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B L Horecker

Publications and source records attributed to B L Horecker.

At least 73 records · Page 4Linked to original sources
PubMed1984

Two cytosolic, Ca2+-dependent, neutral proteinases from rabbit liver: purification and properties of the proenzymes.

Two Ca2+-requiring proteinases have been purified from rabbit liver cytosol and shown to be present in isolated hepatocytes. They differ in relative molecular mass, with the major and minor forms, Mr = 150,000 and Mr = 200,000, accounting for 75 and 18% of the total cytosolic neutral proteinase activity, respectively. Both are recovered as inactive proenzymes that can be converted to the active, low-Ca2+-requiring proteinases by incubation with Ca2+ and substrate [S. Pontremoli, E. Melloni, F. Salamino, B. Sparatore, M. Michetti, and B. L. Horecker (1984) Proc. Natl. Acad. Sci. USA 81, 53-56. Each proenzyme is composed of two subunits, with molecular masses of 80 and 100 kDa, respectively. Activation of the proenzymes was found to correlate with their dissociation into subunits. The optimum pH for conversion of the proenzymes to the active proteinases in the presence of 5 mM Ca2+ and 2 mg/ml of denatured globin was approximately 7.5, and the same pH optimum was observed for the digestion of denatured globin by the activated proteinases. Following activation, each proteinase was observed to undergo autolytic inactivation at rates that were dependent on the concentration of both Ca2+ and the digestible substrate. A model is proposed for the activation of the proenzymes and the subsequent inactivation of the active proteinases.

Animals↗
PubMed1984

Regulation of the Ca2+-dependent neutral proteinases from rabbit liver by an endogenous inhibitor.

An endogenous inhibitor of neutral Ca2+-dependent proteinases has been isolated from rabbit liver cytosol. The inhibitor is a heat-stable, 240-kDa, tetrameric protein. It is dissociated into its 60-kDa subunits by high concentrations of Ca2+ (0.1-1 mM), but not by lower concentrations in the physiological range. Inhibition of the 150-kDa proteinase of rabbit liver [Melloni, E., Pontremoli, S., Salamino, F., Sparatore, B., Michetti, M. and Horecker, B.L. (1984) Arch. Biochem. Biophys. 232, 505-512] requires the monomeric form of the inhibitor, and occurs only at the high concentrations of Ca2+ which also cause dissociation of the dimeric 150-kDa proteinase into its 80-kDa subunits. The molecular weight of the inactive proteinase-inhibitor complex was estimated by the equilibrium gel penetration method to be 140 kDa, suggesting that it contains one subunit of proteinase and one of inhibitor. The mechanism of interaction of the inhibitor with the 200-kDa proteinase at high concentrations of Ca2+ is identical to that observed for the 150-kDa proteinase, namely dissociation of both proteinase and inhibitor into subunits and formation of an inactive 160-kDa proteinase-inhibitor complex. However, unlike the 150-kDa proteinase, which does not interact with the inhibitor at low Ca2+ concentrations, the 200-kDa proteinase is also inhibited at low concentrations of Ca2+. Under these conditions, the high-molecular-weight complex (greater than 400 kDa) formed between the tetrameric inhibitor and the dimeric proteinase prevents conversion of the 200-kDa proenzyme to the active, low-Ca2+-requiring form.

Animals↗
PubMed1984

Interaction of rabbit liver cathepsin M and fructose 1,6-bisphosphatase converting enzyme with their endogenous inhibitors.

The stoichiometry of complex formation between two lysosomal proteinases from rabbit liver, cathepsin M and fructose 1,6-bisphosphatase converting enzyme (CE), and their respective endogenous inhibitors was studied by the equilibrium gel penetration method. In each case the molecular weight of the complex was found to be the sum of the molecular weights of the proteinase and its inhibitor, indicating the formation of 1:1 complexes. From the reappearance of proteinase activity on dilution, it is concluded that complex formation is reversible. Localization of the proteinase activities on the outer surface of the lysosomes was confirmed in these experiments by the inhibition of this proteinase activity on addition of inhibitors to intact lysosomes. The digestion by subtilisin of rabbit liver aldolase and rabbit liver fructose 1,6-bisphosphatase, the endogenous substrates for the lysosomal proteinases, was unaffected by the inhibitors.

