Purification and characterization of an alpha-actinin-like protein from porcine kidney.

An alpha-actinin-like protein was partially purified from the Triton-insoluble cytoskeleton of porcine kidney by 0.6 M MgCl2 treatment, ammonium sulfate fractionation, DEAE-cellulose chromatography and hydroxyapatite chromatography. Apparent purity of the kidney protein was approximately 90% by quantitative densitometry of Coomassie-stained polyacrylamide gels. The kidney alpha-actinin-like protein is very similar to muscle alpha-actinins by the following criteria: (1) both kidney protein and muscle alpha-actinins bind to F-actin at a similar ratio; (2) both proteins demonstrate no difference in the actomyosin turbidity assay and the ATPase assay for alpha-actinin activity; (3) both native proteins contain a large core of identical molecular weight resistant to trypsin; (4) on two-dimensional gels, both kidney protein and muscle alpha-actinins have similar isoelectric points of 5.9-6.1. However, kidney alpha-actinin-like protein is not identical in every respect with muscle alpha-actinins. Electrophoretic mobility of the kidney protein is slightly greater than that of chicken gizzard alpha-actinin and is identical to that of a component of chicken skeletal muscle alpha-actinin. One-dimensional peptide mappings of the kidney protein and muscle alpha-actinins were significantly different from each other. The interaction between kidney alpha-actinin-like protein and F-actin is sensitive to Ca2+. Similar Ca2+-sensitivity was observed with other non-muscle cell alpha-actinins.

Specific effects of spermine on ouabain-sensitive and potassium-dependent phosphatase activity of kidney plasma membranes. Specificity of the potassium sites.

Specific inhibition of ouabain-sensitive and K+-dependent p-nitrophenyl-phosphatase activity of rabbit kidney plasma membranes by spermine (N,N'-bis(3-aminopropyl)-1,4-butanediamine) was characterized kinetically. 1. Inhibition by spermine was competitive with K+. The Ki for spermine was 31 micronM in the presence of 1 mM Mg2+. 2. Excess Mg2+ inhibited the ouabain-sensitive phosphatase activity in competition with K+. The Ki for Mg2+ was 2.6 mM. 3. Increasing Mg2+ concentrations reduced the spermine inhibition. This could be observed at Mg2+ concentrations higher than that of K+. 4. In the absence of inhibition by Mg2+, spermine was noncompetitive with Mg2+ which was essential for the ouabain-sensitive phosphatase activity. This could be observed at Mg2+ concentrations lower than that of K+. 5. Although Ca2+ was a strong inhibitor of the ouabain-sensitive phosphatase activity in the presence of K+, it produced a small stimulation of the activity in the absence of K+. Approximately 0.1 mM Ca2+ gave the maximum stimulation. 6. The observed Ca2+- and Mg2+-dependent phosphatase activity was inhibited strongly by ouabain and by spermine. The half-maximal inhibition concentrations of ouabain and spermine were 0.1 and 63 micronM, respectively. It is likely that Mg2+, Ca2+ and spermine bind to the same site as does K+.

Isolation and characterization of three distinct 34 kDa EDTA-extractable proteins from bovine lens.

EDTA-extractable protein (EEP) is known to be a major lens membrane protein with a molecular mass in the range 32 kDa to 38 kDa, and is also known to bind to the lens membrane and phospholipid-containing liposomes in a calcium-dependent manner. Recent results (Russell, P., Zelenka, P., Martensen, J., and Reid, T.W. (1977) Curr. Eye Res. 6, 533-538) on antibody cross-reactivity have demonstrated that a 34-35 kDa component of EEP is identical to calpactin I (lipocortin II). In this study, we have identified and purified three distinct 34 kDa components of EEP (designated as EEP-34A1, EEP-34A2 and EEP-34B) from bovine lens that inhibit phospholipase A2 activity. These proteins bind to phospholipid-containing liposome and F-actin in a calcium-dependent fashion. Two-dimensional electrophoresis demonstrates that the three proteins were distinct from one another. However, immunochemical studies and one-dimensional peptide mapping indicate that EEP-34A1 and EEP-34B are very similar. Our results also indicate that EEP-34A1 is very similar to calpactin II and that EEP-34A2 corresponds to calpactin I. The bovine lens 34-35 kDa component of EEP is a mixture of proteins rather than a single protein.

