264
Exercise 19
labeled vial with the filtered surface up. Filter the remainder of the samples as
above.
1. Dissolve the filter by adding 1.0 ml of ethylacetate to each vial.
m. Prepare five calibration vials as was described in the tritiated thymidine
incorporation procedures (see p. 258).
3. Radioassay:
a. Add 10 ml of an appropriate scintillant fluor to each vial and shake thoroughly.
b. As the vials are organized for counting, wipe each externally with 70% ethanol.
c. Radioassay with an appropriate spectrometer with a standard tritium program
(e.g., 20 min)
d. Select six vials that have differing quench numbers for internal standardization
(samples with > 1500cpm are preferred). Add an appropriate amount of a
standard (e.g., 3H-hexadecane or 3H 2 0) and radioassay (ca. 10 min).
Calculation of Bacterial Protein Production
1. Convert counts per minute (cpm) to disintegrations per minute (dpm):
dpm = _ cp_m - backgrou_Ild cpm (100)
% efficiency of radioassay
The efficiency is determined from standards of known activity within the LS
spectrometer capabilities. Follow the instructions of the manufacturer.
2. Bacterial protein production (BPP) from 3H-Ieucine incorporation is calculated
as follows (Simon and Azam, 1989):
BPP(g) = (molleucine inc )(100/7.3)(131.2)(ID)
= (molleucine inc )(1797)(ID)
where molleucine inc = moles of exogenous leucine incorporated; 100/7.3 = 100/mol%
of leucine in protein; 131.2 = gram molecular weight of leucine; and ID = intracellular isotope dillution of 3H-Ieucine. If the isotope dilution is not known, the
theoretical minimum estimate of BPP would assume no isotope dilution, i.e., all
leucine of protein would be derived from exogenous sources. Studies ofthis problem
using independent methods demonstrated isotopic dilution to the twofold (Simon
and Azam, 1989). With a twofold isotopic dilution,
BPP(g) = (molleucine inc )(1797)(2)
Alternatively, BPP can be calculated directly from intracellular pool specific
activity (SA):
BPP(g) = (dpminc)(SA -1 )(131.2)
The specific actIVIty of intracellular 3H-leucine (Ci/mmol) reached 42% of
extracellular specific activity within 10 min and a maximum of 61 % of extracellular
specific activity within 30 min of incubation (Simon and Azam, 1989).
The ratios of protein:dry weight and of carbon:dry weight were found to be
quite constant. Thus, BPP measurements for all cell sizes can be converted to
rates of dry weight production simply by multiplying by 1.6, and into bacterial
carbon production by multiplying by 0.86.
Exercise 19
labeled vial with the filtered surface up. Filter the remainder of the samples as
above.
1. Dissolve the filter by adding 1.0 ml of ethylacetate to each vial.
m. Prepare five calibration vials as was described in the tritiated thymidine
incorporation procedures (see p. 258).
3. Radioassay:
a. Add 10 ml of an appropriate scintillant fluor to each vial and shake thoroughly.
b. As the vials are organized for counting, wipe each externally with 70% ethanol.
c. Radioassay with an appropriate spectrometer with a standard tritium program
(e.g., 20 min)
d. Select six vials that have differing quench numbers for internal standardization
(samples with > 1500cpm are preferred). Add an appropriate amount of a
standard (e.g., 3H-hexadecane or 3H 2 0) and radioassay (ca. 10 min).
Calculation of Bacterial Protein Production
1. Convert counts per minute (cpm) to disintegrations per minute (dpm):
dpm = _ cp_m - backgrou_Ild cpm (100)
% efficiency of radioassay
The efficiency is determined from standards of known activity within the LS
spectrometer capabilities. Follow the instructions of the manufacturer.
2. Bacterial protein production (BPP) from 3H-Ieucine incorporation is calculated
as follows (Simon and Azam, 1989):
BPP(g) = (molleucine inc )(100/7.3)(131.2)(ID)
= (molleucine inc )(1797)(ID)
where molleucine inc = moles of exogenous leucine incorporated; 100/7.3 = 100/mol%
of leucine in protein; 131.2 = gram molecular weight of leucine; and ID = intracellular isotope dillution of 3H-Ieucine. If the isotope dilution is not known, the
theoretical minimum estimate of BPP would assume no isotope dilution, i.e., all
leucine of protein would be derived from exogenous sources. Studies ofthis problem
using independent methods demonstrated isotopic dilution to the twofold (Simon
and Azam, 1989). With a twofold isotopic dilution,
BPP(g) = (molleucine inc )(1797)(2)
Alternatively, BPP can be calculated directly from intracellular pool specific
activity (SA):
BPP(g) = (dpminc)(SA -1 )(131.2)
The specific actIVIty of intracellular 3H-leucine (Ci/mmol) reached 42% of
extracellular specific activity within 10 min and a maximum of 61 % of extracellular
specific activity within 30 min of incubation (Simon and Azam, 1989).
The ratios of protein:dry weight and of carbon:dry weight were found to be
quite constant. Thus, BPP measurements for all cell sizes can be converted to
rates of dry weight production simply by multiplying by 1.6, and into bacterial
carbon production by multiplying by 0.86.