Animals↗
PubMed1984

Thymosin beta 11: a peptide from trout liver homologous to thymosin beta 4.

A peptide containing 41 amino acid residues has been isolated from trout liver and identified as a member of the beta-thymosin family. Sequence analysis shows it to be 78% homologous to thymosin beta 4, which is the peptide present in the thymus and other tissues of higher vertebrates, including, as reported here, livers of a species of reptile. Thymosin beta 11 appears to replace the more prevalent thymosin beta 4 in at least two species of bony fish, and represents the sixth structurally characterized member of this widely distributed family of peptides.

Amino Acid Sequence↗
PubMed1984

Cytosolic Ca2+-dependent neutral proteinases from rabbit liver: activation of the proenzymes by Ca2+ and substrate.

Two neutral Ca2+-dependent proteinases, differing in molecular size, have been isolated from rabbit liver. Both are recovered as inactive proenzymes that can be converted to the active forms by high (0.1-1.0 mM) concentrations of Ca2+ in the absence of substrate or, in the presence of a protein substrate, by low (1-5 microM) concentrations of Ca2+. The activated proteinases required only 1-5 microM Ca2+ for maximal activity. Substrates hydrolyzed were denatured globin, globin, casein, and to a lesser extent, several extracellular proteins; no digestion was observed with several intracellular cytosolic enzymes tested. Only those proteins that served as substrates were capable of promoting conversion of the proenzymes to the active low-Ca2+-requiring proteinases.

Animals↗
PubMed1984

A dual role for the Ca2+-requiring proteinase in the degradation of hemoglobin by erythrocyte membrane proteinases.

Binding of hemoglobin chains to erythrocyte membranes is an obligatory step in the conversion of hemoglobin to acid-soluble products by erythrocyte proteinases. This binding requires limited proteolysis of the hemoglobin chains and also modification of the inner surface of the erythrocyte membrane, both of which result from the action of a soluble Ca2+-requiring neutral proteinase. Final digestion of the bound hemoglobin chains in the membrane complex results from the action of intrinsic membrane endopeptidases. Regulation of the activity of the Ca2+-requiring proteinase by the substrate provides a mechanism for the initiation of selective protein turnover.

Calcimycin↗
PubMed1984

Prothymosin alpha: isolation and properties of the major immunoreactive form of thymosin alpha 1 in rat thymus.

A polypeptide containing approximately equal to 112 amino acid residues, with the thymosin alpha 1 sequence at its NH2 terminus, has been isolated from rat thymus by using a radioimmunoassay with an antibody prepared against synthetic thymosin alpha 1. The new polypeptide, named "prothymosin alpha," was found to be the major substance crossreacting with thymosin alpha 1 antiserum in rat thymus extracts; peptides corresponding to thymosin alpha 1 or thymosin alpha 11 were not detected. In gel filtration at pH 2.8, prothymosin alpha emerged as a single symmetrical peak corresponding to an apparent molecular weight of 32,000, approximately 3 times larger than the minimum molecular weight calculated from its amino acid composition. On the same gel filtration columns, synthetic thymosin alpha 1 (calculated Mr = 3108) emerged at a position corresponding to a molecular weight of 10,000-11,000. Thus, both prothymosin alpha and thymosin alpha 1 appear to exist in solution as oligomers, possibly as trimers. Prothymosin alpha and synthetic thymosin alpha 1 also were separated readily in reverse-phase HPLC and in isoelectric focusing; the isoelectric point of prothymosin alpha determined by the latter procedure was found to be 3.55, consistent with an unusually high content of glutamic and aspartic acids based on amino acid analyses. Prothymosin alpha appears to represent the native polypeptide from which thymosin alpha 1 and other fragments are generated during the isolation of thymosin fraction 5.

