Biomedical subjects

J E Mole

Publications and source records attributed to J E Mole.

At least 19 recordsLinked to original sources

PubMed1995

The isolation and comparison of multiple forms of CYP2B from untreated and phenobarbital-treated rabbit liver microsomes.

At low levels of phenobarbital induction two forms of isoenzyme 2 (LM2; CYP2B4) were obtained during purification of cytochrome P450 from rabbit liver microsomes. At high levels of induction only one form (LM2A) was present. Although the two purified forms (LM2A and LM2B) were very similar they differed in: (a) peak elution on CM-Sepharose, (b) wavelength maximum of the reduced P450-CO spectrum, and (c) metabolism of several substrates, where the activities of LM2B ranged from 0.6 to 2.65 times that of LM2A. A third LM2 fraction (2C) was isolated from untreated rabbit liver and, although homogenous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, appeared to be a mixture of LM2B and a form of P450 LM2 other than LM2A. LM2A was not found in the untreated rabbit liver microsomes. On CM-Sepharose the elution of fraction 2C overlapped that of LM2B. The apparent molecular weight and immunoresponse to anti-LM2A IgG were the same for fraction 2C as for LM2A and LM2B. Peptide mapping using trypsin showed no difference between LM2A and LM2B, but consistently revealed at least one extra band with fraction 2C. After CNBr cleavage and high-pressure liquid chromatography separation of the LM2A and LM2B fragments the peptide beginning with Pro(347) of LM2A (peak 4A) eluted 1/2 min later than that of LM2B (peak 4B) indicating a difference in the fragments, although partial NH2-terminal amino acid sequences and molecular masses were the same. The corresponding CNBr fragment of fraction 2C splits into two peaks (4C:1 and 4C:2) with retention times corresponding to 4B and 4A, respectively. The mass of 4C:1 was the same as that of 4B, while the mass of 4C:2 markedly differed from that of 4A and 4B. Both fragments had the same partial NH2-terminal amino acid sequence as 4A and 4B. After comparing the physiochemical properties as well as catalytic activities of these isolated and purified LM2 forms with the cDNA-expressed forms 2B-B0, 2B-B1, 2B-B2, and 2B-Bx [see R. Ryan et al. (1993) Arch. Biochem. Biophys. 304, 454-463], the data suggest that LM2A is the form designated as 2B-B0 (LM2), LM2B is 2B-Bx, and fraction 2C is a mixture containing 2B-B1 and 2B-Bx. This is the first isolation and identification of the three isozymic LM2 proteins from rabbit liver.

Amino Acid Sequence↗

PubMed1994

Role of N-terminal domain of histidine-rich glycoprotein in modulation of macrophage Fc gamma receptor-mediated phagocytosis.

A brief exposure of murine peritoneal inflammatory macrophages to plasma histidine-rich glycoprotein (HRG; 77,000-81,000 MW) for 1-2 hr increased Fc gamma receptor (Fc gamma R) expression and phagocytic function in these cells. However, a continual culture of the cells without the presence of HRG for the next 18-48 hr resulted in down-regulation of Fc gamma R expression and phagocytic function. Similarly, HRG decreased Fc gamma RII expression in less differentiated human THP-1 monocytic cells during treatment for 18 hr, as determined by cellular ELISA and metabolic labelling. The molecular mechanism by which HRG regulates Fc gamma R expression is unknown. However, at a relatively high concentration (> 1 microgram/ml), HRG altered the cellular metabolism by increasing cellular protein synthesis but reducing protein secretion. These observations suggest a likely mechanism for the HRG-mediated reduction of Fc gamma R expression. A degraded HRG (40,000 MW) which possessed an identical N-terminal sequence as that of the native HRG was capable of decreasing macrophage Fc gamma R expression and phagocytosis. The results indicate that the functional domain of HRG responsible for binding to macrophages is localized to the N-terminal half.

Animals↗

PubMed1992

Regulation of complement functional efficiency by histidine-rich glycoprotein.

