Cell and Tissue Reactor Engineering

© 2003 University of Minnesota

Stoichiometry and Kinetics of Cell Growth and Product

Formation
By Wei-Shou Hu

I. BASIC TERMINOLOGY
II. CELL PROPERTIES
A. Composition and Size
B. Variation in Cell Volume in the Course of Cultivation
C. Growth Rate of Mammalian Cells
D. Inoculum cell concentration requirements
III. MATERIAL BALANCE ON CELL GROWTH
A. Balance on Mammalian Cell Growth
IV. QUANTITATIVE DESCRIPTION OF CELL GROWTH & PRODUCT FORMATION
A. Definitions
B. Yields and Stoichiometric Ratios
C. Some complications in specific rate calculation
V. NUTRIENTS AND THEIR RELATION TO TRANSPORT CONSUMPTION
VI. STOICHIOMETRY AND MEDIUM DESIGN
A. Example. Oxygen as a nutrient
1. DEMAND
2. OPTIMAL CONCENTRATION
3. INTERACTION WITH ENERGY METABOLISM
B. Intracellular concentrations of nutrients and lactate
VII. STOICHIOMETRIC RATIO AS METABOLIC INDICATOR
VIII. MEDIUM DESIGN FROM A STOICHIOMETRIC POINT OF VIEW
A. Consumable vs. un-consumable
B. Macromolecules: stoichiometric or catalytic
I. BASIC TERMINOLOGY
Anchorage-dependent Attachment to a suitable substrate is required for multiplication
Anchorage-independent Multiplication occurs in suspension (in either liquid or semi-solid medium)
Anchorage-preferred Attachment to a suitable substrate is not necessary but preferred for
multiplication
Basal medium A mixture defined nutrient dissolved in a buffered physiological saline solution
Chemically defined A medium prepared from reagent grade water and highly purified
medium supplements – trace impurities may be present but at levels too low to
influence cellular growth responses
Conditioning The modification of the cellular environment by product released by cells into
the medium
Growth factor A non-nutrient substance that controls proliferation in a regulative manner
through interaction with specific cellular receptors
Growth medium A mixture of nutrients that promotes cell multiplication
Hormone Chemical substances that are transported through the body and affect target
cells at locations remote from cells that produce them
Maintenance medium A mixture of nutrients and supplements that maintains the structural and
metabolic integrity of the cells (cell viability) but does not promote cell
division.
Nutrient A low molecular weight substance that enters cells and is utilized weather as
substrate for biosynthesis or metabolism or else as a catalyst in those
processes
Peptone Acid or enzyme hydrolysate of fresh animal carcass, muscle or offal
Supplement Any non-nutrient substance needed for cell growth (usually high molecular
weight)
Serum The liquid remaining after allowing freshly collected blood to clot
Serum-free medium A basal medium that lacks whole or processed serum supplementation.
Serum-free medium can and usually does contain one or more protein (or
non-protein) component extracted and partially purified from serum or tissue
II. CELL PROPERTIES
A. Composition and Size
Average Composition of a Cell
Pg/Cell Range Percentage
Wet weight 3500 3000-8000
Dry weight 600 300-1200
Protein 250 200-300 10-20
Carbohydrate 150 40-200 1 -5
Lipid 120 100-200 1-2
DNA 10 8-17 0.3
RNA 25 20-40 0.7
Water 80-85
Volume 4 x 10-9 cm3

Size of Animal Cells

Cell Type Volume Diameter (µm)
(µm3)
Hybridoms 12-20
Endothelial Cells 1400-2500 17
(trypsinized and reattached (2000)
before spreading occur)
Chinese Hamster Ovary Cells 1200-1800 ~14
(suspension)
Chinese Hamster Ovary Cells 1300- 1800
(anchored)
Human foreskin fibroblast 7000
(FS-4)

Typical dry weight

Bacterial 10-12 g/cell
Yeast 10-11 g/cell
Average animal cell 3–6 x 10-10 g/cell
Hybridoma 3.6 x 10-10 g/cell (Frame 1990)
2.4 x 10-10 g/cell (Zhou, 1995)
FS-4 4.3 x 10-10 g/cell (Fleischaker, 1982)
B. Variation in Cell Volume in the Course of Cultivation
AFP-27 hybridana cultivated in DME with 10% horse serum.
♦ Cell volume exhibits a distribution at any time point.
♦ Small cells correspond to dead ones.
♦ The mean and mode varies with growth stage.
♦ Cell volume appears to be skewed and spans over four-fold.

