RESUMEN
Cholesterol, a component of intercellular lipids, is important for stratum corneum (SC) homeostasis, including its barrier function and desquamation. However, cholesterologenesis in the epidermis decreases under basal conditions with aging. We found that the number of horny layers in murine SC increased with the decrease of desquamation in the outermost corneocytes associated with aging. The cholesterol content decreased and the cholesterol sulfate content increased in the horny layer with aging, which resulted in an increase in the ratio of cholesterol sulfate to cholesterol. Moreover, we investigated the effects of accelerated cholesterologenesis on desquamation in aged murine skin following topical application of mevalonic acid. The ratio of cholesterol sulfate to cholesterol in aged murine SC significantly decreased following topical treatment with mevalonic acid, which resulted from an increase in cholesterol content via the acceleration of cholesterologenesis. Treatment with mevalonic acid also significantly reduced the number of cell layers in the SC along with the acceleration of desquamation, as measured by desmoglein I content, corneocyte surface area and proteinase activity. These results indicate that an improvement in the ratio of cholesterol sulfate to cholesterol content by de novo cholesterologenesis may be important for desquamation of the SC in aged epidermis.
Asunto(s)
Envejecimiento/patología , Colesterol/biosíntesis , Epidermis/metabolismo , Envejecimiento/metabolismo , Animales , ADN/biosíntesis , Epidermis/patología , Concentración de Iones de Hidrógeno , Ácido Mevalónico/farmacología , Ratones , Ratones PeladosRESUMEN
Tissue in body must quickly recognize injury to response to the rapid pace of epidermal growth. In skin, the epidermal cells must also react to danger signals from the surrounding extracellular lipid of the stratum corneum spaces and immediately participate by initiating the wound repair process. The topical administration of the lyotropic liquid crystal nanocube to stratum corneum rapidly broke down the lipid lamella structure which would be recognized as a wound without organ-change. This can activate a variety of biological processes. This study set out to determine whether the phase transition of the lipid to a neighbouring different physicochemical structure can stimulate keratinocyte cells and what mechanism is responsible for this response. Using small angle x-ray scattering (SAXS) analysis, a response to the transient structural change of lipid was detected which might result from the diffusion of oil and/or water from nanocube liquid crystal towards the lipid lamella phase. Simultaneously, a significant increase in growth factors and inflammatory cytokines was detected after administration of nanocube. Not only the excess expression of cytokines but also the extent of TEWL as a barrier marker of skin increased. These observations suggest that a structural change in lipid can stimulate and trigger recognition of a slight injury in the wound defence and a repair response as homeostasis. This method actually succeeded in improving photo-induced hyperpigmentation on a human face.
Asunto(s)
Nanotecnología , Regeneración/efectos de los fármacos , Fenómenos Fisiológicos de la Piel/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Emulsiones , Factor de Crecimiento Epidérmico/metabolismo , Humanos , Ácido Hialurónico/metabolismo , Inmunohistoquímica , Interleucina-1/metabolismo , Queratolíticos/farmacología , Masculino , Ratones , Modelos Biológicos , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Dispersión de Radiación , Piel/química , Piel/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Tretinoina/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Rayos XRESUMEN
In order to investigate the mechanism of glycolic acid (GA) function in human stratum corneum, we monitored changes in cathepsin D-like (CD) and chymotrypsin-like (SCCE) proteinases for 3 weeks following topical GA application (50% w/v, pH 0.9) for 30 min to human skin. In the early phase, weakened stratum corneum cohesion in the lower layers was observed on day 2 and the amount of active CD in the upper layer of the stratum corneum was significantly decreased from 30 min until day 2, whereas that in the lower layer remained normal. In contrast, the amount of active SCCE showed no change during the experimental period. The surface pH of the stratum corneum drastically decreased to pH 2 at 30 min and slightly recovered to around pH 3 until 1 day after treatment. From 9 to 19 days, a decrease in corneocyte cell area and a remarkable long-term increase in the amount of active CD in the upper layer were observed. In an in vitro study, the activities of desquamation-regulating proteinases were shown to have remarkably increased at around pH 3, due to activation of CD at its optimal pH. These results suggest that GA functions via at least two different mechanisms, acute activation of CD in the lower layer by acidification around pH 3, along with inactivation of CD in the upper layer, and long-term enhancement of de novo CD production in the few weeks following GA treatment.
