Binding of Amyloid Beta-Protein to Intracellular Brain Proteins in Rat and Human

Amyloid beta-protein (A), in its soluble form, is known to bind several circulatory proteins such as apolipoprotein (apo) E, apo J and transthyretin. However, the binding of A to intracellular proteins has not been studied. We have developed an overlay assay to study A binding to intracellular brain proteins. The supernatants from both rat and human brains were found to contain several proteins that bind to A 1–40 and A 1–42. No major difference was observed in the A binding-proteins from brain supernatants of patients with Alzheimer's disease and normal age-matched controls. Binding studies using shorter amyloid beta-peptides and competitive overlay assays showed that the binding site of A to brain proteins resides between 12–28 amino acid sequence of A. The presence of several intracellular A-binding (AB) proteins suggests that these proteins may either protect A from its fibrillization or alternatively promote A polymerization. Identification of these proteins and their binding affinities for A are needed to assess their potential role in the pathogenesis of Alzheimer's disease.     

Association-dissociation and denaturation-renaturation of high-molecular-weight protein: carmin from safflower seed (Carthamus tinctorius L.) in alkaline solution

The effect of alkalinepH on the association, dissociation, and denaturation of carmin, the high-molecular-weight protein from safflower seed was investigated in thepH range 7–12, using various biophysical techniques. The results indicate that the multimeric protein carmin dissociates atpH 8.0 where denaturation has not set in. The association-dissociation of the protein can be represented schematically as 11S 7S 4S 2S. AbovepH 10, the protein undergoes simultaneous dissociation and denaturation. The denaturation process appears to be complete at pH 12.5. The protein undergoes conformational change and covalent modifications and cleavage during the denaturation process. A reversibility study shows that the process of dissociation is reversible to a large extent, whereas denaturation appears to be irreversible. These results are discussed in terms of association-dissociation, denaturation and alkaline-catalyzed covalent modifications and cleavage of seed proteins.     

Dipeptide frequencies in proteins and the CpG deficiency in vertebrate DNA

Summary Analysis of vertebrate protein sequences totalling 4040 residues shows that amino acids with a high proportion of codons ending in C occur with significantly reduced frequency before amino acids whose codons start with G. This effect is not shown by control bacterial protein sequences. The consequent implication of shortage of XXC. GXX codon pairs in vertebrate messenger RNA is discussed in relation to the extreme rarity of the base doublet CpG in vertebrate DNA.     

Primary structure of the hemoglobin beta-chain of rose-ringed parakeet (Psittacula krameri)

The primary structure of Rose-ringed Parakeet hemoglobin -chain was established, completing the analysis of this hemoglobin. Comparisons with other avian -chains show variations smaller than those for the corresponding -chains. There are 11 amino acid exchanges in relationship to the only other characterized psittaciform -chain, and a total of 35 positions are affected by differences among all avian -chains analyzed (versus 61 for the -chains). At three positions, the Psittacula -chain has residues unique to this species. Three ₁₁ contacts are modified, by substitutions at positions 51, 116, and 125.     

Circadian changes in protein-synthesis rate and protein phosphorylation in cell-free extracts of Gonyaulax polyedra

The polysomal pattern of the dinoflagellate Gonyaulax polyedra, cultured under constant conditions, demonstrates a circadian rhythm. The relative amount of polysomes increases during the phase corresponding to the previous night period (=subjective night phase) when the rate of protein synthesis reaches its maximum (Cornelius et al., 1985, Planta 160, 365–370). Cell-free extracts were isolated at different circadian phase. The rate of protein synthesis in the extracts changed rhythmically in the same manner as the rate of protein synthesis in vivo. Substances in the postribosomal supernatants influenced the protein-synthesis rate of the cell-free system, depending on the phase when they were isolated: night factors stimulated protein synthesis in day extracts whereas day factors inhibited protein synthesis in night extracts. These effects were abolished by heating the postribosomal supernatant. In-vitro phosphorylation in parallel probes showed changes in the pattern of phosphorylated proteins. Phosphorylation of one of the proteins (95 kDa) was decreased after addition of night factor(s) and increased after addition of day factor(s). Cyclic-AMP enhanced the rates of protein synthesis and phosphorylation in the day extracts.Abbreviations cAMP cyclic-AMP - CT circadian time - D (N) subjective day (night)phase     

Single amino acid substitutions producing instability of globular proteins. Calculation of their frequencies in the entire mutational spectra of the α- and β-subunits of human hemoglobin

Summary The frequencies of substitutions resulting in protein instability were calculated by a method estimating changes in stability produced by amino acid substitutions. The method takes into account the accessibility of an amino acid position to a solvent and changes in the specificity of amino acid interactions. When tested on human mutant hemoglobins, the method yielded predictions with a preciseness of 80%. The consideration of the evolutionary homologous proteins in the analysis allowed us to estimate the evolutionary constraints imposed on stability of their spatial structure. With these limitations, approximately 50% of amino acid substitutions in the entire mutational spectra of the - and -subunits of human hemoglobin were found to damage the spatial structure of the globular proteins.     

