A General Method Applicable to the Search for Similarities in the Amino Acid Sequence of Two Proteins

Phosphatidylinositol (Ptd1ns)-glycan-specific phospholipase D was purified from bovine and human serum by phase separation in Triton X-114 and by chromatography on DEAE-cellulose, octylSepharose, concanavalin-A -Sepharose, and hydroxyapatite. The purification of the two enzymes was approximately 1200-fold with a recovery of 3-5%. Bovine serum contained about 40 pgglml of PtdIns-glycan-specific phospholipase D, about 10 times more than the amount determined in human serum. PtdIns-glycan-specific phospholipase D is also present in mammalian cerebrospinal fluid and in mammalian milk but to a much lesser extent than in serum. Enzyme from bovine and human serum displayed amphiphilic properties as revealed by sucrose density gradient centrifugation and gel filtration in the absence and presence of detergent. On density gradient centrifugation, both enzymes sedimented with an apparent sedimentation coefficient of about 6.0 S in the presence of 0.1% Triton X-100, and formed aggregates up to 14.5 S in the absence of detergent. Upon gel filtration, the bovine and human enzymes migrated with a Stokes' radius of 6.5 nm and 6.6 nm, respectively, in the presence of Triton X-100. In the absence of Triton X-100, both enzymes gave a Stokes' radius of 8.8 nm. Serial centrifugation of serum at increasing NaBr concentrations revealed that the majority of the enzyme is contained in the high-density lipoprotein fraction. PtdIns-glycanspecific phospholipase D from bovine and human serum contained 27 and 28 N-acetylglucosamine residues, respectively. Treatment with N-glycosidase F decreased the apparent molecular mass of the bovine and human enzyme from 115 and 123 kDa to 91 and 87 kDa, respectively. Sequence analysis of peptides derived from PtdIns-glycan-specific phospholipase D of bovine serum by CNBr cleavage gave 100% identity to the sequence published for the bovine liver enzyme while there was 83% similarity and 74% identity to the sequence of peptides obtained from the human serum enzyme.

Section: N-glycosidase F-digestion Of Ptdins-glycan-specijic Phospholmentioning

confidence: 99%

Phosphatidylinositol‐glycan‐specific phospholipase D is an amphiphilic glycoprotein that in serum is associated with high‐density lipoproteins

Hoener

Brodbeck

1992

“…All unique pairs of repeats (3828 pairs) were optimally aligned by the Needleman-Wunsch [17] algorithm as programmed in the AMPS package [18] using amino acid substitution scores from Dayhoffs mutation data matrix 1978 (MDM78) [19] with a constant of 8 added, and a fixed-length gap-penalty of 8.…”

