Binding interactons of collagen I, laminin and fibronectin with immobilized Escherichia coli 0157:H7 using a surface plasmon resonance biosensor

Real-time interactions of collagen I, fibronectin, laminin, hyaluronic acid and chondroitin sulfate with immobilized Escherichia coli O157:H7 cells were studied with a surface plasmon resonance biosensor. Results showed that collagen I and laminin bound to the E.coli surface but fibronectin had very low binding while hyaluronic acid and chondroitin sulfate had no detectable interaction. Calcium ion inhibited laminin binding but enhanced collagen I binding. This research provides a model system to study the interactions of bacterial cells with extracellular matrix components. © Rapid Science Ltd. 1998     

Distribution of extracellular matrix proteins type I collagen, type IV collagen, fibronectin, and laminin in mouse folliculogenesis

The extracellular matrix (ECM) plays a prominent role in ovarian function by participating in processes such as cell migration, proliferation, growth, and development. Although some of these signaling processes have been characterized in the mouse, the relative quantity and distribution of ECM proteins within developing follicles of the ovary have not been characterized. This study uses immunohistochemistry and real-time PCR to characterize the ECM components type I collagen, type IV collagen, fibronectin, and laminin in the mouse ovary according to follicle stage and cellular compartment. Collagen I was present throughout the ovary, with higher concentrations in the ovarian surface epithelium and follicular compartments. Collagen IV was abundant in the theca cell compartment with low-level expression in the stroma and granulosa cells. The distribution of collagen was consistent throughout follicle maturation. Fibronectin staining in the stroma and theca cell compartment increased throughout follicle development, while staining in the granulosa cell compartment decreased. Heavy staining was also observed in the follicular fluid of antral follicles. Laminin was localized primarily to the theca cell compartment, with a defined ring at the exterior of the follicular granulosa cells marking the basement membrane. Low levels of laminin were also apparent in the stroma and granulosa cell compartment. Taken together, the ECM content of the mouse ovary changes during follicular development and reveals a distinct spatial and temporal pattern. This understanding of ECM composition and distribution can be used in the basic studies of ECM function during follicle development, and could aid in the development of in vitro systems for follicle growth.     

Expression of human interleukin-2 in recombinant baby hamster kidney, Ltk-, and Chinese hamster ovary cells. Structure of O-linked carbohydrate chains and their location within the polypeptide

The similarity or identity of O-glycosylation in glycoproteins from natural sources or produced in heterologous cell lines, a central problem for the development of many biotechnologically relevant production processes, was examined using interleukin-2 (IL-2) as a model. Human interleukin-2 was constitutively expressed in several mammalian cell lines in high amounts. The recombinant proteins were purified to homogeneity and their carbohydrate structures were analyzed. Only the NeuAc alpha 2-3Gal beta 1-3[NeuAc alpha 2-6]GalNAc oligosaccharide structure or the NeuAc alpha 2-3Gal beta 1-3GalNAc were found in all IL-2 preparations secreted from recombinant Ltk-, Chinese hamster ovary, and baby hamster kidney cell lines. The O-linked chains were exclusively linked to Thr in position 3 of the polypeptide chain which is the carbohydrate attachment site in natural human IL-2. The proportions of O-glycosylated versus nonglycosylated forms of the protein secreted by each recombinant cell line were independent of productivity or of cell culture conditions. Our results show that O-glycosylated human IL-2 can be produced by applying recombinant DNA technology in heterologous cell lines with the same type of post-translational modification that is observed for the protein secreted from natural T lymphocytes.     

Growth conditions for the expression of fibronectin,collagen type I,vitronectin, and laminin binding toEscherichia coli strain NG7C

The ability ofEscherichia coli strains isolated from various human infections to bind fibronectin (Fn), collagen type I (Cn), laminin (Ln), and vitronectin (Vn) was studied. Binding of the proteins was shown to be specific and to be inhibited after protease treatment and heating of bacterial cells at 80°C. Binding of Fn, Cn type I, and Ln was not expressed in CFA broth and Voccani's medium with sodium chloride concentration above 0.5%. Fn binding was specifically enhanced for cells grown in CFA broth and Voccani's medium containing CaCl₂ (up to 10 mM final concentration), whereas binding of Cn type I was decreased. Growth in liquid medium yielded cells with maximal binding of Fn, Cn type I, Vn, and Ln after 20–22 h of growth. The binding of Fn, Cn type I, and Vn to cells of strain NG7C was inhibited for cells preincubated with antisera raised to the homologous strain;E. coli NG7C cells seem to have specific binding sites for Fn, Cn type I, Vn, and possibly also Ln.     

