Phospholipase C delta-type consists of three isozymes: bovine PLCdelta2 is a homologue of human/mouse PLCdelta4

To date, 12 phospholipase C (PLC) isozymes have been identified in mammals, and they are divided into five classes, beta-, gamma-, delta-, epsilon-, and zeta-type. PLCdelta-type is reported to be composed of four isozymes, PLCdelta1-delta4. Here we report that a screening for mouse PLCdelta2 from a BAC library with primers that amplify a specific region of bovine PLCdelta2 resulted in isolation of one clone containing the mouse PLCdelta4 gene. Furthermore, a database search revealed that there is only one gene corresponding to PLCdelta2 and PLCdelta4 in the mouse and human genomes, indicating that bovine PLCdelta2 is a homologue of human and mouse PLCdelta4. However, PLCdelta2 Western blot analysis with a widely used commercial anti-PLCdelta2 antibody showed an expression pattern distinct from that of PLCdelta4 in wild-type mice. In addition, an 80-kDa band, which was recognized by antibody against PLCdelta2, was smaller than an 85-kDa band detected by anti-PLCdelta4 antibody, and the 80-kDa band was detectable in lysates of brain, testis, and spleen from PLCdelta4-deficient mice. We also found that immunoprecipitates from brain lysates with this PLCdelta2 antibody contained no PLC activity. From these data, we conclude that bovine PLCdelta2 is a homologue of human and mouse PLCdelta4, and that three isozymes (delta1, delta3, and delta4) exist in the PLCdelta family.     

Isolation and characterization of alpha1-proteinase inhibitor from common carp (Cyprinus carpio) seminal plasma

Using a three-step procedure, we purified (79 and 51.6-fold to homogeneity) and characterized the two isoforms (a and b) of alpha1-proteinase inhibitor-like protein from carp seminal plasma. The isoforms have molecular masses of 55.5 and 54.0 kDa, respectively. These inhibitors formed SDS-stable complexes with cod and bovine trypsin, chymotrypsin and elastase. The thirty-three amino acids within the reactive loop SLPDTVILNRPFLVLIVEDTTKSILFMGKITNP were identified for isoform b. The same first ten amino acids were obtained for isoform a, and this sequence revealed 100% homology to carp alpha1-proteinase inhibitor (alpha1-PI) from perimeningeal fluid. Both isoforms of alpha1-PI are glycoproteins and their carbohydrate content was determined to be 12.6 and 12.1% for a and b, respectively. Our results indicated that alpha1-PI is one of the main proteins of carp seminal plasma. Using polyclonal anti-alpha1-PI antibodies, alpha1-PI was for the first time localized to the carp testis. The presence of alpha1-PI in testis lobules and in the area surrounding spermatides suggests that this inhibitor may be involved in the maintenance of testis connective tissue integrity, control of spermatogenesis or protection of tissue and spermatozoa against unwanted proteolysis. Since similar alpha1-PI has been identified in rainbow trout semen it can be suggested that the presence of alpha1-PI in seminal plasma is a common feature of cyprinid and salmonid fish.     

Cloning, expression, purification, and characterization of the human Class Ia phosphoinositide 3-kinase isoforms

The Class I phosphoinositide 3-kinases (PI3Ks) are lipid kinases that phosphorylate the 3-hydroxyl group of the inositol ring of phosphatidylinositides. Although closely related, experimental evidence suggests that the four Class I PI3Ks may be functionally distinct. To further study their unique biochemical properties, the three human Class Ia PI3K (alpha, beta, and delta) p110 catalytic domains were cloned and co-expressed with the p85alpha regulatory domain in Sf9 cells. None of the p110 subunits were successfully expressed in the absence of p85alpha. Successful expression and purification of each p85alpha/p110 protein required using an excess of the p110 vector over the p85 vector during co-infection of Sf9 cells. Proteins were purified as the p85alpha/p110 complex by nickel affinity chromatography through an N-terminal His-tag on the p110 subunit using an imidazole gradient. The purification yields were high using the optimized ratio of p85/p110 vector and small culture volumes, with 24mg/L cell culture media for p85alpha/p110alpha, 17.5mg/L for p85alpha/p110delta, and 3.5mg/L for p85alpha/p110beta. The identity of each purified isoform was confirmed by mass spectral analysis and immunoblotting. The activities of the three p85alpha/p110 proteins and the Class Ib p110gamma catalytic domain were investigated using phosphatidylinositol 4,5-bisphosphate (PIP2) as the substrate in a PIP2/phosphatidylserine (PS) liposome. All four enzymes exhibited reaction velocities that were dependent on the surface concentration of PIP2. The surface concentrations that gave maximal activity for each human isoform with 0.5mM PIP2 were 2.5mol% PIP2 for p110gamma, 7.5mol% for p85alpha/p110beta, and 10mol% PIP2 for p85alpha/p110alpha and p85alpha/p110delta. The specific activity of p85alpha/p110alpha was three to five times higher than that of the other human isoforms. These kinetic differences may contribute to the unique roles of these isoforms in cells.     

