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Six major acidic glycosphingolipids were isolated from human amnion using DEAE Sephadex A-25 and silica beads column chromatography. The structures of these glycosphingolipids were determined by methylation analysis, TLC immunostaining and/or negative ion FAB-MS, and were concluded to be II3 alpha NeuAcLacCer(GM3), IV3 alpha NeuAcnLc4-Cer (sialyl[alpha 2-3]paragloboside), IV6 alpha NeuAcnLc4Cer (sialyl[alpha 2-6]paragloboside), IV3 alpha NeuAcIII4 alpha FucLc4Cer (sialyl Lea), VI3 alpha NeuAcnLc6Cer (i-ganglioside) and II3 alpha (NeuAc alpha 2----8NeuAc)LacCer (GD3). In addition, several minor glycosphingolipids were detected with specific monoclonal antibodies, including glycolipids with NeuAc alpha 2-3Gal beta 1-4GlcNAc-beta 1- or NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1- determinant. Our results show that the glycosphingolipids of human amnion are characterized by having mainly type II chain analogues and onco-fetal antigens.

Ganglioside-hydrolyzing sialidase activity was solubilized from rat brain particulate fraction by using Triton X-100 plus sodium deoxycholate. When chromatographed on AH-Sepharose 4B, the solubilized activity was resolved into two peaks, which were designated sialidases I and II in order of elution. The two sialidases were purified by using sequential chromatographies on Octyl-Sepharose CL-4B, Phenyl-Sepharose CL-4B, and Sephadex G-200. Sialidase II was purified further by Mono Q-FPLC. Overall purification was 450- and 2,150-fold, for sialidases I and II, respectively. Purified sialidases I and II were maximally active at near pH 5.0 and exhibited M = 70,000 by gel filtration. Sialidase I hydrolyzed gangliosides but scarcely other substrates including 4-methylumbelliferyl-NeuAc (4MU-NeuAc). Sialidase II hydrolyzed oligosaccharides, glycoproteins, and 4MU-NeuAc although gangliosides appeared to be preferential substrates. Sialidase II cleaved GM2 much faster than sialidase I. An antibody raised in rabbits against sialidase I reacted with only sialidase I and an antibody against sialidase II reacted with only sialidase II. A subcellular distribution study suggested sialidase I in the synaptosomal membrane and sialidase II in the synaptosomal and lysosomal membranes, and this was verified by using the above antibodies.

Blood group A-active glycosphingolipids from human erythrocyte membranes were identified by the combination of thin-layer chromatography and matrix-assisted secondary ion mass spectrometry (TLC/SIMS). Partially purified lipid extracts were chromatographed by TLC and then blood group A-active glycolipids were detected by TLC-immunostaining assay using anti-A antibody. The parts of the plates which contained the same Rf area as anti-A positive spots were cut out and subjected to direct SIMS analysis. The TLC/SIMS spectra were quite similar to those obtained by ordinary SIMS. Detailed information, such as molecular weight, molecular species, ceramide portion, and oligosaccharide sequence, was obtained. Also, peracetylated blood group A-active glycolipids were analyzed in a similar manner. After the position of A-active glycolipids on a TLC plate was confirmed by in situ deacetylation and TLC-immunostaining, acetylated A-active glycolipids were also analyzed by the TLC/SIMS. Enhanced sensitivity was obtained with peracetylated glycolipids. Consequently, small amounts of unpurified bioactive glycolipids can be readily analyzed by TLC/SIMS.

A simple, sensitive, and specific assay method for glycosyltransferase and glycosidase activities has been established by means of an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody, H-11 directed to lactoneotetraosylceramide (nLc4Cer). Enzyme activity was determined by assaying the amount of reaction product, nLc4Cer with the ELISA method. For the assay of galactosyltransferase activity, lactotriaosylceramide (Lc3Cer) immobilized on a 96-well microtiter plate was incubated with bovine milk galactosyltransferase in cacodylate buffer (pH 6.8) containing Triton CF-54, Mn2+, and UDP-galactose. Optimum incubation conditions for the enzyme were determined. Glycosidase activity was also assayed by the ELISA method by using Clostridium perfringens sialidase and neolacto-series gangliosides as substrates, and the substrate specificities towards the gangliosides were examined. By this method, 3-100 pmol of reaction product could be determined. The assay method has several advantages as follows: 1, the method is simple; 2, separation of the reaction product is not required; 3, quantification and identification of the reaction product were done simultaneously; 4, naturally occurring substrates are available (especially for glycosidase); 5, many samples can be assayed in one microplate; 6, sensitivity is very high. The present method was applied for the detection of galactosyltransferase in human sera. Significant elevations of the galactosyltransferase levels were observed in the sera from cancer patients. The formation of nLc4Cer was confirmed by employing the TLC-immunostaining method for bands of Lc3Cer after incubation of the bands with serum and cofactors on an HPTLC plate.

