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K Brew

Publications and source records attributed to K Brew.

At least 73 records · Page 4Linked to original sources
PubMed1986

Evidence for a thiol ester in duck ovostatin (ovomacroglobulin).

The structure and the mechanism for proteinase inhibition of the egg white protein ovostatin (ovomacroglobulin) are similar to those of plasma alpha 2-macroglobulin, but previous studies have shown that chicken ovostatin lacks a reactive thiol ester (Nagase, H., and Harris, E. D., Jr. (1983) J. Biol. Chem. 258, 7490-7498). Here we show that duck ovostatin has conserved such a thiol ester and is capable of inhibiting both metallo- and serine proteinases stoichiometrically. Evidence for thiol esters was established by the following results with duck ovostatin: 1) autolysis into fragments of Mr = 123,000 and 60,000 occurred by heating in sodium dodecyl sulfate, but was prevented by treatment with CH3NH2; 2) covalent linkages were formed with proteinases on complex formation; 3) reaction with CH3NH2 generated 3.6 SH groups/mol, and 3.9 mol of [14C]CH3NH2 were incorporated per mol of protein; and 4) saturation with a proteinase liberated 3.8 SH groups/mol of the inhibitor. Conformational rearrangement of duck ovostatin upon reacting with CH3NH2 or proteinases was demonstrated by an increased mobility of the protein in polyacrylamide gel electrophoresis. CH3NH2-treated duck ovostatin was able to bind and inhibit proteinases without forming covalent bonds, but, unlike unmodified ovostatin, its inhibitory activity was destroyed by freezing and thawing. Complexes formed between CH3NH2-treated duck ovostatin and a proteinase were not dissociable except under denaturing conditions. These results and other evidence indicate that covalent bond formation through reaction with a thiol ester is a separate process from the trapping and inhibition of proteinases by this family of proteins.

Amino Acid Sequence↗
PubMed1986

Composition of the milks of the bottlenose dolphin (Tursiops trucatus) and the Florida manatee (Trichechus manatus latirostris).

Milk samples from four individual bottlenose (Tursiops truncatus) and two Florida manatees (Trichechus manatus latirostris) of known lactation stages were analyzed for protein, carbohydrate and lipid composition, as well as for activity levels of alpha-lactalbumin, the regulatory protein of lactose synthase. The milk from both species had relatively high protein and lipid levels, as reported previously for other marine mammals. The major proportion of the lipid was in the form of triglycerides. Dolphin milk contained an average of 2.2% neutral sugars, which was essentially all in the form of lactose, as determined by several criteria. Manatee milk samples contained 0.6% of neutral sugars, and a larger proportion (about 2%) of amino sugars. Lactose was not detected by enzymatic assay or paper chromatography, but HPLC analysis indicated the presence of low levels of lactose together with two components that were tentatively identified as oligosaccharides. alpha-Lactalbumin activity, determined by assay with bovine galactosyltransferase, was found in both dolphin and manatee milk. The level in dolphin milk was comparable with that found in bovine and other milk, but the level in the manatee was less than 10% of that in the dolphin.

Animals↗
PubMed1986

Cloning and sequencing of cDNA of bovine N-acetylglucosamine (beta 1-4)galactosyltransferase.

