Mordechai Pras

The characterization of soluble amyloid prepared in water

Amyloid was extracted from the spleen of a patient with primary amyloidosis by homogenizing it at high speed with water after preliminary treatments, first to remove proteins soluble in saline, and then to remove salts. The extracts containing amyloid appeared to be clear at concentrations up to 6 mg/ml of protein. The material gave little sediment on being centrifuged up to 20,000 g for 1 hr, but the protein was sedimented at 100,000 g in 1 hr. The amyloid could be precipitated from the extracts by addition of NaCl to 0.0075 mole/liter or of CaCl(2) to 0.0025 mole/liter. The protein-bound Congo red formed a red precipitate and this property was used to estimate recovery and purity of amyloid during extraction. On electronmicroscopy the isolated amyloid proved to be morphologically pure. It existed either as single filaments measuring 60-80 A in diameter or as large aggregates of these filaments.Freshly isolated amyloid in water sedimented as a single homogeneous peak with an s degrees (20,[unk]) of about 45-50S. On standing, the solution became cloudy and more rapidly sedimenting components appeared. On electrophoresis the material migrated as a homogeneous peak towards the anode. The protein had an amino acid composition different from that of all known serum proteins. It was rich in acidic amino acids and had little cysteine and methionine and no hydroxyproline. The total content of carbohydrate was less than 2%.

The Amino Acid Sequence of a Major Nonimmunoglobulin Component of Some Amyloid Fibrils

The complete amino acid sequence of a protein, acid soluble fraction, (ASF) which constitutes up to 50% of amyloid fibrils from a patient with familial Mediterranean fever has been obtained. Partial amino acid sequences of three other proteins from patients with secondary amyloidosis were identical in the regions studied except for an alanine-valine interchange in one. The ASF contains no cysteine, does not resemble any known immunoglobulin, and has not been detected as yet in myeloma-associated amyloid.

Systemic senile amyloidosis. Identification of a new prealbumin (transthyretin) variant in cardiac tissue: immunologic and biochemical similarity to one form of familial amyloidotic polyneuropathy.

Isolated amyloid fibrils from three cases of systemic senile amyloidosis (SSA) contained subunit proteins with molecular masses of 14 (10-20%), 10-12 (60-80%), and 5-6 kD (5-10%) when fractionated under reducing and dissociating conditions. This grouping was identical to that seen in SKO, a case of familial amyloidotic polyneuropathy (FAP) studied earlier. Amino acid sequencing confirmed that SSA subunit proteins were in fact prealbumin (transthyretin). Complete sequence analysis of one SSA preparation revealed the presence of a new variant Pa (TTr) molecule with a single amino acid substitution of isoleucine for valine at position 122. Further studies used an antiserum specific for SKO IV, a subunit protein of SKO previously shown to correspond to carboxy-terminal 78 residues (positions 49-127) of (TTr). Anti-SKO IV reacted with SSA in tissue at equivalent dilutions to anti-Pa (TTr) and with the 10-12-kD fraction of SSA on Western blots; reactivity was blocked by SKO IV, but not by Pa (TTr). SSA is a form of systemic amyloidosis caused by tissue deposition of Pa (TTr) and its fragments, with shared conformational or subunit antigenicity to at least one form of FAP. Identification of a new variant Pa (TTr) molecule in one case suggests further that SSA may be a genetically determined disease expressed late in life.

AA protein in a case of “primary” or “idiopathic” amyloidosis

Amyloidosis constitutes a group of diseases in which extracellular fibrils with a characteristic appearance are deposited in a variety of tissues. Several different proteins have been identified as the major subunits of the fibrils. In the primary and myeloma-associated type, the amyloid fibrils consist of immunoglobulin light chain fragments, whereas in the secondary type and the amyloid associated with familial Mediterranean fever the major component is the AA protein. In this report a 21 year old man of Yemenite extraction with no underlying disease and no family history of amyloidosis was found to have amyloid deposits composed of AA protein. Although clinically this might be classified as primary amyloidosis, the absence of light chain fragments makes that diagnosis unlikely. Therefore, it is suggested that whenever possible the clinical classification be supplemented by a description of the biochemical nature of the fibrils.

The partial amino acid sequence of the major low molecular weight component of two human amyloid fibrils.