We analyzed glycosphingolipids from normal lymph node cells of seven cattle and lymph node cells of eight cattle with enzootic bovine leukosis. The neutral glycosphingolipids and gangliosides were analyzed by thin-layer chromatography. Both normal and tumorous lymph node cells had GlcCer, LacCer, and GbOse3Cer as major neutral glycosphingolipids. In the ganglioside fraction, GM3 was the predominant component in both normal and tumorous lymph node cells, and another component, ganglioside Gx fraction, was also prominent in tumorous lymph node cells. The structure of this ganglioside Gx fraction was elucidated by thin-layer chromatography, sugar analysis, neuraminidase digestion, and permethylation studies. This ganglioside Gx fraction was found to be a mixture of four ganglioside species. The structures of individual gangliosides Gx (1 to 4) were characterized as follows. 1: GD3, NeuAc alpha 2-8NeuAc alpha 2-3Gal1-4Glc-Cer. 2: GD3, NeuAc alpha 2-8NeuGc alpha 2-3Gal1-4Glc-Cer. 3: GD3, NeuGc alpha 2-8NeuAc alpha 2-3Gal1-Glc-Cer. 4: GD3, NeuGc alpha 2-8NeuGc alpha 2-3Gal1-4Glc-Cer. These GD3 species may be formed as a result of the induced synthesis inassociation with malignant transformation.

It is known that ABO blood group substances in human erythrocyte membranes are sphingoglycolipids, but recently several authors have reported that the glycoproteins of the erythrocyte membranes also have ABO blood group activities in addition to MN blood group activities and virus hemagglutination inhibitor activity. We solubilized blood group A erythrocyte membranes with lithium diiodosalicylate and separated the glycoprotein fraction by phenol extraction and ethanol precipitation. This fraction was apparently not contaminated with glycolipid, but it showed weak blood group A activity. The activity of the glycoprotein of the erythrocyte membranes was one-sixth of that of the lgycolipid fraction from the same amount of membranes. The glycoprotein components were purified by Sephadex G-200 gel filtration in SDS. The main component isolated, PAS 1, still showed blood A activity.

Acidic glycosphingolipids were analyzed by field desorption (FD-MS) and secondary ion mass spectrometry (SI-MS) using the primary ion Xe+ with a glycerol matrix. In the analysis of underivatized gangliosides by FD-MS, the fragment corresponding to the asialo residue resulting from the cationized cluster ion (M + Na)+ was the base peak, and ions due to cleavage at the glycosidic linkages were detected, as in the neutral glycosphingolipids. In the case of sulfatide, the ceramide fragment showed the highest intensity in the spectrum. In SI-MS spectra of acidic glycosphingolipids, (M + Na)+, (M + 2Na-H)+, and (M + K)+ were continuously detected as relatively high intensity ions during analysis of gangliosides and sulfatide. Other ions were mostly similar to those obtained by FD-MS. In FD-MS spectra of permethylated gangliosides, the cationized molecular ion (M + Na)+ was the base peak, and fragment ions due to asialo gangliosides were prominent. Other peaks were hard to detect. In SI-MS, molecular ions (M + H)+ and (M + H-32)+ and other ions due to cleavage of the glycosidic linkages were clearly detected. In this case, the sensitivity was greatly improved. Ions due to the non reducing end sugars were clearly detected, because of the relatively low intensity of ion peaks due to the glycerol matrix. It is concluded that the combination with FD-MS and SI-MS is particularly useful for the determination of molecular weight, sugar sequence and ceramide structure with sample amounting to only a few micrograms order.

