Refolding of trypsin-subtilisin inhibitor from marine turtle eggwhite

Trypsin-subtilisin inhibitor from marine turtle eggwhite refolded quantitatively from its fully reduced state atpH 8.5 in the presence of reduced and oxidized glutathione. The refolding process was studied by following the accompanying changes in inhibitory activity, fluorescence, sulfhydryl group titer, and hydrodynamic volume. The refolding process followed second-order kinetics with rate constants of 4.80×10² M^–1 sec^–1 for trypsin-inhibiting domain and 0.77× 10² M^–1 sec^–1 for subtilisin-inhibiting domain of the inhibitor at 30°C and their respective activation energies of the refolding process were 15.9 and 21.6 kcal/mol. Fluorescence intensity of the reduced inhibitor decreased with time of refolding until it corresponded to the intensity of the native inhibitor. The inhibitor contained 1–2%-helix, 40–42%-sheet, and 57–58% random coil structure. Refolded inhibitor gave a circular dichroic spectrum identical to that of the native inhibitor. A number of principal intermediates were detected as a function of the refolding time. Size-exclusion chromatography separated the intermediates differing in hydrodynamic volume (Stokes radius). The Stokes radius ranged from 23 Å (fully reduced inhibitor) to 18.8 Å (native inhibitor). Results indicated the independent refolding of two domains of the inhibitor and multiple pathways of folding were followed rather than an ordered sequential pathway.     

Determination of two sites of automethylation in bovine erythrocyte protein (d -aspartyl/l -isoaspartyl) carboxyl methyltransferase

Protein (d-aspartyl/l-isoaspartyl) carboxyl methyltransferase (PCM, E.C. 2.1.1.77) was previously shown to be enzymatically methyl esterified in an autocatalytic manner at altered aspartyl residues; methyl esters are observed in a subpopulation of the enzyme termed thePCM fraction [Lindquist and McFadden (1994),J. Protein Chem. 13, 23–30]. The altered aspartyl sites serving as methyl acceptors inPCM have now been localized by using proteolytic enzymes and chemical cleavage techniques in combination with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to identify fragments of the [³H]automethylated enzyme that contain a [³H]methyl ester. Methylation was positively identified at positions Asn₁₈₈ and Asp₂₁₇ in the enzyme sequence, a consequence of the spontaneous alteration of these sites tol-isoaspartyl ord-aspartyl sites and their methylation by active PCM molecules. The identification of more than one site of automethylation shows thatPCM is not a homogeneous population of damaged PCM molecules, but rather a complex population of molecules with a variety of age-altered damage sites.Abbreviations PCM protein (d-aspartyl/l-isoaspartyl) carboxyl methyltransferase - EDTA disodium ethylenediaminetetraacetate - PMSF phenylmethylsulfonyl fluoride - TEA trifluoroacetic acid - HPLC high-pressure liquid chromatography     

Computational sequence analysis of matrix metalloproteinases

Matrix metalloproteinases (MMP) play a cardinal role in the breakdown of extracellular matrix involved in a variety of biological and pathological processes. Research on MMPs has classified and characterized these enzymes according to their matrix substrate specificity, gene and protein domain structure, and regulation of activity and expression. However, the discovery of new MMPs has introduced a need for a more comprehensive and systematic method of classification and quantitative comparison of known and newly discovered members. This study compiles a sequence alignment, constructs a dendrogram, and calculates physical data and homology percentage assignments in order to obtain further insight into MMP structure-function relationships. Thorough analysis of MMP primary sequence domains, physical data patterns, and statistical analysis of sequence homology yields higher resolution in the similarities and differences that group MMP members.     

Structural characterization of genes corresponding to cotton fiber mRNA, E6: reduced E6 protein in transgenic plants by antisense gene

Two genes, each corresponding to fiber mRNA E6, were isolated from cotton cultivars Coker 312 (Gossypium hirsutum L.) and Sea Island (G. barbadense L.). E6 is one of the predominant fiber-specific mRNAs present during early fiber development. The distinguishing feature of the nucleotide-derived E6 protein is the presence of a motif where a dimer, Ser-Gly, is repeated several times. Two of the Sea Island genes contained a pentameric motif, Ser-Gly, while one of the Coker genes had one and the other had four motifs each. cDNA clones containing one or five Ser-Gly motifs were also identified. Thus, it appears that the strict conservation of this motif may not be critical to E6 protein function. Sequence characterizations of the genes and cDNAs showed that multiple members of the E6 family are transcribed in fiber and may result in proteins 238 to 246 amino acids long. The 3 ends of the genes and cDNAs showed considerable heterology among them. Transgenic plants containing antisense genes were generated to decipher E6 function. Transgenic cotton with reduced E6 protein levels in the range of 60 to 98% were identified. However, no discernible phenotypic changes in fiber development or properties were apparent. This result leads to the conclusion that E6 is not critical to the normal development or structural integrity of cotton fibers.     