Different expression time of the 105-kDa protein and 90-kDa heat-shock protein in rat testis.

To understand the physiological functions of the 105-kDa protein which is testis-specific and HSP90 (90-kDa heat-shock protein) related protein, the appearance of it in the testis has been followed during the development of rat. On immunoblotting analysis, the 105-kDa protein did not appear until after the age of five weeks, while HSP90 could be detected at three weeks. In the spermatozoa, the 105-kDa protein was much abundant but not in the LC-540 cells (a cell line from Leydig cell tumor in rat testis) cytosol. This finding has attracted much attention to the relationship between this protein and sperm functions.

Expression of pI(Cln) in Escherichia coli gives a strong tolerance to hypotonic stress.

We amplified the coding region DNA sequence from a rat renal pI(Cln) cDNA by PCR and expressed the protein in Escherichia coli cells. The cells were exposed to hypotonic conditions followed by spreading them onto LB plates for subsequent colony survival assay. The present study demonstrated that the cells expressing pI(Cln) exhibit a strong resistance to hypotonic stress. Moreover, the resistance was specifically inhibited by extracellular ATP and some anion channel inhibitors. These findings indicate that the expression of pI(Cln) directly confers tolerance to hypotonic stress, and pI(Cln) is concluded to be an important molecule for cell-volume regulation.

Expression of human beta 1,4-galactosyltransferase in gynecological cancer cell lines.

We examined the expression of beta 1,4-GT gene products in 11 gynecological cancer cell lines. A 4.7 kb mRNA and protein (54,000 Da and 57,000 Da) were detected by Northern blot and Western blot. Immunocytochemical staining revealed that beta 1,4-GT was localized in the Golgi or ER of tumor cells. An intense beta 1,4-GT mRNA signal was detected in ovarian and cervical cancer cells, whereas the level of beta 1,4-GT mRNA was very low in uterine endometrial cancer cells. We also confirmed that expression of beta 1,4-GT mRNA corresponded to expression of beta 1,4-GT protein. These results suggest that expression of the beta 1,4-GT gene products is higher in human cervical and ovarian cancer cells than in uterine endometrial cancer cells.

Simultaneous induction of mitochondrial heat shock protein mRNAs in rat forebrain ischemia.

Several investigations have postulated evidence of the involvement of apoptosis in delayed neuronal death following brief periods of global cerebral ischemia. Apoptosis may be closely linked to mitochondrial dysfunction. Heat shock protein (HSP) 60 and HSP10 are mitochondrial matrix proteins induced by stress and form the chaperonin complex that is implicated in protein folding and assembly within the mitochondria. This study investigated the induction of these mitochondrial stress protein genes in the hippocampal CA1 region and less vulnerable regions following transient forebrain ischemia. In situ hybridization analysis revealed that the induction pattern of HSP60 mRNA was identical to that of HSP10 mRNA throughout the entire ischemic course. No changes occurred in the expression of both mRNAs after 2 min ischemia. Strong induction of both mRNAs occurred in the CA1 region after 10 min ischemia and persisted until 1 d after reperfusion. In contrast, induction of both mRNAs in the less vulnerable regions was terminated by 1 d after reperfusion. These results demonstrate that mitochondrial stress conditions persist concomitantly with cytosolic stress conditions in regions vulnerable to transient forebrain ischemia.

Induction of mitochondrial heat shock protein 60 and 10 mRNAs following transient focal cerebral ischemia in the rat.

Heat shock proteins (HSPs) 60 and 10 are stress-inducible mitochondrial matrix proteins that form a chaperonin complex that is important for mitochondrial protein folding and function. The effect of cerebral ischemia on mitochondrial HSPs is unclear. The topographical and chronological patterns of HSP60 and HSP10 messenger ribonucleic acid (mRNA) expression and induction were investigated in the rat focal cerebral ischemia model. Focal cerebral ischemia was produced by transient middle cerebral artery occlusion for 30 or 90 min. Expression of mRNAs was analyzed using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. RT-PCR analysis showed that both HSP60 and HSP10 mRNA levels increased significantly in the ischemic cortex from 4 to 24 h of reperfusion after 30 min of occlusion. In situ hybridization analysis demonstrated significant induction of both mRNAs in the whole ischemic cortex after 30 min of occlusion and in the dorsomedial border (penumbra) of the ischemic cortex and ipsilateral hippocampus after 90 min of occlusion. Expression patterns and the timing of the induction of both HSP60 and HSP10 mRNAs were identical throughout the experiments. Simultaneous induction of the mRNAs for the mitochondrial chaperonins, HSP60 and HSP10, in various regions in focal cerebral ischemia demonstrates that mitochondrial stress conditions persist concomitantly with cytosolic stress conditions in focal cerebral ischemia.

