Homogenates were prepared from various growth phases of Microsporum gypseum grown on different amino acids as the nitrogen source. When analyzed on 7.5% polyacrylamide disc gels, the water-soluble proteins in these homogenates gave essentially identical banding patterns

Balabanov, V.

Danev, P.

Friedrich, E.

English

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NASA TECHNICAL MEMORANDUM 	 NASA TM-77095
STUDIES OF PROTEINOGRAMS IN DERMATOPHYTES BY DISC ELECTROPHORESIS.
I. PROTEIN BANDS IN RELATION TO GROWTH PHASE AND NITROGEN SOURCE.
P. Danev, E. Friedrich, V. Balabanov
A
F^0 ^`y`
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Translation of "lzsledvaniya vurkhu proteinogramite pri dermatofitite s pomoshtt;^rmp
diskovato elektroforeza. I. Proteinovite ivitsi v zovisimost of fozato i iztochnika na
azot" Dermatologiya i venerologiya (Sofia), Vol. 19, No. 2, 1980, pp. R2-85.
(NkSA-T!l-77095)	 STUDIES OF PROTEINOGRAM S IN	 N84-16755
DERMATOPHYTES BY DISC ELECTROPHORE SIS.	1.
BANDS IN RELATION 10 GROWTH PHASEr a UI  BIN	 Unclas(National Aeronautics and Space
Admiaistratio al 	 8 D HC A02/MF A01 CSCL 06C G3/51 11402
NATIONAL AERONAUTICS AND SPACE ADMINISTRATION
WASHINGTON, D.C. 20546
	 JUNE 1983
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ORIGINAL F';,G^: Zj
OF POOR QUALMY,
STANDARD TITLE PAGE
1.	 Report No. 2. GwprtaenI Accession No. X	 Rocipient'a Catalog No.
NASA TM 77095
d.	 1 Ule and Subtitle S.	 Report Dote
STUDIES OF PROTEINOGRAMS IN DERMATO- June 1983
6. Performing organisation CadePHYTES BY DISC ELECTROPHORESIS. 	 I. PRO-
TEIN BANDS I14 RELATION TO GROWTH PHASE...
7.	 Auth-jr(s) 1. Performing Organization Report No.
P. Danev,	 E.	 Friedrich, V.	 Balabanov
Martin Luther University,	 Halle,. GDR;	 10. Work Unit No.
Dermatovenerology Research Inst.,	 Sofia
0.	 Perform:ng Organi tartan Name and Address	 I). Contract or Grant No.
NASW - 3541Leo Kanner Associates
Type of Report end Period Coverer!Redwood City,  CA 94063	 17.
Translation
12. Sponsoring Agency Name end Address
National Aeronautics and Space Adminis- 1d. sp.n.o:ingAg.nc,cad.tration,	 Washington,	 D.C.	 20546
15.
	
Supplementary Notes
Translation of "Izsledvaniya vurkhu proteinogramite pri der-
matofitite s pomoshtta na diskovata elektroforeza. 	 I. Pro-
teinovite ivitsi v zavisimost of fazata i iztochnika na azot,'
Dermatologiya i venerologiya	 (Sofia) ,	 Vol. 19,	 No.	 2,	 1980,
pp.	 82-85.
16.	 Abstract
Homogenates were prepared from various growth phases of
Microsporum gypseum grown on different amino acids •as the
nitrogen source.
	