Distributions of fibronectin, laminin, type I and IV collagens and carbonic anhydrase isozyme III during bovine ruminal epithelial development

The present study describes the immunohistochemical distributions of extracellular matrices and the carbonic anhydrase isozyme III (CA-3) in the bovine ruminal epithelium during fetal development. Fibronectin (FN), laminin and type I and IV collagens were distributed in the ruminal subepithelial mesenchymal regions with nonspecific regionality during the gestational periods. At stages after about 59 cm crown-rump length (CRL), FN of the epithelial basement membrane disappeared, and CA-3 appeared in the basal epithelial cells of the root of the ruminal papillae, suggesting that the functional differentiation of the ruminal epithelium might start at around 59 cm CRL in bovine fetuses.     

Binding of Griffonia simplicifolia I lectin to rat pulmonary alveolar macrophages and its use in purifying type II alveolar epithelial cells

We report that the isolectin Griffonia simplicifolia I-B4 isolated from G. simplicifolia seeds binds to rat alveolar macrophages present in frozen sections of lung tissue or bronchoalveolar lavage fluid. G. simplicifolia I-B4 does not bind to alveolar epithelial cells. We established that G. simplicifolia I-B4 binds to the macrophages via interaction with terminal alpha-D-galactopyranosyl residues present on these cells. This was substantiated by demonstrating that binding is inhibited either by the haptenic sugar alpha-D-galactopyranoside or by treating the cells with coffee bean alpha-galactosidase. Because murine laminin is known to contain terminal alpha-D-galactopyranosyl end-groups, and because we found that an anti-laminin antiserum binds to rat alveolar macrophages, we suspect that G. simplicifolia I-B4 may be binding to laminin present on the macrophages. To isolate alveolar type II epithelial cells from rat lungs, we developed a method that utilizes the lectin G. simplicifolia I. When proteinase-derived suspensions of pulmonary cells are incubated with G. simplicifolia I, the macrophages agglutinate and can be removed by filtration through nylon mesh. After incubating the resulting cellular suspension in tissue culture, the adherent cells are 94 +/- 2% (S.D.) type II cells. When compared to cells isolated by repeated differential adherence, the lectin-prepared type II cells have similar morphology and staining characteristics, form domes in monolayers and incorporate similar amounts of palmitate into disaturated phosphatidylcholine. We believe that the procedure outlined in this report provides a simple and effective method to isolate type II alveolar epithelial cells from rat lungs.     

Binding of phosphorothioate oligodeoxynucleotides to basic fibroblast growth factor, recombinant soluble CD4, laminin and fibronectin is P-chirality independent.

Antisense oligodeoxynucleotides can selectively inhibit the expression of individual genes and thus have potential applications in anticancer and antiviral therapy. A critical prerequisite to their use as therapeutic agents is the understanding of their non-specific interactions with biological structures, e.g. proteins. In this study we examined the interactions of P-chiral phosphorothioate oligodeoxynucleotides with several proteins. The Rp- and Sp- diastereomers, and racemic machine-made mixtures, or M-oligodeoxynucleotides were used independently as competitors of the binding of a probe, phosphodiester oligodeoxynucleotide bearing a 5' alkylating moiety, to rsCD4, bFGF and laminin. These oligodeoxynucleotides were also used as competitors of the binding of a non-alkylating probe M-phosphorothioate oligodeoxynucleotide, 5'-32P-SdT18 to fibronectin. The average values of and quantitative estimates for the IC50 of competition and the constant of competition (Kc) of Rp-, Sp- and M-stereoisomers of several homo- and heteropolymer oligodeoxynucleotides were determined and compared. Surprisingly, in the proteins we studied, the values of IC50 and Kc for the Rp-, Sp- and M-oligodeoxynucleotides were essentially identical. Thus, the ability of the phosphorothioate oligodeoxynucleotides we employed, to bind to the proteins studied in this work, is virtually independent of P-chirality. Our results also imply that the role of the purine and pyrimidine bases in oligodeoxynucleotide-protein interactions, as well as the nature of the contact points (sulfur versus oxygen) between the oligomer and the protein, may be relatively unimportant.     