Galactosyltransferases constitute a family of enzymes, each member of which transfers galactose from UDPgalactose to a specific acceptor molecule, generating a specific galactose-acceptor linkage. Two synthetic oligonucleotides, 27mer and 21mer, were synthesized, based on the amino acid sequences of two peptides derived from bovine milk N-acetylglucosaminide (beta 1-4)galactosyltransferase (EC 2.4.1.90), and used as hybridization probes to isolate cDNA clones for galactosyltransferase from a bovine mammary gland cDNA library. One of the plasmids, designated pLbGT-1, contains an insert of about 3.7 kilobases that hybridizes to both of the probes and encodes the amino acid sequences of five peptides obtained from bovine milk (beta 1-4)galactosyltransferase. A second plasmid, designated pLbGT-2, contains an insert of about 4.1 kilobases that hybridizes to only the 27mer and that encodes a polypeptide containing the sequence of the carboxyl-terminal 120 residues identical to the peptide encoded by pLbGT-1; the rest of the protein sequence, however, does not contain known sequences from bovine galactosyltransferase. The two cDNAs contain a 3'-untranslated region of about 2.7 kilobases that includes two copies of the Alu-equivalent sequences. pLbGT-1 and pLbGT-2 hybridize to mRNAs of various sizes obtained from the bovine and rat mammary gland and the human mammary tumor cell line MCF-7, with the longest mRNA from each species being around 4.5 kilobases. The results show that pLbGT-1 is a cDNA clone for bovine (beta 1-4)galactosyltransferase, and pLbGT-2 encodes a protein that is structurally and may be functionally related to transferases.

Amino Acid Sequence↗
PubMed1985

Differential trace labeling of calmodulin: investigation of binding sites and conformational states by individual lysine reactivities. Effects of beta-endorphin, trifluoperazine, and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid.

The Ca2+-dependent association of beta-endorphin and trifluoperazine with porcine testis calmodulin, as well as the effects of removing Ca2+ by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) treatment, were investigated by the procedure of differential kinetic labeling. This technique permitted determination of the relative rates of acylation of each of the epsilon-amino groups of the seven lysyl residues on calmodulin by [3H]acetic anhydride under the different conditions. In all cases, less than 0.52 mol of lysyl residue/mol of calmodulin was modified, thus ensuring that the labeling pattern reflects the microenvironments of these groups in the native protein. Lysines 75 and 94 were found to be the most reactive amino groups in Ca2+-saturated calmodulin. In the presence of Ca2+ and under conditions where beta-endorphin and calmodulin were present at a molar ratio of 2.5:1, the amino groups of lysines 75 and 148 were significantly reduced in reactivity compared to calmodulin alone. At equimolar concentrations of peptide and protein, essentially the same result was obtained except that the magnitudes of the perturbation of these two lysines were less pronounced. With trifluoperazine, at a molar ratio to calmodulin of 2.5:1, significant perturbations of lysines 75 and 148, as well as Lys 77, were also found. These results further substantiate previous observations of a commonality between phenothiazine and peptide binding sites on calmodulin. Lastly, an intriguing difference in Ca2+-mediated reactivities between lysines 75 and 77 of calmodulin is demonstrated. In the Ca2+-saturated form of the protein, both lysines are part of the long connecting helix between the two homologous halves of the protein (Babu, Y. S., Sack, J. S., Greenhough, T. G., Bugg, C. E., Means, A. R., and Cook, W. J. (1985) Nature 315, 37-40). Yet, Lys 75 increases in reactivity some 25-fold, compared to only a 2-fold change for Lys 77, in going from EGTA-treated to Ca2+-saturated calmodulin. Thus, the microenvironment of Lys 75 is markedly altered upon Ca2+ binding, and this linker region between the two globular lobes of the protein appears to be quite important in the interaction of calmodulin with inhibitory molecules and perhaps activatable enzymes.

Acetylation↗
PubMed1985

Homology of beta-lactoglobulin, serum retinol-binding protein, and protein HC.

The milk protein beta-lactoglobulin has been extensively studied but its function has not been identified. A clue regarding the function of a protein can be obtained by discovering a genetic relationship with a protein of known function through comparisons of amino acid sequence. Such comparisons revealed that beta-lactoglobulin is similar to human serum retinol-binding protein and to another human protein of unknown function known as complex-forming glycoprotein heterogeneous in charge (protein HC). beta-Lactoglobulins from several species have been found to bind retinol, while the absorption and fluorescence properties reported for the unidentified heterogeneous prosthetic group of protein HC are retinoid-like. The role of serum retinol-binding protein in vitamin A transport in the circulation suggests that the other two homologous proteins may function in the binding and transport of retinoids; beta-lactoglobulin may facilitate the absorption of vitamin A from milk and protein HC may mediate the excretion of retinol-derived metabolites.

