Structural Proteins of Simian Virus 40

Sodium dodecyl sulfate acrylamide gel electrophoresis of the solubilized proteins from purified simian virus 40 (SV40) virions revealed two major and two minor structural polypeptide components. The major components which comprise over 75% of the total virion were shown to be the capsid proteins by immunological and isoelectric focusing fractionation analysis. These two polypeptides have estimated molecular weights of 45,000 daltons as determined by gel electrophoresis. One of the two minor components was identified as the nucleocapsid protein and has an approximate molecular weight of 16,000. The other unidentified minor component has an average molecular weight of 29,000.     

Differential expression of Paragonimus westermani eggshell proteins during the developmental stages

Eggs of trematode parasites are comprised of numerous vitelline cells and one fertilized ovum, and are encapsulated within a protein shell provided by the vitellocytes. In this study, we isolated two full-length cDNA clones that showed substantial levels of sequence identity with trematode-specific eggshell precursor proteins from the human lung fluke, Paragonimus westermani. These cDNAs, designated Pw-Vit20 (868-bp-long) and Pw-Vit36 (883-bp-long), shared a 76% identity with one another at the nucleotide level, and each encoded a 261-amino acid (aa) polypeptide. The deduced aa sequences contained a N-terminal hydrophobic segment, as well as a sequence motif of Gly-Gly-Gly-Tyr-Asp-Asn/Thr-Tyr-Gly-Lys/Gln, which is highly homologous with the eggshell proteins of Fasciola hepatica. With the high frequencies of tyrosine, glycine and lysine, the positions occupied by tyrosine, which has been proved to be converted into dihydroxyphenylalanine, were well preserved. Pw-Vit20 and Pw-Vit36 were found to be monoexonic genes with variably diverged variants scattered into multiple genomic loci. Their protein products were localized in the vitelline follicles and eggshells. Expression of Pw-Vit20 was restricted to the egg and adult stages, thus suggesting a critical involvement of Pw-Vit20 in the parasite's fecundity activity. Conversely, Pw-Vit36 was constitutively expressed in the metacercariae and juvenile stages in the vitelline follicles and ducts, which suggested that the prepositioning of stem or primordial vitelline cells within the juveniles prior to sexual maturation. Pw-Vit36 might acquire a unique or additional function relevant to the maturation and/or development of the vitelline cells/follicles during the evolutionary period of P. westermani. Differential biological implications of multiple eggshell precursor proteins may provide insight into the molecular mechanism of eggshell formation and the developmental process of the vitelline follicles in the parasitic trematode.     

A novel calmodulin-like protein from the liver fluke, Fasciola hepatica

An 18.2 kDa protein from the liver fluke, Fasciola hepatica has been identified and characterised. The protein shows strongest sequence similarity to egg antigen proteins from Schistosoma mansoni, Schistosoma japonicum and Clonorchis sinensis. The protein is predicted to adopt a calmodulin-like fold; it thus represents the third calmodulin-like protein to be characterised in F. hepatica and has been named FhCaM3. Compared to the classical calmodulin structure there are some variations. Most noticeably, the central, linker helix is disrupted by a cysteine residue. Alkaline native gel electrophoresis showed that FhCaM3 binds calcium ions. This binding event increases the ability of the protein to bind the hydrophobic fluorescent probe 8-anilinonaphthalene-1-sulphonate, consistent with an increase in surface hydrophobicity as seen in other calmodulins. FhCaM3 binds to the calmodulin antagonists trifluoperazine and W7, but not to the myosin regulatory light chain binding compound praziquantel. Immunolocalisation demonstrated that the protein is found in eggs and vitelline cells. Given the critical role of calcium ions in egg formation and hatching this suggests that FhCaM3 may play a role in calcium signalling in these processes. Consequently the antagonism of FhCaM3 may, potentially, offer a method for inhibiting egg production and thus reducing the spread of infection.     

A photoactivable phospholipid analogue that specifically labels membrane cytoskeletal proteins of intact erythrocytes

A radioactive photoactivable analogue of phosphatidylethanolamine, 2-(2-azido-4-nitro-benzoyl)-1-acyl-sn-glycero-3-phospho[14C]ethanolamine ([14C]AzPE), was synthesized. Upon incubation with erythrocytes in the dark, about 90% of [14C]AzPE spontaneously incorporated into the cells; of this fraction, about 90% associated with the membrane, all of it noncovalently. Upon photoactivation, 3-4% of the membrane-associated probe was incorporated into protein. Analysis of this fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as well as extraction of labeled membranes with alkali or detergent, showed that the probe preferentially labeled cytoskeletal proteins. [14C]AzPE appears to be a useful tool for the study of lipid-protein interactions at the cytoplasmic face of the plasma membrane of intact cells.     