Note: A four-fold increase in cell volume

corresponds to a 1.6-fold increase in diameter.
Sen et al. (1989), Cytotechnology, 2 : 85-94.
 Doubling Time of Animal Cells in exponential
C. Growth Rate of Mammalian Cells Growth Phase
♦ µ is the specific growth rate; td is the doubling time.
FS-4(human fibroblast) 50-60h
♦ in cell culture µ and td are often calculated with cell
number rather than with dry weight. Vero(monkey kidney epithelial) 24-30h
♦ In principle by reducing the inoculum by two fold, the
Chicken embryo fibroblast 15-24h
time period it take to reach confluence or maximum cell
concentration will increase by a doubling time of the Hybridoma 12-24h
cell. CHO (anchored or suspension) 12-24h
dx Lymphoblastoid 15h
= µx
dt Insect cells 15-30h
x2
ln = µ( t 2 - t 1 ) human lymphocytes (activated) 40h
x1
ln 2 0.693
td = =
µ µ

D. Inoculum cell concentration requirements

♦ Growth rate is often slower at low inoculum cell
Average 3/bead
concentration due to conditioning factor or

Frequency
autocrines.
5
♦ In general, 10 cells/ml (for anchorage Average 6/bead
dependent cells, 104 cells/cm2) is a good number
to start.
♦ Microcarrier cultures need at least an average of
6-8 cell/bead as inoculum. This requirement is
due to cell distribution on microcarriers. At too 0 5 10
low a cell to bead ratio a large fraction of beads Cells/bead
will be empty.
See the section on Microcarriers
III. MATERIAL BALANCE ON CELL GROWTH
Growth of biomass involves
♦ Production of new biomass
♦ Consumption of nutrients
♦ Excretion of products
Growth is autocatalytic (rate of growth of biomass is proportional to the amount of biomass present)
A. Balance on Mammalian Cell Growth
Glucose Biomass
Amino acids Product
Micro-nutrients Carbon dioxide (in addition to
bicarbonate in bulk salts
CELLS
Micro-organic nutrients Water
Bulk salts Lactate, NH3
Trace minerals Excreted amino acids

♦ The composition of cells can be written as a “molecular formula.”

♦ The major items of “inputs” are glucose, glutamine and other amino acids. Fatty acids or lipids also
contribute significantly to biomass formation. Other organic nutrients, such nucleotides and vitamins,
does not contribute a sigficant amount to biomass.
♦ Knowing how much of each “input” is needed to make a certain amount of biomass and product is
important.
♦ The more inhibitory waste products are formed, the less efficient the process.
An example of the “equation” for hybridoma growth:
C 6 H 12 O 6 (glucose) + 0.15 C 6.14 H 12.36 N 1.50 O 2.08 (weighted average of amino acids)
+ 0.34 C 5 H 10 N 2 O 2 (glutamine) + 1.39 O 2
→ 2.37 CH 1.97 N 0.26 O 0.49 (cell mass) + 0.0058 CH 1.83 N 0.14 O 2.06 (antibody) + 1.54 CO 2
+ 1.28 H 2 O + 1.44 C 3 H 6 O 3 (lactate) + 0.16 NH 3 + 0.13 C 4 H 7 NO 2 (alanine)
IV. QUANTITATIVE DESCRIPTION OF CELL GROWTH & PRODUCT FORMATION
Typically a manufacturing process is characterized by its cell growth curve, nutrient consumption curves and
product concentration profile. To describe these curves quantitatively, a number of quantities are frequently
used. Usually, there are three classes of quantities for a complete description of the processes:
concentrations (cell, nutrients, metabolites and product), kinetic parameters (specific rates of growth, nutrient
consumption and product formation, or the physiological state), and stage of the culture (lag, exponential or
decline phase).
A. Definitions
Concentration (amount/L or amount/unit volume)
X: viable cell concentration (number of cells/L or gm cells/L)
S: substrate concentration
P: Product concentration
Normally, the concentration is based on per unit volume of culture (i.e., reactor culture volume) which is
virtually the same as the culture liquid volume because the volume of cells is very small. However, in
some cases (such as high density solid microcarrier culture), the volume occupied by solid beads is
large, and liquid volume and culture volume are not the same. In such cases, the “volume” used for
different kinetic parameters must be well-defined and consistent.