Population genetics ofEulemur macaco macaco (Primates: Lemuridae) on the islands of Nosy-Be and Nosy-Komba and the Peninsula of Ambato (Madagascar)

Analysis for genetic variation of insular and mainland populations ofEulemur macaco has revealed: (1) a different degree of genetic variation between populations; and (2) the phylogenetic relationships between groups, on the islands of Nosy-Be and Nosy-Komba, and in the Peninsula of Ambato (Madagascar). Eleven systems of blood proteins from 157 animals were used as genetic markers. The genetic variation was lower on the island of Nosy-Komba than in the mainland of Ambato. This is consistent with the expectation that genetic variation is lower on islands than on mainlands. In contrast, the genetic variation on the island of Nosy-Be was the highest of the three populations. This finding can best be explained by assuming that the sample of Nosy-Be consists of individuals of several small isolated groups, where genetic drift computation showed the population of Nosy-Be to be distinct, and the populations of Nosy-Komba and Ambato to be close within the same branch of the dendrogram. These findings give an insight into the population history of the island of Nosy-Komba, which might have been populated by mainland groups from Ambato.     

Characterization of a calcium/calmodulin-dependent protein kinase homolog from maize roots showing light-regulated gravitropism

Roots of many species respond to gravity (gravitropism) and grow downward only if illuminated. This light-regulated root gravitropism is phytochrome-dependent, mediated by calcium, and inhibited by KN-93, a specific inhibitor of calcium/calmodulin-dependent protein kinase II (CaMK II). A cDNA encoding MCK1, a maize homolog of mammalian CaMK, has been isolated from roots of maize (Zea mays L.). The MCK1 gene is expressed in root tips, the site of perception for both light and gravity. Using the [³⁵S]CaM gel-overlay assay we showed that calmodulin-binding activity of the MCK1 is abolished by 50 M KN-93, but binding is not affected by 5 M KN-93, paralleling physiological findings that light-regulated root gravitropism is inhibited by 50 M KN-93, but not by 5 M KN-93. KN-93 inhibits light-regulated gravitropism by interrupting transduction of the light signal, not light perception, suggesting that MCK1 may play a role in transducing light. This is the first report suggesting a physiological function for a CaMK homolog in light signal transduction.Abbreviations CaM calmodulin - CaMK (II) Ca²⁺/calmodulin-dependent protein kinase (II) - CBP CaM-binding protein - CDPK Ca²⁺-dependent protein kinase - MCK1 maize homolog of mamalian CaMK This work is supported by the National Aeronautics and Space Administration grant No: NAGW 238.     

Automethylation of protein (d -aspartyl/l -isoaspartyl) carboxyl methyltransferase, a response to enzyme aging

A question that is central to understanding the mechanisms of aging and cellular deterioration is whether enzymes involved in recognition and metabolism of spontaneously damaged proteins are themselves damaged, either becoming substrates for their own activity; or being unable to act upon themselves, initiating cascades of cellular damage. We show here byin vitro experiments that protein (d-aspartyl/l-isoaspartyl) carboxyl methyltransferase (PCM) from bovine erythrocytes does methylate age-dependent amino acid damage in its own sequence. The subpopulation that is methylated, termed thePCM fraction, appears to be formed through age-dependent deamidation of an asparaginyl site to either anl-isoaspartyl ord-aspartyl site because (a) the stoichiometry of automethylation of purified PCM is less than 1%, a value typical of the substoichiometric methylation of many other aged protein substrates, (b)PCM is slightly more acidic than the bulk of PCM, and (c) the methyl esterified site inPCM has the characteristic base-lability of this type of methyl ester. Also, the methyl group is not incorporated into the enzyme as an active site intermediate because the incorporated methyl group is not chased onto substrate protein. The effect of enzyme dilution on the rate of the automethylation reaction is consistent with methylation occurring between protein molecules, showing that the pool of PCM is autocatalytic even though individual molecules may not be. The automethylation and possible self-repair of the PCM pool has implications for maintaining thein vivo efficiency of methylation-dependent protein repair.     

Sequence homology and structure predictions of the creatine kinase isoenzymes

Comparisons of the protein sequences and gene structures of the known creatine kinase isoenzymes and other guanidino kinases revealed high homology and were used to determine the evolutionary relationships of the various guamidino kinases. A CK framework is defined, consisting of the most conserved sequence blocks, and diagnostic boxes are identified which are characteristic for anyone creatine kinase isoenzyme (e.g. for vertebrate B-CK) and which may serve to distinguish this isoenzyme from all others (e.g. from M-CKs and Mi-CKs). Comparison of the guanidino kinases by near-UV and far-UV circular dichroism further indicates pronounced conservation of secondary structure as well as of aromatic amino acids that are involved in catalysis.Abbreviations GuaK guanidino kinase - CK creatine kinase - B-and M-CK brain and muscle cytosolic CK isoenzyme - Mi-CK mitochondrial CK isoenzyme - ArgK arginine kinase - Cr creatine - PCr phosphorylcreatine - PArg phosphorylarginine     

The complete nucleotide sequence and gene organization of carp (Cyprinus carpio) mitochondrial genome

The complete sequence of the carp mitochondrial genome of 16,575 base pairs has been determined. The carp mitochondrial genome encodes the same set of genes (13 proteins, 2 rRNAs, and 22 tRNAs) as do other vertebrate mitochondrial DNAs. Comparison of this teleostean mitochondrial genome with those of other vertebrates reveals a similar gene order and compact genomic organization. The codon usage of proteins of carp mitochondrial genome is similar to that of other vertebrates. The phylogenetic relationship for mitochondrial protein genes is more apparent than that for the mitochondrial tRNA and rRNA genes.Correspondence to: F. Huang