Asunto(s)
Epidermis/efectos de los fármacos , Glicolatos/farmacología , Queratolíticos/farmacología , Administración Cutánea , Adulto , Catepsina D/metabolismo , Quimasas , Epidermis/enzimología , Epidermis/patología , Femenino , Glicolatos/administración & dosificación , Humanos , Concentración de Iones de Hidrógeno/efectos de los fármacos , Queratolíticos/administración & dosificación , Masculino , Persona de Mediana Edad , Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismo , Serina Endopeptidasas/metabolismo , Factores de Tiempo , Agua/metabolismoRESUMEN
BACKGROUND: We previously reported that an ambient aspartic proteinase is crucial to desquamation of the stratum corneum at pH 5. Identification of this aspartic proteinase by using enzyme inhibitors suggested it to be cathepsin D, although we could not exclude cathepsin E. OBJECTIVES: To determine the identity of this aspartic proteinase and its distribution within the stratum corneum. METHODS: We measured enzyme activities of cathepsin D and cathepsin E in the salt and detergent extracts from callus stratum corneum, using a fluorogenic peptide as a substrate and comparing the effect of addition of Ascaris pepsin inhibitor (specific for cathepsin E) with that of pepstatin A (which inhibits both cathepsin D and cathepsin E). Both enzymes were then extracted and purified from plantar stratum corneum samples and identified by Western blotting. Immunofluorescence microscopy was used to investigate the localization of proteinases within human plantar stratum corneum sample sections. RESULTS: We found that 20% of total aspartic proteinase activity could be attributed to cathepsin E, the remainder to cathepsin D. Two subunits of cathepsin D were identified, a mature active form at 33 kDa and an intermediate active form at 48 kDa; cathepsin E was also identified at 48 kDa, although in a stained band 10-fold weaker in the immunoblot. Immunofluorescence microscopy showed the antibody to cathepsin D to be localized in the lipid envelopes of the stratum corneum, whereas that to cathepsin E stained the tissue diffusely. The labelling for cathepsin D was similar to that observed for desmosomes, and immunoelectron microscopy confirmed that cathepsin D was present on desmosomes. On the other hand, cathepsin E occurred intracellularly within the squames. CONCLUSIONS: We conclude that cathepsin D, and not cathepsin E, causes desquamation by degrading desmosomes.
Asunto(s)
Catepsina D/metabolismo , Catepsina E/metabolismo , Desmosomas/metabolismo , Epidermis/metabolismo , Catepsina D/aislamiento & purificación , Catepsina E/aislamiento & purificación , Desmosomas/inmunología , Desmosomas/ultraestructura , Epidermis/inmunología , Epidermis/ultraestructura , Talón , HumanosRESUMEN
Nitric oxide (NO) is a potent intercellular mediator of melanogenesis, whereas metallothionein (MT) is an inducible intracellular antioxidant that has been reported to scavenge NO. We investigated the existence and induction of MT in melanocytes, and its inhibitory effect on NO-induced melanogenesis. The expression of MT was detected in melanocytes, however, at a lower level than in keratinocytes, and its induction was possible by the addition of zinc chloride. Further, an NO-stimulated increase of tyrosinase activity in melanocytes was remarkably suppressed, when MT was induced prior to NO stimulation. Melanogenesis was also suppressed, when dexamethasone was used to induce MT. However, an NO-stimulated increase of tyrosinase expression was not suppressed at the gene and protein level, when MT was induced in melanocytes. The same suppressive effect of melanogenesis was also observed, when alpha-melanocyte-stimulating hormone or endothelin-1 was used as a stimulator. Because these results implied a mechanism other than NO scavenging to explain the suppressive effect of MT induction on melanogenesis, the direct inhibition of tyrosinase by MT was examined. Melanosome fractions were prepared from melanocytes, whose melanogenesis was suppressed by the induction of MT. Tyrosinase suppression was observed in the melanosome fractions, which was neutralized by the addition of anti-MT antibody. These results suggest that MT induction may be effective to suppress melanogenesis stimulated by NO as well as other melanogens, and these suppressive effects might be due to a direct inhibition of tyrosinase activity in melanosome and not a scavenging effect of NO.
Asunto(s)
Melanocitos/metabolismo , Metalotioneína/metabolismo , Metalotioneína/fisiología , Northern Blotting , Western Blotting , Cloruros/metabolismo , Cloruros/farmacología , Dexametasona/farmacología , Endotelina-1/metabolismo , Fibroblastos/metabolismo , Glucocorticoides/farmacología , Células HeLa , Humanos , Queratinocitos/metabolismo , Melanosomas/metabolismo , Monofenol Monooxigenasa/metabolismo , Óxido Nítrico/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Compuestos de Zinc/metabolismo , Compuestos de Zinc/farmacología , alfa-MSH/metabolismoRESUMEN
Ultraviolet (UV) B irradiation evokes erythema and delayed pigmentation in skin, where a variety of toxic and modulating events are known to be involved. Nitric oxide (NO) is generated from L-arginine by NO synthases (NOS). Production of NO is enhanced in response to UVB-stimulation and has an important role in the development of erythema. NO has recently been demonstrated as a melanogen which stimulates melanocytes in vitro, however, no known in vivo data has been reported to support this finding. In this study, we investigated the contribution of NO with UV-induced pigmentation in an animal model using an NOS inhibitor. UVB-induced erythema in guinea pig skin was reduced when an NOS inhibitor, L-NAME (N-nitro-L-arginine methylester hydrochloride), was topically applied to the skin daily, beginning 3 days before UVB-irradiation. Delayed pigmentation and an increased number of DOPA-positive melanocytes in the skin were markedly suppressed by sequential daily treatment with L-NAME. Furthermore, melanin content 13 days after UVB-irradiation was significantly lower in skin treated with L-NAME than in the controls. In contrast, D-NAME (N-nitro-D-arginine methylester hydrochloride), an ineffective isomer of L-NAME, demonstrated no effect on these UV-induced skin responses. These results suggest that NO production may contribute to the regulation of UVB-induced pigmentation.