Histone H1 and H3 phosphorylation during premature chromosome condensation in a temperature-sensitive mutant (tsBN2) of baby hamster kidney cells

The histone phosphorylations of temperature-sensitive mutant cells (tsBN2) were investigated during the induction of premature chromosome condensation (PCC). At the permissive temperature (33.5 degrees C), the histones of the cells were phosphorylated typically as in any other mammalian cell. However, at the nonpermissive temperature (40.5 degrees C), both histone H1 and H3 were phosphorylated extensively as in mitotic cells, and the increase in these phosphorylations throughout S to G2 phase was closely correlated to the frequency of cells showing PCC. The pattern of H1 subtype phosphorylations was quite similar, and the sites of H1 phosphorylation from PCC were the same as those from mitotic cells. Although the degree of phosphorylation was low, H1 and H3 phosphorylations were observed even in G1 phase at the nonpermissive temperature. The effects of metabolic inhibitors on the induction of PCC were parallel in H1 and H3 phosphorylations; actinomycin D failed to inhibit either PCC induction or these phosphorylations, whereas cyclohexamide did, completely inhibiting H3 phosphorylation.     

Comparative study of the sugar chains of factor VIII purified from human plasma and from the culture media of recombinant baby hamster kidney cells.

The asparagine-linked sugar chains of blood coagulation factor VIII preparations purified from human plasma of blood group A donors and from the culture media of recombinant BHK cells were released as oligosaccharides by hydrazinolysis. These sugar chains were converted to radioactive oligosaccharides by reduction with sodium borotritide and separated into neutral and acidic fractions by paper electrophoresis. Most of the acidic oligosaccharides were converted to neutral ones by sialidase digestion, indicating that they are sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were fractionated by serial chromatography on immobilized lectin columns and Bio-Gel P-4 column. Structural study of each oligosaccharide by sequential exo- and endoglycosidase digestion and by methylation analysis revealed that both factor VIII preparations contain mainly high mannose-type and bi-, tri-, and tetra-antennary complex-type sugar chains. Some of the biantennary complex-type sugar chains from human plasma factor VIII contain blood group A and/or H determinant, while those from recombinant product do not. Some of the bi-, tri- and tetra-antennary complex-type sugar chains of the recombinant factor VIII contain the Gal alpha 1----3Gal group. A small number of the triantennary complex-type sugar chains from both preparations was found to contain the Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----4 (Gal beta 1----4GlcNAc beta 1----2)Man group. Studies of pharmacokinetic parameters of the recombinant factor VIII infused into baboons revealed that its half-life in blood circulation is similar to that of plasma derived factor VIII, suggesting that the oligosaccharide structural differences between them do not affect the fate of factor VIII in vivo.     

Fine specificities of two lectins from Cymbosema roseum seeds: a lectin specific for high-mannose oligosaccharides and a lectin specific for blood group H type II trisaccharide

The legume species of Cymbosema roseum of Diocleinae subtribe produce at least two different seed lectins. The present study demonstrates that C. roseum lectin I (CRL I) binds with high affinity to the "core" trimannoside of N-linked oligosaccharides. Cymbosema roseum lectin II (CRL II), on the other hand, binds with high affinity to the blood group H trisaccharide (Fucα1,2Galα1-4GlcNAc-). Thermodynamic and hemagglutination inhibition studies reveal the fine binding specificities of the two lectins. Data obtained with a complete set of monodeoxy analogs of the core trimannoside indicate that CRL I recognizes the 3-, 4- and 6-hydroxyl groups of the α(1,6) Man residue, the 3- and 4-hydroxyl group of the α(1,3) Man residue and the 2- and 4-hydroxyl groups of the central Man residue of the trimannoside. CRL I possesses enhanced affinities for the Man5 oligomannose glycan and a biantennary complex glycan as well as glycoproteins containing high-mannose glycans. On the other hand, CRL II distinguishes the blood group H type II epitope from the Lewis(x), Lewis(y), Lewis(a) and Lewis(b) epitopes. CRL II also distinguishes between blood group H type II and type I trisaccharides. CRL I and CRL II, respectively, possess differences in fine specificities when compared with other reported mannose and fucose recognizing lectins. This is the first report of a mannose-specific lectin (CRL I) and a blood group H type II-specific lectin (CRL II) from seeds of a member of the Diocleinae subtribe.     