A simple and rapid method for the preparation of N-methylamides ( - CONHCH3) of sialic acids in gangliosides and biochemical properties of the modified gangliosides are described. The sialic acid carboxyl groups of gangliosides were esterified with CH3I-dimethylsulfoxide, followed by heating with monomethylamine. The modified gangliosides were chemically identified by TLC, IR spectroscopy, GLC-mass spectrometry and NMR spectroscopy. The N-methylamide derivative of GM1 produced a high titer IgG antibody. The antibody weakly cross-reacted with the methylester of GM1 and its reductive derivative but did not react with the intact GM1. A monoclonal antibody (M2590) specific for GM3 did not react with carboxyl-modified GM3 (methylester, N-methylamide, and reduced GM3), but it reacted with modified GM3 which contains the C7-analog of the sialic acid. Clostridium perfringens and Arthrobacter ureafaciens sialidases did not hydrolyze the N-methylamide derivatives, methylesters or reductive derivatives of the gangliosides and, furthermore, these derivatives did not inhibit the actions of these sialidases.

The gangliosides from the erythrocytes of various strains of inbred mice were analyzed. The ganglioside compositions differed in different strains and the strains could be divided into 4 types on the basis of this difference. The main ganglioside is GM4 or sialosyl galactosyl ceramide in Type 1, and unidentified ganglioside in Type 2, GM4 and GM2 in almost equal amounts in Type 3, and GM2 in Type 4.

Hematoside from dog erythrocyte membrane was previously considered to contain a mixture of N-acetyl- and N-glycolyl- neuraminic acids. However, the hematoside preparation used in the previous study was obtained from pooled blood of several dogs, and individual variation in hematoside was not examined. In this work, hematosides of erythrocytes from 31 mongrel dogs and 108 dogs of 23 breeds were examined individually by thin-layer chromatography, and the component sialic acids were analysed by gas-liquid chromatography. Individual dogs had either NAN-hematoside or NGN-hematoside: dogs with N-glycolyl-neuraminic acid also had a trace of N-acetyl-neuraminic acid, but dogs with N-acetyl-neuraminic acid had no detectable N-glycolyl-neuraminic acid. A few mongrel dogs, some Kai dogs, Kishu dogs, Japanese spaniels and most Shiba dogs had NGN-hematoside, whereas all European dogs had NAN-hematoside and no NGN-hematoside. From pedigrees of some families, inheritance of NGN-hematoside was found to be autosomal dominant. NGN-hematoside is possibly one of dog blood group substances. The sialic acid of delipidized ghost protein of dogs with NGN-hematoside was N-glycolyl-neuraminic acid, and that of dogs with NAN-hematoside was N-acetyl-neuraminic acid. The sialic acid of plasma protein was mainly N-acetyl-neuraminic acid in all dogs.

A novel sulfated glycosphingolipid that belongs to "globo-series" was isolated from human kidney. This lipid was purified from a pooled kidney preparation by chloroform-methanol extraction, mild alkaline treatment, DEAE-Sephadex and silicic acid column chromatographies, and preparative TLC. The structure and the properties were studied by IR spectroscopy, proton NMR spectroscopy, negative secondary ion-mass spectrometry, solvolysis, periodate oxidation, compositional and methylation analyses, monoclonal antibodies, and a sulfatide-binding protein. From the results of the above analyses, the structure of this glycolipid was proposed to be HSO3-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1ceramide. This sulfated lipid reacted with a monoclonal anti-SSEA-3 (stage-specific embryonic antigen-3) (MC-631) (Kannagi, R., Cochran, N.A., Ishigami, F., Hakomori, S., Andrews, P.W., Knowles, B.B., & Solter, D. (1983) EMBO J. 2, 2355-2361), whose epitope is R-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-R', on TLC and solid-phase radioimmunoassay. This lipid also bound to the 125I-labeled sulfatide-binding protein, thrombospondin. The yield of this sulfated glycolipid was 34 pmol/g of tissue, which was about 0.028, 0.16, and 18 mol% of galactosyl- and lactosylceramide sulfates, and globopentosylceramide sulfate (Nagai, K.-i., Roberts, D.D., Toida, T., Matsumoto, H., Kushi, Y., Handa, S., & Ishizuka, I. (1989) J. Biol. Chem. 264, in press), respectively, in human kidney.