Characterization of the protein phosphatase 1 catalytic subunit in endothelium: involvement in contractile responses

We have previously demonstrated the direct involvement of a type 1 Ser/Thr phosphatase (PPase 1) in endothelial cell (EC) barrier regulation [Am. J. Physiol. 269:L99-L108, 1995]. To further extend this observation, we microinjected either the Ser/Thr PPase inhibitor, calyculin, or the PPase 1 inhibitory protein, I-2 into bovine pulmonary artery EC and demonstrated both an increase in F-actin stress fibers and a shift from a regular polygonal shape to a spindle shape with gaps apparent at the cell borders. Northern blot analysis with specific cDNA probes revealed the presence of three major PPase 1 catalytic subunit (CS1) isoforms (alpha, delta, and gamma) in human and bovine EC. To characterize the myosin-associated EC CS1 isoform, myosin-enriched bovine EC fraction was screened with anti-CS1alpha and anti-CS1delta antibodies The anti-CS1delta antiserum, but not anti-CS1alpha antiserum cross reacts with the CS1 isoform present in myosin-enriched fraction and CS1delta was found in stable association with EC myosin/myosin light chain kinase (MLCK) complex in MLCK immunoprecipitates under nondenaturing conditions. Consistent with these data, overexpression of CS1delta-GFP construct in bovine endothelium followed by immunoprecipitation of CS1 with anti-GFP antibody revealed the stable association of CS1delta with actomyosin complex. Finally, screening of a human EC oligo(dT)-primed cDNA library with a probe encoding a rat CS1delta cDNA segment yielding several positive clones that encoded the entire CS1delta open reading frame and partially noncoding regions. Sequence analysis determined a high homology ( approximately 99%) with human CS1delta derived from a teratocarcinoma cell line. Together, these data suggest that CS1delta is the major of PPase 1 isoform specifically associated with EC actomyosin complex and which participates in EC barrier regulation.     

Major pertussis-toxin-sensitive GTP-binding protein of bovine lung. Purification, characterization and production of specific antibodies

Two GTP-binding proteins which can be ADP-ribosylated by islet-activating protein, pertussis toxin, were purified from the cholate extract of bovine lung membranes. Both proteins had the same heterotrimeric structure (alpha beta gamma), but the alpha subunits were dissociated from the beta gamma when they were purified in the presence of AlCl3, MgCl2 and NaF. The molecular mass of the alpha subunit of the major protein (designated GLu, with beta gamma) was 40 kDa and that of the minor one was 41 kDa. The results of peptide mapping analysis of alpha subunits with a limited proteolysis indicated that GLu alpha was entirely different from the alpha of brain Gi or Go, while the 41-kDa polypeptide was identical with the alpha of bovine brain Gi. The kinetics of guanosine 5'-[3-O-thio]triphosphate (GTP[gamma S]) binding to GLu was similar to that to lung Gi but quite different from that to brain Go. On the other hand, incubation of GLu alpha at 30 degrees C caused a rapid decrease of GTP[gamma S] binding, the inactivation curve being similar to that of Go alpha but different from that of Gi alpha. The alpha subunits of lung Gi and GLu did not react with the antibodies against the alpha subunit of bovine brain Go. The antibodies were raised in rabbits against GLu alpha and were purified with a GLu alpha-Sepharose column. The purified antibodies reacted not only with GLu alpha but also with the 41-kDa protein and purified brain Gi alpha. However, the antibodies adsorbed with brain Gi alpha reacted only with GLu alpha, indicating antisera raised with GLu alpha contained antibodies that recognize both Gi alpha and GLu alpha, and those specific to GLu alpha. These results further indicate that GLu is different from Gi or Go. Anti-GLu alpha antibodies reacted with the 40-kDa proteins in the membranes of bovine brain and human leukemic (HL-60) cells. The beta gamma subunits were also purified from bovine lung. The beta subunit was the doublet of 36-kDa and 35-kDa polypeptides. The lung beta gamma could elicit the ADP-ribosylation of GLu alpha by islet-activating protein, increase the GTP[gamma S] binding to GLu and protect the thermal denaturation of GLu alpha. The antibodies raised against brain beta gamma cross-reacted with lung beta but not with lung gamma.     

