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C S Craik

Publications and source records attributed to C S Craik.

137 records · Page 8Linked to original sources
PubMed1985

Redesigning trypsin: alteration of substrate specificity.

A general method for modifying eukaryotic genes by site-specific mutagenesis and subsequent expression in mammalian cells was developed to study the relation between structure and function of the proteolytic enzyme trypsin. Glycine residues at positions 216 and 226 in the binding cavity of trypsin were replaced by alanine residues, resulting in three trypsin mutants. Computer graphic analysis suggested that these substitutions would differentially affect arginine and lysine substrate binding of the enzyme. Although the mutant enzymes were reduced in catalytic rate, they showed enhanced substrate specificity relative to the native enzyme. This increased specificity was achieved by the unexpected differential effects on the catalytic activity toward arginine and lysine substrates. Mutants containing alanine at position 226 exhibited an altered conformation that may be converted to a trypsin-like structure upon binding of a substrate analog.

Amino Acid Sequence↗
PubMed1985

The exocrine protein trypsinogen is targeted into the secretory granules of an endocrine cell line: studies by gene transfer.

The exocrine protein rat anionic trypsinogen has been expressed and is secreted from the murine anterior pituitary tumor cell line AtT-20. We examined which secretory pathway trypsinogen takes to the surface of this endocrine-derived cell line. The "constitutive" pathway externalizes proteins rapidly and in the absence of an external stimulus. In the alternate, "regulated" pathway, proteins are stored in secretory granules until the cells are stimulated to secrete with 8-Br-cAMP. On the basis of indirect immunofluorescence localization, stimulation of release, and subcellular fractionation, we find that trypsinogen is targeted into the regulated secretory pathway in AtT-20 cells. In contrast, laminin, an endogenous secretory glycoprotein, is shown to be secreted constitutively. Thus it appears that the transport apparatus for the regulated secretory pathway in endocrine cells can recognize not only endocrine prohormones, but also the exocrine protein trypsinogen, which suggests that a similar sorting mechanism is used by endocrine and exocrine cells.

Adrenocorticotropic Hormone↗
PubMed1984

Structure of two related rat pancreatic trypsin genes.

A family of approximately 10 trypsin genes was detected in a rat genomic library by hybridization and in vivo recombination techniques using cloned rat pancreatic trypsin I and II cDNAs as probes. Two separate clones containing the entire trypsin I gene and most of the trypsin II gene were sequenced. Four introns split the trypsin I coding sequence. The positions of the first three introns of the trypsin II gene are identical with those in the trypsin I gene (the fourth intron was not present in the trypsin II clone). The coding regions of the two genes are 88% homologous; the 5'-noncoding regions are 92% homologous, whereas the 3'-noncoding regions share 66% identity. In contrast, the proximal 5'-flanking regions from -1 to -500 which may contain the elements controlling gene expression are less than 30% conserved overall, but segments of approximately 70% homology can be discerned in this region. Some of these sequences are homologous to sequences found in the chymotrypsin and elastase genes. More distal upstream sequences (-500 to -2500) and the intervening sequences show no evident sequence homology (less than 20%). Unique sequences containing homopolymeric purine/pyrimidine repeats are found 2.5 kilobases upstream from the start of transcription of the trypsin I gene and within the second and third introns of the trypsin II gene. The nucleotide homologies as well as the similarities of intron positions of the two trypsin genes to those of other serine protease genes clearly support an evolutionary relationship between members of this gene family.

Animals↗
PubMed1984

Structure of the two related elastase genes expressed in the rat pancreas.

We have isolated and characterized rat genomic DNA fragments bearing the two secretory elastase genes that are expressed in the exocrine pancreas. The complete exonic sequences for each of the genes as well as considerable intronic and flanking sequences are reported. Each elastase gene is interrupted by seven intervening sequences which are located at corresponding positions within the two genes, with one exception: the third intron of the elastase II gene has shifted one codon in the 5' direction. The placement of introns within the amino acid coding domains in part may reflect the formation of the progenitor serine protease gene by the duplication of an exon encoding a characteristic polypeptide structure comprising three beta sheets. The activation peptides of the zymogens and the signal peptides, which form discrete functional domains in the protein precursors, are encoded by separate exons. In addition to the TATAA box, the two genes share considerable sequence similarity in the 5'-proximal flanking regions (up to approximately 450 base pairs upstream); however, a number of gaps must be introduced to optimize the sequence alignment. The similarities are largely confined to six oligonucleotide regions with greater than 70% sequence conservation. The elastase I gene has a perfect repeating copolymer (GT)24 located 427-379 nucleotides upstream from the start of transcription. The elastase II gene has a similar GT-rich region (52/55 G or T) located 384-330 nucleotides upstream. Comparison of the 5'-flanking regions of the two elastase genes with those of pancreatic chymotrypsin and trypsin I and II reveals that one of the six conserved oligonucleotide regions is generally conserved for these genes as well. This conserved region contains putative enhancer core sequences.

Amino Acid Sequence↗
PubMed1984

Isolation and sequence of a rat chymotrypsin B gene.

A cDNA clone encoding part of chymotrypsin B was isolated from a cDNA library prepared from rat pancreatic mRNA and used as a probe to isolate the chymotrypsin B gene. The nucleotide sequence of this gene is presented. The 4709-base pair transcribed portion of the isolated gene was inferred from the cDNA and gene sequence, and the 5' border was determined by primer extension on pancreatic polyadenylated RNA. The coding portion of the gene is interrupted by six introns. The active site residues His 57, Asp 102, and Ser 195 are encoded by separate exons. Moreover, two regions of the enzyme which form the substrate-binding pocket are also encoded by separate exons. Thus, the substrate specificity and catalytic activity of the enzyme are produced by joining several exons encoding protein segments that are intrinsically catalytically inactive. The number and location of the intron/exon junctions of the chymotrypsin gene as compared to those of other serine protease genes, as well as the location of the genes on separate chromosomes, suggest that the duplication that resulted in the formation of the chymotrypsin gene was an ancient evolutionary event.

