76
3 The Structural Variety and Metabolism of Proteins
kinase a-subunit and are insensitive to the two
inhibitors; however, they are very different in
molecular size, substrate specificity and dependence on divalent cations. The different protein
phosphatases, together with their inhibitors and
several protein kinases, make up an interlocking
regulatory network: protein phosphatase 1 is
only inhibited by inhibitor 1 when the latter is
phosphorylated by a cAMP-dependent protein
kinase; the dephosphorylation of inhibitor 1,
which in muscle is mainly carried out by protein
phosphatase 2B, leads to the inactivation of the
inhibitor and hence to the activation of phosphatase 1. Some protein phosphatase 1 is present in
muscle as an inactive complex with inhibitor 2.
This complex dissociates, and phosphatase 1
thereby becomes activated, when inhibitor 2 is
phosphorylated by the glycogen synthase kinase.
Thus, the glycogen synthase kinase not only is
responsible for the phosphorylation of glycogen
synthase but also promotes its dephosphorylation
via protein phosphatase 1. Protein phosphatase 2B, the only one that is activated by Ca 2 +calmodulin, is found mainly in the brain and in
skeletal muscle and is identical to the Ca 2 +binding protein calcineurin in the brain; it possesses a high-affinity Ca 2 + -binding site [36, 118].
Proteins isolated and characterized from the head
of Drosophila are very similar to the protein
phosphatases 1, 2A and 2B and the inhibitors l'
and 2. Here also the phosphatase 1 forms an
inactive complex with the corresponding inhibitor 2. The inhibition of protein serine/threonine
phosphatases by inhibitors 1 and 2 is also a common feature of organisms like baker's yeast, and
the regulation of the protein phosphatases thus
appears to be similar in all eukaryotes [36,
57,201].
3.1.3 Analysis and Comparison
of Protein Sequences
Whilst Sanger had to work for 10 years to determine the order of the 51 amino acids of insulin,
and required several grams of the substance, the
sequence analysis of far bigger proteins has
become a routine affair since the 1960s, and can
be carried out on milligram amounts. By the
beginning of 1977, 1250 protein sequences were
known, not including the anomalous human haemoglobins and other alleloproteins [51, 126]; the
number has since increased by several-fold.
Nowadays, the nucleotide sequence of structural
genes or of mRNA-complementary DNA
(cDNA) is far easier to determine than the amino
acid sequence, the more so as the amount of substance for analysis can be amplified by cloning.
Thus, amino acid sequences are now mostly
determined indirectly from the gene or cDNA
sequences. Whilst direct analysis of amino acid
sequences is mostly carried out on proteins of
known function, the analysis of DNA sequences
often leads to the question of the biological function of the encoded polypeptide. Comparison
with consensus sequences of particular functional
regions or motifs in proteins of known function
can often be helpful in such cases [3, 19].
The coincidence or similarity of protein
sequences can be assessed with relatively little
effort and without previous isolation using immunological and electrophoretic methods, although
the room for error is here quite large. Since it was
discovered in 1897 that the antigen-antibody reaction can take place as a "cross-reaction" with
other (heterologous) antigens as well as with the
protein used for immunization (the homologous
antigen), there have been innumerable investigations of the serological relationships between proteins. Protein comparisons were almost always
carried out like this until the invention in 1937 of
paper electrophoresis. It is important to realize
that reactions of proteins of different origin with a
given antibody do not provide evidence of the
identicalness of either the proteins or reactive
sites on the molecules, but only the recognition of
similar structural features (epitopes) by the antibody. If antibodies are to be used for protein
identification, the highest possible specificity for
a particular protein, or for a particular immunological determinant, is essential. This can be
achieved by the production of monoclonal antibodies. Immunodiffusion and immunoelectrophoresis can be used to examine possible reactions
between proteins of interest and antibodies, and
this can be quantified, e.g. using microcomplement fixation, enzyme inhibition by antibodies,
and both radio- and enzyme-immunoassays.
Immunological methods are used mainly in the
biochemical analysis of relationships and will be
thoroughly discussed in the relevant sections.