Alpha-Globulins↗
PubMed1984

Evolution of alpha-lactalbumins. The complete amino acid sequence of the alpha-lactalbumin from a marsupial (Macropus rufogriseus) and corrections to regions of sequence in bovine and goat alpha-lactalbumins.

alpha-Lactalbumin was purified from a whey protein fraction of the milk of the red-necked wallaby (Macropus rufogriseus). The complete amino acid sequence was determined from the results of automatic sequenator analyses of the intact protein, the three cyanogen bromide fragments, and of peptides generated from the larger, COOH-terminal CNBr fragment by digestion with trypsin or staphylococcal protease. This is the first sequence to be determined of an alpha-lactalbumin from a marsupial and differs from known eutherian alpha-lactalbumins in size and locations of deletions in alignments with the homologous type c lysozymes, as well as in having amino acid substitutions at 8 sites that are invariant in known eutherian proteins. Some corrections are also reported for two regions of sequence in both bovine and goat alpha-lactalbumins. The new and previously published information on alpha-lactalbumin sequences is analyzed in relation to the evolutionary history of the alpha-lactalbumin line as well as the relationship of structure to function in these proteins.

Amino Acid Sequence↗
PubMed1983

Ovostatin: a novel proteinase inhibitor from chicken egg white. I. Purification, physicochemical properties, and tissue distribution of ovostatin.

A proteinase inhibitor which has strong anti-collagenase activity was found in chicken egg white. The inhibitor (pI = 4.9) was purified by poly(ethylene glycol) (5.5-10%) precipitation and chromatography on Ultrogel AcA 34, DEAE-cellulose, and Sephacryl S-300. The final product was homogeneous on 5% polyacrylamide gel electrophoresis. Stoichiometric inhibition was observed with the inhibitor and rabbit synovial collagenase and thermolysin (1:1 molar ratio with thermolysin). The inhibitor ran on sodium dodecyl sulfate-gel electrophoresis with reduction as a single protein band of Mr = 165,000. The molecular weight of the native inhibitor was estimated to be 780,000 by sedimentation equilibrium centrifugation. Centrifugation analysis in 6 M guanidine hydrochloride and of the reduced sample gave M omega = 380,000 and M omega = 195,000, respectively, where M omega is the weight-average molecular weight determined by equilibrium ultra-centrifugation. The results indicated that the inhibitor molecule is a tetramer of identical subunits linked in pairs by disulfide bonds. Since the molecular weight and the quaternary structure of the inhibitor were similar to those of alpha 2-macroglobulin (alpha 2M) in plasma, chicken alpha 2M was isolated and compared with the inhibitor. The inhibitor was not sensitive to methylamine, whereas chicken alpha 2M was. No immunocross-reactivity was observed between the inhibitor and chicken alpha 2M. The NH2-terminal sequence of the egg white inhibitor is Lys-Glu-Pro-Glu-Pro-Gln-Tyr-Val-Leu-Met-Val-Pro-Ala. The sequence of chicken alpha 2M is Ser-Thr-Val-Thr-Glu-Pro-Gln-Tyr-Met-Val-Leu-Leu-Pro-Phe. Considerable homology was found between the two sequences and to the NH2-terminal sequence of human alpha 2M. Monospecific antibody raised against the egg white inhibitor was employed to examine the tissue distribution of the inhibitor. The inhibitor was found only in oviduct and egg white, but not in other tissues or serum of chickens.

Amino Acid Sequence↗
PubMed1983

The primary structure of human serum transferrin. The structures of seven cyanogen bromide fragments and the assembly of the complete structure.

