Mark A. Hermodson

The major proteins of human and monkey amyloid substance: Common properties including unusual N-terminal amino acid sequences

Amyloid substance is a complex proteinaceous material found in the tissues of patients with the disease amyloidosis. Recently we have presented evidence that there are two chemically distinct kinds of amyloid substance, one associated with the classical inflammation-related amyloidosis and another, frequently designated atypical or paramyloid, occurring with tumors such as multiple myeloma or without evident pre-existing disease [l] . The classical, or inflammation-associated, substance is distinguished by: a) instability to alkali of its characteristic Congo red binding capacity; b) its amino acid composition and c) the presence of a major protein constituent, amyloid protein A. The family of proteins comprising amyloid protein A has a molecular weight range of 6000-8000 and a characteristic amino acid composition [2] ; in addition, human amyloid protein A has the capacity to bind Congo red and exhibits the characteristic hyperchromism and spectral changes previously described for amyloid substance [ 1] . In this communication we compare the chromatographic and electrdphoretic properties, the amino acid composition and the 24 amino acid N-terminal sequence of the humanand monkeyderived protein A of amyloid substance.

Reversed-phase supports for the resolution of large denatured protein fragments☆

Large pore (much greater than 300 A), spherical partical silica stationary phases possessing either C18 or C8 hydrocarbon ligands out-performed small pore (60-100A), irregular shaped silicas for the purification of large denatured peptides. Since columns 5 cm in length appeared to be as effective in separating peptides as columns 5 times longer, it is likely that large peptides absorb to the matrix rather than partition between the stationary and mobile phases.

Evidence for a hetero-oligomeric structure of the chloroplast cytochrome b-559

A second nonhomologous polypeptide in the thylakoid membrane cytochrome b‐559 has been suggested by the finding of a smaller reading frame just slightly downstream from that corresponding to the 9 kDa cytochrome polypeptide that is dominant on a Coomassie‐stained gel. This reading frame encoded a 39‐residue polypeptide that was similar in having a central hydrophobic domain of 25–26 residues and a single His residue at the same position in the hydrophobic domain. The smallest polypeptide seen on SDS gels of the cytochrome was isolated by high‐performance liquid chromatography (HPLC). The NH2‐terminal sequence matched that of the downstream gene. The stoichiometry of the 2 gene products separated by HPLC was approx. 1:1, based on the molecular masses of 9.16 and 4.27 kDa calculated from the nucleotide sequence. It is concluded that the cytochrome contains both the 9.16 kDa (α) and 4.27 kDa (β) polypeptides. These data, the single His residue on each polypeptide, and the previous finding of a bis‐histidine coordination, imply that the unit heme binding structure of the cytochrome is a heme cross‐linked dimer. If the cytochrome contains a single heme, the dimer structure would be (αβ). If there are 2 hemes/cytochrome, the more likely structure would be (αβ)2, a tetramer consisting of 2 heme cross‐linked hetero‐dimers.

Subunit interactions in southern bean mosaic virus

The structures of the three quasi-equivalent subunits A, B and C of southern bean mosaic virus have been carefully built in accordance with an electron density map that had been refined with three cycles of molecular replacement averaging. The map was interpreted with the aid of the amino acid sequence and a computer graphics system. The three quasi-equivalent subunits were compared in pairs and found to have almost the same conformation except for five residues in the carboxy-terminal vestigial “hinge”. Contacts between subunits in the capsid were tabulated and analyzed. The largest number of interactions was between the 5-fold-related subunits. although the quasi-3-fold or quasi-6-fold-related subunits had nearly the same number of contacts. The interactions across the 2-fold axes were far fewer. A number of charge interactions and numerous hydrogen-bonding possibilities exist in the subunit interactions. The icosahedral 5-fold interactions between A subunits (AA 5 ) and the quasi-6-fold interactions between C and B subunits without the interposed βA arm (CB 5 ) are essentially identical. The quasi-6-fold interactions containing the interposed ordered βA arm of the C subunit (B 2 C) are quite different. There is a “vector of rotation” or line of contact about which the C and B subunits rotate to form either CB 5 or B 2 C interactions. A similar situation pertains to the relationship between the quasi-2-fold contacts AB 5 and the icosahedral 2-fold contacts CC 2 . The amount of rotation about the hinge is 36° to 39° in both cases. The quasi-3-fold contacts BA and AC are essentially identical but differ slightly but significantly from the BC contacts. The latter have an additional 5° rotation between subunits. It is proposed that the “relaxed” AB 5 conformation of subunit dimers might self-assemble into 10mer caps composed of five dimers about a 5-fold axis with RNA binding to the basic random domain and the basic interior surface of the shell domain. This 10mer might nucleate the assembly of a T =1 or T =3 capsid, depending on the charge state of carboxyl group clusters in the subunit contact region. In the low charge state, T =1 capsids are formed. Alternatively, in the high charge state, dimers must be “tensed”, as in the CC 2 contacts of the native virus, for binding to the 10mer cap, creating a hydrophobic channel to induce the ordering of the βA arm and formation of the β-annulus. The two-state condition of dimers in a viral coat protein extends the range of structures originally envisaged by Caspar & Klug (1962) . Appendix I considers a possible manner in which viral RNA might interact with the coat protein. A pattern of basic residues on the southern bean mosaic virus coat protein surface facing the RNA is able to dock a nine base-pair double-helical A -RNA structure with surprising accuracy. The basic residues are each associated with a different phosphate and the protein can make interactions with five bases in the minor groove. It is proposed that this is one of a small number of ways in which the RNA interacts with southern bean mosaic virus coat protein.