Asunto(s)
Óxido Nítrico/metabolismo , Pigmentación de la Piel/efectos de la radiación , Animales , Dihidroxifenilalanina/metabolismo , Inhibidores Enzimáticos/farmacología , Eritema/diagnóstico por imagen , Cobayas , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Cintigrafía , Piel/metabolismo , Piel/patología , Rayos UltravioletaRESUMEN
Ultraviolet light (UV) radiation causes skin-tanning, which is thought to be mediated by stimulating the release of melanogenic factors from keratinocytes as well as other cells. Nitric oxide (NO) has been reported to be generated after UV radiation and to stimulate melanocytes as one of the melanogens. In a previous experiment by another group on melanogenesis induced by NO, increases in both tyrosinase activity and tyrosinase protein levels were observed after daily stimulation of NO for 4 days. In the present study, we investigated tyrosinase gene expression within the first 24 hr of NO-induced melanogenesis. Tyrosinase mRNA expression was found to be induced 2 hr after a single treatment with S-nitroso-N-acetyl-L-arginine. An increase of tyrosinase activity was also detected time-dependently within the 24-hr period, accompanied by an increase of tyrosinase protein levels. The induction of mRNA expression was suppressed by a cyclic guanosine 3',5'-monophosphate (cGMP)-dependent protein kinase (cGMP/PKG) inhibitor. These results suggest that the enhancement of tyrosinase gene expression via the cGMP pathway may be a primary mechanism for NO-induced melanogenesis.
Asunto(s)
Carbazoles , Indoles , Melanocitos/enzimología , Monofenol Monooxigenasa/genética , Óxido Nítrico/farmacología , Penicilamina/análogos & derivados , Alcaloides/farmacología , Células Cultivadas , GMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Melanocitos/efectos de los fármacos , Monofenol Monooxigenasa/metabolismo , Óxido Nítrico/metabolismo , Penicilamina/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , S-Nitroso-N-Acetilpenicilamina , Tirosina 3-Monooxigenasa/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Extracellular lipids of the stratum corneum, which are composed of cholesterol, fatty acid, and ceramides, are essential for the epidermal permeability barrier function. With damage to the barrier, a decreased capacity for epidermal lipid biosynthesis in aged epidermis results in an impaired repair response. Mevalonic acid is an intermediate after the rate-limiting step in cholesterol biosynthesis, which is catalyzed by 3-hydroxy-3-methylglutaryl coenzyme A reductase. In the present study, we investigated the effect of topical mevalonic acid on the murine epidermal permeability barrier function, comparing it with that of cholesterol. Topical treatment with acetone caused linear increases in transepidermal water loss, in proportion to the number of treatments more rapidly in aged mice than in young mice. Administration of mevalonic acid on aged murine epidermis enhanced its resistance against damage and the recovery rate of barrier function from acute barrier disruption. In contrast, although cholesterol also had the same effect, it required a much higher amount than mevalonic acid. In young mice, neither mevalonic acid nor cholesterol had any effect on resistance against acetone damage nor the recovery rate from acetone damage. In the skin of mice topically administered with mevalonic acid, stimulation of cholesterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme A reductase activity were both observed, whereas none was seen with stimulation by equimolar cholesterol. These data indicate that a topical application of mevalonic acid enhances barrier recovery in aged mice, which is accompanied by not only acceleration of cholesterol synthesis from mevalonic acid but also stimulation of the whole cholesterol biosynthesis.