Testis-specific β2 tubulins are identical in Drosophila melanogaster and D. hydei but differ from the ubiquitous β1 tubulin

In Drosophila as in many organisms tubulins are encoded by a gene family. We have determined the complete nucleotide sequences coding for the 1 and 2 tubulins of Drosophila melanogaster and the 2 tubulin of D. hydei, and found these insect tubulins to be highly conserved and like tubulins of other organisms. This is discussed with reference to the possible functional domains of these proteins. — The 1 tubulin gene of Drosophila is constitutively expressed, whereas the 2 tubulin is expressed specifically in the testes. In D. melanogaster the amino acid sequences of these proteins are 95% homologous, differing at only 25 positions. In the testes the 2 tubulin participates in different microtubules as shown by genetic analysis (Kemphues et al. 1982). Interestingly, all of the amino acids characteristic of the testis-specific 2 tubulin are also present in the corresponding gene of D. hydei. Of special interest is the high degree of conservation of the carboxy-terminal domain in these functionally equivalent tubulins.     

Primary structure of hemoglobin β-chain fromColumba livia (gray wild pigeon)

Primary structure of -chain of pigeon is presented. It was determined by amino acid sequence analysis of intact -chain and its peptides obtained by the enzymatic and chemical cleavage. Comparison of amino acid sequence of the chain with other available data shows 14 Ile, 61 Lys, and 113 Ile as residues specific to pigeon. One important replacement at ₁₁ contact is 55 MetSer.     

The protein sequence homology of gamma-crystallins among major vertebrate classes and their DNA sequence homology to heat-shock protein genes

A systematic characterization of lens crystallins from five major classes of vertebrates was carried out by exclusion gel filtration, cation-exchange chromatography and N-terminal sequence determination. All crystallin fractions except that of -crystallin were found to be N-terminally blocked. -Crystallin is present in major classes of vertebrates except the bird, showing none, or decreased amounts, of this protein in chicken and duck lenses, respectively. N-Terminal sequence analysis of the purified -crystallin polypeptides showed extensive homology between different classes of vertebrates, supporting the close relatedness of this family of crystallin even from the evolutionarily distant species. Comparison of nucleotide sequences and their predicted amino acid sequences between -crystallins of carp and rat lenses and heat-shock proteins demonstrated partial sequence homology of the encoded polypeptides and striking homology at the gene level. The unexpected strong homology of complementary DNA (cDNA) lies in the regions coding for 40 N-terminal residues of carp -II, rat 2-1, and the middle segments of 23,000- and 70,000-M _r heat-shock proteins. The optimal alignment of DNA sequences along these two segments shows about 50% homology. The percentage of protein sequence identity for the corresponding aligned segments is only 20%. The weak sequence homology at the protein level is also found between the invertebrate squid crystallin and rat -crystallin polypeptides. These results pointed to the possibility of unifying three major classes of vertebrate crystallins into one // superfamily and corroborated the previous supposition that the existing crystallins in the animal kingdom are probably mutually interrelated, sharing a common ancestry.     

Beta-tubulins are encoded by at least four genes in the brown algaEctocarpus variabilis

Complementary DNA clones of two mRNA species that encode -tubulin in the brown algaEctocarpus variabilis have been isolated. Sequence analysis revealed that the encoded proteins are very similar in primary structure to homologues in other eukaryotes, and differ from each other at six of 447 amino acid residues. The 6 message shows a preference for C-or G-terminated codons, using only 49 codons. The 5 message has a lesser codon bias, and makes a minor contribution to the -tubulin mRNA pool. Southern analysis ofE. variabilis DNA demonstrated a -tubulin gene family of at least four members.     

Identification of amino acids involved in the binding of hMIP-1α to CC-CKR1, a MIP-lα receptor found on neutrophils

Human macrophage inflammatory protein-1 (hMIP-1) and human macrophage inflammatory protein-1 (hMIP-1) are chemokines involved in a diverse range of immunological effects. Both hMIP-1 and hMIP-1 are involved in the activation of monocytes and THP-1 cells probably through a common receptor(s). However, only hMIP-1 can bind to neutrophils with high affinity, presumably through CC-CKR1 (CKR1). Since the structure of these two proteins is highly conserved, non-conserved amino acids must define the disparate binding patterns that these two proteins exhibit. Measurements of binding, chemotaxis and calcium influx conducted with hMIP-1 and hMIP-1 chimeric proteins and mutants show that two amino acids (37K and 43L) are important in the binding and signaling of hMIP-1 through CKR1. Furthermore, we also show that mutations of the three charged amino acids at the C-terminus of hMIP-1 and hMIP-1 (amino acids 61, 65 and 67), do not adversely affect the binding to THP-1 cells.     