When analyzed on 7.5% polyacrylamide disc
gels, the water-soluble proteins in these homogenates gave
essentially identical banding patterns.
17. Key words (Selected by Authar(t)) 11.	 Distribution Statement
Unclassified-Unlimited
19.	 Security Cleseil. (of this rgwt) 20.	 Security Clessil. (of this page) 21. Me. of Pages 22.
Unclassified Unclassified 6
41
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STUDIES OF PROTEINOGRAMS IN DERMATOPHYTES BY DISC
ELECTROPHORESIS. I. PROTEIN BANDS IN RELATION TO GROWTH
PHASE AND NITROGEN SOURCE
P. Danev, E. Friedrich, V. Balabanov1
Dermatophytes are among the most narrowly specialized fungi on
	 /82*
man and animals. The physiological manifestations of their patho-
genicity are based on the fact that they are keratinophilic. As
opposed to saprophytic fungi, they are capable of attacking intact,
native keratin in vivo.
There are a variety of difficulties causing the defi;:it, even
today, of a natural system of dermatophytes, and especially their
pathogenic representatives, which comprise the majority. First we
have their morphological similarity (e.g., convergence), resulting
t
from the close conditions of their existence. Also important
is their simplified morphology in parasitism, e.g., morphological
reduction. We have found yet another physiological and biological
reduction with parasitism, which, together with the specified mor-
phological alterations during biological differentiation, complicates
diagnosing the pathogenic strains. We should note here the lack of
antigen test specificity and the inability to make a genetic deter-
mination.
Despite these limitations, the general characteristics of derma-
tophytic species make possible a single classification, close to
natural, which is useful in epidemiology and the battle against der-
4
	 matomycoses. This classification was achieved through a complex
1	 determination of the quality of the pathogenic species, by a combin-
ation of all diagnostic signs -- ecological and biological, in connec-
tion with the variability and dimorphism,versus ontophylogenetic,
1. Institute of Physiological Chemistry, Dermatology Clinic, Martin
E	 Luther University, Halle, GDR; Dermatovenerology Research Institute,
Sofia, Bulgaria.
*Numbers in the margin indicate pagination in the foreign text.
R
^	 1
(F
!
f	 ^1
parasitologic, and, not least of all, biochemical-enzymatic [2, 3, 61.
Disc electrophoresis, the subject of this series of studies, is /83
t	 of particular interest for investigating the relationships between
t	 dermatophytes and explaining their taxonomy.
1
Disc electrophoresis on polyacrylamide gels is a method first
introduced at the beginning of the 1960's by Davis and Ornstein [9,
10, 11, 12, 17, 181. It is used to separate electrically charged
biological macromolecules, such as proteins and nucleic acids. The
separation is accomplished on one hand by molecular weight, on the
other according to charge in an electric field. The molecular sieve
effect depends on the gel's pores. Distinct protein fraction sepa.-a-
tion is achieved with this method.
We used this method to separate water-soluble mycelium protons
from various dermatophytes consisting of keratinophilic fungi, and
c	 used them to compare the electrophoretic patterns of the various
'	 dermatophytes.
f	 Since corresponding studies have established a relationship
between dermatophyte enzyme activity and growth phase, as well as
nutrient medium nitrogen source, we also set out to establish whether
or not dermatophyte protein fractions are affected by these para-
meters.
The results of our preliminary studies will be described below,
wh_ . the protein band locations of the various dermatophyte
species vill be given in another publication.
Ii
Method. As the experimental fungus we used strain No. 243 of
Microsporum gypseum, isolated in 1961.
i
Culture conditions. The preliminary culturing method of preparing
the suspension of the experimental fungus has been described in previ-
ous publications [4, 5, 6, 6a].
2
6
t
1
0.585 g/k
0.136 g/k
0.087 g/k
0.247 g/k
0.216 g/k
9.910 g/k
NaCl
KH2PO4
K2HPO4
MgSO4.7H2O
CaC12.6H2O
Glucose
The nutrient medium (culture medium) was a solution consisting
of salts, amino acids, and glucose in the following composition:
t
i
The nitrogen source was varied as follows:
Series 1. One liter of nutrient medium contained glutamic acid (0.294
g), lysine (0.182 g), leucine (0.262 g), phenylalanine (0.330 g). The
total nitrogen content put liter was 0.14 g.
Series 2. The nutrient medium contained double the amount of nitrogen,
0.24 q/k, consisting to equal parts glutamic acid and cystine.
Series 3. One liter cf culture medium contained 0.14 g of nitrogen,
consisting entirely in glutamic acid.
The initial acidity (pH) of each nutrient medium was 4.8. After
autoclaving it fell to 4.0.
200 ml Erlenmeyer flasks we re filled with 50 ml of nutrient
medium. After 20 min of autoclaving in these flasks at 120°C, 0.5 ml
of fungus culture suspension was pipetted out, providing 0.08 g of
mycelium dry weight per flask.
The tests were completed by reading the results at room tem-
perature.
Mycelium processing and handling. The dry weight of material
contained in 3 (three) flasks was determined on the day of the
experiments. Mycelia from two or three other flasks were used to
obtain the cell contents.
3
VDry weight determination. A fine-porosity paper filter,
90 mm in diameter, of a known weight was placed in a Buchner funnel
with an internal diameter of 90 mm, after which the flask contents
F
	 were poured over the filter. A water-jet pump was used to draw out
the liquid, then the mycelia were washed with 5 ml each of buffer
solution (pH 8.0). The filtrate and washed mycelium were placed in	 /84
a previously weighed Petri dish filled with two ml of acetone, then
t	
left three hours in an air-ventilation chamber.
The mycelia were then dried for 16 hours at 95°C. After cooling
to room temperature, dry weight was determined. The mycelia were
opened, using a previously described method [4, 5, 6, 6a], to obtain
the cell contents. Lowry's method was used to determine the soluble
part of the cell homogenate. The amount containing 50 micrograms of
protein was determined by disc electrophoresis.
Three parallel flasks with mycelium, designated a, b, and c, were
prepared for each experiment. Then identical samples of mycelium
homogenate from the same flask were designated with the same letters.
The experiments done on different days were sequentially numbered
for each series.
Disc electrophoresis conditions. 7.5% polyacrylamide, containing
2.6% N,N'-methylene-bis-acrylamide and kept in test tubes, served as
the matrix.
50 micrograms from the tipper mycelium homogenate residue were
pipetted over the gel. The pH of the separation medium was 9.5, and
the pH of the buffer solution was 8.3; 220V and 4-5mA of current were
applied to the test tubes. The fractions were fixed on the gel with
12^ trichloracetic acid and stained with brilliant blue G-250
(iii 12% trichloracetic acid). Clear, narrow bands resulted.
4
Results
The mycelium dry weight values are presented in supplement 1,
and the growth curve is given for M. gypseum.
From it we can see that fungi are present in the three experi-
mental series at the first study, i.e., five days after culturing,
in a logarithmic growth phase. The end of the Logarithmic phase was
reached in the first series after 15 days, in the second after 14
days. Since research on the last two series was done until the 23rd
day of culturing, it was impossible to determine the end of the
logarithmic phase for the third series, with glutamic acid as the
nitrogen source, in which growth was slower.
-1
	 Supplements 2 to 7 illustrate the proteinograms from series
t:
	