Amino Acid Sequence↗
PubMed1983

Splice junctions: association with variation in protein structure.

A comparison between eukaryotic gene sequences and protein sequences of homologous enzymes from bacterial and mammalian organisms shows that intron-exon junctions frequently coincide with variable surface loops of the protein structures. The altered surface structures can account for functional differences among the members of a family. Sliding of the intron-exon junctions may constitute one mechanism for generating length polymorphisms and divergent sequences found in protein families. Since intron-exon junctions map to protein surfaces, the alterations mediated by sliding of these junctions can be effected without disrupting the stability of the protein core.

Amino Acid Sequence↗
PubMed1982

Intron-exon splice junctions map at protein surfaces.

There have been several suggested explanations for the presence of noncoding intervening sequences in many eukaryotic structural genes. They may be examples of 'selfish DNA, conferring little phenotypic advantage, or they may have some importance in gene expression and/or evolution. It has been suggested that each exon (coding sequence) may represent a structural or functional unit of the encoded protein, for which there is good evidence in the case of immunoglobulin and haemoglobin genes. Exon modification, duplication and recombination may thus be general mechanisms for the rapid evolution of eukaryotic structural genes. In many cases, however, it is not apparent that an exon corresponds to some specific feature of the encoded protein. We describe here evidence that intron-exon junctions usually map to amino acid residues located at the protein surface, suggesting a restriction on the permitted positions of introns within a gene.

Amino Acids↗
PubMed1981

O2 binding properties of the product of the central exon of beta-globin gene.

The hypothesis that the exons of eukaryotic structural genes code for functional domains and that the partitioned arrangement of coding information may thus serve to mediate the rapid evolution of new and unique proteins from pre-existing exons is also supported by our recent studies which demonstrate that the product of the central exon of the human beta-globin gene is a complete functional domain capable of binding haem tightly and specifically. Moreover, an analysis of the structure/function changes induced by mutations in different parts of the haemoglobin molecule suggests that each of the three exon-encoded segments is primarily associated with different functions of haemoglobin (for example, haem-binding, haem-haem interaction). We have now extended our studies to determine whether the central fragment is sufficient for maintenance of a stable complex of ferrous haem with molecular oxygen and, if not, what are the minimal requirements for the expression of this activity. The results of our reconstitution experiments indicate that the isolated central exon peptide is unable to maintain a ferrous haem-dioxygen complex unless the side exon products and the complementary haem-containing subunit are present. A conformational change which accompanies the noncovalent association of fragments may account for the restoration of reversible oxygen binding.

Ferrous Compounds↗
PubMed1980

Characterization of globin domains: heme binding to the central exon product.

We have prepared and isolated the peptide fragments coded for by the three exons of the human beta-globin gene, using the arginine-specific protease clostripain (EC 3.4.22.8). The region encoded by the central exon (amino acid residues 31-104) contains an arginine at position 40. This site was less susceptible to cleavage than the two sites that correspond to the exon-intron boundaries, and the isolated central fragment was an approximately equimolar mixture of the entire central fragment, beta(o) (31-104), and the somewhat smaller fragment contained within it, beta(o) (41-104). This central fragment mixture bound heme stoichiometrically and tightly at micromolar concentrations, generating a strong Soret absorption band as well as a characteristic absorption band in the visible spectrum. The Soret band occurred at the same wavelength and had the same shape as in hemoglobin, exhibiting an intensity greater than (2/3) that achieved when native intact beta globin is reconstituted with heme. Nearly the full intensity was regained when an equivalent of heme was added to the unfractionated digest, suggesting that the noncovalently associated side fragments add precision to the fit of the heme pocket. Three controls were used in establishing the specificity of heme binding to the central fragment mixture. Similar, but preliminary, experiments have also been undertaken with alpha globin. A clostripain digest containing alpha(o) (1-31) and alpha(o) (32-141) bound heme, yielding a Soret band identical to that observed in alpha subunits reconstituted from the native globin chains and heme. Measurements of circular dichroism spectra as indices of secondary structure suggested a role for the side exon products in the acquisition of the native three-dimensional structure of hemoglobin. These experiments confirm a prediction of W. Gilbert that the product of the central exon of the globin gene is a complete functional domain that binds heme tightly and specifically.

Amino Acid Sequence↗
PubMedDate not supplied

Site-directed mutagenesis shows that tyrosine 248 of carboxypeptidase A does not play a crucial role in catalysis.

The residue Tyr 248 of carboxypeptidase A (CPA) is thought to play a role in catalysis by contributing a proton to the incipient amine anion generated during cleavage of peptide substrates. To test this hypothesis we have modified the rat CPA cDNA by site-directed mutagenesis so that the codon for Tyr 248 is replaced by that for Phe. Here, we report the expression of the cDNAs for proCPA and its Tyr-to-Phe variant in yeast via the alpha-factor system. Following zymogen activation by trypsin, wild-type CPA (CPA-WT) and variant CPA (CPA-Phe 248) were purified to homogeneity and characterized enzymatically. CPA-Phe 248 displays essentially undiminished values for the catalytic constant (kcat) towards various peptide and ester substrates. However, the Michaelis constants (Km values) of peptide substrates and the inhibition constant (Ki) of the potato carboxypeptidase inhibitor are increased 6-fold and 70-fold, respectively. These data suggest that the phenolic hydroxyl of Tyr 248 does not act as the requisite general acid catalyst but participates in ligand binding.

Amino Acid Sequence↗