A protein that accumulates during starvation in Tetrahymena nuclei

Tetrahymena pyriformis was starved in 50 mM Tris-HCl, pH 7.5, at 28 degrees C. The number of cells did not change appreciably under the starvation conditions. Nuclear proteins of unstarved cells and cells starved for 1, 2, 4, and 7 d were analyzed by SDS-polyacrylamide gel electrophoresis. Most of the large amount of nonhistone proteins present in the unstarved cell nucleus disappeared with the starvation time. However, the relative amounts of the high mobility group protein and histones did not change appreciably. On the other hand, a protein with a molecular weight of ca. 16,000 gradually accumulated in the nucleus on starvation. This protein was extracted with 0.25 M HCl, but was not soluble in 0.5 M perchloric acid. The amino acid composition and molecular weight of this protein were similar to those of HMG protein LG-2 of T. thermophila. Some lysyl endopeptidase peptides of this protein were found to have amino acid sequences present in LG-2, thus we tentatively named it an LG-2-like protein.     

A novel approach to identify bovine sperm membrane proteins that interact with receptors on the vitelline membrane of bovine oocytes

At fertilization, the sperm triggers intracellular calcium oscillations, which are pivotal to oocyte activation and development. A working hypothesis for the interaction between the sperm and the oocyte is that disintegrin ligands on the inner acrosomal membrane of the sperm bind to integrin receptors on the oocyte vitelline membrane. The aim of these experiments was to find and identify the sperm protein ligands involved in bovine sperm-oocyte interactions. In situ fluorescent labeling of proteins and 2-D gel electrophoresis were used to identify specific sperm membrane proteins that interact with proteins in the oocyte vitelline membrane. Sperm were labeled with a fluorescent dye and used to fertilize zona-free oocytes. Sperm-oocyte complexes were either lysed immediately, or following covalent cross-linking of proteins with dibromobimane. The cross-linking reagent serves the critical function of covalently linking proteins together so that they will remain as a unit through lysis of the cells and 2-D gel analysis, and which can be subsequently identified by mass spectrometry. Lysates were electrophoretically run on the same 2-D gel. The comparison of uncross-linked and cross-linked protein spots revealed that some proteins shifted position based on binding. These spots were picked and proteins identified by mass spectrometry. These results provide a list of specific sperm proteins that interact with oocyte membrane proteins and establish a group of candidate ligands, one or more of which may be responsible for induction of outside-in signaling resulting in oocyte activation and fusion of the gametes.     

Characterization of the apolipoproteins of rat plasma lipoproteins.

Purified fractions of three major rat high-density lipoproteins (HDL) and one rat very low-density lipoprotein (VLDL) were isolated by Sephadex gel chromatography or preparative sodium dodecyl sulfate gel electrophoresis. These proteins were characterized by amino acid analysis, end-group analysis, molecular-weight determination, polyacrylamide gel electrophoresis, and circular dichroism. One of these rat proteins, of molecular weight 27 000, appears to be homologous with the human A-I protein. However, rat HDL possesses two additional major components not reported in human HDL - an arginine-rich protein of molecular weight 35 000 and a protein of molecular weight 46 000. The arginine-rich protein of the rat is similar in size and amino acid analysis to the arginine-rich protein reported in human VLDL. A major component of rat VLDL of 35 000 molecular weight appears similar or identical to the arginine-rich protein in rat HDL by every criterion employed for their characterization.     

Isolation from human erythrocytes of a new membrane protein which inhibits the formation of complement transmembrane channels

A protein which inhibited complement channel formation was isolated from extracts of papain-digested human erythrocyte membranes using DEAE-Sephacel, Bio-Gel A0.5m column chromatographies, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer to nitrocellulose paper and elution with 2% NP-40 solution. The purified protein showed a molecular weight of 18 kDa, and efficiently inhibited hemolysis of EC5-7 cells with C8 and C9, but did not show any decay-accelerating activity to C5 convertase. Immunochemical analysis of native membranes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis using the antibody against this protein gave a single band having the same mobility as this protein; papain did not eliminate a significant portion of this protein.     