Growth rate (G) (change in cell concentration per unit time, number of cells/L-hr; or gm of cells/L-hr)
dx
G= x: viable cell concentration
dt
Specific Growth Rate (µ) (number of cells/cell-hr or gm cell/gm cell-hr)
dx 1 dx
= µx µ=
dt x dt
Specific Death Rate
dx dxd
= µx − αx = αx x d = dead cell concentrat ion
dt dt
Specific Nutrient consumption Rate (gm nutrient/gm cell-hr)
− 1 ds
qs = s: substrate (nutrient) concentration
x dt
Specific Product Formation Rate
1 dp
qp = p: product concentration
x dt
Specific Rate vs. Volumetric Rate
For growth G. vs. µ
ds 1 ds
for nutrient consumption, Q = vs. q =
dt x dt
Yield coefficient for biomass (Yx/s) (gm cells/gm nutrient)
∆x dx
or
∆s ds
Yield coefficient for product (YP/S)(gm product/gm nutrient)
∆p dp
or
∆s ds
 B. Yields and Stoichiometric Ratios
Yield: Output/Input
e.g., no cells produced/g glucose consumed
g antibody produced/g total amino acids consumed
Stoichiometric Ratio:
e.g., g lactate produced/g glucose consumed
mole NH3 produced/mole glutamine consumed

Example: Growth of hybridoma cells

Specific Antibody Production

Specific Growth Rate (1/HR)

Rate (µG/HR/106 Cells

Cell Conc. (10 /ml)

Antibody Conc..
5

T im e
Time (Hours)
Specific Glucose Consumption

Specific Lactate Production

Rate (MG/HR/106 Cells)
Glucose Conc. (MG/ML)

Lactate Conc. (MG/ML)

Rate (MG/HR/106 Cells)

Time (Hours)
Time (Hours)
For batch culture, the specific rates are estimated with difference equations instead of with differential
equations. The four curves in the two figures at left are obtained by measurement. The slope of the curve
at a time point divided by the mean cell concentration at the same time points gives the specific rate. The
four calculated specific rates are shown in the two figures at the right. This calculation is easily performed
with a spreadsheet program.
Material balance:
∆X = 7 x 108/l = 0.5 g/109 cell x 108 cell/l = 0.05 g cell/l
∆P = 50 mg/l = 0.05 g/l
∆X = ∆X = ∆P on a weight basis
Rate analysis: Maximum specific rates occur in late exponential growth before cell concentration
reaches its peak
 MAK MAK SP0 VO 208 6H2 Anti.HT AB2-143.2 Murine Hybridoma SF-9
(L/G=1.4) (L/G=0.22) Hybridoma 167.4G5.3
Serum Free
Serum Free Serum Free Serum Free W/FBS W/FBS W/FBS W/FBS W/FBS
(SF900II)
9
(mmol/10 cells/hr)
Glucose 0.185 0.046 0.159 0.05
Oxygen 0.356 0.276 0.318 0.22
Lactate -0.259 -0.010 -0.287 0
Ammonia -0.048 -0.019 -0.049 0
IgG1(mg/
-0.440 -0.485 -0.876
109cells/hr)

amino acids (mmol/1012cells/hr)

ALA -22.70 -0.73 -33.51 -23.71 -19.15 -34 -13.301 -33.63 -23.5
ARG* 23.98 4.97 3.54 2.89 12.18 6 2.08 2.71 2.8
ASN 2.73 0.75 0.50 3.9 4.49 -1 0.75 0.85 1.6
ASP 2.78 -0.04 -1.28 1.2 0.67 1.45 4.1
CYS 3.63 2.08 0.3
GLU 2.99 0.52 0.52 -5.78 -10.88 13.01 11.041 13.431 2.6
GLN* 100.80 27.65 37.69 61.6 47.6 79 35.41 45.8 2.4
GLY 14.96 -4.62 -2.72 -6.09 -2.83 -0.78 0.2
HIS 8.95 3.30 1.62 1.01 1.76 1 1.58 0.94 0.7
ILE* 12.61 6.57 5.90 6.84 9.93 9 4.17 5.9 2.8
LEU* 18.76 6.26 7.56 7.69 11.7 10 4.46 6.77 8.3
LYS* 26.03 8.51 3.14 4.22 6.81 6 2.67 2.65 2.2
MET* 5.87 1.71 1.63 1.71 2.48 2 0.96 2.65 1.7
PHE* 6.08 1.96 0.75 2.03 3.04 6 1.33 0.85 0.7
PRO 0.48 -2.48 1.9
SER 12.13 3.36 0.45 0.53 8.25 4 1.04 -2.26 9.5
THR* 20.14 1.59 1.73 2.99 4.89 4 2.38 1.36 2.5
TRP 0.53 1.12 1.4
TYR 4.84 1.74 1.29 2.03 3.6 2 0.96 2.65 1.9
VAL* 11.92 5.19 5.49 3.85 6.49 4 3.71 4.45 3.4
1. Negative sign indicates production of the compound
2. “*” denotes essential amino acids
3. The two columns of MAK cells represent two different metabolic states with different stoichiometric ratios of
glucose to lactate.
 Specific rate and yield coefficients for the same compound vary widely among cells.
♦ It is also affected by the medium composition and metabolic states of cells.
♦ In a typical culture, the fraction of carbon atoms going into metabolites (CO2, lactate and excreted
non-essential amino acids) is much higher than that incorporated into biomass and product.
♦ That fraction (or various stoichiometric ratios) changes because of different cell metabolism.