The amino acid sequences of seven cyanogen bromide fragments of human serum transferrin have been determined, and the primary structure of transferrin established by determining the order of these and three additional fragments (Sutton, M. R., MacGillivray, R. T. A., and Brew, K. (1975) Eur. J. Biochem. 51, 43-48) in the polypeptide chain. The order of the fragments was deduced from peptides that overlap methionyl residues which were obtained by thermolysin digestion of performic acid-oxidized transferrin or by partial peptic hydrolysis of unmodified transferrin, together with other evidence. The polypeptide chain of transferrin contains 679 amino acid residues, which together with the two N-linked oligosaccharide chains gives a calculated molecular weight of 79,570. Transferrin consists of two homologous domains (residues 1-336, 337-679), each associated with a single Fe-binding site, with both sites of glycosylation in the carboxyl-terminal domain at positions 413 and 611. Consideration of the primary structure in relation to previously published results provides information concerning the evolutionary development of transferrins and related proteins, and the locations of metal-binding residues in the transferrin molecule.

Amino Acid Sequence↗
PubMed1982

The complete amino acid sequence of human serum transferrin.

The complete amino acid sequence of human serum transferrin has been determined by aligning the structures of the 10 CNBr fragments. The order of these fragments in the polypeptide chain is deduced from the structures of peptides overlapping methionine residues and other evidence. Human transferrin contains 678 amino acid residues and--including the two asparagine-linked glycans--has an overall molecular weight of 79,550. The polypeptide chain contains two homologous domains consisting of residues 1-336 and 337-678, in which 40% of the residues are identical when aligned by inserting gaps at appropriate positions. Disulfide bond arrangements indicate that there are seven residues between the last half-cystine in the first domain and the first half-cystine in the second domain and therefore, a maximum of seven residues in the region of polypeptide between the two domains. Transferrin--which contains two Fe-binding sites--has clearly evolved by the contiguous duplication of the structural gene for an ancestral protein that had a single Fe-binding site and contained approximately 340 amino acid residues. The two domains show some interesting differences including the presence of both N-linked glycan moieties in the COOH-terminal domain at positions 413 and 610 and the presence of more disulfide bonds in the COOH-terminal domain (11 compared to 8). The locations of residues that may function in Fe-binding are discussed.

Amino Acid Sequence↗
PubMed1981

Characteristics of the binding of Ca2+ and other divalent metal ions to bovine alpha-lactalbumin.

Removal of the tightly bound Ca2+ ion from bovine alpha-lactalbumin (Hiraoka et al. (1980) Biochem. Biophys. Res. Commun. 95, 1098-1104) produces a pronounced conformational change, as indicated by fluorescence and absorbance changes. These changes closely resemble the changes that occur on acid denaturation of the native protein. The binding of ions to apo-alpha-lactalbumin at pH 7.4 has been examined by monitoring fluorescence changes and by direct binding measurements with 45CaCl2. The results indicate the presence of two Ca2+ binding sites on apo-bovine alpha-lactalbumin, a stronger binding site (Ka of 2.7 X 10(6) M-1) and a weaker site (Ka of 3.1 X 10(4) M-1); the fluorescence changes on Ca2+ rebinding correlate with saturation of the stronger site. Mn2+ can also bind to restore a "native" structure but with a lower affinity(Ka of 3.5 X 10(5) M-1). Zn2+ and Co2+ do not produce this change, but Zn2+ (1 mM) greatly inhibits the binding of 45Ca2+ in the direct binding assay and produces a time-dependent displacement of Ca2+ from the native protein to an apo-protein-like conformation. Co2+ does not produce these effects. Studies with metal-depleted galactosyltransferase activated with Zn2+ or Co2+ and apo-alpha-lactalbumin or Ca2+-saturated alpha-lactalbumin show that the Ca2+, Zn2+, and apo-alpha-lactalbumin are all able to bind with galactosyltransferase to produce an active lactose synthase complex.