A potential diagnostic reagent for bovine cysticercosis

A fraction of larval Taenia hydatigena cyst fluid was shown to have high sensitivity and specificity in the enzyme-linked immunosorbent assay (ELISA) for the detection of bovine antibodies to the heterologous parasite Taenia saginata. This antigenically active lipoprotein fraction was isolated by ultracentrifugal density flotation using either ammonium sulfate (specific gravity = 1.231 g per ml) or NaCl/KBr (specific gravity = 1.225 g per ml), followed by ion-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that this fraction was composed of high molecular weight (65,000 to 77,000 Mr) and low molecular weight (9,500 to 16,000 Mr) proteins. Electrophoresis under non-denaturing conditions in either acrylamide (5%) or agarose (1%) resulted in 1 major diffuse band staining for both protein and lipid. The high and low molecular weight proteins observed on SDS-PAGE under reducing conditions could not be resolved by gel filtration chromatography and emerged as a single lipoprotein peak. This T. hydatigena cyst fluid fraction appears promising as a diagnostic reagent in the ELISA for bovine cysticercosis.     

Binding characteristics and affinity labeling of protein constituents of the human IM-9 lymphoblast receptor for substance P

The neuropeptide substance P (SP) stimulates human T-lymphocyte function in vitro. Human blood T-lymphocytes and cultured human IM-9 B-lymphoblasts express 7,000-10,000 and 25,000-30,000 substance P receptors per cell, respectively. The specific binding of 125I-SP is retained in IM-9 lymphoblast membranes solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) at a detergent-to-protein ratio of 1.0. In addition, specific and reversible SP binding to soluble IM-9 cell membrane proteins is demonstrated by gel filtration. The saturation of binding of 125I-SP to both intact and solubilized IM-9 cell membranes attained a steady state after 40-50 min at 4 degrees C. Scatchard analysis of the concentration dependence of 125I-SP binding to IM-9 cell membranes revealed a KD of 0.87 +/- 0.8 nM (mean +/- S.D., n = 4), which is similar to that observed in intact cells, and a density of receptors of 21 +/- 3 fmol/mg of membrane protein (mean +/- S.D.). Binding of 125I-SP to solubilized membranes demonstrated a KD of 0.75 +/- 0.33 nM (mean +/- S.D., n = 3) and a density of receptors of 3.7 +/- 1.5 fmol/mg of membrane protein (mean +/- S.D., n = 3). Affinity cross-linking of 125I-SP by disuccinimidyl suberate to intact IM-9 cells and membranes revealed specifically labeled proteins of Mr 58,000 and 33,000 in cells, and 58,000, 33,000, and 16,000 in membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. Competitive effects of substituent peptides of SP on cross-linking and 125I-SP binding to membranes demonstrated that the SP receptor recognized the carboxyl-terminal domain of the peptide. Membranes from cells preincubated in vitro for 12 h at 37 degrees C with 10(-8) M SP demonstrated a decrease in SP receptor density to 13 +/- 2 fmol/mg (mean +/- S.D., n = 2), and a parallel diminution in the specific labeling of membrane proteins of Mr 58,000 and 33,000. These observations suggest that solubilization in CHAPS preserves the binding characteristics of the IM-9 lymphoblast receptor for SP, and that affinity cross-linking techniques identify by sodium dodecyl sulfate-polyacrylamide gel electrophoresis membrane proteins that are specifically labeled by SP.     

Proteins and glycoproteins of the erythrocyte membrane

Erythrocyte membranes from several species were prepared by three different methods of hypotonic hemolysis and examined for variations in protein and glycoprotein content by acrylamide gel electrophoresis in sodium dodecyl sulfate. Significant variations were noted in morphology of the membranes prepared by the different methods without attendant variations in protein patterns of the major membrane proteins for most cases observed, which show a similar pattern of nine common bands for all of the species observed. The significant difference in protein pattern which was noted was attributed to proteolytic digestion of membranes which were fragmented during preparation. Failure to remove white blood cells from membrane preparations was shown to be a significant source of the problem with proteolytic digestion. Glycoproteins were analyzed by acrylamide gel electrophoresis or by column chromatography. Each species appears to have a different major glycoprotein (or group of closely related glycoproteins). Molecular weights of glycoproteins calculated from acrylamide gel electrophoresis were found to vary with the percentage of acrylamide in the gel, indicating that these proteins do not behave in a normal fashion in this electrophoresis system. The molecular weight calculated from gel filtration data for the human membrane glycoproteins (26,000) was quite disparate from those calculated from gel electrophoresis (88,000 to 62,000 in 5 to 10% gels).     