Amino Acid Sequence↗
PubMed1984

Distribution of prothymosin alpha in rat tissues.

A radioimmunoassay, using a rabbit antiserum directed against thymosin alpha 1, was employed to detect the presence of crossreacting peptides in rat tissues. Highest concentrations were present in thymus, but thymosin alpha 1 cross-reacting material was also detected in brain, liver, kidney, lung, and spleen, in amounts ranging from 15% to 65% of the quantities found in thymus. In each case, the major immunoreactive peptide, after extraction and purification by a procedure that avoids proteolytic modification, was identified as prothymosin alpha, a peptide containing approximately equal to 112 amino acid residues. Prothymosin alpha is believed to be the endogenous peptide from which thymosin alpha 1 and other fragments are formed by proteolytic modification during the preparation of thymosin fraction 5. No peptides corresponding in size and chromatographic behavior to thymosin alpha 1 were detected with the extraction procedure employed.

Amino Acids↗
PubMed1984

Cloning and sequence analysis of cDNA for rat spleen thymosin beta 4.

Molecular cloning of a cDNA has established the sequence of the translated portion of the mRNA for rat spleen thymosin beta 4. The presence of a methionyl initiator codon immediately preceding the codon for the first seryl residue of mature thymosin beta 4 is consistent with previous results indicating the absence of a signal peptide in the product translated in vitro from rat spleen mRNA. The cDNA sequence analysis also established the presence of two terminator codons immediately following the codon for the COOH-terminal seryl residue. Thymosin beta 4 is thus synthesized as a 5100-dalton peptide containing 44 amino acid residues. Removal of the initiator methionyl residue and acetylation of the NH2-terminal serine residue would yield mature thymosin beta 4 containing 43 amino acids. The absence of a signal peptide makes it unlikely that thymosin beta 4 is a secreted peptide.

Amino Acid Sequence↗
PubMed1983

Thymosin beta arg10, a major variant of thymosin beta 10 in rabbit tissues.

Two homologous peptides, designated thymosin beta 4 and thymosin beta 10, respectively, have been shown to be widely distributed in mammalian cells and tissues (S. Erickson-Viitanen, S. Ruggieri, P. Natalini, and B.L. Horecker (1983) Arch. Biochem. Biophys. 221, 570-576; S. Erickson-Viitanen, S. Ruggieri, P. Natalini, and B.L. Horecker, (1983) Arch. Biochem. Biophys. 225, 407-413). In the rabbit, thymosin beta 4 is replaced by a variant, thymosin beta ala4, that contains alanine in place of serine at the blocked NH2-terminus. It is reported that in rabbit tissues thymosin beta 10 is also replaced by a variant, designated thymosin beta arg10, that contains an additional amino acid, arginine, inserted following lysine-38. The rabbit tissues analyzed also differ from those of other mammals in the relative quantities of thymosin beta ala4 and beta arg10, which are nearly equal, compared to tissues from other mammals where the quantities of thymosin beta 10 are only one-third to one-tenth those of thymosin beta 4.

Amino Acid Sequence↗
PubMed1983

Thymosin beta 10, a new analog of thymosin beta 4 in mammalian tissues.

A new analog of thymosin beta 4 has been isolated from tissues of several mammalian species. This peptide, designated thymosin beta 10, is composed of 42 amino acid residues and shows 75% sequence homology with thymosin beta 4. It occurs together with thymoxin beta 4 in a variety of tissues including spleen, liver, and thymus and also in several cultured cell lines. In the spleen of rat, mouse, cat, and man, the new peptide accounts for approximately 0.02% by weight of the total protein. In the calf it is replaced by another homologous peptide, designated thymosin beta 9, whose structure has been reported.

Amino Acid Sequence↗
PubMed1983

Distribution of thymosin beta 4 in vertebrate classes.