Reported and calculated specific protein production rates by different animal cell types

Cell Type Product Specific productivity (pg protein cell-1 h-1)

MouseXMouse IgG 1.21
Hybridoma 0 48-0.962
0 5, 1.5-2.53
0 48,0.744
1.15
Transfected myeloma IgG 1.1-4.06
CHO HepBSAg 0.6 -1.27
Lymphocyte IgG 1.8 (estimated)8
Hepatocyte Albumin 3 4 - 20 (in vivo)9
0 04 - 0.4 (in vitro)l°
1 (in vitro)l1

1. Frame (1988) Ph.D Thesis, cell line AFP-27
2. Hiller et al. (1991) Continuous culture,
serum-free medium
3. Ray et al (1989) Continuous culture
4. Reuveny et al. (1986b) Batch and spin filter
perfusion
5. Schneider & Lavoix, (1990) Semi-continuous
culture in serum- and protein-free medium
6. Hassell et al., (1992) Estimated from
presented data with serum-free, fed-batch
culture
7. Spier (1989) "The ideal cell"
8. Murakami (1990) Data from Waller, N. (1974)
based on plaque assay
9. Figure calculated on the basis that 10-60% of
hepatocytes synthesise 12 g albun~in/day and
that hepatocytes comprise 80% of a 1.8 };g liver
(Arias et al., 1982 and Wilson, J.D. et al., (1991)
10. Peterson (1976). Clonal variants of hepatoma
lines (estimated protein content was 490 pg
cell~
11. Ijima et al., (1992) Cultured hepatocytes in high
density bioreactor
C. Some complications in specific rate calculation
*In general, in cases with low viability, use viable cell concentration instead of total cell concentration
dxv
=µx v - αxv
dt
dx d
= αx v
dt
xt = x v + x d
− 1 ds X d : dead cell concentration
qs =
x v dt α : Specific death rate
1 dχ t
µ=
χ v dt
1 dχ d
α=
χv dt

This assumes that dead cells are metabolically inactive, furthermore it neglects cell lysis which
may occur in both viable and dead cells. To account for cell lysis, either from apoptosis or from
neucrosis, one more term should be added,

dxv
= µxv - αxv - λv xv
dt
λ v : Dead Cell Lysis Rate
dxd
= αx v − λ d x d
dt

Another complication is that almost all calculations are based on cell number instead of cell mass
or other quantitative measurement of cell materials. The size of cells varies with cell line, medium,
growth stage, pH, osmolality etc.
For anchorage-dependent cells, especially normal diploid cells, the size is also dependent on cell
density.
V. NUTRIENTS AND THEIR RELATION TO TRANSPORT CONSUMPTION
The uptake of nutrients is regulated and most is mediated
Pro
by transporters. Gly Transporter A
J
pro
J
♦ The transport rate of a compound across membrane is Ala
Km: 1 mM gly
J
roughly the same as its metabolic rate. Ser
J
ala
Met Transporter ASC
♦ Provided that everything else is kept constant, increasing ser
Gln Km: 50-150 µm m J cys
the concentration of a nutrient species increases its Cys J
Thr
uptake, until the concentration reaches a saturation level. Thr J
Transporter L Gln
♦ In many cases, the transport of a nutrient is mediated by Leu J
leu
a number of transporters, and a transporter is shared by Phe J
phe
many nutrients Small J Met
♦ The uptake of nutrients is therefore highly interactive. zwitterionic a.a Glucose Metabolism
Transporter
The uptake rate of a compound is dependent not only on
its own concentration, but also the concentrations of Glucose JGlucose
Glucose
species competing for shared transporters. Transporter 4

