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N K Thomsen

Publications and source records attributed to N K Thomsen.

10 recordsLinked to original sources
PubMed1999

Structure and interactions of NCAM modules 1 and 2, basic elements in neural cell adhesion.

The structure in solution of the second Ig-module fragment of residues 117-208 of NCAM has been determined. Like the first Ig-module of residues 20-116, it belongs to the I set of the immunogloblin superfamily. Module 1 and module 2 interact weakly, and the binding sites of this interaction have been identified. The two-module fragment NCAM(20-208) is a stable dimer. Removal of the charged residues in these sites in NCAM(20-208) abolishes the dimerization. Modeling the dimer of NCAM(20-208) to fit the interactions of these charges produces one coherent binding site for the formation of two antiparallel strands of the first two NCAM modules. This mode of binding could be a major element in trans-cellular interactions in neural cell adhesion.

Amino Acid Substitution↗
PubMed1993

Low energy of activation for amide hydrogen exchange reactions in proteins supports a local unfolding model.

Hydrogen exchange reactions of amides in hen egg white lysozyme that are pH dependent and have a low energy of activation have been shown to be in accordance with a reaction mechanism in two steps, an equilibrium step and an exchange step. These results are not in agreement with the model, proposed by C.K. Woodward & B.D. Hilton, known as the penetration model. Therefore our results suggest that this model should be revised. The amide hydrogen/deuterium exchange rates in hen egg white lysozyme were measured at 4 degrees C, 10 degrees C, 15 degrees C and 25 degrees C at pH 7.0 by 1H nuclear magnetic resonance spectroscopy. Activation energies of the exchange reactions in the range from 20 kJ mol-1 to 333 kJ mol-1 were obtained for 32 of the 129 residues in the protein. The amides of lysozyme studied here could be divided into two groups, one group of amides are characterized by an observed amide exchange rate (ko) in the range 10(-4) to 10(-6) s-1, an equilibrium constant k1/k2 close to 10(-5), a low energy of activation (20 to 50 kJ mol-1) and a distance less than 6 A from solvent. The other group of amides are characterized by a ko less than 10(-6) s-1, a k1/k2 close to 10(-7), higher energies of activation (40 to 330 kJ mol-1) and a distance more than 4 A from solvent. In terms of structure the amides of the last group are from the core of the protein. They are typically involved in a hydrogen bond and form part of the secondary structure either as interior alpha-helices or central strands of beta-sheets. The first group consists of amides that are in the shell of the protein between the core and the surface. These amides are typically hydrogen bonded and involved in secondary structure such as external alpha-helices or outer strands of beta-sheets and turns.

Amides↗
PubMed1993

Determination of the rate constants k1 and k2 of the Linderström-Lang model for protein amide hydrogen exchange. A study of the individual amides in hen egg-white lysozyme.

The pH dependence of the amide/solvent hydrogen exchange of individual amide groups in hen egg-white lysozyme has been studied by nuclear magnetic resonance spectroscopy. Lysozyme has been used here as a model for a globular protein to re-examine the hypothesis for the amide/solvent hydrogen exchange reaction proposed by K. Linderstrøm-Lang and described in detail by Hvidt and Nielsen. The work has been focused on the most slowly exchanging amide at the temperature of 21 degrees C and in the pH range between 4 and 8. Exchange rates have been measured for 64 of the 126 amide protons and the pH dependence has been determined for 52 of these. The amides examined represent a sample that includes all the types of secondary structure and they are placed in the globular structure in a range of 3.2 A to 8.5 A from the closest water molecule on the surface. The measured exchange rates at pH 6 have been compared to these structural parameters and the results suggest that the rate constants are determined partly by the distance to the surface and partly by the type of secondary structure the amide is engaged in. Near the surface and in the very interior the distance to the surface seems to be rate-determining. Between the extremes the type of secondary structure is rate determining. The pH dependent exchange of the examined amides was shown to be in agreement with the Linderstrøm-Lang model. For each of the amides examined the rate constants for the opening and the closing reaction in the first reaction step of the Linderstrøm-Lang model has been calculated and compared to structural parameters.

Amides↗
PubMed1993

alpha-Macroglobulin domain structure studied by specific limited proteolysis.

Limited proteolysis was used to probe the domain structures of rat alpha 1-inhibitor 3, human pregnancy zone protein, and rat alpha 1-macroglobulin representing monomeric, dimeric, and tetrameric members, respectively, of the alpha-macroglobulin family. Specific limited digestion with trypsin, chymotrypsin, elastase, subtilisin, or Staphylococcus aureus V8 proteinase produced well-defined fragments as monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fragments were electroblotted to polyvinylidene difluoride membranes and subjected to NH2-terminal sequence analysis to locate the cleavage sites by comparison with the known primary structures of these proteins. Based on these results and the sizes of the generated fragments the 180-kDa alpha-macroglobulin subunit is proposed to contain seven relatively large domains resistant to proteolytic digestion, constituted by approximate residues 1-200 (I), 290-400 (II), 415-660 (III), 710-860 (IVa), 920-1160 (IVb), 1203-1305 (Va), and 1314-1451 (Vb) (human alpha 2-macroglobulin numbering). The overall domain organization is similar to that recently proposed by D. S. Rubenstein, J. J. Enghild, and S. V. Pizzo (1991, J. Biol. Chem. 266, 11252-11261) from studies of rat alpha 1-inhibitor 3, but the present results suggest that the large domains IV and V proposed by these authors are each composed of two domains. The present study emphasizes that domain Vb contains the determinants necessary for receptor recognition.