A peptide containing 43 amino acid residues, rich in glutamic acid and lysine, was originally isolated from calf thymus and designated thymosin beta 4 [T.L.K. Low, S. -K. Hu, and A. L. Goldstein (1981) Proc. Nat. Acad. Sci. USA 78, 1162-1166]. However, thymosin beta 4 was also shown to be present in other tissues of rats and mice, with highest concentrations in spleen and peritoneal macrophages [E. Hannappel, G.-J. Xu, J. Morgan, J. Hempstead, and B.L. Horecker (1982), Proc. Nat. Acad. Sci. USA 79, 2172-2175]. We have now identified the same peptide in tissues of other mammalian species and other vertebrate classes, including birds and amphibia. Exceptions are the rabbit and bony fish, where thymosin beta 4 is replaced by different peptides, similar in size and in amino acid sequence. None of these peptides was detected in several invertebrates or in the protozoan, Tetrahymena pyriformis. In subcellular fractionation of rat spleen, thymosin beta 4 was recovered in the cytosol.

Animals↗
PubMed1983

Thymosin beta 4 in cultured mammalian cell lines.

Thymosin beta 4, originally isolated from calf thymus [Low et al., Proc. Nat. Acad. Sci. USA 78, 1162-1166 (1981)] is present in a number of cell lines unrelated to the reticuloendothelium, including myoblasts and fibroblasts. It is also actively synthesized by these cell lines. Its content and rate of synthesis in the cell lines examined appear to be correlated with their ability to adhere and their motility.

Amino Acid Sequence↗
PubMed1983

Thymosin alpha 11: a peptide related to thymosin alpha 1 isolated from calf thymosin fraction 5.

Two peptides related to thymosin alpha 1 have been isolated from preparations of calf thymosin fraction 5. One, lacking four amino acid residues at the COOH terminus, is designated des-(25-28)-thymosin alpha 1. The other, named thymosin alpha 11, contains seven additional amino acid residues at the COOH terminus. The sequence of this peptide is: AcSer-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu- Lys-Glu-Lys- Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-Gly-Arg-Glu-Ala-Pro-Ala-AsnOH. Thymosin alpha 11, in doses of less than 300 ng per mouse, protects susceptible inbred murine strains against opportunistic infections with Candida albicans. It is approximately equal to 30 times as potent as thymosin fraction 5 and approximately equal in potency to thymosin alpha 1.

Amino Acid Sequence↗
PubMed1983

Endogenous inhibitors of lysosomal proteinases.

Specific inhibitors of three lysosomal proteinases are present in the cytosolic and lysosomal compartments of rabbit liver. The cytosolic inhibitors, purified by chromatography on DEAE-Trisacryl and Sephadex G-75, show specificities toward cathepsin M, cathepsins B and L, and fructose 1,6-bisphosphatase converting enzyme (CE), respectively, and are designated IM, IB/L, and ICE. Inhibitors with similar specificities have been isolated from the intralysosomal compartment. Two of these inhibitors, IM and ICE, are also present in the lysosomal membranes. The lysosomal distribution parallels that of the respective proteinases. The inhibitors are polypeptides with molecular weights of 5,000-10,000 for the two forms of IB/L, 12,500 for IM, and 10,000-40,000 for the ICE species.

Animals↗
PubMed1983

In vitro synthesis of thymosin beta 4 encoded by rat spleen mRNA.

Thymosin beta 4, containing 43 amino acids and acetylated at the NH2 terminus, is synthesized in vitro in a rabbit reticulocyte lysate or in a yeast protein-synthesis system in the presence of mRNA from rat spleen. The product formed was identified as beta 4 by immunoprecipitation by a specific anti-beta 4 antiserum, comigration with authentic beta 4 in NaDodSO4/polyacrylamide gel electrophoresis and in HPLC, and identity of peptide fragments. The immunoprecipitable product generated in the wheat germ protein-synthesizing system emerged slightly ahead of beta 4 in HPLC and appeared to lack the NH2-terminal acetyl group. There was no evidence for formation of a larger polypeptide precursor of beta 4 in any of the three systems used. In sucrose density gradient centrifugation, the mRNA coding for beta 4 was recovered in the 7-8S mRNA fraction.

Animals↗