3 research outputs found
Rat basophilic leukaemia (RBL) cells overexpressing Rab3a have a reversible block in antigen-stimulated exocytosis
Monotreme whey proteins: isolation, characterization and comparative aspects
Lactation is a specific mammalian adaptation. Milk is the principal source of
nutrition for the newborn mammal. Both its needs and the physiology of lactation vary
among the different mammalian species. These factors are reflected in the differing milk
compositions, in particular the protein compositions. Thus, comparative studies of the
protein composition of eutherian, metatherian and prototherian mammals are of great
importance in their own right. Furthermore, these proteins exhibit an extraordinary array
of conformations and protein-protein interactions. Thus, their study will also throw
light on basic aspects of protein structure and physiological function.
This thesis is concerned with a study of the milk proteins of the egg-laying mammals
(monotremes), the echidna and platypus. At the time of commencement of this
study little was known of the protein composition of their milk. Thus, it was necessary
to establish initially the overall protein composition of the limited number and volume
of milk samples available. This was achieved by electrophoretic and immunological
studies, fractionation of the proteins and determination of certain properties, e.g.
molecular weight, amino acid composition and partial amino acid sequence. Efforts were
concentrated on the whey proteins and where possible they were identified in terms of
known proteins, the bovine whey proteins being used as a point of reference.
In addition, two groups of whey proteins were chosen for more intensive study:
the lysozymes and iron (III) binding proteins.
The main echidna whey proteins have been resolved into eleven bands on starch
gel electrophoresis at pH 7.G. These were designated A through K on the basis of
decreasing electrophoretic mobility. Nine bands, designated 1 through 9, have been
resolved in a sodium dodecyl sulphate-gradient polyacrylamide gel system. The relationship
between the bands of the two gel systems has been determined and proteins corresponding
to Bands A, B, C, E, G-J have been isolated. Band A protein has an apparent molecular weight of ca. 17,800 and appears to
have no counterpart in echidna blood serum. Twenty-five residues of the TV-terminal sequence
have been determined. It bears no simple relationship to any known protein, but
has ca. 30% homology with an acidic whey protein precursor found in rat and mouse
milk.
Band B protein has an apparent molecular weight of ca. 55,000 and a counterpart
is not detected in the blood serum. The sequence of the first fifteen TV-terminal residues
was determined. Sequence homology with any known protein is not evident.
Band C protein occurs in the milk and blood and is echidna serum albumin. Both
milk and blood protein have the. same electrophoretic mobility and apparent molecular
weight. The determination of the TV-terminal sequence indicates 63% homology with
human and bovine serum albumin over the first 30 residues.
Band E protein occurs only in the milk, and has an apparent molecular weight of
ca. 21,000. The sequence of the first 68 TV-terminal residues indicates that it is a novel
protein.
Bands G-J and a minor band (G ) evident in some milk samples are echidna
transferrins (milk). The electrophoretic patterns vary depending on the sample, but all
exhibit 3-4 of the bands. Bands H-J are evident in the electrophoretic patterns of the
blood serum. The milk bands are identified as transferrins on the basis of their
electrophoretic mobilities, ,r,^Fe(III) binding capacity, molecular size and A-terminal sequences.
The multiple banding reflects differences in the numbers of sialic acid residues
(0-4) present. The principal cathodic electrophoretic band in the milk is lysozyme. Echidna milk
is relatively rich in this protein, containing ca. 2 mg mf*. Previous observations that
there is a lysozyme, echidna lysozyme 1, occurring'in the milk of Tachyglossus aculeatus
rnultiaculeatus and another, echidna lysozyme II, of different electrophoretic mobility at
pH 5.3, occurring in Tachyglossus aculeatus aculeatus, have been confirmed. Each enzyme
has been isolated and the complete amino acid sequences determined. They are both c-type lysozymes, M 13,988 and 13,861 respectively. They differ in three amino
acid residues. Both proteins have a number of unusual features, e.g. some residues considered
important in a-lactalbumins for lactose synthase activity are conserved.
However, contrary to previous findings in this laboratory, echidna lysozyme I isolated
during the course of the present study, does not appear to be active in the lactose synthase
system. Furthermore, evidence is obtained for the presence of very low concentrations
of a conventional o-lactalbumin. Proposals for further work on this particular
problem are presented.
Seven electrophoretic bands are observed on starch gel electrophoresis (pH 7.6) of
platypus whey proteins. These are designated A through G. Their apparent molecular
weight range from 19.000-79,000. Proteins corresponding to Bands A, C, D, F and G
have been isolated and preliminary characterization made.
Bands F and G proteins are shown to be transferrins. The major blood serum iron
(III) binding protein has a similar electrophoretic mobility to Band G of the milk.
Platypus arid echidna transferrins exhibit 80% homology over the first 30 A-terminal
residues.
Only trace levels of lysozyme activity and weak lactose synthase activity were
detected in the 18 platypus milk samples examined.
The significance of the work in the overall framework of comparative studies of
milk proteins is discussed