Section: Pairwise Sequence Comparison Of the 88 Repeatsmentioning

confidence: 99%

Amino acid sequence analysis of the annexin super‐gene family of proteins

Barton

Newman

Freemont³

et al. 1991

147

The annexins are a widespread family of calcium-dependent membrane-binding proteins. No common function has been identified for the family and, until recently, no crystallographic data existed for an annexin. In this paper we draw together 22 available annexin sequences consisting of 88 similar repeat units, and apply the techniques of multiple sequence alignment, pattern matching, secondary structure prediction and conservation analysis to the characterisation of the molecules. The analysis clearly shows that the repeats cluster into four distinct families and that greatest variation occurs within the repeat 3 units. Multiple alignment of the 88 repeats shows amino acids with conserved physicochemical properties at 22 positions, with only Gly at position 23 being absolutely conserved in all repeats. Secondary structure prediction techniques identify five conserved helices in each repeat unit and patterns of conserved hydrophobic amino acids are consistent with one face of a helix packing against the protein core in predicted helices a, c, d, e. Helix b is generally hydrophobic in all repeats, but contains a striking pattern of repeat-specific residue conservation at position 31, with Arg in repeats 4 and Glu in repeats 2, but unconserved amino acids in repeats 1 and 3. This suggests repeats 2 and 4 may interact via a buried saltbridge. The loop between predicted helices a and b of repeat 3 shows features distinct from the equivalent loop in repeats I, 2 and 4, suggesting an important structural and/or functional role for this region. No compelling evidence emerges from this study for uteroglobin and the annexins sharing similar tertiary structures, or for uteroglobin representing a derivative of a primordial one-repeat structure that underwent duplication to give the present day annexins. The analyses performed in this paper are re-evaluated in the Appendix, in the light of the recently published X-ray structure for human annexin V. The structure confirms most of the predictions and shows the power of techniques for the determination of tertiary structural information from the amino acid sequences of an aligned protein family.The annexins are a family of proteins that share the common features of binding both membranes and phospholipids in a Ca2 +-dependent manner. The annexins are found in many tissues, and their membrane binding activity has been claimed to be involved in cytoskeletal interactions [I] Abbreviations. AI-AX, annexins I-X; AI, annexin I; AII, annexin 11; AIII, annexin 111; AVI, annexin VI; AVII, annexin VII; AIX, annexin IX; AX, annexin X; AIH etc., human annexin I etc.; AIP etc., porcine annexin I etc.; AIR etc., rat annexin I etc.; AIIH etc., human annexin I1 etc.; AIIM etc., murine annexin I1 etc.; AIIB etc., bovine annexin 11 etc.; AIXD etc., Drosophila annexin IX etc.;AIH2 etc., human annexin I repeat 2; AIIxl, human, murine and bovine annexin I1 repeat 1 ; AIVx2, human, porcine and bovine annexin IV repeat 2; AIVx4, AIVH4, AIVP4 and AIVB4 together; AVIx58, human and murine annexin VI, from...

“…Cyanophora paradoxa (Evrard et al, 1990); Mca, Mycoplasma capricolum (Ohkubo et al, 1987); Ym, Saccharomyces cerevisiae, mitochondrial (Graack et al, 1992; YmL9 is the equivalent to the L3 protein family from yeast mitochondrial ribosomes); Yea, Saccharomyces cerevisiae, cytoplasm (Schultz and Friesen, 1983); Hma, Haloarcula marismortui (Orcutt et al, 1982). Percentage identities have been ascertained by the computer program GAP (Needleman and Wunsch, 1970). Bst, Bacillus stearothermophilus; Eco, Escherichia coli (Kimura and Wittmann-Liebold, 1980); Mca, Mycoplasma capricolum (Ohkubo et al, 1987); Yea, Saccharomyces cerevisiae, cytoplasm (Presutti et al, 1988; YeaLl is the equivalent to the L4 protein family from cytosolic yeast ribosomes); Hma, Haloarcula marismortui 1987).…”

Section: Gene Organizationmentioning

confidence: 99%

Primary structures of ribosomal proteins L3 and L4 from Bacillus stearothermophilus

Herwig¹,

Kruft²,

Wittmann‐Liebold³

1992

Ribosomal proteins L3 and L4 were purified to homogeneity from total protein isolated from the 50s subunit of Bacillus stearothermophilus by reversed-phase high-performance liquid chromatography (RP-HPLC). Amino acid sequences of both proteins were determined by automated N-terminal sequence analysis and sequencing of internal peptides. Using oligonucleotides deduced from the Nterminal region of protein L3 as hybridization probes, a DNA fragment coding for proteins L3, L4 and the N-terminal part of protein L23 has been identified, cloned and sequenced. The organization of the genes is identical to that found in the SIO operon of Escherichia coli. Comparison of the sequences of proteins L3 and L4 with those of other organisms revealed that all proteins of the L3 family are highly conserved. On the other hand, the archaebacterial L4 proteins show no significant sequence similarity to the E. coli L4 protein whereas the L4 protein of B. stearothermophilus is significantly similar to all of the L4 proteins and thus justifies the membership of all the L4 proteins in one protein family. The results are discussed with respect to the phylogenetic relationship between eubacteria, archaebacteria and eukaryotes and possible functional domains of proteins L3 and L4.