DIFFERENTIATION OF KIDNEY MESENCHYME
267
amounts of RNA observed some hours earlier. In addition, our objective
is to correlate the morphogenetic events to any new proteins possibly
appearing and to locate the site where they appear, either in the aggregates and tubules, or in the loose mesenchyme.
Because we do not know any protein specifically related to the early
tubules, such as a distinct tubule-specific structural protein or secretory
product, we have had to confine ourselves to studies of more generally
occurring protein species and analyze their synthesis in relation to tubule
differentiation. A biochemically well-defined group of proteins, lactate
dehydrogenase (LDH)isozymes, was chosen as the first object of study.
Lactate dehydrogenase is an enzyme known to occur in five multiple
molecular forms or isozymes. These isozymes are tetramers composed of
two different types of polypeptide subunits referred to either as A and B
or as muscle (M) and heart-type (H) subunits (Appella and Markert,
1961; Markert, 1963; Cahn et al, 1962). The polypeptide subunits of
LDH combine at random in all possible combinations to form LDH
molecules with enzyme activity. Thus the compositions of the five isozymes can be written: LDH-1 = A°B
4
, LDH-2 = A
X
B
3 , LDH-3 = A
2 B
2 ,
LDH-4 = AT}
1 , LDH-5 = A
4 B°. These isozymes differ in electrophoretic
mobility, LDH-1 being the fastest- and LDH-5 the slowest-moving
molecular form. Analyses of the subunits have revealed that they are
different in respect to their amino acid composition, immunological reactivity, and physical properties (Cahn et al, 1962). From these data
and from studies of mutations of the genes governing LDH synthesis
(Shaw and Barto, 1963), it has become clear that the polypeptides A and
B are products of separate nonallelic genes.
It has been postulated that a change in LDH isozyme pattern reflects
switching on and off of the corresponding genes (Markert, 1963). The
proportions of the LDH isozymes in various cells and tissues are known
to change during embryonic and postnatal development (e.g., Markert
and Ursprung, 1962; Latner and Skillen, 1964). These changes are very
regular and thus LDH isozymes can be used as markers of differentiation.
Both the precise knowledge of the structure of LDH isozymes and the
knowledge of the gene-polypeptide relationship makes LDH isozymes
especially suitable as markers for study of the differentiation of kidney
mesenchyme in vitro.
Undifferentiated kidney mesenchyme exhibits an "embryonic" LDH
isozyme composition, LDH-5 and LDH-4 being the strongest bands observed in electrophoresis (Koskimies and Saxen, 1966). Thus the mesenchyme cells mainly produce A polypeptides of LDH. The first change in
267
amounts of RNA observed some hours earlier. In addition, our objective
is to correlate the morphogenetic events to any new proteins possibly
appearing and to locate the site where they appear, either in the aggregates and tubules, or in the loose mesenchyme.
Because we do not know any protein specifically related to the early
tubules, such as a distinct tubule-specific structural protein or secretory
product, we have had to confine ourselves to studies of more generally
occurring protein species and analyze their synthesis in relation to tubule
differentiation. A biochemically well-defined group of proteins, lactate
dehydrogenase (LDH)isozymes, was chosen as the first object of study.
Lactate dehydrogenase is an enzyme known to occur in five multiple
molecular forms or isozymes. These isozymes are tetramers composed of
two different types of polypeptide subunits referred to either as A and B
or as muscle (M) and heart-type (H) subunits (Appella and Markert,
1961; Markert, 1963; Cahn et al, 1962). The polypeptide subunits of
LDH combine at random in all possible combinations to form LDH
molecules with enzyme activity. Thus the compositions of the five isozymes can be written: LDH-1 = A°B
4
, LDH-2 = A
X
B
3 , LDH-3 = A
2 B
2 ,
LDH-4 = AT}
1 , LDH-5 = A
4 B°. These isozymes differ in electrophoretic
mobility, LDH-1 being the fastest- and LDH-5 the slowest-moving
molecular form. Analyses of the subunits have revealed that they are
different in respect to their amino acid composition, immunological reactivity, and physical properties (Cahn et al, 1962). From these data
and from studies of mutations of the genes governing LDH synthesis
(Shaw and Barto, 1963), it has become clear that the polypeptides A and
B are products of separate nonallelic genes.
It has been postulated that a change in LDH isozyme pattern reflects
switching on and off of the corresponding genes (Markert, 1963). The
proportions of the LDH isozymes in various cells and tissues are known
to change during embryonic and postnatal development (e.g., Markert
and Ursprung, 1962; Latner and Skillen, 1964). These changes are very
regular and thus LDH isozymes can be used as markers of differentiation.
Both the precise knowledge of the structure of LDH isozymes and the
knowledge of the gene-polypeptide relationship makes LDH isozymes
especially suitable as markers for study of the differentiation of kidney
mesenchyme in vitro.
Undifferentiated kidney mesenchyme exhibits an "embryonic" LDH
isozyme composition, LDH-5 and LDH-4 being the strongest bands observed in electrophoresis (Koskimies and Saxen, 1966). Thus the mesenchyme cells mainly produce A polypeptides of LDH. The first change in