VI. STOICHIOMETRY AND MEDIUM DESIGN

A key medium design issue is thus a stoichiometric consideration: key nutrients must be maintained at
an optimal range of concentration and at an acceptable ratio to other nutrients that will support their
consumption at a desired rate to sustain growth and production.
Therefore, we need to know:
♦ The demand (specific consumption rate)
♦ The optimal concentration
♦ Interactions with other nutrients
For calculation of stoichiometric ratio, see video tutorial and Fedbatch culture
A. Example. Oxygen as a nutrient
1. DEMAND
In general, for animal cells
q0 0.5 - 5 × 10-10 mmole 02 /cell hr
2

Spodoptera frugiperda (insect cell)

Exp growth 1.5 x 10-10 mmole/cell-hr
Stationary phase 0.5 x 10-10
Viral infection 1.5 x 10-10 mmole/cell-hr
 Hu & Oberg (1990) In: Large Scale Cell
Culture Technology Lubiniekie ed.

2. OPTIMAL CONCENTRATION
♦ It appears that DO > 30% satn. is okay.
♦ Too high of a DO is not desirable in a stirred tank.
Specific Oxygen Uptake
Rate x 1014

Dissolved Oxygen
vs. Specific Oxygen
3. INTERACTION WITH ENERGY METABOLISM
♦ The consumption of oxygen is affected by glucose
concentration. The stoichiometric ratios of
lactate/glucose, oxygen/glucose vary with glucose Energy Metabolism is also
concentration. affected by sugar concentration.
 B. Intracellular concentrations of nutrients and lactate
Intracellular concentrations of nutrients and lactate
in exponentially growing hybridoma cells
(mM) DME DMEpyr1 DMElac2

Glucose <0.05 <0.05 <0.05

Lactate 19.52 37.08 42.73
† 0.29 <0 05 4.02
Gln
Ala 8.95 21.90 9.29

Asp 0.69 0.42 0.76

Glu 0.53 0.66 0.38
Asn 0.36 044 0.76
Ser 1.73 3.51 1.29
† <0.05 <0.05 <0.05
His Selected steady-state intracellular amino acid concentrations as
a function of the glutamine concentration in the feed medium.
Gly 6.52 12.51 7.20
† 1.35 3.95 2.11
Thr
† <0.05 <0.05 <0.05
Arg
Tyr
† 0.28 0.39 0.30 Note:
Met < 0.05 < 0.05 < 0.05 • Intracellular concentrations of amino acid are much higher
than the km for t-RNA (1 ~ 100 µM)
† 0.29 0.64 0.33
Val
† 0.31 0.52 0.32
• Intracellular concentrations of glucose are tightly regulated.
Phe
†
Ile 0.29 0.49 0.35 • The intracellular concentrations of essential amino acids are in
Leu
† 0.36 0.42 0.33 the same order of magnitude of the extracellular
Lys
† 0.68 N.A. 0.58 concentrations.
† • The intracellular non-essential amino acid concentration is
Denotes essential amino acids
1: DMEM with 6 mM pyruvate initially
affected by glutamine concentration in medium.
2: DMEM with 50 mM lactate

VII. STOICHIOMETRIC RATIO AS METABOLIC INDICATOR

♦ A combination of stoichiometric ratios can reflect the metabolic states of cells.
♦ Typical stoichiometric ratios under different metabolism for hybridoma cells.
 Comparison of results of a fed-batch hybridoma culture with on-line control of nutrient feeding with those of a
batch culture.
Batch Fed-batch 1 Fed-batch 2 Fed-batch 3

