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Biomedical subjects

F Wold

Publications and source records attributed to F Wold.

At least 55 records · Page 3Linked to original sources
PubMed1983

The amino acid sequence of Escherichia coli cyanase.

The amino acid sequence of the enzyme cyanase (cyanate hydrolase) from Escherichia coli has been determined by automatic Edman degradation of the intact protein and of its component peptides. The primary peptides used in the sequencing were produced by cyanogen bromide cleavage at the methionine residues, yielding 4 peptides plus free homoserine from the NH2-terminal methionine, and by trypsin cleavage at the 7 arginine residues after acetylation of the lysines. Secondary peptides required for overlaps and COOH-terminal sequences were produced by chymotrypsin or clostripain cleavage of some of the larger peptides. The complete sequence of the cyanase subunit consists of 156 amino acid residues (Mr 16,350). Based on the observation that the cysteine-containing peptide is obtained as a disulfide-linked dimer, it is proposed that the covalent structure of cyanase is made up of two subunits linked by a disulfide bond between the single cystine residue in each subunit. The native enzyme (Mr 150,000) then appears to be a complex of four or five such subunit dimers.

Amino Acid Sequence↗
PubMed1981

The isolation and characterization of a root lectin from soybean (Glycine max (L), cultivar Chippewa).

A lectin has been isolated from the roots of 5-day soybean (Glycine max (L) cultivar Chippewa) seedlings, and its properties have been compared to those of the soybean seed lectin. The sugar-binding activities of the two lectins, both in terms of specific hemagglutinating activity and sugar specificity, are indistinguishable. Molecular properties of the two lectins, measured as relative molecular weights, isoelectric and electrophoretic patterns, amino acid compositions, immunochemical cross-reactivity, and chromatographic behavior on Sepharose-concanavalin A adsorbents suggest that the seed and the root lectin are very similar but not identical. On the basis of these comparisons, we conclude that models regarding biological functions of soybean lectin derived from studies using the seed lectin can be extended to include the root lectin in this cultivar. Studies on the distribution of the lectin in the root tissue suggest that it is associated with the outer surface of the root and is concentrated in the segments of the root at which hair and early secondary roots are observed. Since this is the region at which Rhizobium binding occurs and at which nodulation probably is initiated, all the reported observations on the root lectin are consistent with its proposed role in the specific interaction of the developing soybean with its symbiont.

Acetylgalactosamine↗
PubMed1981

Detection of alpha-amylase activity in unprocessed preamylase produced in the cell-free translation of porcine pancreatic RNA.

Preamylases, synthesized in the RNA-dependent rabbit reticulocyte lysate translation system supplemented with porcine pancreatic RNA were identified by their specific immunoprecipitation with anti-amylase. The preamylases have apparent Mr = 55,000 and 58,000 as compared to 52,000 and 55,000 for the purified, secreted alpha-amylase isozymes. In order to establish whether the unprocessed precursors may assume enzymatically active conformations, we have explored a highly sensitive activity gel electrophoresis technique, by which picogram quantities of enzyme can be detected. When standard alpha-amylase and translation products are subjected to electrophoresis on polyacrylamide gel containing 0.01% starch and CaCl2, active amylase which binds tightly to starch can only migrate as the starch is hydrolyzed. When the gel is subsequently stained with I2, the appearance of clear tracks, the lengths of which are roughly proportional to the logarithm of amylase concentration, signifies the presence of amylase activity. By this approach, we were able to detect amylase activity in a range corresponding to about 100 pg of pure amylase/10 microliters of translation mixture. This value agrees well with an estimate from radioactivity incorporation of total preamylase in the translation mixture, and we consequently conclude that unprocessed preamylase can assume the appropriate conformation to give enzymatic activity.

Amylases↗
PubMed1981

The amino acid sequence of yeast enolase. Preparation and characterization of peptides produced by chemical and enzymatic fragmentation.