The modulation of complement functional efficiency by serum histidine-rich glycoprotein (HRG) was investigated. Addition of exogenous HRG to prewarmed diluted serum, followed immediately by sensitized sheep erythrocytes (EA), resulted in enhanced hemolysis. However, when HRG was incubated with diluted serum for 10 minutes at 37 degrees C, inhibition of hemolysis occurred. The biphasic modulation of complement function was also obtained with the complement alternative pathway when HRG was added to diluted serum for hemolysis of rabbit erythrocytes. Partial reduction of complement functional activity was shown when serum was absorbed by an HRG-Sepharose 6MB column. Western blot analysis showed that complement C8, C9, factor D, and S-protein in diluted serum were bound by nylon membrane-immobilized HRG. However, by immunoprecipitation of relatively undiluted serum with anti-HRG IgG beads, HRG was found to coprecipitate with S-protein and plasminogen, which suggested that HRG may complex with these proteins in serum. In functional tests, HRG inhibited C8 hemolytic activity, probably by preventing C8 binding to EAC1-7 cells. HRG also enhanced polymerization of purified C9 as well as the generation of a 45-Kd C9 fragment. Such an effect was even more pronounced in the presence of divalent cations with the reaction mixtures of C9 and HRG. Partial dimerization of C9 was shown when exogenous HRG was added to normal serum. In contrast, polymerization of serum C9 was inhibited by exogenous HRG during poly I:C activation of serum or incubation under low ionic strength conditions. HRG was further shown to inhibit factor D-mediated cleavage of factor B when bound by cobra venom factor. The molecular basis by which HRG regulates serum complement function is not clear. Hypothetically, the tandem repetitions of a consensus histidine-rich penta-peptide sequence in HRG may provide a highly charged area that interacts with complement components.

Animals↗

PubMed1992

Characterization of a factor VIII immunogenic site using factor VIII synthetic peptide 1687-1695 and rabbit anti-peptide antibodies.

A 9 amino acid peptide, Ser-Pro-Arg-Ser-Phe-Gln-Lys-Lys-Thr, corresponding to the clotting factor VIII (FVIII) sequence Ser1687-Thr1695, was synthesized in order to analyze a site on FVIII to which antibody inhibitors of FVIII may be directed. This sequence contained a thrombin cleavage site. It was predicted to be immunogenic because a Hopp-Woods hydrophilicity analysis of the amino acid sequence of FVIII showed it to be very hydrophilic, and it contained a proline. The HPLC-purified peptide was cleaved by thrombin at Arg1689-Ser1690, as determined by amino acid sequencing of the cleavage product. Thrombin which had been treated with a specific chloromethyl ketone inhibitor, did not cleave the peptide. Two rabbits immunized with the peptide/keyhole limpet hemocyanin conjugate generated FVIII inhibitory sera with titers of 5.4 and 4.8 Bethesda units. These rabbit anti-peptide antibodies reacted with a peptide/-BSA conjugate on immunodot blot analyses and with native, affinity-purified FVIII in Western blots. In competitive immunoradiometric assays, cryosupernatants of 38/82 patients with FVIII inhibitors reacted with the synthetic peptide. We conclude that FVIII peptide Ser1687-Thr1695 is cleaved by thrombin at the same peptide bond which is cleaved in FVIII, and the peptide contains a site to which patients' inhibitory antibodies can be directed.

Amino Acid Sequence↗

PubMed1992

Regulation of macrophage Fc receptor expression and phagocytosis by histidine-rich glycoprotein.

Regulation of macrophage Fc receptor (Fc gamma R)-mediated phagocytic function by histidine-rich glycoprotein (HRG) was investigated. Pretreatment of oil-elicited inflammatory mouse peritoneal macrophages with HRG for 1-3 hr increased their Fc gamma R-mediated binding and phagocytosis of IgG-opsonized sheep erythrocyte conjugates (EA). A significant reduction of Fc gamma R-dependent EA binding and phagocytosis occurred after pretreatment of macrophages with HRG for more than 8 hr. These results indicate that HRG is capable of modulating Fc gamma R expression in a biphasic fashion, which directly affects the overall efficiency of phagocytosis. HRG differentially regulated the functions of Fc gamma R subclasses. For example, HRG reduced the efficiency of Fc gamma RII (Fc gamma 2b/gamma 1R)-dependent phagocytosis of erythrocytes conjugated with monoclonal IgG2b or IgG1 by macrophages pretreated with HRG for 24 hr. However, when similar studies were performed using erythrocytes coated with monoclonal IgG2a, HRG was less effective in inhibiting Fc gamma RI (Fc gamma 2aR)-dependent phagocytosis. As an HRG-binding glycosaminoglycan, heparin failed to block the regulatory function of HRG on macrophages. Similarly, interferon-gamma (IFN-gamma) was not capable of blocking the functional activity of HRG. These studies suggest that HRG regulates macrophage function via a novel pathway different from that of heparin or IFN-gamma.