A simple and rapid method for the analysis of lipids on a thin layer chromatography (TLC) plate by matrix-assisted secondary ion mass spectrometry (SI-MS) is reported. Analysis was performed without elution of the sample from the TLC plate. Mass spectra obtained by this method are free from interference due to the TLC plate absorbent and reagents used for the detection of the spots. About 1 micrograms of lipids applied on a TLC plate can be analyzed by this method. On scanning the plate, mass chromatograms of each lipid were obtained based on its migration distance along the plate.

A quantitative consecutive method was developed for analysis of sphingoglycolipids in biological materials by high performance liquid chromatography (HPLC). Crude lipid extracts were separated into neutral and acidic fractions on a DEAE-Sephadex column. Glycolipid fractions were obtained by acetylation and Florisil column chromatography, and the acetylated glycolipids were N-p-nitrobenzoylated by treatment with p-nitrobenzoyl chloride in pyridine at 60 degrees C for 6 h. Excess reagent and by-products were removed by solvent partition and gel filtration. The glycolipid derivatives were analyzed by their absorption at 254 nm on Zorbax SIL, a silica gel column, with a gradient of 0.5--7% isopropanol in hexane-chloroform (2 : 1, v/v) at a flow rate of 0.5 ml/min. The detector response was linear with up to 60 nmol of injected glycolipids. The practical lower limit of detection was about 50 pmol. The derivatives were separated on the basis of their sugar chains. Effluents corresponding to each peak were collected and analyzed further on the basis of their lipid portion on mu-Bondapak C18, a reversed phase column. This combined procedure was applied to the analysis of erythrocyte glycolipids. Samples containing as little as 20 micrograms of glycolipids could be analyzed by this method.

Neutral and acidic glycosphingolipids of Friend cells were characterized in 1) undifferentiated Friend cells (745A), 2) differentiated Friend cells induced with dimethyl-sulfoxide, and 3) solid tumors grown in mice after subcutaneous implantation of Friend cells. The structures of the isolated glycosphingolipids were determined by means of compositional analysis, methylation analysis and enzyme treatment. Gangliosides GD1a and N-acetylgalactosaminyl-GD1a, followed by GM1a and GM2, were the main gangliosides in undifferentiated Friend cells. GD1a and N-acetylgalactosaminyl-GD1a accounted for 45 and 25% of the total gangliosides, respectively. On differentiation, ganglioside GM2 decreased significantly, from 10% to a trace amount. In solid tumors, GD1a was the major ganglioside, whereas in contrast to the situation in the cultured cells, N-acetylgalactosaminyl-GD1a was almost completely absent, and ganglioside GM1b, but not GM1a, was detected. In addition, ganglioside GD1 alpha was detected in the solid tumors. Galactosylceramide, glucosylceramide, and lactosylceramide were the main neutral components in both types of cells, while globotetraosylceramide (globoside), IV3-N-acetyl-galactosaminyl globotetraosylceramide (Forssman glycolipid) and gangliotetraosylceramide (GA1) were major in solid tumors grown in vivo.

Ganglioside GM1(NeuAc), labeled at the C-3 position of sphingosine with tritium, was injected into C3H/He, C57BL/10, B10.AQR mice intraperitoneally. The incorporation and the distribution of the radioactivity in various organs were examined. The injected [3H]GM1(NeuAc) was mainly incorporated in the liver and hydrolyzed sequentially. Sialic acid of ganglioside GM1(NeuAc) and metabolites was converted to N-glycolyl type from N-acetyl type. An appreciable amount of the sphingosine moiety in the administered GM1(NeuAc), moreover, was reutilized, being converted to sphingomyelin, and incorporated into alkyl chain of the ether lipid in phosphatidylethanolamine. The distributions of radioactivity in the metabolites of GM1(NeuAc) administered to the three strains of mice were different from each other. In other organs, GM1(NeuAc) was incorporated and metabolized only slightly. The N-methylamide, at the carboxyl group of the sialic acid, of the labeled ganglioside GM1(GM1(NeuAc)-NMe) was injected into C3H/He mice. Most of the administered [3H]GM1(NeuAc)-NMe was incorporated in the liver, and was metabolized to GM3(NeuAc)-NMe, via GM2(NeuAc)-NMe, within 24 h. GM3(NeuAc)-NMe was the only radioactive compound in the subsequent 10 weeks, but disappeared from the liver gradually. N-Methylamide-modified gangliosides were resistant to hydrolysis by mouse hepatic sialidase, to elongation by glycosyltransferase and to N-glycolylation at N-acetylneuraminic acid by monooxygenase.