New genetic variants identified in donkey's milk whey proteins

Novel genetic variants for donkey milk lysozyme and -lactoglobulins I and II have been identified by the combined use of peptide mass mapping and sequencing by tandem mass spectrometry in association with database searching. The novel donkey lysozyme variant designated as lysozyme B (Mr 14,631 Da) differed in three amino acid exchanges, N⁴⁹ D, Y⁵² S, and S⁶¹ N, from the previously published sequence. Three novel genetic variants for donkey -lactoglobulins were identified. One of them is a type -lactoglobulin I with three amino acid exchanges at E³⁶ S, S⁹⁷ T, and V¹⁵⁰ I (-lactoglobulin I B, Mr 18,510 Da). The two others are type -lactoglobulins II with two amino acid exchanges at C¹¹⁰ P and M¹¹⁸ T (-lactoglobulin II B, Mr 18,227 Da) and with three amino acid exchanges at D⁹⁶ E, C¹¹⁰ P, and M¹¹⁸ T (-lactoglobulin II C, Mr 18,241 Da). All these primary structures are closely related to those of homologous proteins in horse milk (percent identity >96%).     

Peptide insertions in β-galactosidase activating interface can improve binding in TPEG-Sepharose affinity chromatography

Summary Chimeric -galactosidase fusion proteins containing foreign peptides inserted either at the amino terminus or at inner sites have been studied regarding their purification properties. Whereas fusions at the amino terminal are retained less on TPEG-Sepharose columns than native -galactosidase, the insertion in a specific site of the activating interface increases the binding of the modified -galactosidase. This offers a way to construct more powerful -galactosidase purification tags.     

Differential accumulation of four phaseolin glycoforms in transgenic tobacco

An intron-less phaseolin gene [15] was used to express phaseolin polypeptides in transgenic tobacco plants. The corresponding amounts of phaseolin immunoreactive polypeptides and mRNA were similar to those found in plants transformed with a bean genomic DNA sequence that encodes an identical -phaseolin subunit. These results justified the use of the intron-less gene for engineering of the phaseolin protein by oligonucleotide-directed mutagenesis. Each and both of the two Asn residues that serve as glycan acceptors in wild-type phaseolin were modified to prevent N-linked glycosylation. Wild-type (wt^i–) and mutant phaseolin glycoforms (dgly ₁, dgly ₂ and dgly _1,2) were localized to the protein body matrix by immunogold microscopy. Although quantitative slot-blot hybridization analysis showed similar levels of phaseolin mRNA in transgenic seed derived from all constructs, seed from the dgly ₁ and dgly ₂ mutations contained only 41% and 73% of that expressed from the wild-type control; even less (23%) was present in seed of plants transformed with the phaseolin dgly _1,2 gene. Additionally, the profile of 25–29 kDa processed peptides was different for each of the glycoforms, indicating that processing of the full-length phaseolin polypeptides was modified. Thus, although targeting of phaseolin to the protein body was not eliminated by removal of the glycan side-chains, decreased accumulation and stability of the full-length phaseolin protein in transgenic tobacco seed were evident.Abbreviations bp base pair(s) - DAF days after flowering - GUS -glucuronidase - kb kilobase - kDa kilodalton     

Transformation of some hydroxy amino acids to other amino acids

It has been observed that -hydroxy--amino acids are transformed into other amino acids, when heated in dilute solutions with phosphorous acid, phosphoric acid or their ammonium salts. It has been shown that as in the case of previously reported glycine-aldehyde reactions, glycine also reacts with acetone to give -hydroxyvaline under prebiologically feasible conditions. It is suggested, therefore, that the formation of -hydroxy--amino acids and their transformation to other amino acids may have been a pathway for the synthesis of amino acids under primitive earth conditions.     

Design of superactive and selective integrin receptor antagonists containing the RGD sequence

Summary Integrins play a major role in cell-cell and cell-matrix interactions. The majority of the different types of integrins recognize the tripeptide sequence arginine-glycine-aspartic acid (RGD). To explore the spatial requirements of the pharmacophore for receptor selectivity and high activity, a new procedure, spatial screening, was used. The procedure is based on the experience that the conformation of small cyclic peptides is mainly determined by the chirality of the amino acids (and glycine or proline). For example, cyclic pentapeptides with one d and four l amino acids prefer a II'/ conformation. The sequence RGDFV was shifted around this spatial II'/ template by synthesis of five peptides in which one of the amino acids was used in d-configuration. It turned out that cyclo(-RGDfV-) is a selective inhibitor for the _v3 integrin, which is strongly expressed in cancer cells. Systematic variations with different turn mimetics, retro-inverso structures, modified peptide bonds and sugar amino acids are discussed.     