	 (1 and 2, M. gypseum) in the early logarithmic phase (5th day of
study), late logarithmic phase (15th and 11th days, respectively), and
in the stationary, corresponding to autolytic phase (33rd day for
series 1, 23rd day fol series 2). The good reproducibility of the
results can be seen first of all in each figure. The mycelium homo-
genates from the second and third flasks (a, b, c) show the same
banding patterns as the two tests of the same homogenate.
l
Comparison of the pherograms from the different growth phases
established relatively corresponding protein banding for both series
1 (supplements 2, 3, and 4) and series 2 (supplements 5, 6, and 7).
A considerable qualitative difference is seen with one protein moving
rapidly under the conditions of our studies. `this appears especially
clearly in the autolytic phase (first band in figs. 4 and 7, but
slightly or very s.ligntly in the logarithmic phase -- figs. 2 and 3).
It was necessary to see if these results could have been reproduced.
Supplement 2 and [illegible) from series 1 (glutamic acid, gly-
cine, leucine, phenylalanine, and lysine) and supplements 5 to 7 of
series 2 (glutamic acid, cystine) serve as a comparison of protein
fractions from M. gypseum grown on various nitrogen. sources. Further-
5
AO
more, fig. 8 shows the protein bands from series 3 at the beginning
and fig. [illegible] during the logarithmic phase after growth of
M. gypseum with glutamic acid as the nitrogen source.
A qualitative difference is seen with the slowly moving proteins
(bands between one and two cm). After growth with glutamic acid and
cystine (series 2) these bands were more clearly distinguished in each
of the five studies than in series land 2. The minor qualitative
differences result to some extent from the photographic reproduction.
Thus we can state that the M. gypseum protein bands do not display
major differences at different growth phases with differing amino
acids as a nitrogen scurce. They agree strongly in each figure.
Generalization
y
	 Disc electrophoresis was used to separate water-soluble proteins
from an M. gypseum mycelium homogenate at various growth phases and
after growth with differing amino acids as a nitrogen source. The
obtained pherograms are very much the same and reveal no substantial
differences.
2
References are given at the end of the second report.
(References are contained on pp 86-89 of this publication) .
2. We wish to express our special gratitude to Charlotte Rikus and
Ingrid Zernal for their technical assistance, to Berbel Daneva for
preparing the figures, and to Ingrid Senger for the photographs.
6
r


Studies of proteinograms in dermatophytes by disc electrophoresis. 1. Protein bands in relation to growth phase

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