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Biomedical subjects

B A Cunningham

Publications and source records attributed to B A Cunningham.

At least 109 records · Page 6Linked to original sources
PubMed1980

Liver aldolase anomeric specificity.

Stopped-flow kinetic studies of liver aldolase and of mixed liver-muscle aldolase catalyzed reactions of fructose 1,6-bisphosphate (FBP) have been carried out and interpreted by computer simulation. These experiments indicate no utilization or binding of the alpha anomer by the liver enzyme unlike the findings for either the muscle aldolase which binds the alpha anomer nonproductively or the yeast aldolase which catalyzes its cleavage. Both beta-fructose 1,6-bisphosphate and its acyclic keto form may serve as substrates, necessitating the spontaneous anomerization of the alpha anomer before its utilization. Thus, liver aldolase cleaves 100% of the substrate present in the millisecond time scale because of the inability to bind alpha-FBP, allowing rapid spontaneous anomerization. This result fulfills earlier predictions of the differing specificities and substrate binding properties for aldolases from yeast, muscle, and liver.

Animals↗
PubMed1980

Structure of the heavy chain of the H-2Kk histocompatibility antigen.

We have used radiochemical techniques to characterize the heavy chain (Mr 46,000) of the murine H-2Kk histocompatibility antigen in terms of six fragments (I-VI) obtained after cleavage of the polypeptide chain with CNBr. The tentative order of the fragments, which account for more than 90% of the heavy chain, was assigned by radiochemical sequence analysis of the intact heavy chain and of each purified CNBr fragment and by analysis of the CNBr fragments obtained from the large papain fragment of the heavy chain. Treatment of cells with tunicamycin yielded H-2 molecules with heavy chains of molecular weight 40,000, suggesting that the carbohydrate moieties have a combined molecular weight of approximately 6000. CNBr cleavage of H-2Kk heavy chains labeled with [3H]fucose indicated that the carbohydrate moieties are located on fragments II and IV.. Incubation of cells with 32PO4 gave H-2 molecules with radioactive phosphoserine in the carboxyl-terminal CNBr fragment (VI) of the heavy chain and in the fraction containing beta 2-microglobulin. Sequence analysis of each CNBr fragment intrinsically labeled with 3H- and 35S-labeled amino acids identified a total of 87 residues in the H-2Kk heavy chain. The sequence closely resembles that of the H-2Kb molecule, and the 11 differences are scattered throughout the polypeptide chain. Comparison with HLA sequences indicates that the two allelic H-2 sequences are more closely related to each other (88% identity) than either is to the HLA-B7 or A2 antigens (approximately 70%). Similarly, the nonallelic HLA antigens are more closely related to each other (83%) than either is to the H-2Kk or H-2Kb molecules.

Amino Acid Sequence↗
PubMed1979

The chemical characterization of favin, a lectin isolated from Vicia faba.

We have determined the subunit structure of the glucose- and mannose-binding lectin favin, from Vicia faba. The molecule is composed of two nonidentical polypeptide chains held together by noncovalent interactions. We have determined the complete amino acid sequence of the smaller alpha chain (Mr = 5,571) and shown that it is homologous to the alpha chain of the lectins from lentil and pea and to residues 72 to 120 of concanavalin A (Con A). The larger beta chain (Mr = 20,000) contains carbohydrate and is homologous to the beta chain of lentil, pea, soybean, peanut, and red kidney bean lectins and is homologous to a portion of the Con A molecule beginning at residue 122. Favin also contains a minor component, beta' (Mr = 18,700), that closely resembles the beta chain but lacks carbohydrate and may, on the basis of apparent molecular weight, lack some part of the COOH-terminal region of the polypeptide chain. Although favin is similar to Con A, it, like the lentil and pea lectins, appears to lack residues corresponding to positions 1 to 71 of Con A. Because these residues contribute significantly to the carbohydrate binding site of Con A, the lack of this region in the otherwise homologous lectin favin suggests that the carbohydrate binding site of favin differs from that of Con A or that the region represented by residues 1 to 71 of Con A is located in a different portion (i.e. in the beta chain) of the favin molecule.

Amino Acid Sequence↗
PubMed1979

Investigation of the pre-steady-state kinetics of fructose bisphosphatase by employment of an indicator method.

