The amino acid sequence of the beta subunit of allophycocyanin

The complete amino acid sequence of the beta subunit of Anabaena variabilis allophycocyanin is: H2N-Ala-Gln-Asp-Ala-Ile-Thr-Ala-Val-Ile-Asn-Ser-Ala-Asp-Val-Gln-Gly-Lys-Tyr-Leu-Asp-Thr-Ala-Ala-Leu-Glu-Lys-Leu-Lys-Ala-Tyr-Phe-Ser-Thr-Gly-Glu-Leu-Arg-Val-Arg-Ala-Ala-Thr-Thr-Ile-Ser-Ala-Asn-Ala-Ala-Ala-Ile-Val-Lys-Glu-Ala-Val-Ala-Lys-Ser-Leu-Leu-Tyr-Ser-Asp-Ile-Thr-Arg-Pro-Gly-Gly-Asn-Met-Tyr-Thr-Thr-Arg-Arg-Tyr-Ala-Ala-Cys-Ile-Arg-Asp-Leu-Asp-Tyr-Tyr-Leu-Arg-Tyr-Ala-Thr-Tyr-Ala-Met-Leu-Ala-Gly-Asp-Pro-Ser-Ile-Leu-Asp-Glu-Arg-Val-Leu-Asn-Gly-Leu-Lys-Glu-Thr-Tyr-Asn-Ser-Leu-Gly-Val-Pro-Val-Gly-Ala-Thr-Val-Gln-Ala-Ile-Gln-Ala-Ile-Lys-Glu-Val-Thr-Ala-Ser-Leu-Val-Gly-Ala-Asp-Ala-Gly-Lys-Glu-Met-Gly-Ile-Tyr-Leu-Asp-Tyr-Ile-Ser-Ser-Gly-Leu-Ser-COOH Phycocyanobilin is attached though a thioether linkage to cysteinyl residue 81, indicated by an asterisk. Comparison of this sequence with those of C-phycocyanins shows that there are 60 identities between corresponding subunits of these two biliproteins. Of the region between residues 79 and 120, 29 residues are identical in the beta subunits of allophycocyanin and phycocyanin. The character of all 10 charged residues in this region of the beta subunit sequences is completely conserved.     

Primary structures of MCP-1 and MCP-2, natural peptide antibiotics of rabbit lung macrophages

The microbicidal peptides, MCP-1 and MCP-2, of rabbit alveolar macrophages were purified by an improved procedure that employed preparative gel electrophoresis and high performance liquid chromatography. The peptides were arginine- and cystine-rich and lacked free sulfhydryl groups and detectable levels of carbohydrate. Complete sequence determinations revealed that MCP-1 differed from MCP-2 only by the substitution of arginine for leucine at residue 13 from the NH2 terminus and that the molecules were each single chain polypeptides of 33 amino acid residues containing three intramolecular disulfide bonds. The complete amino acid sequences of MCP-1 and MCP-2 are: (sequence in text)     

Phycoerythrin fluorescence-based assay for peroxy radicals: a screen for biologically relevant protective agents

Under the conditions of this assay, antioxidants that react rapidly with peroxy free radicals (e.g., ascorbate, vitamin E analogs, urate), protect phycoerythrin completely from damage by such radicals generated by thermal decomposition of 2,2'-azobis(2-amidinopropane); other compounds provide partial concentration-dependent protection. Change in phycoerythrin fluorescence emission with time provides a measure of the rate of free radical damage. The assay exploits the unusual reactivity of phycoerythrin toward these peroxy radicals. On a molar basis, phycoerythrin reacts with these radicals over 100-fold slower than do ascorbate or vitamin E analogs, but over 60-fold faster than other proteins. Applications of this assay to the estimation of the peroxy radical scavenging capacity of human plasma are described, and to the comparison of the scavenging properties of several proteins and of DNA, of vitamins and their derivatives, of catecholamine neurotransmitters, and of a variety of other low molecular weight biological compounds.     

Females of the sea urchin Strongylocentrotus purpuratus differ in the structures of their egg jelly sulfated fucans

The egg jelly coats of sea urchins contain sulfated fucans which bind to a sperm surface receptor glycoprotein to initiate the signal transduction events resulting in the sperm acrosome reaction. The acrosome reaction is an ion channel regulated exocytosis which is an obligatory event for sperm binding to, and fusion with, the egg. Approximately 90% of individual females of the sea urchin Strongylocentrotus purpuratus spawned eggs having only one of two possible sulfated fucan electrophoretic isotypes, a slow migrating (sulfated fucan I), or a fast migrating (sulfated fucan II) isotype. The remaining 10% of females spawned eggs having both sulfated fucan isotypes. The two sulfated fucan isotypes were purified from egg jelly coats and their structures determined by NMR spectroscopy and methylation analysis. Both sulfated fucans are linear polysaccharides composed of 1-->3-linked alpha-L-fucopyranosyl units. Sulfated fucan I is entirely sulfated at the O -2 position but with a heterogeneous sulfation pattern at O -4 position. Sulfated fucan II is composed of a regular repeating sequence of 3 residues, as follows: [3-alpha-L-Fuc p - 2,4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)- 1]n. Both purified sulfated fucans have approximately equal potency in inducing the sperm acrosome reaction. The significance of two structurally different sulfated fucans in the egg jelly coat of this species could relate to the finding that the sperm receptor protein which binds sulfated fucan contains two carbohydrate recognition modules of the C-type lectin variety which differ by 50% in their primary structure.      