Animals↗

PubMed1989

Amino-terminal sequence analysis of rat heart and muscle glycogen synthase: homology to the rabbit enzyme and the implications for hormonal control.

Glycogen synthase was purified from rat heart and muscle and electroblotted from sodium dodecyl sulfate polyacrylamide gels to polyvinylidene difluoride, and the NH2-terminal amino acid sequence was determined. The NH2-terminal amino acid sequence of the enzymes was identical. Further, phosphorylation site 2, a major cyclic AMP-dependent protein kinase recognition site in the rabbit muscle isozyme, is conserved in the rat isozymes suggesting that it serves an important function in hormonal regulation. However, two potentially important differences were observed. Threonine-5 and valine-8 of the rabbit muscle enzyme are serine and methionine residues, respectively, in the rat isozyme, the latter being critical in the analysis of phosphopeptides produced by cyanogen bromide cleavage. These variations may provide a partial explanation for previously observed differences in rat and rabbit phosphopeptide maps.

Amino Acid Sequence↗

PubMed1988

Isolation and analysis of murine serum amyloid P component cDNA clones.

In contrast to other animals, the biosynthesis of serum amyloid P component in mice is regulated as an acute-phase protein. As a first step in studying the regulation and biosynthesis of serum amyloid P component in the mouse, cDNA clones have been isolated from a liver cDNA library and sequenced. The largest of these clones was 960 bp in length, and contained an open reading frame encoding a protein of 224 amino acids. Comparison of the mouse cDNA sequence to that published for humans (Mantzouranis, E. C., S. B. Dowton, A. S. Whitehead, M. D. Edge, G. A. P. Bruns, and H. R. Colten, 1985. J. Biol. Chem. 260:7752.) revealed 74% identity for nucleotides in the translated region. Northern-blot analysis demonstrated that murine serum amyloid P component synthesis in the liver is directed by a 1.2-kb mRNA that is elevated in high responder (C57BL/6J) mice after thioglycollate-induced inflammation.

Amino Acid Sequence↗

PubMed1988

Further characterization of RLM2 and comparison with a related form of cytochrome P-450, RLM2b.

We have extended the characterization of RLM2, a constitutive form of rat liver cytochrome P-450, using immunochemical means to quantitate its presence in microsomes, to follow its development in maturing male and female rats, and to determine its response to prototypical P-450 inducers. In addition, RLM2 is compared to RLM2b, a form of P-450 with similar migration on SDS-PAGE and NH2-terminal amino acid sequence. RLM2b is expressed in both sexes at a level of 0.08 nmol/mg microsomal protein at 2 weeks of age. In female rats, this level is unchanged with maturation. However, in the male, the level declined with maturation to reach 0.02 nmol/mg protein by 12 weeks of age. RLM2 is a male-specific form of cytochrome P-450. Originally absent in the 2-week-old rat, it reached a level of 0.03 nmol/mg protein in the adult male, its appearance and increase coinciding with the onset of puberty. Both phenobarbital and 3-methylcholanthrene induced microsomal levels of RLM2b in the adult male and female rat. RLM2, however, was suppressed in the male rat, 58 and 42%, respectively, by the same treatments. RLM2b and RLM2 each catalyze a unique spectrum of hydroxytestosterone metabolites. RLM2b is highly site specific. In contrast, RLM2 produces several isomeric products in the same region of the testosterone molecule. Substitution of the acetyl group of progesterone for the 17-hydroxy group of testosterone did not alter the site specificity of RLM2b, but did alter it for RLM2, indicating, further, a difference in the active site conformation of the two enzymes. Although RLM2b and RLM2 responded differently to inducers and to a changing physiology during maturation, and were functionally quite distinct, the proteins showed a high degree of immunologic relatedness which is suggestive of significant structural similarities. Structural differences do exist, however, as alpha-chymotryptic digestion formed a number of peptide fragments that differed between the two proteins.

Amino Acid Sequence↗

PubMed1988

Purification and characterization of two trypsin inhibitors from the hemolymph of Manduca sexta larvae.