A series of underivatized sulfoglycolipids (SM4g, lyso-SM4g, SM4s, SM3, SM2, SB2, and SB1a) from various tissues were analyzed by both positive (POS-SI-MS) and negative (NEG-SI-MS) secondary ion mass spectrometry. By POS-SI-MS were detected the molecular ions of sulfoglycolipids in the form with sodium or potassium together with some fragment ions useful for the carbohydrate sequence determination. The analysis of monosulfogangliotriaosyl- or monosulfogangliotetraosylceramide and bis-sulfoglycolipid was difficult due to noise in the high mass region. On the other hand, NEG-SI-MS of sulfoglycolipids gave more intense signals from molecular ion of (M-H)- for monosulfoglycolipids and [M-H+Na)-H)- for bis-sulfoglycolipid. Many fragment ions useful for the elucidation of the carbohydrate sequences were also obtained with significant intensities. The fragmentation was assessed to occur at the glycosidic linkages to form ions of the oligosaccharides with or without ceramide. These ions were useful for sugar sequencing and also for distinguishing the differences in the position of the sulfate group. The intensities of saccharide ions without sulfate were lower than those with sulfates. In the case of SB2 and SB1a, containing 2 mol of sulfate ester groups, the molecular ion was detected as [M-H+Na)-H)-. Also, fragment ions with 2 mol of sulfate were detected as the sodium-additive form. It was concluded that NEG-SI-MS is a very useful technique for the structural elucidation of higher sulfoglycolipids.

Liposomes were applied to the immunization with GgOse4Cer and screening for production of monoclonal antibody to GgOse4Cer. Four-week-old and 22-week-old Balb/c mice were immunized with GgOse4Cer and Salmonella minnesota R595 lipopolysaccharides incorporated liposomes which were composed of dipalmitoyl-phosphatidylcholine and cholesterol. Since antibody response to GgOse4Cer was higher in 22-week-old than 4-week-old Balb/c mice after immunization, 22-week-old Balb/c mice were used for the immunization prior to generation of the monoclonal antibodies to GgOse4Cer. The screening of monoclonal antibodies was performed by complement-dependent liposome immune lysis assay using GgOse4Cer-containing liposomes. Six kinds of monoclonal antibodies, AG-1, -2, -3, -4, -5, and -6, of the IgM class were established. The specificities of the monoclonal antibodies obtained were defined by complement-dependent liposome immune lysis assay using various glycosphingolipids incorporated in liposomes and by thin-layer chromatography (TLC) with immunostaining. All of the monoclonal antibodies reacted only with GgOse4Cer in the liposome immune lysis assay. In addition, the monoclonal antibodies reacted only with GgOse4Cer in the TLC immunostaining. However, none of the monoclonal antibodies obtained was capable of removing natural killer activity from C3H/He mice spleen cell suspensions in vitro. Liposomes may be useful in the procedures of immunization and screening for generation of antiserum and monoclonal antibody to GSLs.

Gangliosides were isolated from rat liver and erythrocytes by chromatography on columns of DEAE-Sephadex and Iatrobeads, and finally purified by preparative TLC. The chemical structures of the purified components were studied by carbohydrate analysis, methylation analysis, sialidase treatment, fatty acid analysis and direct mass spectrometry. In rat liver, gangliosides GM3, GM1, GD3, GD1a, GD1b, and GT1b were identified. Gangliosides in rat erythrocytes were characterized as GM1, fucosyl-GM1, and GD1a. Sialic acid was the N-acetyl type only and lignoceric acid was the main fatty acid in all components of rat liver and erythrocytes.

Simple molecular species of intact ceramide mono-, di-, tri-, tetra-, and pentasaccharides purified by reversed phase high-performance liquid chromatography (HPLC) were analyzed by field desorption mass spectrometry (FD-MS). The analysis of sphingoglycolipids without chemical derivatization by FD-MS not only provides molecular information but also significant characteristic fragments for structural determination due to the cleavage of glycosidic bonds. These ions, therefore, give information on the molecular species of sphingoglycolipids and sugar sequences of their oligosaccharides. Intact GL1a, GL2a, and an equimolar mixture of GL1a and GL2a were also analyzed by FD-MS. In the spectra, the ions, (M + H)+, (M + Na)+, and (M + H- H2O)+, were observed as high intensive ions and different molecular species ions thereafter could be identified in all spectra. The FD-MS method is particularly useful in structural studies of glycolipids from natural sources.