Overexpression and DNA-binding properties of the mer-encoded regulatory protein from plasmid NR1 (Tn21).

In plasmid NR1 the expression of genes involved in mercury resistance (Tn21) is regulated by the trans-acting product of the merR gene. An in vivo T7 RNA polymerase-promoter overexpression system was used to detect a protein of approximately 16,000 daltons encoded by the merR reading frame. Overexpressed MerR constituted about 5% of labeled proteins. An in vitro MerR-mer-op (mer-op is the mer operator and promoter region) gel electrophoresis binding assay established that the binding site for MerR was located between the putative -35 and -10 sequences of the promoter for the mer structural genes. A nonsense mutation in the carboxyl half of MerR resulted in the loss of biological function and the loss of in vitro mer-op binding properties.     

Studies on the biogenesis of an enzymatically active complex III of the respiratory chain from yeast mitochondria

Complex III isolated from yeast mitochondria catalyzed an antimycin A and Diuron-sensitive coenzyme QH₂-cytochrome c reductase activity with a turnover number of 15.7 sec^?1 and contained 10 nmoles of cytochrome b and 4.6 nmoles of cytochrome c₁ per mg of protein. Electrophoresis in sodium dodecyl sulfate acrylamide gels resolved Complex III into 10 bands with apparent molecular weights of 50,000, 40,000, 30,000, 29,000, 24,000, 17,000, 16,000, 12,000, 8,400, and 5,800. Yeast cells were labeled under nongrowing conditions with (³⁵S)-methionine in the absence or presence of inhibitors of cytoplasmi? or mitochondrial protein synthesis. Labeled Complex III was isolated by immunoprecipitation from detergent-solubilized mitochondria using antiserum raised against the purified complex. Analysis of the immunoprecipitates by polyacrylamide gel electrophoresis revealed that a 30,000-dalton protein, cytochrome b, as well as 16,000-dalton protein were labeled in the presence of cycloheximide, indicating that they are products of mitochondrial protein synthesis. Immunoprecipitates from mitochondria obtained from cells labeled in the presence of chloramphenicol contained a new radioactive peak with a molecular weight of 100,000. In addition, significant decreases in the labeling of the proteins with molecular weights of 50,000, 40,000, 30,000, and 16,000 were observed. When Complex III was isolated by immunoprecipitation from intact spheroplasts after a 5-minute pulse with (³⁵S)-methionine, the 100,000-dalton protein was labeled in the immunoprecipitate whether or not chloramphenicol was present; however, after a 1-hour chase with unlabeled methionine, decreased labeling of the 100,000-dalton protein was observed concomitant with an increased labeling of the 50,000- and 40,000-dalton proteins. These results suggest that a protein with a molecular weight of 100,000 may either be a precursor or a partially assembled form of other proteins of Complex III, most probably the two largest polypeptides.     

Yeast plasma membrane ghosts. An analysis of proteins by two-dimensional gel electrophoresis

We have examined yeast cell ghost preparations to assess their value in obtaining plasma membrane proteins. Ghosts prepared by two methods involving stabilization of spheroplast envelopes had similar protein patterns by two-dimensional gel electrophoresis, and approximately 200 proteins were resolved. Spheroplasts were lactoperoxidase iodinated, and recovery of label in ghost preparations was greater than 60%. Spheroplasts appeared to be impermeable to the lactoperoxidase reagents as judged by an examination of two-dimensional gel electrophoretic patterns of ghost proteins that had been iodinated in spheroplasts or in unsealed ghosts. Spheroplasts were also impermeable to pronase proteases. Surface iodination and surface proteolysis allowed us to identify exposed ghost proteins; the major ghost glycoprotein was exposed in spheroplasts.Two-dimensional patterns of ghost proteins were not heavily contaminated (?25% of all proteins) by proteins present in soluble or promitochondrial fractions, and estimates of surface label and total cell protein recovery suggested that the ghost fraction represents a cell envelope enrichment of 8–10 fold over whole cells.Resolution of ghost proteins by two-dimensional gel electrophoresis appears to be a powerful aid toward identifying membrane proteins.     