Animals↗
PubMed1981

A label selection procedure for determining the location of protein-protein interaction sites by cross-linking with bisimidoesters. Application to lactose synthase.

A procedure is described that is designed to identify the primary site of cross-linking by bisimidoesters of a component of an interacting protein system. It is based on the mutually exclusive nature of acetylation and amidination. The procedure has been applied to the regulatory protein of lactose synthase, alpha-lactalbumin. A sample of bovine alpha-lactalbumin was acetylated with a trace amount of high specific activity [3H]acetic anhydride to produce a population of protein molecules essentially all of which contain 0 to 1 acetyl group; partial labeling of all 13 amino groups was obtained. This material was mixed with bovine colostrum galactosyltransferase in the presence of Mn2+, UDP-glucose, and N-acetylglucosamine, at pH 8.0, to promote complex formation and was cross-linked with dimethyl 3,3'-dithiobispropionimidate. Covalently cross-linked alpha-lactalbumin-galactosyltransferase (1:1) complex with characteristic enzymic and other properties was purified from the reaction mixture, and the distribution of [3H]acetyl label on each amino group of the alpha-lactalbumin component was determined, using procedures similar to those described in previous differential labeling studies (Richardson, R., and Brew, K. (1980) J. Biol. Chem. 255, 3377-3385). In comparison with the original labeled sample used for cross-linking, the specific activity of tritium label in 10 amino groups showed little change, whereas the labeling of three groups was changed markedly. The acetyl moiety on the epsilon-amino groups of lysines 5 and 108 showed major decreases in specific activity while that of lysine 114 was greatly increased. Similar results were obtained when the cross-linking was performed under different conditions of temperature, and cross-linker concentration. As the changes in lysines 5 and 114 are similar to those observed in differential labeling, they are attributed to alterations in the affinity for galactosyltransferase resulting from acetylation of these groups. In contrast, lysine 108, which is not sufficiently close to the interaction site to be perturbed in differential labeling studies but is greatly decreased in tritium content in the cross-linked complex, appears to represent the major site through which alpha-lactalbumin is cross-linked to galactosyltransferase as a result of the exclusion of protein molecules acetylated in this position from covalent cross-linking. Studies with a homologous series of bisimidoesters indicate that lysine 108 is situated 6.1 to 7.3 A degrees from an amino group on galactosyltransferase in the cross-linked complex. The general utility of the procedure and the nature of the interaction site in lactose synthase are discussed.

Acetylation↗
PubMed1980

Synthesis of 4-deoxy-D-xylo-hexose and 4-azido-4-deoxy-D-glucose and their effects on lactose synthase.

Syntheses are reported of 4-deoxy-D-xylo-hexose and 4-azido-4-deoxy-D-glucose as potential inhibitors for lactose synthase [uridine 5'-(alpha-D-galactopyranosyl pyrophosphate):D-glucose 4-beta-D-galactopyranosyltransferase, EC 2.4.1.22]. These syntheses involved SN2 displacement of the 4-methylsulfonyloxy group of methyl 2,3,6-tri-O-benzoyl-4-O-methylsulfonyl-alpha-D-galactopyranoside by iodide and azide ions. In both cases, inversion in configuration was observed. The resulting intermediates, methyl 2,3,6-tri-O-benzoyl-4-deoxy-4-iodo-alpha-D-glucopyranoside and methyl 4-azido-2,3,6-tri-O-benzoyl-4-deoxy-alpha-D-glucopyranoside, were obtained in crystalline form. Both 4-deoxy-D-xylo-hexose and 4-azido-4-deoxy-D-glucose were found to be inhibitors for lactose synthase in the presence of alpha-lactalbumin, but had no effect in the absence of alpha-lactalbumin. Both D-glucose analogues bind to the enzyme system far more weakly than D-glucose, suggesting that the recognition of the 4-OH group of the acceptor substrate is an important factor in binding.

Animals↗