Trypsin inhibitory activity from the hemolymph of the tobacco hornworm (Manduca sexta) was purified by affinity chromatography on immobilized trypsin and resolved into two fractions with molecular weights of 14,000 (M. sexta hemolymph trypsin inhibitor (HLTI) A) and 8,000 (HLTI B) by molecular sieve chromatography on Sephadex G-75. Electrophoresis of these inhibitors under reducing conditions on polyacrylamide gels gave molecular weight estimates of 8,300 for HLTI A and 9,100 for HLTI B, suggesting that HLTI A is a dimer and HLTI B is a monomer. Isoelectrofocusing on polyacrylamide gels focused HLTI A as a single band with pI 5.7, whereas HLTI B was resolved into two components with pI values of 5.3 and 7.1. Both inhibitors were stable at 100 degrees C and pH 1.0 for at least 30 min. HLTIs A and B inhibited serine proteases such as trypsin, chymotrypsin, and plasmin, but did not inhibit elastase, papain, pepsin, subtilisin BPN', and thermolysin. In fact, subtilisin BPN' completely inactivated both inhibitors. Both inhibitors formed low-dissociation complexes with trypsin in a 1:1 molar ratio. The inhibition constant for trypsin inhibition by HLTI A was estimated to be 1.45 x 10(-8) M. The HLTI A-chymotrypsin complex did not inhibit trypsin; similarly, the HLTI A-trypsin complex did not inhibit chymotrypsin, indicating that HLTI A has a common binding site for both trypsin and chymotrypsin. The amino-terminal amino acid sequences of HLTIs A and B revealed that both these inhibitors are homologous to bovine pancreatic trypsin inhibitor (Kunitz).

Animals↗

PubMed1988

A single receptor binds both insulin-like growth factor II and mannose-6-phosphate.

Amino acid sequences deduced from rat complementary DNA clones encoding the insulin-like growth factor II (IGF-II) receptor closely resemble those of the bovine cation-independent mannose-6-phosphate receptor (Man-6-P receptorCI), suggesting they are identical structures. It is also shown that IGF-II receptors are adsorbed by immobilized pentamannosyl-6-phosphate and are specifically eluted with Man-6-P. Furthermore, Man-6-P specifically increases by about two times the apparent affinity of the purified rat placental receptor for 125I-labeled IGF-II. These results indicate that the type II IGF receptor contains cooperative, high-affinity binding sites for both IGF-II and Man-6-P-containing proteins.

Amino Acid Sequence↗

PubMed1988

Identification of a cDNA clone for the beta-subunit of the pyruvate dehydrogenase component of human pyruvate dehydrogenase complex.

We report the isolation of a 1.5 kb cDNA clone for the beta subunit of human pyruvate dehydrogenase (E1) from a human liver lambda gt11 cDNA library using anti-E1 serum. We generated a peptide sequence of 24 amino acids starting from the N-terminus of bovine heart mature E1 beta. The identity of the E1 beta cDNA clone was confirmed by the similarity between the amino acid sequence deduced from the cDNA nucleotide sequence and the known amino acid sequence of bovine heart E1 beta. In Northern analysis of total RNA extracted from human heart, the E1 beta cDNA clone hybridized to a major 1.6 kb and a minor 5.2 kb RNA species.

Amino Acid Sequence↗

PubMed1988

Heterogeneous expression of protein and mRNA in pyruvate dehydrogenase deficiency.

Deficiency of pyruvate dehydrogenase [pyruvate:lipoamide 2-oxidoreductase (decarboxylating and acceptor-acetylating), EC 1.2.4.1], the first component of the pyruvate dehydrogenase complex, is associated with lactic acidosis and central nervous system dysfunction. Using both specific antibodies to pyruvate dehydrogenase and cDNAs coding for its two alpha and beta subunits, we characterized pyruvate dehydrogenase deficiency in 11 patients. Three different patterns were found on immunologic and RNA blot analyses. (i) Seven patients had immunologically detectable crossreactive material for the alpha and beta proteins of pyruvate dehydrogenase. (ii) Two patients had no detectable crossreactive protein for either the alpha or beta subunit but had normal amounts of mRNA for both alpha and beta subunits. (iii) The remaining two patients also had no detectable crossreactive protein but had diminished amounts of mRNA for the alpha subunit of pyruvate dehydrogenase only. These results indicate that loss of pyruvate dehydrogenase activity may be associated with either absent or catalytically inactive proteins, and in those cases in which this enzyme is absent, mRNA for one of the subunits may also be missing. When mRNA for one of the subunits is lacking, both protein subunits are absent, suggesting that a mutation affecting the expression of one of the subunit proteins causes the remaining uncomplexed subunit to be unstable. The results show that several different mutations account for the molecular heterogeneity of pyruvate dehydrogenase deficiency.

Acidosis, Lactic↗

PubMed1987

Responses to insulin by two forms of rat hepatic microsomal cytochrome P-450 that undergo major (RLM6) and minor (RLM5b) elevations in diabetes.