Identification of exposed and buried determinants of the membrane-bound acetylcholine receptor from Torpedo californica

The ability of five rabbit anti-acetylcholine receptor antisera to recognize the membrane-bound receptor from Torpedo californica has been investigated. Two antisera, raised against affinity-purified native receptor, react extensively with purified receptor-rich membrane vesicles. Since the membrane vesicles are impermeable to macromolecules and are oriented right side out, these two antisera recognize predominantly extracellular determinants. Two antisera against sodium dodecyl sulfate denatured receptor and one against purified delta subunit react poorly with the membrane-bound receptor. Only 10-20% of the determinants recognized by these antisera are accessible to antibodies when the receptor is membrane bound. Many of the latent sites can be exposed by permeabilizing the vesicles with saponin, by alkaline extraction of the membranes to remove peripheral proteins, or by a combination of these two treatments. These treatments neither solubilize the receptors nor interfere with their ability to undergo agonist-induced affinity changes. Subunit analysis of the sites on the membrane-bound receptor that are accessible to antibodies indicates that the alpha, beta, and delta chains possess extracellular determinants. Buried sites are present on all four of the subunits. Saponin permeabilization makes latent sites accessible on alpha and delta while alkaline extraction uncovers determinants on alpha, gamma, and delta. Treatment of membranes by both procedures reveals sites on beta, gamma, and delta that are not uncovered by either treatment alone. This study, in conjunction with results from other laboratories demonstrating that the gamma chain is extracellularly exposed, suggests that all four subunits are transmembrane proteins.     

Mammalian protein geranylgeranyltransferase. Subunit composition and metal requirements.

An enzyme capable of specifically modifying, with a geranylgeranyl isoprenoid, candidate proteins containing a consensus prenylation sequence ending in leucine has been purified from bovine brain. This protein geranylgeranyltransferase (PGGT), isolated using affinity chromatography on an immobilized peptide column, contains two subunits with molecular masses of 48 and 43 kDa, designated alpha and beta, respectively. An antiserum raised to the alpha subunit of the related enzyme, protein farnesyltransferase (PFT), also recognizes this chromatographically identical alpha-subunit of the PGGT by immunoblot analysis. The PGGT and PFT enzymes from bovine brain are shown to be dependent on both Mg2+ and Zn2+ for optimal activity. Demonstration of the Zn2+ dependence of the enzymes requires prolonged incubation or purification in the presence of a chelating agent; we therefore propose that these enzymes be placed into the category of metalloenzymes. Under optimal assay conditions, these enzymes show high specificity toward their prenyl diphosphate substrates, with only a weak competition observed with farnesyl diphosphate in the PGGT reaction or geranylgeranyl diphosphate in the PFT reaction. The two enzymes are differentially sensitive to several detergents tested to determine suitable ones for product stabilization in the reactions. These results confirm previous predictions on the subunit structure of the PGGT and provide an avenue to initiating a molecular analysis of the geranylgeranyl modification of many mammalian proteins.     

Effects of gelsolin on human platelet cytosolic phosphoinositide-phospholipase C isozymes.