Changes in Casein and Egg Albumin due to Reactions with Oxidizing Methyl Linoleate in Dehydrated Systems

Casein and egg albumin were allowed to react with methyl linoleate (ML) at a relative humidity (RH) of 0% or 80% at 50°C for 10 days (protein: ML= 1:0.2 or 1:1, w/w). Changes in the molecular sizes of the reacting proteins were examined by gel filtration and gel electrophoresis. Both proteins showed similar changes, whereas the reaction at RH 80% (protein: ML= 1:1) resulted in insolubilization because of polymerization. Changes in the amino acid residues of the reacting proteins were investigated after acid (6 n HC1) and enzymatic (pepsin-pancreatin, followed by aminopeptidase-prolidase) hydrolyses. Insignificant changes were observed in the amino acid composition of proteins reacted at RH 0%. After reaction at RH 80% (protein: ML =1:1), Lys, His and Met were the only amino acids affected. The percentage loss of these amino acids after acid hydrolysis was Lys (22%), His (41%), Met (9%) for casein and Lys (22%), His (31%), Met (1%) for egg albumin. This percentage loss after enzymatic hydrolysis was Lys (41%), His (49%), Met (94%) for casein and Lys (37%), His (42%), Met (88%) for egg albumin. Some differences between our results and other researchers were also discussed.     

Monoclonal antibody analysis of human pancreatic juice components related to the pancreatic calculi protein

A CaCO3-crystal growth inhibitor has recently been isolated from the calculi of patients affected by pancreatic lithiasis. It is a phosphoglycoprotein, with a molecular weight of 14,000, whose probable physiological role is the stabilization of exocrine pancreatic secretion which is supersaturated with respect to CaCO3. In order to isolate this inhibitor from human pancreatic juice, monoclonal antibodies to the protein were prepared and an immunoadsorbent column was developed. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of proteins fixed by the immunoadsorbent reveals the form having a molecular weight of 14,000, in addition to other protein bands which have higher molecular weights (16,000, 16,800, 18,000, and 18,800). All of these different proteins are also recognized by a monospecific polyclonal antibody to the 14,000 molecular weight form. Using the same monospecific polyclonal antibody only one messenger RNA coding for this inhibitor has been demonstrated. Thus, this heterogeneity might be explained by post-translational modifications.     

Prolactin-inducible proteins in human breast cancer cells

The mechanism of action of prolactin in target cells and the role of prolactin in human breast cancer are poorly understood phenomena. The present study examines the effect of human prolactin (hPRL) on the synthesis of unique proteins by a human breast cancer cell line, T-47D, in serum-free medium containing bovine serum albumin. [35S]Methionine-labeled proteins were analysed by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and fluorography. Treatment of cells with hPRL (1-1000 ng/ml) and hydrocortisone (1 microgram/ml) for 36 h or longer resulted in the synthesis and secretion of three proteins having molecular weights of 11,000, 14,000, and 16,000. Neither hPRL nor hydrocortisone alone induced these proteins. Of several other peptide hormones tested, only human growth hormone, a hormone structurally and functionally similar to hPRL, could replace hPRL in causing protein induction. These three proteins were, therefore, referred to as prolactin-inducible proteins (PIP). Each of the three PIPs was purified to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and specific antibodies were generated to them in rabbits. By immunoprecipitation and immunoblotting (Western blot) of proteins secreted by T-47D cells, it was demonstrated that the three PIPs were immunologically identical to one another. In addition, the 16-kDa and 14-kDa proteins (PIP-16 and PIP-14), and not the 11-kDa protein (PIP-11), incorporated [3H]glycosamine. Furthermore, 2-deoxyglucose (2 mM) and tunicamycin (0.5 micrograms/ml), two compounds known to inhibit glycosylation, blocked the production of PIP-16 and PIP-14, with a concomitant increase in the accumulation of PIP-11. These results indicate PIP-16 and PIP-14 are glycosylated variants of PIP-11. Finally, in vitro translation of poly(A)+ messenger RNA followed by immunoprecipitation revealed a 12.5-kDa protein, possibly the precursor form of PIPs. In addition, T-47D cells treated with hPRL plus hydrocortisone contained 10-fold more mRNA for PIPs than control cells, suggesting that the hormones' action is at the level of gene expression. Our finding represents a first demonstration of prolactin regulation of gene expression in human target cells. The human breast cancer cells, T-47D, appear to be an excellent model to afford future studies on the molecular action of prolactin and on the possible role of prolactin in human breast cancer.     