Sequence variations in small-subunit ribosomal RNAs of Hartmannella vermiformis and their phylogenetic implications

Evidence of associations between free-living amoebas and human disease has been increasing in recent years. Knowledge about phylogenetic relationships that may be important for the understanding of pathogenicity in the genera involved is very limited at present. Consequently, we have begun to study these relationships and report here on the phylogeny of Hartmannella vermiformis, a free-living amoeba that can harbor the etiologic agent of Legionnaires' disease. Our analysis is based on studies of small-subunit ribosomal RNA genes (srDNA). Nucleotide sequences were determined for nuclear srDNA from three strains of H. vermiformis isolated from the United Kingdom, Germany, and the United States. These sequences then were compared with a sequence previously obtained for a North American isolate by J. H. Gunderson and M. L. Sogin. The four genes are 1,840 bp long, with an average GC content of 49.6%. Sequence differences among the strains range are 0.38%-0.76%. Variation occurs at 19 positions and includes 2 single-base indels plus 14 monotypic and 3 ditypic single-base substitutions. Variation is limited to eight helix/loop structures according to a current model for srRNA secondary structure. Parsimony, distance, and bootstrap analyses used to examine phylogenetic relationships between the srDNA sequences of H. vermiformis and other eukaryotes indicated that Hartmannella sequences were most closely related to those of Acanthamoeba and the alga Cryptomonas. All ditypic sites were consistent with a separation between European and North American strains of Hartmannella, but results of other tests of this relationship were statistically inconclusive.      

The amino acid sequence of fragment A, an enzymically active fragment of diphtheria toxin. III. The chymotryptic peptides, the peptides derived by cleavage at tryptophan residues, and the complete sequence of the protein.

Fragment A (21,145 daltons in its longest known form) may be derived from diphtheria toxin (60,000 daltons) by mild tryptic digestion and reduction. Purified Fragment A consists of a mixture of 3 molecules of 190, 192, and 193 residues; the first 190 residues are in common and correspond to the NH2-terminal region the toxin. All three species of Fragment A are active in catalyzing ADP ribosylation of elongation factor 2, an essential component of protein synthesis. This reaction inactivates the factor and is responsible for the toxin's action in inhibiting protein synthesis in animal cells. It is believed that Fragment A or similar enzymically active fragments released into the cytosol of toxin-treated cells mediate this inhibition. The complete amino acid sequence of Fragment A has been determined from 32 chymotryptic peptides, three peptides derived by chemical cleavage of Fragment A at its 2 tryptophan residues, five cyanogen bromide peptides, and six tryptic peptides from the maleylated protein.     

The amino acid sequence of fragment A, an enzymically active fragment of diphtheria toxin. II. The cyanogen bromide peptides.

Cyanogen bromide cleavage of Fragment A from diphtheria toxin at the four methionines present in each molecule resulted in five major peptides which were isolated and studied by sequence methods. These five peptides of 4, 11, 14, 63, and 101 residues account for all 193 residues in Fragment A and provide overlaps for the tryptic peptides from the maleylated protein. Two additional peptides were isolated and shown to be shorter forms (8 and 10 residues) of the COOH-terminal cyanogen bromide peptide (11 residues).     

Probing intramolecular orientations in rhodopsin and metarhodopsin II by polarized infrared difference spectroscopy.

The light-induced conformational changes of rhodopsin, which lead to the formation of the G-protein activating metarhodopsin II intermediate, are studied by polarized attenuated total reflectance infrared difference spectroscopy. Orientations of protein groups as well as the retinylidene chromophore were calculated from the linear dichroism of infrared difference bands. These bands correspond to changes in the vibrational modes of individual molecular groups that are structurally active during receptor activation, i.e., during the rhodopsin to metarhodopsin II transition. The orientation of the transition dipole moments of bands previously assigned to the carboxyl (C=O) groups of Asp83 and Glu113 has been determined. The orientation of specific groups in the retinylidene chromophore has been inferred from the dichroism of the bands associated with the polyene C-C, C=C, and hydrogen-out-of-plane vibrations. Interestingly, the use of polarized infrared light reveals several difference bands in the rhodopsin to metarhodopsin II difference spectrum which were previously undetected, e.g., at 1736 and 939 cm(-1). The latter is tentatively assigned to the hydrogen-out-of-plane mode of the HC(11)=C(12)H segment of the chromophore. Our data suggest a significant change in orientation of this group in the late phase of rhodopsin activation. On the basis of available site-directed mutagenesis data, bands at 1406, 1583, and 1736 cm(-1) are tentatively assigned to Glu134. The main features in the amide regions in the dichroic difference spectrum are discussed in terms of a slight reorientation of helical segments upon receptor activation.