The effective resolution of human platelet cytosolic phosphoinositide-phospholipase C (PLC) revealed five distinct activity peaks by Q-Sepharose and heparin-Sepharose column chromatographies when assayed using phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2). The results of Western blotting analysis with various antibodies against PLC isozymes showed that peak-Ia (PLC-delta type), peak-Ib (PLC-gamma 1 type), and peak-IIc (PLC-beta type) and two unidentified activity peaks (PLC-IIa and PLC-IIb) were present in human platelet cytosol. A protein with guanosine 5'-3-O-(thio)triphosphate-binding activity was coeluted with the PLC-IIa and was purified to homogeneity. It exhibited 86- and 42-kDa polypeptide bands upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis which were identified as gelsolin and actin by immunostaining, respectively. Large amounts of gelsolin/actin (1:1) complex "gelsolin complex" were detected in the PLC-delta and PLC-gamma 1 fractions. The PLC-gamma 1 and the gelsolin complex were co-immunoprecipitated by the antibody raised against PLC-gamma 1. Furthermore, the partially purified bovine brain PLC-gamma 1 fraction also was found to be associated with the gelsolin complex and the association was released by the addition of 1% sodium cholate. This finding has prompted us to examine effects of the gelsolin complex and the free gelsolin on activities of the above PLC isoforms from platelet cytosol. The gelsolin complex did not affect the PIP2 hydrolyzing activities of all PLC isoforms. In contrast, the purified gelsolin inhibited distinctly PIP2 hydrolyses by PLC-Ia (delta), PLC-Ib (gamma 1), and PLC-IIa (unidentified), whereas the inhibitory effects for PLC-IIb (unidentified) and PLC-IIc (beta) were moderate. The inhibitory effect of gelsolin on PIP2-hydrolysis by PLC-gamma 1 was diminished by a large amount of PIP2 substrate. These results suggested that the inhibition of PLC by gelsolin is due to sequestration of substrate PIP2 by its competitive binding.     

Bovine γ-Aminobutyric AcidA Receptor Sequence-Specific Antibodies: Identification of Two Epitopes Which Are Recognised in Both Native and Denatured γ-Aminobutyric AcidA Receptors

Polyclonal antibodies have been raised against synthetic peptides whose sequences correspond to the N-terminal 15 amino acids and the C-terminal 17 amino acids of the bovine gamma-aminobutyric acidA (GABAA) receptor alpha 1 subunit. These antibodies were shown to react with the denatured GABAA receptor alpha subunit, Mr 53,000, in Western blots with both purified receptor and brain membranes as antigens. Also, both antibodies recognised both the purified and detergent-solubilised GABAA receptor as demonstrated by dose-dependent specific immunoprecipitation of the GABA and benzodiazepine binding sites from solution. Evidence is also presented to show brain-regional distribution of the expression of the alpha 1 subunit.     

Different Glycosylation in Acetylcholinesterases from Mammalian Brain and Erythrocytes

Acetylcholinesterases (EC 3.1.1.7, AChE) have varying amounts of carbohydrates attached to the core protein. Sequence analysis of the known primary structures gives evidence for several asparagine-linked carbohydrates. From the differences in molecular mass determined on sodium dodecyl sulfate-polyacrylamide gel before and after deglycosylation with N-glycosidase F (EC 3.2.2.18), it is seen that dimeric AChE from red cell membranes is more heavily glycosylated than the tetrameric brain enzyme. Furthermore, dimeric and tetrameric forms of bovine AChE are more heavily glycosylated than the corresponding human enzymes. Monoclonal antibodies 2E6, 1H11, and 2G8 raised against detergent-soluble AChE from electric organs of Torpedo nacline timilei as well as Elec-39 raised against AChE from Electrophorus electricus cross-reacted with AChE from bovine and human brain but not with AChE from erythrocytes. Treatment of the enzyme with N-glycosidase F abolished binding of monoclonal antibodies, suggesting that the epitope, or part of it, consists of N-linked carbohydrates. Analysis of N-acetylglucosamine sugars revealed the presence of N-acetylglucosamine in all forms of cholinesterases investigated, giving evidence for N-linked glycosylation. On the other hand, N-acetylgalactosamine was not found in AChE from human and bovine brain or in butyrylcholinesterase (EC 3.1.1.8) from human serum, indicating that these forms of cholinesterase did not contain O-linked carbohydrates. Despite the notion that within one species, the different forms of AChE arise from one gene by different splicing, our present results show that dimeric erythrocyte and tetrameric brain AChE must undergo different postsynthetic modifications leading to differences in their glycosylation patterns.     