The pre-steady-state kinetics for the hydrolysis of fructose 1,6-bisphosphate by rabbit liver fructose bis-phosphatase have been investigated by stopped-flow kinetics utilizing an acid-base indicator method that permits the continuous monitoring of the inorganic phosphate product. The reaction sequence is characterized by two successive first-order steps followed by establishment of the steady-state rate. The first exponential process results from a conformational change in the protein that is dye sensitive owing to a perturbation of an acidic residue on the protein. A second process reflects the rapid initial turnover of all four subunits of the enzyme with the concomitant release of inorganic phosphate followed by the rate-limiting step of the catalytic cycle. This latter step may involve a product release (fructose 6-phosphate) or a second conformational change. The catalytic cycle ends with decay of the enzyme to its initial unreactive resting state.

Animals↗
PubMed1979

Favin versus concanavalin A: Circularly permuted amino acid sequences.

We have determined the tentative amino acid sequence of the beta chain (M(r) 20,000) of the lectin favin. In previous studies, we have shown that the alpha chain (M(r) 5600) of this lectin is homologous to a region in the middle of the concanavalin A (Con A) sequence (residues 70-119). Now we present evidence that the beta chain is homologous to two discrete segments of Con A. The homology begins at residue 120 of Con A, extends to the COOH terminus (residue 237) and continues without interruption through the NH(2)-terminal 69 residues of Con A. Together, the alpha and beta chains of favin account for a polypeptide chain equivalent in size to that of Con A. The comparison of the two proteins thus reveals a circular permutation of extensive homologous sequences. The favin molecule contains residues identical to many of the residues postulated to be involved in sugar binding by Con A, and contains all of the direct metal ligands as well as residues homologous to most of the residues that form the beta-pleated sheets of Con A. These homologies suggest that the three-dimensional structures of the two lectins are likely to be very similar. Moreover, favin appears to be even more closely related in primary structure and sugar specificity to the lectins from pea and lentil, raising the possibility that all of these lectins may have structures that resemble Con A. Some of these similarities may also extend to the lectins from soybean, peanut, and red kidney bean, which have different sugar specificities but share sequence homologies with the favin beta chain.

Journal Article↗
PubMed1979

Cell-free synthesis and segregation of beta 2-microglobulin.

beta2-Microglobulin has been synthesized in vitro by using a rabbit reticulocyte lysate system and mRNA from the mouse tumor cell line EL4. The molecule is synthesized as a precursor with an NH2-terminal extension of 19 amino acids: Ser-X-Ser-Val-X-Leu-Val-Phe-Leu-Val-Leu-Val-Ser-Leu-X-Gly-Leu-Tyr-X. The processing and segregation of this peripheral membrane protein are directly comparable to those of secretory proteins and integral membrane proteins: addition of dog pancreas microsomal membranes during translation caused conversion to the processed chain, but addition of membranes after synthesis did not; only the processed chain sedimented with the membrane vesicles and was protected from proteolysis by the vesicles; and processing of nascent beta 2-microglobulin was blocked by competitive inhibitors that prevent processing and segregation of secretory and integral membrane proteins. These results suggest that the signal sequences of secretory proteins, integral membrane proteins, and peripheral membrane proteins have a common function and a common receptor on the cytoplasmic face of dog pancreas microsomal membranes. This system also provides a means for studying in vitro the expression and function of the major histocompatibility antigens that are associated with beta 2-microglobulin on cell surfaces.

Amino Acid Sequence↗
PubMed1977

Biochemical characterization of six trisomics of grain sorghum, Sorghum bicolor (L.) Moench.

To determine protein differences of grain sorghum disomics and trisomics, we analyzed leaf extracts from six trisomics and a disomic control by disc gel, gel isoelectric focusing, and SDS gel electrophoresis. Based on the number and position of protein bands revealed by Coomassie blue staining, the disomic control could be differentiated from the trisomics, and trisomics could be shown to differ among themselves in most cases. SDS gel revealed the most protein bands, followed by isoelectric focusing and disc gel. However, disc gel electrophoresis was the simplest technique of the three and was just as effective in identifying trisomics and differentiating trisomics from the disomic control.

Edible Grain↗
PubMed1977

Crystallographic studies of bovine beta2-microglobulin.

Crystals of the bovine milk protein lactollin yield x-ray diffraction data extending to a resolution of 2.8 A. Lactollin is a bovine analogue of beta2-microglobulin, a protein that is homologous in amino acid sequence to the constant domains of immunoglobulins and is the light chain of the human and murine major histocompatability antigens. The protein crystallizes in the orthorhombic space group P2(1)2(1)2(1) with a = 77.4, b = 47.9, and c = 34.3 A. The unit cell parameters and physical chemical solution studies indicate that the molecule exists in the crystal and in solution as a single polypeptide chain of 12,000 daltons.

