2؉sensitivity of the myofilament.It is suggested that Ser-43/Ser-45 and Ser-23/Ser-24 in cardiac TnI are important for normal Ca 2؉ sensitivity of the myofilament, and that phosphorylation of Ser-43/ Ser-45 and Ser-23/Ser-24 is primarily involved in the protein kinase C regulation of the activity and Ca 2؉ sensitivity, respectively, of actomyosin S-1 MgATPase.In cardiac myocytes, the activation of several types of receptors, such as ␣ 1 -adrenergic (1-5), muscarinic (1, 6), and purinergic (6) dynorphin A (7), endothelin-1 (8, 9), and angiotensin (14 -18). However, the complex molecular events mediated by PKC (or more precisely, by the individual isozymes) that are responsible for cardiac contractility regulation, for example, remain largely unclear. It has been reported that phenylephrine (␣ 1 -adrenergic receptor agonist) elicited transient negative inotropy followed by sustained positive inotropy (3, 19 -21), endothelin-1 caused monotonic positive inotropy (22), whereas dynorphin A (-opioid receptor agonist) induced negative inotropy (7). All three of these distinct receptor agonists are believed to act, at least in part, through PKC activation. Furthermore, phorbol esters (such as TPA), potent and long-acting PKC activators, produced predominantly negative inotropic effects in various cardiac preparations (23-27). These seemingly paradoxical observations might reflect certain opposing factors of PKC activation which include the net effects of intracellular pH change, the size of intracellular Ca 2ϩ transient, and the states and species of cellular proteins being phosphorylated.One target for PKC in the heart is the contractile apparatus itself. Cardiac TnI and TnT from the thin filament have been shown to be effective substrates for PKC (28), and some of the phosphorylation sites in these proteins have been determined (29,30). Phosphorylation of TnI and/or TnT by PKC resulted in an inhibition of Ca 2ϩ -stimulated MgATPase of the reconstituted actomyosin complex (31-33) or in native myofibril preparations (32, 34), an effect associated with altered interactions among the contractile protein components (32,33). PKC also phosphorylated MLC2 (34 -36) and C-protein (34 -36) in myofibrillar and thick filament preparations. Phosphorylation of

A cDNA clone that hybridizes to an mRNA (lA10) that accumulates to a substantial level in the axis of the germinating soybean seed was sequenced. The amino acid sequence of the clone indicates an almost perfect repeat of Pro-Pro-Val-Tyr-Lys resulting in a protein containing 40% proline and lacking serine and histidine. On the likelihood that such a protein might be a hydroxyproline-rich cell-wall-glycoprotein (HRGP), cell walls of a soybean cell culture were extracted by procedures used to obtain soluble basic cell-wall glycoproteins, and the proteins were fractionated and purified. A 33-kDa protein (and possibly a 28-kDa protein) was obtained that has an amino acid distribution similar to that of the cDNA clone. The protein lacks histidine and serine and contains 20% hydroxyproline and 20% proline. The HRGP is thus distinct both in its amino acid content and in its pentameric repeat of Pro-Pro-Val-Tyr-Lys, with half of the prolines being hydroxylated. (4) was maintained on a 14-day cycle with a 1:12 dilution of cells (7 ml and 77 ml of medium in a 250-ml flask) every 2 weeks.In the preparation described in Results,25 ,uCi (1 Ci = 37 GBq) of [14C]proline was added to 8-day cells, and the cells were allowed to continue growing for 4 hr, after which time they were filtered on a coarse scintered funnel and frozen in liquid N2. In more recent preparations, 8-to 9-day cultures have been collected directly without labeling and the purification steps have been monitored solely by following the absorbance at 280 nm. Frozen cells (4.6 g) were homogenized in a Polytron in 0.25 M sucrose/3 mM EDTA/40 mM Tris-HCI, pH 8/0.5 mM phenylmethylsulfonyl fluoride/5 mM dithiothreitol (2.2 ml/g), and centrifuged for 2 min at 750 x g. The pellet was recentrifuged for 10 min at 23,500 x g and then washed one time in 14 ml of 5 mM potassium phosphate (pH 6.8) and six times with 15 ml of deionized water, centrifuging each time for 2 min at 750 x g. The pellet was extracted twice with 12 ml of 0.2 M CaC12 (5, 6), each extraction lasting 45 min. After centrifuging for 5 min at 2000 x g, the supernatant was adjusted with 68% trichloroacetic acid to a final concentration of 10% trichloroacetic acid, and the suspension was kept at 0C overnight. After centrifuging for 15 min at 24,000 x g, the supernatant was dialyzed against five changes of water over a 5-hr period and then overnight against 25 mM Tris HCI (pH 8.0). The dialyzer tubing had a Mr 3000 cutoff (Thomas 3787-H45). A typical preparation yielded 1.9 mg of protein based on 4 A280 units for a solution of 1 mg/ml, a figure calculated from the presence of 13 mol % tyrosine (see Table 1) in comparison with bovine serum albumin, which has 3.3% tyrosine (7) and 1 A280 unit for a solution of 1 mg/ml. The sample was chromatographed on a column of CM-cellulose (1.2 x 10 cm) equilibrated with 25 mM Tris-HCl (pH 8.0) and eluted with 100 ml of a linear gradient of 0.4 M NaCl in the same buffer. The 33-kDa protein, eluting at ""0.14 M NaCI, was pooled (tubes 17-27 in Fig. 3), concentra...

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