Effects of the expression of bovine growth hormone on the testes and male accessory reproductive glands in transgenic mice

The effects of physiological and excessive levels of growth hormone (GH) on reproductive functions are poorly understood, and impairment of fertility is frequently observed in transgenic animals overexpressing GH genes. The present study was undertaken to determine the effects of chronic exposure to heterologous bovine GH (bGH) on the testes and accessory reproductive glands in transgenic mice. Endocrine function of the testes was evaluated by measuring the activities of two steroidogenic enzymes, 5-3-hydroxysteroid dehydrogenase (5-3-HSD) and 17-hydroxysteroid dehydrogenase (17-HSD). The activities of acid phosphatase, alkaline phosphatase and -glucuronidase, important hydrolytic enzymes of lysosomal origin, were measured in testes, seminal vesicles and ventral prostates in normal and transgenic mice. Testicular 5-3-HSD activity was higher in transgenic than in normal mice, while testicular 17-HSD activity in transgenic mice was not altered. Acid phosphatase activity was elevated in both seminal vesicles and ventral prostates of transgenic mice, while alkaline phosphatase activity was increased only in the prostate. The activity of -glucuronidase was elevated in the testes, seminal vesicles and ventral prostate gland of transgenic mice. These results suggest that chronic exposure to bGH is associated with significant stimulation of some hydrolytic enzymes in the testes and in the accessory reproductive glands of transgenic mice.     

Subcellular localization of phospholipase C isoforms in vascular smooth muscle

The phospholipase C (PLC) isoform most important during agonist-activated IP(3) production in vascular smooth muscle is still unknown. When PLC activity in rat tail artery homogenate was determined, this activity was shown to be inhibited by an antibody directed against PLCbeta2. Antibodies directed against the gamma1, beta1, beta3 and delta1 isoforms of PLC failed to inhibit PLC activity in this tissue. Both PLCbeta2 and PLCgamma1 were isolated from rat tail artery by DEAE column chromatography and PLCbeta2 activity was shown to be 3-fold greater than PLCgamma1 activity. When rat tail artery was treated with norepinephrine (10 mM), PLCbeta2 was shown to translocate from cytosol to membranes. When subcellular fractions of rat tail artery were isolated by sucrose density gradient centrifugation, including nuclei, plasma membrane, and cytosol, PLCbeta2 was detected in the plasma membrane and the cytosol but not in the nuclei. PLCdelta1 and PLCgamma1 were found only in cytosol. This evidence is consistent with the model wherein an agonist such as norepinephrine can activate smooth muscle contraction via interaction with a plasma membrane receptor which can easily interact with a plasma membrane-associated isoform of PLC, such as PLCbeta2.     

Effects of unilateral orchidectomy on rat epididymal delta 4-5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase

The effects of unilateral orchidectomy on the adult rat epidiymal testosterone metabolizing enzymes, delta 4-5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase, are investigated. Five weeks following unilateral orchidectomy, it is found that the activity of 3 alpha-hydroxysteroid dehydrogenase per organ is not altered, whereas delta 4-5 alpha-reductase activity decreased by more than 80% on the side of the orchidectomy. Neither accessory sex tissue weights, ventral prostate and seminal vesicles, nor the concentration of circulating testosterone, luteinizing hormone, follicle-stimulating hormone, or prolactin is altered by unilateral orchidectomy. These data indicate that (1) epididymal 3 alpha-hydroxysteroid dehydrogenase activity can be maintained by circulating androgens and that (2) the major factor regulating delta 4-5 alpha-reductase activity is not a substance secreted by the testes into the peripheral circulation. It is suggested that a substance directly secreted into the epididymis by the testis regulates epididymal delta 4-5 alpha-reductase activity.     

Characterization of two 85 kd proteins that associate with receptor tyrosine kinases, middle-T/pp60c-src complexes, and PI3-kinase.

Affinity-purified bovine brain phosphatidylinositol 3-kinase (PI3-kinase) contains two major proteins of 85 and 110 kd. Amino acid sequence analysis and cDNA cloning reveals two related 85 kd proteins (p85 alpha and p85 beta), which both contain one SH3 and two SH2 regions (src homology regions). When expressed, these 85 kd proteins bind to and are substrates for tyrosine-phosphorylated receptor kinases and the polyoma virus middle-T antigen/pp60c-src complex, but lack PI3-kinase activity. However, an antiserum raised against p85 beta immunoprecipitates PI3-kinase activity. The active PI3-kinase complex containing p85 alpha or p85 beta and the 110 kd protein binds to PDGF but not EGF receptors. p85 alpha and p85 beta may mediate specific PI3-kinase interactions with a subset of tyrosine kinases.     