Initial glucose conc. (mM) 17.0 2.78 1.4 1.4

Initial glutamine conc.(mM) 4.0 0.7 0.3 0.3

Glucose conc./set point (mM) 2.50 0.55 0.55

Glutamine conc./set point (mM) 4 0 ~0.5 → 3.0 ~0.5 0.2

Maximal viable cell conc. (106 cells/mL) 2.4 7.2 12.0 10.5

Antibody conc. (Mg/L) 8 35 60 200

Duration of Growth stage (hr) 70 100 145 140

Glucose consumption (mmole/L) 12.0 46.0 46.50 27.0

Glutamine consumption(mmole/L) 4.0 15.6 17.0 10.1

Oxygen consumption (mmole/L) 24.0 90.0 143.0 122.0

Lactate production (mmole/L) 18.0 70.0 37.4 17.0

Ammonium production (mmole/L) 3.0 6.5 9.7 4.5

Lactate/Glucose (mmole/mmole) 1.5 1.47 1.60 → 0.16 0.90 → 0.05

Oxygen/Glucose (mmole/mmole) 1.85 2.27 1.9 →6.0 2.7 → 4.7

Ammonium/Glutamine (mmole/mmole) 0.75 0.78 0.5 0.31 → 0.10

Alanine/Glutamine (mmole/mmole ) 0.35 0.37 0.34 → 1.35 0.08 → 0.42

Osmolality (mosm/kg) ~350 455 410 400

♦ Stoichiometric ratio is typically calculated from cumulative consumption data
VIII. MEDIUM DESIGN FROM A STOICHIOMETRIC POINT OF VIEW
A. Consumable vs. un-consumable
Consumables: glucose, amino acids, vitamins, Approximate Concentrations in Cellular
nucleotides, lipids, fatty acids, some growth factor, Environment
some salts
Interstitial Intracellular
Unconsumables: Most bulk salts, albumin, some (mM) (mM)
growth factor Na+ 140 14
Strictly speaking, almost every component of the medium K+ 4.0 140
is consumable. However, many of them, especially bulk Ca++ 1.2 0
salts and trace salts, are consumed so little by cells (in Mg++ 0.7 20
other words, their concentration in the medium is to Cl- 108 4
HCO3 28.3 10
provide the appropriate uptake rate, not to prevent
HPO4--. H2PO4 2 11
depletion) that they are practically “unconsumable.” SO4 0.5 1
However, as cells grow to a higher concentration, Phosphocreatine
significant quantities of these salts will be taken up and Camosine 14
become constituents of cellular fluid. They will also need Amino acids 2 8
to be replenished. Creatine 0.2 9
Lactate 1.2 1.5
Example:
Adenosine
PO4-3 concentration in medium is typically 1 mM, triphosphate
while its cellular concentration is 11 mM. A cell line Hexose
with an average volume of monophosphate
2 x 10-12 ℓ Glucose 56
and take up about Protein 02 4
Urea 4 4
11 mmole/ℓ x 2 x 10-2 = 0.22 mmole/ℓ of PO4.
Others 3.9 11
It will certainly cause PO4 concentration to TOTAL (mmole/ℓ) 301.8 302.2
decrease. corrected 281.3 281.3
B. Macromolecules: stoichiometric or catalytic osmolar activity
(mM)
Some are internalized and degraded (consumed), others Total osmotic 5430 5430
are recycled. So, some need to be replenished while pressure at 37 °C
others don’t.
Below right: An example of consumable macro-molecule fate of LDL particles and LDL receptors
after endocytosis. The same pathway is followed by other ligands that are internalized by
receptor-mediated endocytosis and degraded in the Iysosome, such as asialoglycoproteins.
After an LDL particle binds to an LDL receptor on the plasma membrane, the receptor-ligand
complex is internalized in a clathrin-coated pit that pinches off to become a coated vesicle. Then the
clathrin coat depolymerizes to triskelions, resulting in an uncoated (smooth-surfaced) vesicle, often
called an endosome. This then fuses with an uncoupling vesicle called CURL Compartment of
uncoupling of receptor and ligand) that is characterized by an internal pH of about 5.0. The low pH
causes the LDL particles to dissociate from the LDL receptors. A receptor-rich region buds off to form
a separate vesicle that recycles the LDL receptors back to the plasma membrane. The vesicles
containing the LDL particles fuse with Iysosomes, forming a large secondary Iysosome. In this
Iysosome the apo-B protein is degraded to amino acids, and the cholesterol esters are hydrolyzed to
fatty acids and cholesterol. Cholesterol is incorporated into cell membranes. Abundant imported
cholesterol inhibits synthesis by the cell of both cholesterol and LDL receptor protein.

An example of “catalytic” macromolecules: the

transferrin cycle. After endocytosis, iron is released
from the transferrin-receptor complex in the acidic
CURL compartment. The transferrin protein remains
bound to its receptor, and they cycle to the cell surface
together. When the receptor-transferrin complex
encounters the neutral pH of the exterior medium, the
iron-free transferrin is released.
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