Yeast enolase was subjected to chemical and enzymatic fragmentation, and the individual peptides produced were isolated by gel filtration and ion exchange chromatography. The chemical fragmentation was achieved by cleavage at the single cysteine residue with 2-nitro-5-thiocyanobenzoic acid, or at the 5 methionine residues with cyanogen bromide. The assignment of the two 2-nitro-5-thiocyanobenzoic acid fragments to the NH2-terminal or COOH-terminal regions (designated C1 and C2, respectively) of the enolase subunit could be done unequivocally on the basis of NH2-terminal and COOH-terminal analysis, and the same was the case for the NH2-terminal and COOH-terminal cyanogen bromide peptides (designated M1 and M6, respectively). From a comparison of the CNBr peptides from enolase with those from Fragment C1, the identity of methionine peptides M4, of which only the NH2-terminal half is present in C1, and M5, which along with M6 is missing in C1, could also be established. The major enzymatic fragmentation was achieved by tryptic cleavage at the 14 arginine residues after acetylation of the lysine residues. Based on overlaps with methionine peptides, most of the arginine peptides could be ordered in proper sequence during the early phases of the work. Because of the size of several of the primary fragments, secondary cleavages were required for optimal sequencing data. These secondary cleavages were accomplished by digestion with Staphylococcus aureus protease, or by tryptic cleavage at cysteine after aminoethylation.

Amino Acid Sequence↗
PubMed1981

The amino acid sequence of yeast enolase.

Automatic sequencing of yeast enolase and of its chemically and enzymatically produced peptide fragments has established the sequence of 416 of the 436 residues in the enolase subunits. The missing segments have been provided from results from sequencing the DNA of the yeast enolase genes (Holland, M. J., Holland, J. P., Thill, G. P., and Jackson, K. A. (1981) J. Biol. Chem. 256, 1385-1395). The reported enolase sequence thus represents the results of two completely independent studies, which yielded identical results for 404 of the 436 residues, and which on re-examination are consistent with the reported sequence in all but nine positions. The availability of the entire yeast enolase sequence has permitted a reassessment of structure-function parameters available for the enzyme, and some implications of the sequence information on the secondary, tertiary, and quarternary structure and on the active site components of yeast enolase have been summarized and discussed.

Amino Acid Sequence↗
PubMed1979

Isolation and characterization of two homoserine dehydrogenases from maize suspension cultures.

Homoserine dehydrogenase in unpurified extracts of maize (Zea mays L.) cell suspensions is inhibited 73% by the feedback regulator threonine; the remaining 27% of the total activity is not affected even by high concentrations of threonine. The threonine-resistant and threonine-sensitive homoserine dehydrogenase activities were separated by affinity chromatography on Blue Sepharose columns, and the two distinct homoserine dehydrogenases were purified. The threonine-resistant enzyme is an Mr = 70,000 dimer of two Mr = 38,000 subunits and the threonine-sensitive enzyme is an Mr = 190,000 dimer containing two apparently different subunits with molecular weights of 89,000 and 93,000. The threonine-resistant enzyme exhibits normal Michaelis-Menten kinetics and its activity is not affected by any of the amino acid end products of the aspartate pathway. The threonine-sensitive enzyme exhibits positive cooperative kinetics with respect to NADPH and is inhibited by threonine and stimulated by isoleucine. All attempts to affect interconversion of the two purified enzymes have been unsuccessful. Because the purified enzymes correspond to activities present in crude extracts of various maize tissues, it is concluded that the two types of homoserine dehydrogenase are natural in vivo constituents of maize.

Alcohol Oxidoreductases↗
PubMed1977

Posttranslational covalent modification of proteins.

A search for derivatized amino acids in proteins has shown that the extent of posttranslational modification of proteins is quite substantial. While only 20 primary amino acids are specified in the genetic code and are involved as monomer building blocks in the assembly of the polypeptide chain, about 140 amino acids and amino acid derivatives have been identified as constituents of different proteins in different organisms. A brief consideration of the questions about where and when the derivatization reactions occur, how the specificity of the reactions is established, and how the posttranslational modifications can facilitate biological processes, reveal a need for more information on all these points. Answers to these questions should represent significant contributions to our understanding of biochemistry and cell biology.

Amino Acid Sequence↗
PubMed1977

Introduction of artificial crosslinks into proteins.

In this chapter, we present a brief overview of the current status of protein crosslinking technology. An attempt is made to compare the natural crosslinks and natural crosslinking agents to the artificial ones, and a brief section is devoted to the potential use of enzymes (transglutaminase and peroxidase) as crosslinking agents in vitro. Homobifunctional (x-R-x) and heterobifunctional (x-R-y) reagents are considered in terms of the kinds of functional groups and R-groups that have been used in protein crosslinking, and some examples of reagents and applications from the recent literature are tabulated.

Chemical Phenomena↗