Characterization of rat muscle fructose 1,6-bisphosphatase.

Fructose 1,6-bisphosphatase has been purified from rat muscle. Although the specific activity of the enzyme in the crude extract of rat muscle was extremely low, purification by the present procedure is highly reproducible. The purified enzyme showed a single band in SDS-polyacrylamide gel electrophoresis. The subunit molecular weight of the muscle enzyme was 37,500 in contrast to 43,000 in the case of the liver enzyme. Immunoreactivity of the muscle enzyme to anti-muscle and anti-liver fructose 1,6-bisphosphatase sera was clearly distinct from that of the liver enzyme. All one-dimensional peptide mappings of the muscle enzyme with staphylococcal V8 protease, chymotrypsin, and papain showed different patterns from those of the liver enzyme. When incubated with subtilisin, the extent of activation of muscle fructose 1,6-bisphosphatase at pH 9.1 was smaller than that of the liver enzyme. The subtilisin digestion pattern of the muscle enzyme on SDS-polyacrylamide gel electrophoresis was distinct from that of the liver enzyme. The AMP-concentration giving 50% inhibition of the muscle enzyme was 0.54 microM, whereas that of the liver enzyme was 85 microM. The concentrations of fructose 2,6-bisphosphate that gave 50% inhibition of rat muscle and liver enzymes were 6.3 and 1.5 microM, respectively. Fructose 1,6-bisphosphatase protein was not detected in soleus muscle by immunoelectroblotting with anti-muscle fructose 1,6-bisphosphatase serum.

Control of rabbit liver fructose-1, 6-diphosphatase activity by magnesium ions.

EDTA at a concentration of 1 muM produced a threshold effect in the activation of purified rabbit liver fructose-1, 6-diphosphatase [EC 3.1.3.11] in the presence of 5 mM Mg2+ at pH 7.2. Without EDTA, biphasic activation curves were produced by Mg2+. A double-reciprocal plot of the data gave the Km values corresponding to the two linear regions. They were 0.19 and 0.83 mM at pH 7.5, and 0.055 and 0.83 mM at pH 9.1. In the presence of 5muM EDTA a sigmoidal curve was obtained for Mg2+ activation in the range of noninhibitory Mg2+ concentrations at pH 7.2. The apparent Km value for Mg2+ was 0.15 mM, and the Hill coefficient was 2.0. At pH 9.1 cooperativity among the Mg2+ sites disappeared, and the apparent Km value for Mg2+ was 0.055 mM. These Km values at pH 7.2 or 9.1 corresponded to the smaller of the biphasic Km values obtained without EDTA. In the absence of EDTA, no inhibition by Mg2+ was observed in the Mg2+ concentration range below 10 mM. In the presence of EDTA, the enzyme was inhibited markedly by Mg2+ at concentrations above 0.5 mM at pH 7.2, and was more sensitive to inhibition at pH 9.1. The effects of pH on the Km value for Mg2+ activation and on the Mg2+ inhibition contributed to an apparent shift of the pH optimum for activity induced by EDTA. Cooperative interaction among fructose-1, 6-diphosphate sites was observed for the enzyme in the presence of EDTA. The Hill coefficient was approximatley 1.8, and the apparent Km value for the substrate was 0.74 muM. EDTA appears to make liver fructose-1, 6-diphosphatase very sensitive to various effectors. It is suggested that Mg2+ serves as a regulator for the enzyme activity.

Fructose-1,6-biphosphatase of the small intestine. Purification and comparison with liver and muscle fructose-1,6-bisphosphatases.

The occurrence of specific fructose-1,6-bisphosphatase [D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11] (Fru-1,6-P2ase) in the small intestine was confirmed. 1. Fru-1,6-P2ase was isolated from mouse small intestine by a simple method. The isolated enzyme preparation was an electrophoretically homogeneous protein. 2. The molecular weight and subunit molecular weight were 140,000 and 38,000, respectively. 3. The intestinal enzyme was electrophoretically distinct from the liver enzyme. 4. The kinetic properties of the purified intestinal enzyme were compared with those of the mouse liver and muscle enzymes. 5. Mouse intestinal and muscle Fru-1,6-P2ases hydrolyzed ribulose-1,5-bisphosphate in addition to fructose-1,6-bisphosphate and sedoheptulose-1,7-bisphosphate.