Amino Acid Sequence↗
PubMed1992

Interaction between bovine trypsin and a synthetic peptide containing 28 residues of the bait region of human alpha 2-macroglobulin.

The time course of the interaction between trypsin and a synthetic peptide corresponding to a segment (residues 676-703) of the bait region (residues 666-706) of human alpha 2-macroglobulin (alpha 2M) was studied by measuring the generation of cleavage products as a function of time by HPLC. Three primary cleavage sites for trypsin were present in the synthetic peptide. The fastest cleavage occurred at the bond corresponding to Arg696-Leu in alpha 2M with an estimated kcat/Km = 1-2 x 10(6) M-1.s-1. This value is of the same magnitude as that characterizing the interaction of alpha 2M and trypsin when taking into account the fact that alpha 2M is a tetramer, kcat/Km = 5 x 10(6) M-1.s-1 [Christensen, U. & Sottrup-Jensen, L. (1984) Biochemistry 23, 6619-6626]. The values of kcat/Km for cleavage at bonds corresponding to Arg681-Val and Arg692-Gly in alpha 2M were 1.5 x 10(5) M-1.s-1 and 1.3 x 10(5) M-1.s-1, respectively. Cleavage of intermediate product peptides was slower, with kcat/Km in the range 13-1.3 x 10(6) M-1.s-1. The value of Km determined for fast cleavage in the synthetic peptide was 8-10 microM. 1H-NMR spectroscopy indicated no ordered structure of the peptide. Hence, the very fast cleavage of the peptide is compatible with a loose structure that readily adopts a conformation favorable for recognition and cleavage by trypsin.

Amino Acid Sequence↗
PubMed1980

Influence of similarly flavored milk replacers and starters on calf starter consumption and growth.

One of three commercial feed flavors, butter, milk arome, or maple, was added to a milk replacer and a starter to determine if a flavor association by calves would increase starter consumption and growth. Eighty-five calves were randomized completely to 1 of 16 treatment combinations arranged in a 4 X 4 factorial experiment. Calves received milk replacer from day 6 to weaning at 5 wk. Starter was offered ad libitum from day 6 to 8 wk. Butter and milk arome flavor were added to milk replacer at 500 mg/kg and starter at 750 mg/kg. Maple was added to milk replacer at 990 mg/kg and starter at 1980 mg/kg., Data were subjected to analysis of covariance with sex, breed, date of birth, and birth weight as covariables. There was no effect of flavor in milk replacer on starter consumption or growth as compared to the control milk replacer. Calves fed maple starter consumed more starter to 8 wk and from 6 to 8 wk than did calves fed the control starter. Average daily gain to 5 wk and from 6 to 8 wk was greater for calves fed maple starter, and to 8 wk for calves fed either maple starter or milk arome starter than for calves fed the control starter. Least squares mean for starter consumption (kg) and average daily gain (g) to 8 wk for treatments control, butter, milk arome, and maple starters were 42.2, 367; 46.2, 427; 47.7, 431; and 48.8, 469. Starter consumption (kg) and average daily gain (g) to 8 wk for treatment combinations involving the same flavor were 45.2, 415 for control replacer-control starter; 45.4, 428 for butter replacer-butter starter; 48.9, 484 for milk arome replacer-milk arome starter; and 46.3, 427 for maple replacer-maple starter.

Animal Feed↗
PubMed1979

Mastitis history, California Mastitis Test, and somatic cell counts for identifying cows for treatment in a selective dry cow therapy program.

Mastitis history, California Mastitis Test scores, and filter-deoxyribonucleic acid cell counts from 232 cows were used to project the effectiveness of criteria which could be used to determine which cows to treat in a selective dry cow therapy program. Bacteriological analyses of quarter milk samples were used to identify infected cows. Effects of month, lactation parity, stage of lactation, and interaction of stage of lactation with lactation parity on somatic cell numbers in milk were determined. The various criteria singly or in combinations correctly identified from 50 to over 92% of the cows with one or more infected quarters at drying off. However, from 25 to 80% of the cows free of infection would have been treated as well. The most discriminating criteria were two monthly cell counts exceeding 500,000 cells per milliliter where 53% of the cows with infected quarters were identified correctly and only 25% of the uninfected cows would be treated, or the two monthly cell counts exceeding 500,000 cells per milliliter plus a California Mastitis Test score of +2 or +3 at drying off. Here 89% of the cows with infected quarters would be treated, and 56% of the uninfected cows would be treated.

Animals↗