Amyloid flbrils from several patients with secondary amyloidosis or amyloidosis associated with Familial Mediterrean Fever (FMF), when treated with dilute acid yield a major low molecular component which appears to be similar in all, and unrelated to any known immunoglobulin. In an effort to separate the protein subunit present in amyloid fibrils from mucopolysaccharides, amyloid fibrils isolated by the method of Pras et al. [ 1 ] were extracted with dilute acid. This procedure yielded a low molecular weight substance which migrated as a single band on disc electrophoresis, had a molecular weight of around 6,000 daltons, bound Congo red, and could be made to assume again a fibrillar appearance which however differed from that of typical amyloid fibrills [2] . In the present study we present the partial amino acid sequences of this component from two patients and show them to be identical.

Amyloidosis associated with renal cell carcinoma of the AA type.

Amyloid fibrils were found at postmortem examination in a 70 year old woman with generalized amyloidosis associated with renal carcinoma (hypernephroma). Clinically, her amyloid disease presented as nephrotic syndrome. It was demonstrated by electrophoretic and amino acid sequence analysis studies that the amyloid fibrils contained AA protein identical to that found in amyloidosis associated with chronic inflammatory and infectious diseases as well as in the genetic form of familial Mediterranean fever.

Studies of lysosomes—VIII: The effect of polyene antibiotics on lysosomes

Abstract Polyene antibiotics were added to suspensions rich in lysosomes from rabbit liver, kidney, and leukocytes. Filipin, etruscomycin, and pimaricin and nystatin released acid phosphatase, aryl sulfatase, and β-glucuronidase from lysosomes in rabbit liver and kidney granule suspensions at near-neutral and at acid pH (4.6). They did not release malic dehydrogenase from mitochondria present in the same suspension. Filipin and etruscomycin released β-glueuronidase from rabbit peritoneal leukocyte lysosomes and changed the turbidity of such suspensions. Amphotericin B induced the release of lysosomal enzymes from rabbit kidney granules only at acid pH, but did not induce leakage of malic dehydrogenase. None of the polyenes affected mitochondria, as judged by optical studies of mitochondrial swelling and/or lysis under conditions which led to mitochondrial changes induced by substrates, vitamin A, or thyroxine. These studies suggest that lysosomes from different tissues vary in their susceptibility to a given pharmacologic agent and that they are much more sensitive to polyene antibiotics than mitochondria, which are more responsive to excess of vitamin A. Subcellular organelles can be disrupted by polyenes; this finding supports the hypothesis that polyene antifungal antibiotics act, indiscriminately in a sense, upon the membranes which bound many types of cells and organelles.

Characterization of amyloid deposits and P component from a patient with factor X deficiency reveals proteins derived from a lambda VI light chain

Amyloid fibrils were isolated from a spleen obtained at surgery from a 58-year-old white man with primary amyloidosis presenting with factor X deficiency and responding dramatically to splenectomy. Gel filtration on Ultragel ACA 54 in 5 M guanidine 1 M acetic acid yielded components with molecular weights between 17,000 and 13,000. Two of them (17K and 15K) were studied in detail. Antigenic and amino acid sequence analysis showed that these proteins were related to lambda VI immunoglobulin light chain. The predominant protein subunits of the amyloid fibril of the deposits (17K) was processed at the carboxy terminus in the same section of the constant region as the only other lambda VI amyloid protein previously reported. Amino terminal sequence of the 15K protein revealed not only degradation at the C terminal, but also minor degradation at the amino terminal (three residues difference from the 17K species). P component was also isolated from the spleen and characterized. This represents the first antigenic and sequence analysis of tissue amyloid proteins and P component from a patient presenting with factor X deficiency and another example of amyloid proteins derived from the newly discovered amyloidogenic lambda VI light chain subgroup.

Genetic Heterogeneity of Familial Amyloid Polyneuropathies of Jewish Type

The Jewish SKO amyloid protein is a prealbumin monomer with two amino acid substitutions. Glycine is substituted for Threonine at position 49 and Isoleucine for Phenylalanine at position 33. The distribution of these components is different in thyroid and spleen suggesting tissue specificifity in amyloid disposition. The SKO variant differs from the prealbumin proteins found in the Portuguese, Japanese and Swedish Familial Amyloid Polyneuropathies. Additional prealbumin variants will be expected when the amyloid proteins of the facial and upper limb amyloid neuropathies as well as the Van Allen kinship can be studied.