Maize Storage Proteins: Characterization and Biosynthesis

Developing seeds provide plant molecular biologists with useful model systems for studying the physiological and genetic mechanisms regulating the synthesis of specific plant proteins, i.e. seed storage proteins. These studies have perhaps even greater significance considering the importance of seed proteins in human and livestock nutrition. Maize storage proteins are interesting from both these aspects, since maize is an economically important crop and mutations affecting both the quantitative and qualitative synthesis of maize storage proteins have been identified (Mertz et al., 1964; Nelson et al., 1965). Our research in the last several years has been devoted to the characterization of these storage proteins and the reactions regulating their biosynthesis.

Hemoglobinopathic Erythrocytosis due to a New Electrophoretically Silent Variant, Hemoglobin San Diego (β109(G11)Val→Met)

Examination of 13 members of a Filipino family revealed that 6 had erythrocytosis inherited as a simple autosomal dominant trait. Application of several electrophoretic and chromatographic tests failed to reveal the presence of an abnormal hemoglobin in hemolysates from affected individuals. However, measurements of oxygen dissociation curves using whole bloods, dialyzed hemolysates, and 2,3-diphosphoglyceric acid-stripped hemolysates clearly showed that affected persons had an abnormal hemoglobin characterized by a high affinity for oxygen. Compositional analyses of all tryptic peptides from the beta-chains of the proband revealed a valyl-methionyl ambiguity in betaT12a. Blockage of lysyl residues and subsequent tryptic hydrolysis at arginyl residues permitted the isolation of fragments containing residues 105 through 146. Automatic sequence analysis of the fragments demonstrated the presence of both valine and methionine in nearly equal proportions at position beta109. This new hemoglobin variant is designated Hb San Diego (beta109(G11) Val-->Met).

Identification of phosphorylation sites in peptides using a gas-phase sequencer☆

A simple procedure is described for determining the location of phosphorylation sites in phosphopeptides. The method employs measurement of 32P-labeled inorganic phosphate release during Edman degradation cycles using a gas-phase sequencer. The procedure is based on extracting peptides and inorganic phosphate from portions of the sample filter at strategic cycles in the sequence analysis followed by determination of the relative amounts of phosphate and phosphopeptide. One advantage of this technique is the very high recovery of the phosphate associated with the peptide, 80-97% in this study. In the course of this work, it was also found that phosphoserine residues themselves caused reduced efficiency of the Edman degradation as compared with unesterified serine residues. The present procedure has the merit of being simple and easy to apply.

A novel cereal storage protein: molecular genetics of the 19 kDa globulin of rice

A λgt11 cDNA library, constructed from poly(A)+ RNA isolated from immature rice seed endosperm, was screened with affinity-purified antibodies against the rice storage protein called α-globulin (previously), or the 19 kDa globulin (our term). A positive clone was isolated and sequenced and shown to encode a 21 kDa precursor for the 19 kDa globulin, based on the identity of portions of the inferred amino acid sequence and the sequence of three cyanogen bromide peptides of the 19 kDa globulin. Analysis of genomic DNA by Southern blotting using the cDNA clone probe revealed one hybridizing band inEco RI,Hind III, andBam HI digests. This strongly suggests that the 19 kDa globulin is encoded by a single-copy gene. Because of its single-copy nature and its abundance of Arg and lack of Lys, the 19 kDa rice globulin appears to be a particularly attractive target for genetically engineering increased Lys content in rice seeds.

Comparison of the amino terminal sequences of bovine, dogfish, and lungfish trypsinogens

Trypsin and trypsin-like enzymes have been isolated from phyla and species which extend over the whole range of evolution from bacteria to mammals [l] . Purified trypsinogen has been obtained from a more limited number of species. In fact, there are only two lower vertebrates from which this zymogen has been isolated viz. the dogfish (Squalus QCQ&Z~US) [2] and the African lungtish (Protoprerus aethiopicus) [3]. In all known cases, the activation of mammalian trypsinogens involves the cleavage of a lysyl bond in the invariant amino terminal sequence X-Asp-Asp-Asp-Asp-Lys-Be-Y , with the concomitant libertation of an activation peptide having the general structure X-(As~)~-Lys [4]. The availability of purified dogfish and lungfish trypsinogen has enabled us to determine the amino terminal sequences of these zymogens and to compare them to those of bovine trypsinogen and trypsin. Extensive homology between bovine and lungfish trypsinogens has been previously documented [2]. The present investigation is part of a general study of the evolution and phylogenetic variation of proteolytic enzymes.

HUMAN PHOSPHOGLYCERATE MUTASE: ISOZYME MARKER FOR MUSCLE DIFFERENTIATION AND FOR NEOPLASIA

ABSTRACT. Tissue-specific isozymes of the glycolytic enzyme phosphoglycerate mutase can be distinguished by electrophoresis, chromatography, heat stability, and amino acid composition. Phosphoglycerate mutase (PGAM) from skeletal muscle migrates slowly, while PGAM from brain and other tissues migrates rapidly toward the anode in pH 7.5 Tris-citrate or pH 8.6 Tris-EDTA-borate horizontal starch gels. Striking developmental and neoplastic transitions for PGAM expression in man have been identified. Human fetal skeletal muscle at 50-60 days gestation contains the single-banded type B PGAM pattern, as in adult brain. The appearance of a band of intermediate mobility signifies production of type M PGAM subunits and formation of MB hybrid enzyme molecules. By 166 days gestation and thereafter, the skeletal muscle contains almost exclusively the type M PGAM phenotype. Fetal human brain has the same phenotype as adult brain for PGAM, as do relatively benign brain tumors, such as meningioma or a grade II astrocytoma. However, highly malignant grade III and IV astrocytomas and a hemangioblastoma gave electrophoretic patterns with prominent hybrid and muscle type bands. PGAM promises to be a useful isozyme marker for differentiation in vivo and in cultured systems in vitro.