Purification and properties of mouse liver fructose 1,6-bisphosphatase.

A simple procedure has been developed for the purification of mouse liver and kidney fructose-1,6-bisphosphatase. In addition to the conventional method, including substrate elution from phosphocellulose, Blue Sepharose column chromatography made the purification procedure highly reproducible. The enzyme from rabbit liver was also purified by this method with a small modification. The isolated preparation was electrophoretically homogeneous. The mouse liver enzyme was identical with the kidney enzyme, and different from the rabbit liver enzyme electrophoretically. The structural properties and the amino acid composition were similar to those of this enzyme from other mammalian livers; the molecular weight was 143,000, subunit size was 37,500, S20, w was 7.0, and partial specific volume was 0.74. Cysteine and methionine residues amounted to 5-6 mol per subunit. Tryptophan was not detected. The Km value for fructose-1,6-bisphosphate was 1.3 microM. The Ki value for AMP was 19 microM. EDTA strongly activated the activity of the mouse liver enzyme at neutral pH. A partial proteolytic digestion of the mouse liver enzyme decreased the activity at neutral pH, and increased it at alkaline pH.

The 30 kDa abnormal protein in avian dystrophic muscle is indistinguishable from carbonic anhydrase III by physicochemical, immunological, and enzymological criteria.

In the accompanying paper, we described the existence, molecular characterization, and ontogeny of a 30 kDa abnormal protein in chicken dystrophic muscles. In this study, we have purified chicken carbonic anhydrase III and the 30 kDa protein and directly compared them. In terms of its enzymological features, the 30 kDa protein is a typical carbonic anhydrase III. Like carbonic anhydrases, it contains one mole zinc per mole of protein. The protein selectively cross-reacted with a chicken carbonic anhydrase III antibody. Antibody to the 30 kDa protein cross-reacted with chicken skeletal muscle carbonic anhydrase III. Moreover, the distribution of the abnormal protein is exactly identical to that of carbonic anhydrase III; however, there is a possibility that the 30 kDa protein is a variant of carbonic anhydrase III. Slight differences were found in antigenicities and in the apparent molecular weights of the two proteins. We have compared the two proteins by 125I-labeled two-dimensional peptide mapping. Tryptic maps have shown that the two proteins are highly homologous. Combined, these results strongly indicate that the 30 kDa protein and carbonic anhydrase III are similar, if not identical.

Purification and some properties of porcine kidney tropomyosin.

A tropomyosin has been purified from porcine kidney and its properties compared with those of rabbit skeletal muscle tropomyosin. Kidney tropomyosin was separated from contaminating vascular smooth muscle tropomyosin by hydroxylapatite chromatography. Kidney tropomyosin resembles tropomyosin from other non-muscle cells in regard to subunit size, mobility on SDS-polyacrylamide gels in the presence and absence of 6 M urea, amino acid composition and morphology. The binding of tropomyosin to F-actin is strongly dependent on the Mg2+ concentration. With kidney tropomyosin, binding begins at 1 mM Mg2+ and is complete at about 4-5 mM, while with muscle tropomyosin, binding is initiated at 1 mM Mg2+ and reaches saturation at 2-3 mM Mg2+. Both kidney and muscle tropomyosins bind to actin in a similar ratio of 1 tropomyosin/6-7 actin monomers at saturation. Both kidney and skeletal muscle tropomyosins prevent "severing" of F-actin filaments induced by gelsolin/villin-like protein purified from kidney. These results suggest that, in non-muscle cells, tropomyosin may protect microfilaments from Ca2+-dependent solation at the site where they may interact with myosin.

Effects of dibutyryl adenosine 3':5'-cyclic monophosphate and other agents on induction of alkaline phosphatase activity in monkey kidney cells.