Heymann antigen GP330 demonstrates affinity for fibronectin, laminin, and type I collagen and mediates rat proximal tubule epithelial cell adherence to such matrices in vitro.

We have utilized monoclonal antibodies directed against glycoproteins on the surface of proximal tubule epithelial cells (PTEC) to study their interaction with matrix components. PTEC exposed to monoclonal antibodies directed against a 330-kDa cell surface glycoprotein exhibited a significant epitope-specific inhibition of attachment and proliferation on type I collagen-, fibronectin-, laminin-, and gelatin-coated tissue culture surfaces. This effect was not due to antibody toxicity since such cells did not exhibit metabolic dysfunction in suspension cultures and the inhibition could be reversed upon removal of the antibody from the cell surface. Furthermore, detergent-solubilized gp330 demonstrated specific affinity for fibronectin, laminin, and type I collagen which was not inhibited by Arg-Gly-Asp-containing peptides. A monoclonal antibody directed against the receptor epitope was capable of promoting PTEC adherence and growth when such an antibody was immobilized on cell culture dishes. Although gp330 acted as a receptor for matrix proteins in primary cultures of freshly isolated PTEC, this effect was not demonstrable in established cultures. These results suggest that freshly isolated PTEC depend on gp330 for their attachment to matrix molecules while in vitro-adapted PTEC rely on other receptors activated by culture conditions. The affinity of gp330 for matrix molecules may be of pathogenic relevance in the persistence of gp330-containing immune complexes formed in the glomerular capillary wall in experimental membranous nephropathy (Heymann nephritis).     

Structures of N-glycans of a ricin-resistant mutant of baby hamster kidney cells. Synthesis of high-mannose and hybrid N-glycans

The asparagine-linked glycopeptides (N-glycans) of a ricin-resistant mutant of baby hamster kidney (BHK) cells, RicR21, have been isolated and fractionated from a Pronase digest of disrupted cells by concanavalin A (Con A)-Sepharose chromatography, ion-exchange chromatography, and lentil lectin chromatography. The structures of all the major N-glycans have been determined by 500-MHz H NMR spectroscopy. RicR21 synthesizes only hybrid and high-mannose N-glycans. All the hybrid structures contain only three mannose residues. The major hybrid glycopeptide has the following structure: (Formula: see text). There is also about 15% of the nonfucosylated species present. Only a small amount (less than or equal to 5%) of the asialo hybrid is produced. Branched hybrid N-glycans are also present in RicR21 cells, containing two complex antenna linked beta 1----2 and beta 1----4 to the Man alpha 1----3 arm; about 70% of this species is core fucosylated. Man6GlcNAc2 glycopeptide is the most abundant (about 70%) of the high-mannose N-glycans. These studies account for the very poor ricin binding property of this mutant, as the sialic acid residues of the major hybrid N-glycan are exclusively linked alpha 2----3 to galactose and ricin is unable to bind to alpha 2----3-substituted galactosyl residues [Baenziger, J. U., & Fiete, D. (1979) J. Biol. Chem. 254, 9795-9799].     

Changing patterns of fibronectin, laminin, type IV collagen, and a basement membrane proteoglycan during rat Mullerian duct regression

Antibodies to type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin were used to study the regression of the rat Mullerian duct. All four of these matrix constituents are located at the perimeter of the Mullerian duct within the ductal basement membrane. As the Mullerian duct regresses, the staining of all of these basement membrane constituents becomes irregular and discontinuous. Fibronectin, which is also present in the interstitium, becomes undetectable in the mesenchyme which condenses around the regressing Mullerian duct. These data indicate that degradation of the extracellular matrix around the male Mullerian duct is a central event in the regression of this structure.     

