Intracellular Step

Cell Line or Tissue Research Problem Reference 

Adrenal Y-1 Cells Role of microfilaments in steroid release Considine et a/., 1992  

HIT-T15Cells Role of actin filaments in insulin secretion Li etal., 1994 

Ileum G/F-actin transition in smooth muscle contraction Mauss et al., 1989 

Lymphocytes Receptor-mediated cell activation Melamed et al., 1991 

---

Bugg TDH and Walsh CT (1992) Intracellular steps of bacterial cell wall peptidoglycan biosynthesis enzymology, antibiotics, and antibiotic resistance. Nat Prod Rep 9, 199-215. 

One can distinguish between structured and unstructured models, the latter neglecting intracellular phenomena. On the contrary, structured models account for intracellular processes and states in different compartments of the cell or include explicit kinetics for various intracellular steps of virus replication. 

Fig. 9. Maturation pathway of a class II non-lantibiotic bacteriocin presented as a 4-step process. The HPK and RR constitute a protein complex involved in signal transduction of external stimuli (step 1), resulting in transcription and translation of the bacteriocin precursor (step 2). The inactive precursor peptide is targeted to a membrane anchored ATP dependent translocation complex and processing at the double-glycine consensus site occurs intracellularly (step 3) and is followed by secretion (step 4). The role of the accessory protein in this mechanism is not yet understood... 

Maximum specific growth rate rx,max h meant as a state, where no external substrate is limiting, but a—in most cases not known— intracellular step. The metabolism of the cell consists of physical and enzymatically catalyzed steps, which may differently react to different temperatures. Especially for microalgae, the photosynthetic apparatus down to proton transport through the thylakoid membrane as a transport step could react differently from the anabolic reactions. Explanations and first model approaches for combined temperature/Hght effects are given in Bechet et al (2013), Bernard and Remond (2012), and Yun and Park (2003). 

Techniques used in bioseparations depend on the nature of the product (i.e., the unique properties and characteristics which provide a handle for the separation), and on its state (i.e., whether soluble or insoluble, intra- or extracellular, etc.). All early isolation and recovery steps remove whole cells, cellular debris, suspended solids, and colloidal particles, concentrate the product, and, in many cases, achieve some degree of purification, all the while maintaining high yield. For intracellular compounds, the initial harvesting of the cells is important... 

You should note that the figure in SAQ 5.1 is a simple outline as fermentations generally have more steps than indicated for example many have a multiple purification step. If the product were the whole cell (for example in single cell protein processes) then purification of the cell biomass would be necessary. If the required product were an intracellular compound then some stage of cell breakage would be essential. 

Fermentation broths are complex, aqueous mixtures of cells, comprising soluble extracellular, intracellular products and any unconverted substrate or unconvertible components. Recovery and extraction of product is important in bioprocess engineering. In particular separation is a useful technique it depends on product, its solubility, size of the process, and product value. Purification of high-value pharmaceutical products using chromatography such as hormones, antibody and enzymes is expensive and difficult to scale up.1 Tire necessary steps to follow a specific process depend on the nature of the product and the characteristics of the fermentation broth. There are a few steps for product recovery the following processes are discussed, which are considered as an alternative for product recovery from fermentation broth. 

It is often desirable to recover product and to choose a suitable strain of microorganism which produces extracellular rather than intracellular product. If the product stays inside the cells, the cells must be ruptured, so freeing intracellular enzyme, after which extraction or purification is performed to recover the valuable product. The fermentation broth has to be processed, and pass through several stages for separation and purification. The product requites a sequence of operations for high purification. Hie usual steps to follow are as follows. 

During the purification of intracellular proteins, cell disruption by mechanical or biochemical means is the first step required in the process. However, it commonly initiates cellular and... 

The catalytic cycle of the Na+/K+-ATPase can be described by juxtaposition of distinct reaction sequences that are associated with two different conformational states termed Ei and E2 [1]. In the first step, the Ei conformation is that the enzyme binds Na+ and ATP with very high affinity (KD values of 0.19-0.26 mM and 0.1-0.2 pM, respectively) (Fig. 1A, Step 1). After autophosphorylation by ATP at the aspartic acid within the sequence DKTGS/T the enzyme occludes the 3 Na+ ions (Ei-P(3Na+) Fig. la, Step 2) and releases them into the extracellular space after attaining the E2-P 3Na+ conformation characterized by low affinity for Na+ (Kq5 = 14 mM) (Fig. la, Step 3). The following E2-P conformation binds 2 K+ ions with high affinity (KD approx. 0.1 mM Fig. la, Step 4). The binding of K+ to the enzyme induces a spontaneous dephosphorylation of the E2-P conformation and leads to the occlusion of 2 K+ ions (E2(2K+) Fig. la, Step 5). Intracellular ATP increases the extent of the release of K+ from the E2(2K+) conformation (Fig. la, Step 6) and thereby also the return of the E2(2K+) conformation to the EiATPNa conformation. The affinity ofthe E2(2K+) conformation for ATP, with a K0.5 value of 0.45 mM, is very low. 

Once the intracellular Ca " concentration begins to rise, calmodulin-calcium binding also rises and MLCK, which is dependent on calmodulin activation, rises in turn. The next step in this cascade is the phosphorylation of myosin. Finally, the phosphorylation of myosin results in the activation of the crossbridges and the accompanying transduction of ATP energy into mechanical work. Despite its differences in regulation, smooth muscle behaves mechanically much like other muscles. 

---