Total cytochrome P-450 levels rise in diabetic rats. Two specific forms of cytochrome P-450 that are elevated have been isolated from liver microsomes of streptozotocin-induced idabetic male rats. One enzyme, termed RLM6, metabolizes aniline and acetol, but not testosterone, in a reconstituted system with NADPH-cytochrome P-450 reductase. RLM6 is isolated as a high spin cytochrome with a minimum molecular weight of 53,500. It has a unique amino-terminal amino acid sequence lacking methionine at the amino-terminal position. Polyclonal antibodies to RLM6 recognized most other forms of cytochrome P-450 in Western blots, but could be made monospecific by adsorption to cross-reacting proteins coupled to Sepharose 4B. Using the monospecific antibodies, RLM6 was estimated to be present in microsomes of untreated male rats at 0.04 nmol/mg protein (5% of total P-450). In chronically diabetic rats this level rose to 0.35 nmol/mg protein and 24% of the P-450 content. Immunoreactive protein of molecular weight identical to RLM6 was elevated in microsomes of non-diabetic rats treated with ethanol, acetone, or isoniazid as well as in rats starved for 48 h. Insulin treatment of diabetic rats for 1 week lowered the immunologically detectable levels of RLM6 to levels found in the untreated rat. The other form of cytochrome P-450, RLM5b, does not metabolize aniline and only poorly metabolizes acetol and testosterone. This 52.5-kDa protein is isolated as a predominantly (60%) high spin enzyme. It has a unique NH2-terminal amino acid sequence with methionine as the terminal residue, and is present in untreated male rat liver microsomes at 0.16 nmol/mg protein. It is elevated in diabetes, like RLM6, but treatment with insulin for 1 week does not completely restore the microsomal content to that of the non-diabetic rat.

Amino Acid Sequence↗

PubMed1987

The transforming growth factor-beta system, a complex pattern of cross-reactive ligands and receptors.

A new homodimer form of transforming growth factor-beta (TGF-beta), TGF-beta 2, has been identified in porcine blood platelets. TGF-beta 2 is homologous to ordinary TGF-beta (TGF-beta 1), which is also present in platelets. TGF-beta 1.2, a heterodimer containing one TGF-beta 1 chain and one TGF-beta 2 chain, has also been isolated. TGF-beta 1 and TGF-beta 2 interact differently with a family of receptors in target cells. A 280 kd receptor displays high affinity for both TGF-beta 1 and TGF-beta 2. Occupancy of this receptor by TGF-beta 1 or TGF-beta 2 correlates with the ability of these TGF-beta s to inhibit cell proliferation. In contrast, 65 kd and 85 kd receptors have high affinity for TGF-beta 1 but lower affinity for TGF-beta 2. The existence of distinct forms of TGF-beta that interact differently with a family of TGF-beta receptors could provide flexibility to the regulation of tissue growth and differentiation by the TGF-beta system.

Amino Acid Sequence↗

PubMed1987

Sequence homologies between type 1 and type 2A protein phosphatases.

The Mr = 33,000 catalytic fragment of rabbit skeletal muscle type 1 protein phosphatase was digested with trypsin after reduction and alkylation. The resulting peptides were isolated, subjected to automated Edman degradation, and their sequences compared to the deduced peptide sequence of the bovine type 2A protein phosphatase cDNA. Of 10 tryptic peptides from the type 1 phosphatase that were sequenced, nine showed a high degree of homology with the type 2A phosphatase. This provides the first direct sequence comparison suggesting that the type 1 and type 2 protein phosphatases, distinguished functionally by their substrate specificities and sensitivity to inhibitors, make up part of a family of closely related gene products with similar structures.

Amino Acid Sequence↗

PubMed1986

Purification and amino-terminal sequence of an insulin-like growth factor-binding protein secreted by rat liver BRL-3A cells.