Strict specificity for high-mannose type N-glycans and primary structure of a red alga Eucheuma serra lectin

We have elucidated the carbohydrate-binding profile of a non-monosaccharide-binding lectin named Eucheuma serra lectin (ESA)-2 from the red alga Eucheuma serra using a lectin-immobilized column and a centrifugal ultrafiltration-high performance liquid chromatography method with a variety of fluorescence-labeled oligosaccharides. In both methods, ESA-2 exclusively bound with high-mannose type (HM) N-glycans, but not with any of other N-glycans including complex type, hybrid type and core pentasaccharides, and oligosaccharides from glycolipids. These findings indicate that ESA-2 recognizes the branched oligomannosides of the N-glycans. However, ESA-2 did not bind with any of the free oligomannoses examined that are constituents of the branched oligomannosides implying that the portion of the core N-acetyl-D-glucosamine (GlcNAc) residue(s) of the N-glycans is also essential for binding. Thus, the algal lectin was strictly specific for HM N-glycans and recognized the extended carbohydrate structure with a minimum size of the pentasaccharide, Man(alpha1-3)Man(alpha1-6)Man(beta1-4)GlcNAc(beta1-4) GlcNAc. Kinetic analysis of binding with a HM heptasaccharide (M5) showed that ESA-2 has four carbohydrate-binding sites per polypeptide with a high association constant of 1.6x10(8) M-1. Sequence analysis, by a combination of Edman degradation and mass analyses of the intact protein and of peptides produced by its enzymic digestions, showed that ESA-2 is composed of 268 amino acids (molecular weight 27950) with four tandemly repeated domains of 67 amino acids. The number of repeats coincided with the number of carbohydrate-binding sites in the monomeric molecule. Surprisingly, the marine algal lectin was homologous to hemagglutinin from the soil bacterium Myxococcus xanthus.     

Interactions of rat hepatocytes with type IV collagen, fibronectin and laminin matrices. Distinct matrix-controlled modes of attachment and spreading

We have examined the interaction of adult rat hepatocytes in primary culture, to type IV collagen, fibronectin, and laminin, the major basement membrane proteins of normal rat liver. Culture substrata consisted of glass coverslips, which were covalently derivatized with individual purified basement membrane constituents at varying densities of protein. The attachment of freshly prepared hepatocytes was examined after incubation at 37 degrees C for 30 min as a function of the amount of protein on the coverslips. For each of the three types of substratum under study, distinct modes of cell attachment were observed, with the apparent affinity of hepatocytes for type IV collagen being three-fold greater than for fibronectin and ten-fold greater than for laminin. Cell attachment exhibited saturation on all substrata. Hepatocyte spreading was measured by scanning electron microscopy of cells incubated at 37 degrees for 2 h on similarly prepared coverslips. A five-fold greater surface density of type IV collagen was required for maximal spreading compared with attachment. For cells on fibronectin or laminin the maximal cell spreading reached on type IV collagen did not occur even at coverslip protein densities 10 to 20 times those providing for maximal cell attachment. A very similar qualitative pattern of cell proteins was secreted within a few hours of plating on the various substrata and further studies failed to reveal any evidence that attachment and spreading was mediated by endogenously produced matrix molecules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modular autonomy, ligand specificity, and functional cooperativity of the three in-tandem fibronectin type II repeats from human matrix metalloproteinase 2

Matrix metalloproteinase 2 (MMP-2) contains three fibronectin type II (col) modules that contribute to its collagen specificity. We observed that the CD spectra of the separate col modules account for the CD and temperature profiles of the in-tandem col-123 construct. Thus, to the extent of not significantly perturbing the secondary structure and thermal stability characteristics of the neighboring units, the domains within col-123 do not interact. Via NMR, we investigated ligand binding properties of the three repeats within col-123: col-123/1 (the col-1 domain within col-123), col-123/2, and col-123/3. Interactions of col-123 with the collagen mimic peptide (Pro-Pro-Gly)6 (PPG6) and propeptide segment PIIKFPGDVA (p33-42) were studied. While col-123/1 and col-123/2 bound PPG6, they interacted more weakly with p33-42. In contrast, col-123/3 exhibited a higher affinity for p33-42 than for PPG6. Thus, despite their structural homology, the col repeats of MMP-2 differ in substrate specificity. Furthermore the binding affinities toward the three in-tandem col repeats were close to those determined for the individual isolated domains or for col-12/1, indicating that vis-a-vis these ligands each module interacts essentially as an autonomous unit. Interestingly the domains within col-123 exhibited enhanced affinities for Hel3, a construct that contains ((Gly-Pro-Pro)12)3 in triple helical configuration. Nevertheless the affinities were significantly higher for col-123/1 and col-123/2 relative to col-123/3 in line with their behaviors toward PPG6. This hints at a cooperative participation toward Hel3, which is a closer mimic of collagen, a hypothesis that is supported by the detected lower affinities of col-12/1, col-12/2, col-2, col-23/2, col-3, and col-23/3 for Hel3.     

