A. Feinstein

ISOLATION OF AMYLOID P COMPONENT (PROTEIN AP) FROM NORMAL SERUM AS A CALCIUM-DEPENDENT BINDING PROTEIN

The P component of amyloid (protein AP, pentagonal unit) has been isolated from normal serum by using its hitherto undescribed calcium-dependent affinity for agarose. The presence of P component in all forms of amyloid may be due to this calcium-dependent binding to certain polyanions.

PHYLOGENETIC ASPECTS OF C-REACTIVE PROTEIN AND RELATED PROTEINS*

C-reactive protein (CRP) was discovered by Tillett and Francis’ in the sera of patients with various infectious and inflammatory diseases as a material which precipitated pneumococcal C-polysaccharide (CPS). Subsequently Abernethy and Ave

C-reactive protein and serum amyloid P component in the plaice (Pleuronectes platessa L.), a marine teleost, are homologous with their human counterparts

characterized CRP as a protein and identified the requirement for calcium ions in its interaction with CPS, while they and established that the appearance of CRP in the serum is a nonspecific response to infection, inflammation and tissue damage. Abernethy and Averf also introduced the term “acute phase sera” to designate samples obtained from patients in the acute phase of infectious diseases. CRP was called the “acute phase protein” and this term was subsequently applied to the large number of other plasma proteins, the concentrations of which are raised in acute phase sera. At an early stage Abernethy’ reported the presence of a precipitin comparable to CRP in acute phase monkey serum and, although he had been unable to find any in mouse or rabbit sera, Anderson and McCarty’later described the existence

Calcium-dependent aggregation of human serum amyloid p component

C-reactive protein and serum amyloid P component were isolated from serum of the plaice (Pleuronectes platessa L.), a murine teleost. The isolation was based on their calcium-dependent binding affinity for pneumococcal C-polysaccharide and for agarose, respectively. These specificities are the same as those of human C-reactive protein and serum amyloid P component, respectively, and we have previously reported that the plaice molecules resemble human C-reactive protein and serum amyloid P component in their electron microscopic appearance. We describe here estimation of the molecular weights of plaice C-reactive protein and serum amyloid P component and their subunits, and analysis of their amino acid composition, glycosylation and partial amino-terminal amino acid sequences. The results establish that plaice C-reactive protein and serum amyloid P component are homologous with each other and with their human counterparts and indicate that there has been stable conservation of this protein family throughout vertebrate evolution.

Isolation and characterization of goat C-reactive protein

Normal human serum or isolated human amyloid P component was ultracentrifuged on density gradients containing either 10 mM EDTA or different concentrations of Ca2+ between 0.15 and 2.15 mM. In the presence of Ca2+ concentrations of 1 mM or more human P component sedimented more rapidly than it did in the presence of lower Ca2+ levels or of EDTA. This phenomenon was due to Ca2+-dependent aggregation of P component molecules and did not require the presence of any other serum constituents. It was completely inhibited by incorporating a physiological concentration (40 mg/ml) of serum albumin in the gradients, suggesting that free ionized Ca2+ is required to promote aggregation of the P component. P component from the mouse and the plaice (Pleuronectes platessa L.), a marine teleost, did not undergo the same Ca2+-dependent aggregation as human P component. These observations resolve a discrepancy existing in the literature concerning the sedimentation rate of human P component in density gradient ultracentrifugation and shed new light on its behaviour with respect to Ca2+ which may be relevant to the deposition of P component in amyloidosis.

Analogues in other mammals and in fish of human plasma proteins, C-reactive protein and amyloid P component

A pentraxin was isolated from acute phase goat serum by its calcium-dependent affinity for agarose, and although it did not bind to phosphorylcholine immobilised on Sepharose, its binding to agarose was reversed by exposure to fluid phase phosphorylcholine. It was identified as goat C-reactive protein on the basis of its immunochemical cross-reactivity with human and bovine C-reactive protein. The molecule was composed of five identical, glycosylated, non-covalently associated subunits, each of molecular weight approx. 24,000. Acute phase serum levels in a small number of samples were not significantly different from normal levels (means 72 and 55 micrograms/ml, respectively), suggesting that goat C-reactive protein is not a major acute phase reactant. No other pentraxin was detected in goat serum.