A protein preparation that specifically binds insulin-like growth factors (IGFs) I and II was purified from medium conditioned by rat liver BRL-3A cells using molecular sieve chromatography in 1 M acetic acid followed by affinity chromatography on IGF-II-agarose. The affinity-purified IGF-binding protein exhibits a single major band with apparent Mr = 36,300 under reducing conditions on sodium dodecyl sulfate-polyacrylamide gels. The IGF-binding protein is efficiently and specifically cross-linked to either 125I-IGF-I (human) or 125I-IGF-II (rat) using disuccinimidyl suberate. An IGF-binding protein of similar apparent molecular weight was also affinity purified from rat hepatoma H-35 cell conditioned medium and found to differ from the BRL-3A protein such that potent polyclonal antisera prepared in rabbits against the purified BRL-3A IGF-binding protein exhibited a much lower titer for the H-35 protein in an enzyme-linked immunosorbent assay and upon immunoblotting. In order to determine whether a single BRL-3A IGF-binding protein is present in the affinity-purified preparation, the protein was prepared for sequencing on a Sephacryl S-300 column in 6 M guanidine HCl after reduction and alkylation. The amino acid composition (expressed in percentages) of this IGF-binding protein was determined to be: Cys = 5.5, Lys = 4.8, His = 2.8, Arg = 7.8, Asx = 10.2, Thr = 5.1, Ser = 3.9, Glx = 15.7, Gly = 17.4, Ala = 7.3, Val = 4.6, Met = 1.4, Ile = 2.4, Leu = 8.3, Tyr = 1.0, Phe = 1.9. Sequencing of the NH2-terminal portion of this protein led to the identification of 31 amino acids in the following order: Phe-Arg-Cys-Pro-Pro-Cys-Thr-Pro-Glu-Arg-Leu-Ala-Ala-Cys-Gly-Pro-Pro-Pro- Asp-Ala-Pro-Cys-Ala-Glu-Leu-Val-Arg-Glu-Pro-Gly-Cys. We conclude that rat liver BRL-3A cells secrete a single major IGF-binding protein capable of binding both IGF-I and IGF-II.

Amino Acid Sequence↗

PubMed1986

Identification of serine 24 as the unique site on the transferrin receptor phosphorylated by protein kinase C.

Addition of tumor-promoting phorbol diesters to [32P]phosphate-labeled A431 human epidermoid carcinoma cells caused an increase in the phosphorylation state of the transferrin receptor. The A431 cell transferrin receptor was also found to be a substrate for protein kinase C in vitro. Tryptic phosphopeptide mapping of the transferrin receptor resolved the same two phosphopeptides (X and Y) after either protein kinase C phosphorylation in vitro or treatment of labeled A431 cells with phorbol diesters. [32P]Phosphoserine was the only labeled phosphoamino acid detected. Phosphopeptide X was shown to be an incomplete tryptic digestion product which could be further digested with trypsin to generate the limit tryptic phosphopeptide (Y). Radiosequence analysis of [32P]phosphopeptide Y demonstrated that the [32P]phosphoserine was the second residue from amino terminus of the peptide. This receptor phosphopeptide was found to co-migrate with the synthetic peptide Phe-Ser(P)-Leu-Ala-Arg (where Ser(P) is phosphoserine) during reverse-phase high pressure liquid chromatography and two-dimensional thin layer electrophoresis and chromatography. The peptide Phe-Ser(P)-Leu-Ala-Arg is an expected tryptic fragment of the cytoplasmic domain of the transferrin receptor corresponding to residues 23-27. We conclude that the major site of protein kinase C phosphorylation of the transferrin receptor in vivo and in vitro is serine 24. This phosphorylation site is located within the intracellular domain of the transferrin receptor, 38 residues away from the predicted transmembrane domain.

Amino Acid Sequence↗

PubMed1985

Isolation and properties of two actin-binding domains in gelsolin.

Gelsolin is a Ca2+-sensitive 90-kDa protein which regulates actin filament length. A molecular variant of gelsolin is present in plasma as a 93-kDa protein. Functional studies have shown that gelsolin contains two actin-binding sites which are distinct in that after Ca2+-mediated binding, removal of free Ca2+ releases actin from one site but not from the other. We have partially cleaved human plasma gelsolin with alpha-chymotrypsin and identified two distinct actin-binding domains. Peptides CT17 and CT15, which contain one of the actin-binding domains, bind to actin independently of Ca2+; peptides CT54 and CT47, which contain the other domain, bind to actin reversibly in response to changes in Ca2+ concentration. These peptides sequester actin monomers inhibiting polymerization. Unlike intact gelsolin, neither group of peptides nucleates actin assembly or forms stable filament end caps. CT17 and CT15 can however sever actin filaments. Amino acid sequence analyses place CT17 at the NH2 terminus of gelsolin and CT47 at the carboxyl-terminal two-thirds of gelsolin. Circular dichroism measurements show that Ca2+ induces an increase in the alpha-helical content of CT47. These studies provide a structural basis for understanding the interaction of gelsolin with actin and allow comparison with other Ca2+-dependent actin filament severing proteins.

Actins↗