Identification of a novel cytosolic poly-phosphoinositide-specific phospholipase C (PLC-86) as the major G-protein-regulated enzyme.

Activation of phosphoinositide-specific phospholipase C (PLC) generates two intracellular signals which play major roles in many cellular processes including secretion, proliferation and contraction. PLC activation by many receptors occurs via a guanine nucleotide regulatory protein, Gp. PLCs are found predominantly in the cytosolic fraction though some activity is membrane-associated. At least four families of iso-enzymes of PLC (alpha, beta, gamma and delta) have been identified, but there is only scant evidence to indicate that any of the mammalian cytosolic activities are involved in G-protein-regulated signalling. In this study we demonstrate that the PLC activity from rat brain cytosol can be regulated in a G-protein-dependent manner in a reconstituted system using pre-permeabilized HL60 cells. We identify two enzymes, PLC-beta and a novel 86 kDa enzyme (designated PLC-epsilon) as the G-protein-regulated enzymes. PLC-epsilon was found to be the major G-protein-regulated enzyme.     

Regulation of signal transfer from beta 1-adrenoceptor to adenylate cyclase by beta gamma subunits in a reconstituted system

The beta gamma subunits of guanine nucleotide binding proteins from bovine brain and bovine rod outer segments have different structural and immunochemical properties. In spite of these structural differences, beta gamma subunits from these sources have been found to be fully interchangeable in terms of their interaction with alpha subunits of pertussis-toxin-sensitive G proteins. In contrast, however, there are striking differences between these beta gamma subunits with regard to their ability to deactivate fluoride-stimulated Gs. These profound differences were also observed when the interaction of the purified components of the adenylate cyclase system was studied after reconstitution into phospholipid vesicles. Addition of beta gamma purified from bovine brain to vesicles containing beta-receptor and Gs results in a biphasic effect on receptor-stimulated GTPase activity, whereas addition of transducin beta gamma was virtually without any effect. Likewise, beta gamma from bovine brain, but not transducin beta gamma, affected adenylate cyclase activity of a reconstituted system consisting of three purified components (R, Gs, C). Thus, the alpha subunit of Gs, but not the alpha subunits of pertussis-toxin-sensitive G proteins discriminate between structurally different beta gamma subunits.     

Identification of two calcineurin alpha isoforms in bovine brain by two different monoclonal antibodies.

Calcineurin (calcium- and calmodulin-stimulated phosphatase) alpha subunit purified from bovine brain was found to be composed of two polypeptides, 61 KDa (alpha 1) and 59 KDa (alpha 2). The two peptides were separated and extracted from polyacrylamide gel. The immuno-peptide mapping of the purified peptides by partial proteolysis showed that the 59-KDa polypeptide was not a degradative product of the 61-KDa polypeptide. The interaction of the enzyme with two monoclonal antibodies, Vj6 and Vd3, raised against bovine brain calcineurin revealed that the 61-KDa polypeptide was recognized by both Vj6 and Vd3, whereas the 59-KDa one was recognized only by Vj6. These results indicate that there are at least two isoforms of calcineurin alpha subunits in bovine brain.     

An antibody against 100- to 116-kDa polypeptides in coated vesicles inhibits triskelion binding

There is considerable evidence that the 100- to 116-kDa polypeptides in calf brain coated vesicles are involved in the assembly of clathrin triskelions to form coated vesicles. We have raised polyclonal antibodies against these polypeptides. By Western blot analysis, these antibodies bind to a distinct subset of the six polypeptides in the region 100-116 kDa. Whole cell homogenates from calf brain, calf liver, and rat liver also show immunoreactivity in the 100-kDa region with no other cross reactivity. Isolated coated vesicles from calf liver, rat brain, and soybean roots also cross-react. Stripped coated vesicles, which are depleted of clathrin but which retain the 100- to 116-kDa polypeptides, quantitatively rebind 125I-triskelions. This binding is inhibited in a dose-dependent manner by 100- to 116-kDa antibody but not by nonimmune serum or by anti-clathrin polyclonal antibody. These studies indicate that (1) specific sites on the 100- to 116-kDa polypeptides are required for assembly of coated vesicles, and (2) this antibody will be useful in clarifying more precisely the role of the 100- to 116-kDa polypeptides in coated vesicle recycling.