Inhibition of type I and type II iodothyronine deiodinase activity in rat liver, kidney and brain produced by selenium deficiency.

Selenium deficiency for periods of 5 or 6 weeks in rats produced an inhibition of tri-iodothyronine (T3) production from added thyroxine (T4) in brain, liver and kidney homogenate. This inhibition was reflected in plasma T4 and T3 concentrations, which were respectively increased and decreased in selenium-deficient animals. Although plasma T4 levels increased in selenium-deficient animals, this did not produce the normal feedback inhibition on thyrotropin release from the pituitary. Selenium deficiency was confirmed in the animals by decreased selenium-dependent glutathione peroxidase (Se-GSH-Px) activity in all of these tissues. Administration of selenium, as a single intraperitoneal injection of 200 micrograms of selenium (as Na2SeO3)/kg body weight completely reversed the effects of selenium deficiency on thyroid-hormone metabolism and partly restored the activity of Se-GSH-Px. Selenium administration at 10 micrograms/kg body weight had no significant effect on thyroid-hormone metabolism or on Se-GSH-Px activity in any of the tissues studied. The characteristic changes in plasma thyroid-hormone levels that occurred in selenium deficiency appeared not to be due to non-specific stress factors, since food restriction to 75% of normal intake or vitamin E deficiency produced no significant changes in plasma T4 or T3 concentration. These data are consistent with the view that the Type I and Type II iodothyronine deiodinase enzymes are seleno-enzymes or require selenium-containing cofactors for activity.     

Localization of binding sites for laminin, heparan sulfate proteoglycan and fibronectin on basement membrane (type IV) collagen

Rotary shadowing electron microscopy was used to examine complexes formed by incubating combinations of the basement membrane components: type IV collagen, laminin, large heparan sulfate proteoglycan and fibronectin. Complexes were analyzed by length measurement from the globular (COOH) domain of type IV collagen, and by examination of the four arms of laminin and the two arms of fibronectin. Type IV collagen was found to contain binding sites for laminin, heparan sulfate proteoglycan and fibronectin. With laminin the most frequent site was centered approximately 81 nm from the carboxy end of type IV collagen. Less frequent sites appeared to be present at approximately 216 nm and approximately 291 nm, although this was not apparent when the sites were expressed as a fraction of the length of type IV collagen to which they were bound. For heparan sulfate proteoglycan the most frequent site occurred at approximately 206 nm with a less frequent site at approximately 82 nm. For fibronectin, a single site was present at approximately 205 nm. Laminin bound to type IV collagen through its short arms, particularly through the end of the lateral short arms and to heparan sulfate proteoglycan mainly through the end of its long arm. Fibronectin bound to type IV collagen through the free end region of its arms. Using a computer graphics program, the primary laminin binding sites of two adjacent type IV collagen molecules were found to align in the "polygonal" model of type IV collagen, whereas with the "open network" model, a wide meshed matrix is predicted. It is proposed that basement membrane may consist of a lattice of type IV collagen coated with laminin, heparan sulfate proteoglycan and fibronectin.     

Endogenous substrates of rat heart protein kinase C type I, II, and III isozymic forms in cardiac sarcolemma.

The endogenous substrate proteins of rat cardiac protein kinase C type I, II, and III isozymic forms were studied in rat cardiac sarcolemma. The 19-, 21-, 29-, 35-, and 95-kDa proteins were phosphorylated by both types II and III, but not type I. The extent of phosphorylation by individual protein kinase C isozymic forms was additive and equal to the extent of phosphorylation observed when a mixture of isozymic forms was employed. The extent of phosphorylation of the 21-kDa protein by type III was much higher than that by type II. These results suggest that the protein kinase C isozymes have preferences for specific endogenous substrate proteins. The phosphorylation of these endogenous substrate proteins by protein kinase C isozymes probably plays a role in cardiac cell functions.