drug metabolism and disposition: the biological fate of chemicals. Retrieved November 29, 2022, from https://pubmed.ncbi.nlm.nih.gov/31699809/


See also

*
Cytochrome P450 Cytochromes P450 (CYPs) are a Protein superfamily, superfamily of enzymes containing heme as a cofactor (biochemistry), cofactor that functions as monooxygenases. In mammals, these proteins oxidize steroids, fatty acids, and xenobiotics, and are ...
*
List of biological development disorders References Bibliography * Reece JB, Urry LA, Cain ML, Wasserman SA, Minorsky PV, Jackson RB. Campbell Biology (10th ed.). Addison Wesley Longman; 2014. {{DEFAULTSORT:Biological development disorders Lists of diseases Disability-related l ...
*
S9 fraction The S9 fraction is the product of an organ tissue homogenate used in biological assays. The S9 fraction is most frequently used in assays that measure the metabolism of drugs and other xenobiotics. It is defined by the U.S. National Library of ...
*
Cell-free protein synthesis Cell-free protein synthesis, also known as '' in vitro'' protein synthesis or CFPS, is the production of protein using biological machinery in a cell-free system, that is, without the use of living cells. The ''in vitro'' protein synthesis environ ...
* Pulse-Chase experiments *
Differential Centrifugation In biochemistry and cell biology, differential centrifugation (also known as differential velocity centrifugation) is a common procedure used to separate organelles and other sub-cellular particles based on their sedimentation rate. Although o ...
*
Microsomal Triglyceride Transfer Protein Microsomal triglyceride transfer protein large subunit is a protein that in humans is encoded by the ''MTTP'' gene. MTP encodes the large subunit of the heterodimeric microsomal triglyceride transfer protein. Protein disulfide isomerase (PDI) comp ...


References


External links

* Membrane biology {{biochemistry-stub