Deduced Peptide Sequence Research Articles

The alpha 4(IV) chain of basement membrane collagen. Isolation of cDNAs encoding bovine alpha 4(IV) and comparison with other type IV collagens.

Renal basement membranes are believed to contain five distinct type IV collagens. An understanding of the specific roles of these collagens and the specificities of their interactions will be aided by knowledge of their comparative structures. Genes for alpha 1(IV), alpha 2(IV), alpha 3(IV), and alpha 5(IV) have been cloned and the deduced peptide sequences compared. A fifth chain, alpha 4(IV), has been identified in glomerular and other basement membranes. Using a polymerase chain reaction-based strategy and short known peptide sequences from the noncollagenous domain (NC1), we have cloned and characterized partial bovine cDNAs of alpha 4(IV). Sequence analysis shows that this molecule has characteristic features of type IV collagens including an NH2-terminal Gly-X-Y domain which is interrupted at several points and a COOH-terminal NC1 domain with 12 cysteine residues in positions identical to those of other type IV collagens. Within the NC1 domain bovine alpha 4(IV) has 70, 59, 58, and 53% amino acid identity with human alpha 2(IV), alpha 1(IV), alpha 5(IV), and alpha 3(IV), respectively. Alignment of the peptides also shows that alpha 4(IV) is most closely related to alpha 2(IV). Nevertheless, in the extreme COOH-terminal region of the NC1 domain there are structural features that are unique to alpha 4(IV). Cloning of the region of alpha 4(IV) that encodes the NC1 domain allows comparison of all five type IV collagens and highlights certain regions that are likely to be important in the specificities of NC1-NC1 interactions and in other discriminant functions of these molecules.

A residue substitution in phosphoribulokinase of Synechocystis PCC 6803 renders the mutant light-sensitive.

We have isolated a light-sensitive mutant (BRLS) of the photosynthetic cyanobacterium Synechocystis 6803 (S. 6803) that does not survive exposure to bright light: 70% of BRLS cells die upon exposure to light of greater than 3,000 lux for 2 h. A complementing DNA fragment from wild-type cells and the corresponding DNA from the BRLS cells have been cloned and sequenced. An open reading frame is found to encode phosphoribulokinase, a key enzyme in the enzyme system for photosynthetic carbon reduction (ES-PCR). The deduced peptide sequence of this enzyme is highly homologous to eukaryotic phosphoribulokinases but is not similar to known prokaryotic phosphoribulokinases. The mutation responsible for the phenotype of BRLS is a single nucleotide change that results in substitution of phenylalanine for Ser-222 in the phosphoribulokinase. The catalytic activity and the apparent affinity for ATP of the mutated kinase are about one-tenth and one-seventh those of the wild-type kinase, respectively. Furthermore, the mutated kinase is selectively degraded in BRLS cells in bright light. Degradation of the mutated kinase and cell death in bright light can be suppressed by inhibiting photosynthetic electron flow (PS-EF) with 3-(3,4-dichlorophenyl)-1,1-dimethylurea. The data indicate that PS-EF is not impeded by an impaired ES-PCR although the ES-PCR activity is controlled by the rate of PS-EF. Continued PS-EF in the absence of the normal substrates for carbon reduction appears to result in damage to cellular components essential for life or in the generation of lethal components.

The andropin gene and its product, a male-specific antibacterial peptide in Drosophila melanogaster.

In our study of the cecropin locus in Drosophila we have found a gene for a new peptide, andropin, with antibacterial properties. Transcripts from this gene, Anp, could be detected in newly eclosed males and reached steady-state levels after 1 day. Transcription was strongly induced in response to mating and is strictly confined to the ejaculatory duct of adult males. The deduced peptide sequence reveals a hydrophobic amino terminus with striking similarity to the signal peptide of the cecropins. The sequence of the predicted mature andropin shows no direct homology with the cecropins, but the two peptides may have similar secondary structures. We have synthesized the predicted gene product and shown it to be antibacterial. Crude extracts from male genital tracts show a potent bactericidal activity, and electrophoretic separation revealed at least three antibacterial components, one with the same mobility as the synthetic peptide. It appears that insects have evolved a mechanism for the protection of the seminal fluid and the male reproductive tract against microbial infections.

Complementary DNA sequence and deduced peptide sequence for CD59/MEM-43 antigen, the human homologue of murine lymphocyte antigen Ly-6C

Complementary DNA sequence and deduced peptide sequence for CD59/MEM-43 antigen, the human homologue of murine lymphocyte antigen Ly-6C

A C-terminal, calmodulin-like regulatory domain from the plasma membrane Ca2+-pumping ATPase.

A cDNA that encodes what appears to be the inhibitory domain of the plasma membrane calcium-pumping ATPase (Ca2+-ATPase) has been isolated by screening a lambda gt11 bovine brain cDNA library with antibodies prepared against the human erythrocyte membrane Ca2+-ATPase. This screening resulted in isolation of a bacteriophage containing a 1.5-kilobase cDNA insert encoding a 71-residue polypeptide, the remainder being a large 3' terminal noncoding region. A portion of this deduced peptide sequence was identical to that of a peptide isolated from a V8 protease digest of the human erythrocyte Ca2+-ATPase except for 1 residue. Antibodies purified by immunoabsorption to the fusion protein containing this cDNA-encoded polypeptide reacted only with those fragments of a limited trypsin digest of the human erythrocyte Ca2+-ATPase that contain the inhibitory domain. Moreover, these antibodies were able to partially stimulate basal enzyme activity and block further activation by calmodulin. The encoded polypeptide bears homology to the glutamic acid-rich regions N-terminal to the Ca2+-binding loops of calmodulin and to a lesser extent with the loops themselves. This encoded polypeptide also represents the C terminus of the Ca2+-ATPase. Portions of the isolated cDNA were homologous to the 3' noncoding region of the sarcoplasmic reticulum Ca2+-ATPase cDNA, indicating a possible mechanism for the evolution of these distinct membrane Ca2+ pumps.

Molecular cloning of a cDNA for the E1 alpha subunit of rat liver branched chain alpha-ketoacid dehydrogenase.

We have isolated a cDNA encoding the branched chain alpha-ketoacid dehydrogenase E1 alpha subunit. A rat liver lambda gt11 expression library was screened with antibody reactive with the 2-oxoisovalerate dehydrogenase (lipoamide) component. A positive clone, lambda BZ304, contains a 1.7-kilobase pair cDNA insert with a 1323-base pair open reading frame. Translation of the open reading frame predicts the 24 residues of the previously reported phosphorylation sites 1 and 2 for the bovine kidney and rabbit heart enzymes. The N-terminal sequence of purified E1 alpha was determined, and this sequence was found 40 residues from the beginning of the deduced peptide sequence. Northern blots of rat liver and muscle RNA demonstrate a single mRNA species of approximately 1.8 kilobase pairs in each tissue, suggesting that this cDNA is nearly full length.

Sequence homologies between type 1 and type 2A protein phosphatases.

The Mr = 33,000 catalytic fragment of rabbit skeletal muscle type 1 protein phosphatase was digested with trypsin after reduction and alkylation. The resulting peptides were isolated, subjected to automated Edman degradation, and their sequences compared to the deduced peptide sequence of the bovine type 2A protein phosphatase cDNA. Of 10 tryptic peptides from the type 1 phosphatase that were sequenced, nine showed a high degree of homology with the type 2A phosphatase. This provides the first direct sequence comparison suggesting that the type 1 and type 2 protein phosphatases, distinguished functionally by their substrate specificities and sensitivity to inhibitors, make up part of a family of closely related gene products with similar structures.