The induction of alkaline phosphatase (ALP) by dibutyryl adenosine 3':5'-cyclic monophosphate (Bt2cAMP) was investigated in strain JTC-12 . P3 cells derived from monkey (Maccaca irus) kidney cortex. ALP activity was increased by Bt2cAMP in a dose-dependent manner, reaching a plateau at concentrations higher than 5 mM with the activity being about 4 times that of the controls. The concentration of Bt2cAMP required for half-maximal induction of ALP activity was about 0.8 mM. ALP activity was increased rapidly by Bt2cAMP for the first 5 days and then continued to increase gradually towards a plateau level. Removal of Bt2cAMP from the medium caused a rapid decrease in the activity, suggesting that the induction of ALP activity by Bt2cAMP is reversible. ALP activity was induced synergistically in the presence of 1 mM sodium butyrate together with Bt2cAMP at concentrations from 0.01 to 1 mM. It was also found that in the presence of 1 mM Bt2cAMP, sodium butyrate increased ALP activity in the same manner as Bt2cAMP did in the presence of 1 mM sodium butyrate. Although dexamethasone, a potent glucocorticoid, had no effect on ALP activity in control cells, the hormone suppressed the ALP activity induced by Bt2cAMP in a dose-dependent manner. At concentrations above 0.2 mM, two xanthine derivatives, theophylline and 3-isobutyl-1-methyl-xanthine (IBMX), also inhibited the induction of ALP activity by 1 mM Bt2cAMP. Inhibitors of protein synthesis, cycloheximide (1.5 micrograms/ml) and pactamycin (10 micrograms/ml), as well as inhibitors of RNA synthesis, actinomycin D (2 micrograms/ml) and alpha-amanitin (50 micrograms/ml), suppressed the induction of ALP activity.

Detection and isolation of a 30 kDa abnormal protein in avian dystrophic muscle.

We have studied the protein composition of the pectoralis superficialis muscle of genetically dystrophic (New Hampshire line 413) and normal control (line 412) chickens by one- and two-dimensional gel electrophoresis. A protein, referred to hereafter as the 30 kDa abnormal protein, was specifically detected in the affected muscle. It was purified to homogeneity, and its molecular properties were studied. It is a monomer with a molecular mass of approximately 30 kDa and an isoelectric point of about pI 8.4. We have screened by Western blotting a variety of muscles from line 412 and line 413 chickens for the presence of the 30 kDa protein. While the pattern of total protein is very similar in all cases, the 30 kDa protein was not detected in the pectoralis superficialis muscle of line 412 chickens. However, the immunoreactive bands were detected in the sartorius muscle and the tensor fasciae latae muscle from dystrophic and normal chickens. Interestingly, the immunoreactive bands of normal skeletal muscles are smaller in molecular weight than those of dystrophic skeletal muscles. To determine the early time sequence of the appearance of the abnormal protein, we studied muscles from embryos and post-hatched chickens at various ages. The abnormal protein was detected in dystrophic muscles as early as 15 days ex ovo and occurred throughout development up to six months ex ovo. Although the implication of the dystrophy-associated appearance of the 30 kDa protein in the affected muscle is not clear at present, it would be of particular interest to elucidate the biochemical functions of the 30 kDa protein in the affected muscle (pectoralis superficialis muscle) of genetically dystrophic chicken.

Specific effects of spermine on Na+,K+-adenosine triphosphatase.

Specific effects of spermine on Na+,K+-ATPase were observed using an enzyme partially purified from rabbit kidney microsomes by extraction with deoxycholate. 1. Spermine competed with K+ for K+-dependent, ouabain-sensitive nitrophenylphosphatase. The K1 for spermine was 0.075 mm in the presence of 1 mM Mg2+ and 5 mM p-nitrophenylphosphate at pH 7.5. 2. spermine activated Na+,K+-ATPase over limited concentration ranges of K+ and Na+ in the presence of 0.05 mM ATP. The spermine concentration required for half maximal activation was 0.055 mM in the presence of 1 mM K+, 10 mM Na+, 1 mM Mg2+, and 0.05 mM ATP. 3. The activation of Na+,K4-ATPase was not due to substitution of spermine for K+, Na+, or Mg2+. 4. When the concentration of K+ or Na+ was extremely low, or in excess, spermine did not activate Na+,K+-ATPase, but inhibited it slightly. 5. Plots of 1/v vs. 1/[ATP] at various concentrations of spermine showed that spermine decreased the Km for ATP without changing the Vmax. 6. Plots of 1/v vs. 1/[ATP] at concentrations of K+ from 0.05 mM to 0.5 mM showed that K+ increased the Km for ATP with increase in the Vmax in the presence of 0.2 mM spermine similarly to that in the absence of spermine. The contradictory effects of spermine on this enzyme system suggest that the K+-dependent monophosphatase activity does not reflect the second half (the dephosphorylation step) of the Na+,K+-ATPase catalytic cycle.