Depending upon the system used, electrophoretic methods, concern charge differences (gel
electrophoresis, isoelectric focusing) or molecular
size differences (SDS gel electrophoresis). 1\vodimensional electrophoresis makes use of two different separation criteria to achieve quite amazing separation, after which the protein spots can
be detected by highly sensitive silver staining or
by autoradiography. Using these methods, it
3 The Structural Variety and Metabolism of Proteins
kinase a-subunit and are insensitive to the two
inhibitors; however, they are very different in
molecular size, substrate specificity and dependence on divalent cations. The different protein
phosphatases, together with their inhibitors and
several protein kinases, make up an interlocking
regulatory network: protein phosphatase 1 is
only inhibited by inhibitor 1 when the latter is
phosphorylated by a cAMP-dependent protein
kinase; the dephosphorylation of inhibitor 1,
which in muscle is mainly carried out by protein
phosphatase 2B, leads to the inactivation of the
inhibitor and hence to the activation of phosphatase 1. Some protein phosphatase 1 is present in
muscle as an inactive complex with inhibitor 2.
This complex dissociates, and phosphatase 1
thereby becomes activated, when inhibitor 2 is
phosphorylated by the glycogen synthase kinase.
Thus, the glycogen synthase kinase not only is
responsible for the phosphorylation of glycogen
synthase but also promotes its dephosphorylation
via protein phosphatase 1. Protein phosphatase 2B, the only one that is activated by Ca 2 +calmodulin, is found mainly in the brain and in
skeletal muscle and is identical to the Ca 2 +binding protein calcineurin in the brain; it possesses a high-affinity Ca 2 + -binding site [36, 118].
Proteins isolated and characterized from the head
of Drosophila are very similar to the protein
phosphatases 1, 2A and 2B and the inhibitors l'
and 2. Here also the phosphatase 1 forms an
inactive complex with the corresponding inhibitor 2. The inhibition of protein serine/threonine
phosphatases by inhibitors 1 and 2 is also a common feature of organisms like baker's yeast, and
the regulation of the protein phosphatases thus
appears to be similar in all eukaryotes [36,
57,201].
3.1.3 Analysis and Comparison
of Protein Sequences
Whilst Sanger had to work for 10 years to determine the order of the 51 amino acids of insulin,
and required several grams of the substance, the
sequence analysis of far bigger proteins has
become a routine affair since the 1960s, and can
be carried out on milligram amounts. By the
beginning of 1977, 1250 protein sequences were
known, not including the anomalous human haemoglobins and other alleloproteins [51, 126]; the
number has since increased by several-fold.
Nowadays, the nucleotide sequence of structural
genes or of mRNA-complementary DNA
(cDNA) is far easier to determine than the amino
acid sequence, the more so as the amount of substance for analysis can be amplified by cloning.
Thus, amino acid sequences are now mostly
determined indirectly from the gene or cDNA
sequences. Whilst direct analysis of amino acid
sequences is mostly carried out on proteins of
known function, the analysis of DNA sequences
often leads to the question of the biological function of the encoded polypeptide. Comparison
with consensus sequences of particular functional
regions or motifs in proteins of known function
can often be helpful in such cases [3, 19].
The coincidence or similarity of protein
sequences can be assessed with relatively little
effort and without previous isolation using immunological and electrophoretic methods, although
the room for error is here quite large. Since it was
discovered in 1897 that the antigen-antibody reaction can take place as a "cross-reaction" with
other (heterologous) antigens as well as with the
protein used for immunization (the homologous
antigen), there have been innumerable investigations of the serological relationships between proteins. Protein comparisons were almost always
carried out like this until the invention in 1937 of
paper electrophoresis. It is important to realize
that reactions of proteins of different origin with a
given antibody do not provide evidence of the
identicalness of either the proteins or reactive
sites on the molecules, but only the recognition of
similar structural features (epitopes) by the antibody. If antibodies are to be used for protein
identification, the highest possible specificity for
a particular protein, or for a particular immunological determinant, is essential. This can be
achieved by the production of monoclonal antibodies. Immunodiffusion and immunoelectrophoresis can be used to examine possible reactions
between proteins of interest and antibodies, and
this can be quantified, e.g. using microcomplement fixation, enzyme inhibition by antibodies,
and both radio- and enzyme-immunoassays.
Immunological methods are used mainly in the
biochemical analysis of relationships and will be
thoroughly discussed in the relevant sections.
Depending upon the system used, electrophoretic methods, concern charge differences (gel
electrophoresis, isoelectric focusing) or molecular
size differences (SDS gel electrophoresis). 1\vodimensional electrophoresis makes use of two different separation criteria to achieve quite amazing separation, after which the protein spots can
be detected by highly sensitive silver staining or
by autoradiography. Using these methods, it