Neutral and acidic glycosphingolipids were purified from porcine pancreas by chromatography on columns of DEAE-Sephadex and Iatrobeads. The chemical structures of the purified glycolipids were determined by carbohydrate analysis, methylation analysis, enzyme treatment, fatty acid analysis, NMR and IR. The major glycolipid of porcine pancreas was Gal(alpha,1-4)Gal(beta,1-)ceramide. Gangliosides GM3 and GD3 were major acidic components and galactosylceramide 3-sulfate was also found.

A simple and quantitative method for the modification of sialic acid carboxyl group in ganglioside is described. Methyl iodide was added to the ganglioside in dimethylsulfoxide. The reaction was completed quickly at room temperature, giving the methyl ester with practically no by-products. Reduction of the methyl ester was achieved by sodium borohydride. These modified gangliosides were chemically characterized. Reduced GM1 has a strong antigenicity compared with the original GM1, and it raised high titer antisera which did not crossreact with the original GM1 nor with its methyl ester. Peanut agglutinin, which binds strongly to asialo GM1 but weakly to GM1, bound to the methyl ester of GM1 and reduced GM1. Cholera toxin, which is specific for GM1, also reacted with the methyl ester of GM1 and reduced GM1 to a certain extent.

Neutral and acidic glycolipids from the bovine pineal organ and neutral glycolipids from the bovine retina were characterized. The chemical structures of the isolated glycolipids were determined by means of carbohydrate analysis, methylation analysis, enzyme treatment, fatty acid analysis, long chain base analysis, mass spectrometry, NMR spectroscopy, and IR spectroscopy. GM3, GD3, and GT1 were the major bovine pineal organ gangliosides, GD3 accounting for 75% of the total gangliosides. Galactosylceramide, glucosylceramide, and lactosylceramide were found in both the bovine pineal organ and retina. Sulfatide was also present in both tissues. It had already been reported that the major bovine retina ganglioside was GD3 (Handa, S. & Burton, R.M. (1969) Lipids 4, 205-208). The glycolipid patterns of the two tissues were very similar to each other and quite different from those of other tissues.

The effects of glycolipids on the interaction of the MIF (migration inhibition factor) with rat macrophages were examined using a migration inhibition assay system. MIF activity was specifically blocked by fucosylated Glycolipid RM [Gal alpha 1-3Gal(2-1 alpha Fuc) beta 1-3GalNAc beta 1-3Gal beta 1-4Glc beta 1-1ceramide, (1978) J. Biochem. 83, 85-90], but not by Cytolipin R, hematoside, or blood group B active glycolipid [Gal alpha 1-3Gal(2-1 alpha Fuc) beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1ceramide]. Inhibition of MIF activity was proportional to the concentration of Glycolipid RM. These findings suggest that Glycolipid RM acts as a receptor for MIF.

The ganglioside patterns of erythrocytes from individual horses and cattle were examined. Variations in the ganglioside patterns were found in both horses and cattle. In the erythrocytes of most horses examined, NeuGc-Gal-Glc-ceramide (NeuGc-GM3) of 25 horses examined had only NeuGc-GM3 with no 4-O-Ac-NeuGc-GM3. The erythrocytes of various breeds of cattle had a characteristic ganglioside pattern, but they could be divided into 4 types on the basis of the composition of their gangliosides.

Pure myofibrils were isolated from bovine heart by sucrose layer ultracentrifugation. Cardiac myofibrils thus prepared contained more protein as insoluble stroma than skeletal muscle. The insoluble stroma largely consisted of connectin, an elastic protein of muscle. The connectin content in cardiac myofibrils was about 18% of the total myofibrillar protein and was three times that in skeletal myofibrils. In view of the role of connectin as an elastic component of muscle, the abundance of connectin in cardiac myofibrils may be responsible for keeping myofibrils short at rest. This would account for the more effective tension generation in cardiac muscle on passive stretching due to blood inflow (Stirling's law).