Identical properties of aldosterone and corticosterone binders and their presence in rat brain and kidney.

The [3H]corticosterone binders from rat brain and kidney were characterized by binding affinity and chromatographies, and compared with the binders for [3H]aldosterone and [3H]triamicinolone acetonide. Corticosterone-binding globulin-like molecules at very high concentrations in crude extracts were completely eliminated by a DEAE-gel adsorption procedure. [3H]Aldosterone binder in the renal, DEAE-treated fraction was recovered in a single peak by gel-filtration chromatography and by ultracentrifugation in linear sucrose gradients, independent of hormone-binding and tungstate, a stabilizer of the binder. The Stokes' radius and sedimentation coefficient of the renal aldosterone binder were 6.6 nm and 9.3S, respectively, indicating an apparent molecular weight of 263,000. Corticosterone-preferring binder also existed in the DEAE-treated fraction. Both aldosterone and corticosterone binders were found in the brain and kidney preparations. Comparison among the binders showed identical values of Stokes' radius and elution pattern from DEAE-Toyopearl in a linear salt gradient regardless of the organ and the hormones. Scatchard analyses of [3H]aldosterone and [3H]corticosterone binding showed for each ligand only one group of high-affinity sites with the equivalent dissociation constants, 4-7 nM. The orders of steroids in competing for the two high-affinity sites were equivalent: corticosterone greater than or equal to aldosterone much greater than triamcinolone acetonide, and that for the triamcinolone acetonide binding was triamcinolone acetonide much greater than aldosterone greater than or equal to corticosterone. Hydroxyapatite column chromatography separated the aldosterone and corticosterone binders from the triamcinolone acetonide binder, but not the aldosterone binder from the corticosterone binder. It is concluded that aldosterone and corticosterone binders distinct from triamcinolone acetonide binder exist in rat brain and kidney.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of glucocorticoids on induction of fructose bisphosphatase and glucose-6-phosphatase in fetal mouse liver.

Glucocorticoids significantly affected the developmental appearance of fructose-1,6-bisphosphatase [EC 3.1.3.11] and glucose-6-phosphatase [EC 3.1.3.9] in fetal mouse liver. In fragments of 15- or 16-day-old fetal livers maintained in organ culture in the absence of serum, induction of the bisphosphatase by dibutyryl cyclic AMP was repressed completely when the tissue was treated with 10(-7)M dexamethasone for 24 h during the second day of culture. The induction of the glucose phosphatase was greatly stimulated after a lag of 1 to 2 days. The glucocorticoid action continued over a period of 2 days even though the steroid had been washed out. The dose response curve of hydrocortisone with the half-maximally effective concentration of roughly 2 X 10(-8)M is in the physiological range. The corticoid action was specific for glucocorticoids, and aldosterone or progesterone was ineffective. When the tissue was cultured for 4 days before addition of dexamethasone, the bisphosphatase induction became insensitive to the steroid. Glucose-6-phosphatase induction, however, remained sensitive, but the long latent period required for the appearance of the hormone action disappeared. These results indicate the involvement of glucocorticoids in the developmental appearance of glucose-6-phosphatase in fetal liver.

pI(Cln) and cytosolic F-actin constitute a heteromeric complex with a constant molecular mass in rat skeletal muscles.

To elucidate the function of pI(Cln), its localization in subcellular organellae was investigated. A specific polyclonal anti-pI(Cln) antibody detected the soluble 38-kDa pI(Cln) exclusively in the cytosols of rat heart, lung, liver, spleen, skeletal muscle, testis, and brain, but not rat kidney. pI(Cln)-associated proteins in skeletal muscle were also analyzed. Native-gradient PAGE showed a single 340-kDa protein band reactive to anti-pI(Cln) antibody. This band also stained with anti-actin antibody. Two-dimensional PAGE and immunoprecipitation analysis indicated that all of the pI(Cln) was present in association with actin of a constant length: the molecular ratio of pI(Cln) to actin was roughly 1:7. In addition, all actin in the cytosol fractions was found in association with pI(Cln). These results suggest the possibility that skeletal muscle pI(Cln) controls the length of cytosolic F-actin.