Lokalisierung von Aminos?ureaustauschen bei Nitritmutanten des Tabakmosaikvirus

Summary The positions of amino acid exchanges within eleven mutants of tobacco mosaic virus (TMV) isolated after treatment with nitrous acid have been localized. The protein chemical results are given in Tables 1–11 in detail and summarized in Table 12.In the discussion the localized amino acid exchanges of 36 mutants which arose spontaneously or were isolated after nitrite treatment or fluorouracil incorporation and whose primary protein structure have been described in this and earlier papers were compared with the current codon dictionary. All of the amino acid exchanges can be explained by the postulation that alteration of only one of the three nucleotides within a codon (Table 13) has occurred. Of the 26 amino acid exchanges localized in mutants isolated after treatment with nitrous acid all but two (isoleucinmethionine and glutamic acidaspartic acid) are compatible with the nucleotide alterations CU and AG postulated for the action of nitrous acid on TMV-RNA.The distribution of the localized amino acid exchanges along the protein chain of TMV is shown in Fig. 1.

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Herrn Prof.G. Melchers zum 60. Geburtstag gewidmet.     

Structure of the cDNA coding for conglutin γ, a sulphur-rich protein from Lupinus angustifolius

The sequence of cDNA coding for a sulphur-rich storage protein from Lupinus angustifolius L., conglutin , was determined. The coding region contained an N-terminal leader peptide of 28 amino acids which directly preceded subunits of M _r 28 239 and 16 517. Extensive sequence homology between the protein encoded by conglutin cDNA and basic 7S globulin from soybean was observed. Sequence homology to proteins from other classes of storage proteins, 11S, 7S and 2S, was limited to short and highly fragmented sequences. The amino acid sequence, Asn-Gly-Leu-Glu-Glu-Thr, characteristic of the primary site for post-translational cleavage of the precursors of 11S proteins, was absent from the sequence predicted for prepro-conglutin . It is concluded that conglutin is a representative of a fourth type of storage protein in legumes, distinct from the 11S, 7S and 2S storage protein families.     

Aldimine to ketoamine isomerization (Amadori rearrangement) potential at the individual nonenzymic glycation sites of hemoglobin a: Preferential inhibition of glycation by nucleophiles at sites of low isomerization potential

The relative roles of the two structural aspects of nonenzymic glycation sites of hemoglobin A, namely the ease with which the amino groups could form the aldimine adducts and the propensity of the microenvironments of the respective aldimines to facilitate the Amadori rearrangement, in dictating the site selectivity of nonenzymic glycation with aldotriose has been investigated. The chemical reactivity of the amino groups of hemoglobin A forin vitro reductive glycation with aldotriose is distinct from that in the nonreductive mode. The reactivity of amino groups of hemoglobin A toward reductive glycation (i.e., propensity for aldimine formation) decreases in the order Val-1(), Val-1(), Lys-66(), Lys-61(), and Lys-16(). The overall reactivity of hemoglobin A toward nonreductive glycation decreased in the order Lys-16(), Val-1(), Lys-66(), Lys-82(), Lys-61(), and Val-1(). Since the aldimine is the common intermediate for both the reductive and nonreductive modification, the differential selectivity of protein for the two modes of glycation is clearly a reflection of the propensity of the microenvironments of nonenzymic glycation sites to facilitate the isomerization reaction (i.e., Amadori rearrangement). A semiquantitative estimate of this propensity of the microenvironment of the nonenzymic glycation sites has been obtained by comparing the nonreductive (nonenzymic) and reductive modification at individual glycation sites. The microenvironment of Lys-16() is very efficient in facilitating the rearrangement and the relative efficiency decreases in the order Lys-16(), Lys-82(), Lys-66(), Lys-61(), Val-1(), and Val-1(). The propensity of the microenvironment of Lys-16() to facilitate the Amadori rearrangement of the aldimine is about three orders of magnitude higher than that of Val-1() and is about 50 times higher than that of Val-1(). The extent of nonenzymic glycation at the individual sites is modulated by various factors, such as thepH, concentration of aldotriose, and the concentration of the protein. The nucleophiles—such as tris, glycine ethyl ester, and amino guanidine—inhibit the glycation by trapping the aldotriose. The nonenzymic glycation inhibitory power of nucleophile is directly related to its propensity to form aldimine. Thus, the extent of inhibition of nonenzymic glycation at a given site by a nucleophile directly reflects the relative role ofpK _a of the site in dictating the glycation at that site. The nonenzymic glycation of an amino group of a protein is an additive/synergestic consequence of the propensity of the site to form aldimine adducts on one hand, and the propensity of its microenvironment to facilitate the isomerization of the aldimines to ketoamines on the other. The isomerization potential of microenvironment plays the dominant role in dictating the site specificity of the nonenzymic glycation of proteins.