Antigenic, molecular and functional heterogeneity of Clara cell secretory proteins in the rat

In an earlier publication we had reported the preparation of a rabbit antiserum specific for rat Clara cell secretory proteins. This rabbit anti-rat Clara cell serum was found to react with two proteins in rat lung lavage by crossed-immunoelectrophoresis. Immunoblotting of rat lung lavage proteins, after sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis, disclosed three bands of reactivity with anti-Clara cell serum. The relative molecular masses of these three proteins were about 200 (protein A) 55 (protein B) and about 12 kDa (protein C). Anti-Clara cell antibodies eluted from Sepharose-4B-linked protein C (as well as the antiserum raised by immunizing rabbits with protein C) reacted with proteins A and C. Anti-Clara cell antiserum unbound to proteins A and C (as well as antiserum raised by immunizing rabbits with protein B) reacted with protein B only. In non-SDS polyacrylamide gel electrophoresis, protein B migrated as a single band, slightly cathodic to albumin; protein C resolved into three bands, all anodic to albumin. Immunoblots of isoelectric focusing gels showed three bands (pI 5.2-5.7) that reacted with antibody to protein C, and four bands corresponding to protein B were seen in the pI range 4.6-5.0. As determined by immunoperoxidase staining of paraformaldehyde fixed methacrylate embedded 1 micron thick sections of rat lung, protein(s) A (and protein C) and protein B were present in the same cells and in the same granules. Protein B was resistant to trypsin digestion, whereas proteins A and C were readily degraded by trypsin. Rat Clara cell secretory proteins consist of at least two antigenic types that appear to be functionally distinct, and each antigenic type displays charge microheterogeneity.     

Studies on the brush border membrane of the mouse duodenum

Summary Brush border membranes have been isolated from villus epithelial cells of the adult Swiss mouse duodenum. Preparations of these membranes are not contaminated by other organelles as judged from electron-micrographs of sectioned pellets of brush borders. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins from brush borders solubilized in Tris-sodium dodecyl sulfate buffer reveals a reproducible Coomassie Brilliant Blue pattern of 17 bands. By comparing the brush border protein band positions with those of standard proteins run concurrently on sodium dodecyl sulfate-polyacrylamide gel slabs it is estimated that the 17 brush border proteins and subunits have molecular weights ranging from over 250,000 to around 16,000. Periodate-fuchsin sulfite staining shows that the five more slowly migrating, high molecular weight proteins are glycoproteins. The two proteins of smallest molecular size react positively with Oil Red O but have very small amounts of lipophilic amino acid residues, which indicates that the lipid extractable from the gels in these areas is a contaminant and is not bound to the proteins.     

Comparison of black coral skeleton and insect cuticle by a combination of carbon-13 NMR and chemical analyses.

Cross-polarization, magic-angle spinning 13C NMR spectra of skeletal components of individual colonies of the New Zealand black coral, Antipathes fiordensis, have a marked similarity to spectra of the sclerotized exoskeleton of the adult tobacco hornworm, Manduca sexta. NMR analysis estimates the organic content of the load-bearing skeletal base of A. fiordensis as 70% protein, 10% chitin, 15% diphenol, and 5% lipid by weight, and that of M. Sexta moth cuticle as 60% protein, 20% chitin, 15% diphenol, and 5% lipid. The younger pinnules or tips of A. fiordensis are less than 3% diphenol by weight. The only diphenols extracted from coral skeleton by hydrochloric acid are 3-(3,4-dihydroxyphenyl)-DL-alanine (DOPA) and 3,4-dihydroxybenzaldehyde (DOBAL), while the predominant diphenols in acid extracts of insect cuticles are N-acyldopamines. More DOPA is found in the base than in the tips of A. fiordensis and it appears to be a peptidyl component of coral skeletal protein. The oxidation of DOPA and DOBAL to quinones may provide mechanical stabilization of the coral skeleton by cross-linking of structural proteins to other proteins or to chitin.