Amino Acid Sequence↗
PubMed1976

Subunit structure, cell surface orientation, and partial amino-acid sequences of murine histocompatibility antigens.

Detergent and papain solubilized murine histocompatibility (H-2) antigens have been compared by gel exclusion chromatography, ultracentrifugation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and amino-acid sequence analysis. From these data, we propose a molecular model for the H-2 antigens that includes the size and arrangement of the subunits on the cell surface and in solution, and we provide evidence for the orientation of these molecules on the cell surface. Detergent solubilized H-2 antigens (molecular weight 116,000) consist of two disulfide-linked heavy chains (46,000 daltons) and two monocovalently associated light chains (12,000 daltons). Alkylation with iodoacetamide prior to extraction prevented the formation of a disulfide linkage between the two heavy chains. A water-soluble 51,000-dalton molecule (Fs) consisting of a 39,000-dalton fragment (FH) of the heavy chain and one intact light chain was obtained by papain digestion of cells or detergent extracts. Therefore, the disulfide linkage between the heavy chains is located in the remaining membrane-associated portion (Fm). Amino-acid sequence analysis of the FH fragment of H-2Kb by radiochemical techniques showed that it is identical to the detergent solubilized H-2Kb heavy chain in eight positions for the three amino acids tested. These data indicate that the fragment FH derives from the amino-terminus of the heavy chain and suggest that it projects outward from the cell surface, while the carboxyl-terminal region is associated with the plasma membrane. The described amino-terminal sequence data have been found constant in H-2Kb, H-2Kd, H-2Kk, H-2Db, and H-2Dd gene products. These data support the hypothesis that the K and D products of the major histocompatibility antigen complex have evolved by gene duplication.

Amino Acid Sequence↗
PubMed1976

Structure and significance of beta2-microglobulin.

beta2-Microglobulin shares many structural features with the homology regions of the immunoglobulins. Particularly significant is the fact that its amino acid sequence is homologous to the sequences of the constant regions of both classes of light chains (kappa and lambda) and to the constant homology regions of at least three classes (gamma, mu and epsilon) of heavy chains, especially the carboxyl-terminal regions Cgamma3 Cmu4 and Cepsilon4. Molecules similar to human beta2-microglobulin have been found in other vertebrate species. The properties of beta2-microglobulin suggest that the gene for this protein may have evolved from a precursor gene that by duplication gave rise to immunoglobulin light and heavy chains. Furthermore, the observation that beta2-microglobulin is synthesized by and appears on the surfaces of a variety of cell types, including nonlymphoid cells, suggests that the concepts derived from analysis of the immune system may be applicable to other areas of cell biology. In particular, the close association of this immunoglobulin-like molecule with the histocompatibility antigens has a number of implications for the origin, structure, and function of these as well as other cell surface glycoproteins.

Amino Acid Sequence↗
PubMed1975

Partial amino acid sequence of rabbit beta2-microglobulin.

The amino acid sequence of the first 35 residues of a low-molecular-weight protein obtained from the urine of rabbits treated with sodium dichromate was determined and shown to be identical with human beta2-microglobulin in 30 positions. Rabbit beta2-microglobulin, like the human protein, is strikingly homologous to the constant regions of rabbit immunoglobulin G, particularly the CH3 region.

Amino Acid Sequence↗
PubMed1975

The covalent and three-dimensional structural of concanavalin A. I. Amino acid sequence of cyanogen bromide fragments F1 and F2.

Concanavalin A is a lectin composed of identical subunits, each containg 237 amino acid residues. The complete amino acid sequence of the first 129 residues of the polypeptide chain has been determined by analysis of peptides obtained from digests of CNBr Fragments F1 (residues 1 to 42) and F2 (residues 40 to 129). Correlation of the chemical sequence with x-ray crystallographic results indicates that Fragment F1 contains all of the protein ligands for the binding of 2 metal ions, Mn2+ and Ca2+, and that Fragment F2 contains many of the residues involved in the interactions of the subunits to form dimers and tetramers. The site of cleavage of the polypeptide chain to yield the naturally occurring Fragments A1 and A2 has also been identified as the peptide bond between residues 118 and 119.

Amino Acid Sequence↗