Asunto(s)
Envejecimiento/fisiología , Permeabilidad de la Membrana Celular/fisiología , Colesterol/biosíntesis , Ácido Mevalónico/farmacología , Piel/citología , Administración Tópica , Animales , Ácidos Grasos no Esterificados/análisis , Homeostasis , Hidroximetilglutaril-CoA Reductasas/metabolismo , Ratones , Ratones Pelados , Piel/químicaRESUMEN
Even though the skin surface is acidic (about pH 5), most in vitro studies on desquamation have been performed at alkaline pH. We demonstrate that the standard in vitro model system, which achieves squame shedding upon incubation of plantar stratum corneum for 1 day in an alkaline buffer that must include a chelating agent, can be extended to a more realistic model in which the incubation is for 4 days, at varying pHs from 5 to 8, without exogenous chelators. Desmoglein I from stratum corneum was degraded by the squames shed at pH 5 as well as at pH 8. Squame shedding was inhibited to varying extents by the addition of proteinase inhibitors, whose specificity suggested that the crucial enzymatic activity at pH 8 was a chymotrypsin-like serine proteinase, while a similar activity at pH 5 was accompanied by an aspartic proteinase activity of comparable strength. Four degradation peaks were observed when the insulin B chain was reacted with shed squames at pH 5. Two of these peptides were suppressed by the addition of phenylmethylsulphonyl fluoride, the other two by pepstatin A; chymostatin inhibited all four, but E-64 and leupeptin showed no effect. The implied specificity was confirmed by reacting the insulin (without squames) with the standard enzymes human liver cathepsin D and pancreatic chymotrypsin, reproducing the expected degradation products. These results suggest that epidermal desquamation at acidic pH requires two proteolytic activities, one of which is an analogue of chymotrypsin and the other of cathepsin D. Endogenous proteinases corresponding to these activities have been previously identified, namely the stratum corneum chymotryptic enzyme and the mature active form of cathepsin D.
Asunto(s)
Catepsina D/metabolismo , Quimotripsina/metabolismo , Epidermis/metabolismo , Piel/citología , Western Blotting , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Humanos , Insulina/metabolismoRESUMEN
We have characterized the subcellular distribution of S100A3, a cysteine-rich calcium binding protein, in human scalp hair shaft. This was accomplished using rapid-freezing immunocytochemistry, a technique that combines rapid-freezing, freeze-substitution fixation without chemical fixatives, and subsequent electron microscopic detection of immunocytochemical labeling. This technique preserves both the antigenicity and the ultrastructural integrity of fully keratinized tissues, which are highly unmanageable when prepared for immunoelectron microscopy. In the hair shaft, S100A3 was primarily identified in the endocuticle and was also present in the intermacrofibrillar matrix surrounding macrofibril bundles of intermediate filament keratins in cortex cells. Double immunolabeling of S100A3 and hair keratins revealed the in situ spatial relationship between them. In the endocuticle, S100A3 was present on the inner portion of the endocuticle adjacent to the cell membrane complex, whereas hair keratins were present on the outer portion. These results provide the first ultrastructural evidence that an S100 protein is localized in specific subcompartments in human hair cells. (J Histochem Cytochem 47:525-532, 1999)
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Cabello/metabolismo , Proteínas S100 , Adolescente , Western Blotting , Niño , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Substitución por Congelación , Folículo Piloso/metabolismo , Humanos , Queratinas/metabolismo , Microscopía Inmunoelectrónica , Manejo de EspecímenesRESUMEN
We have previously identified a cysteine-rich calcium binding protein S100A3 present in the cuticle of human hair fiber. In this study, we cloned a cDNA for mouse S100A3, identified its gene location, and elucidated the expression profile throughout hair follicle development. The mouse S100A3 gene was clustered with other S100 family members on chromosome 3, and specifically expressed in dorsal skin containing hair follicles. The level of S100A3 mRNA was elevated during the anagen phase of the hair growth cycle, and sharply declined from the regression phase on. In situ hybridization revealed that the S100A3 gene was prominently expressed in cuticular cells of the hair follicle, and mRNA levels were highest in the keratogenous zone over the entire cuticular layer. Expression was also observed to a lesser extent in differentiated cortical cells; however, expression was not observed in any other component of the hair follicle or dorsal tissues. Immunohistochemical analysis showed that the S100A3 protein accumulated in cuticular and cortical cells undergoing terminal differentiation. These results indicate that the S100A3 gene is exclusively expressed, and the translation product retained, in follicular cells differentiating into major components of the hair shaft. It seems likely that S100A3 plays an important role in calcium-dependent processes leading to hair shaft formation.
Asunto(s)
Proteínas de Unión al Calcio/genética , Folículo Piloso/metabolismo , Osteonectina/genética , Proteínas S100 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Ciclo Celular/genética , Femenino , Expresión Génica , Cabello/citología , Humanos , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos ICR , Datos de Secuencia Molecular , Familia de Multigenes , Embarazo , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
Analyses on sodium dodecyl sulfate-polyacrylamide gel electrophoreses showed that the human hair cuticle extracts mainly consist of a 7-kDa component and keratin proteins. The S-carboxymethylation of the cuticle extracts made the 7-kDa band shift to the 15-kDa position. After electroblotting of the S-carboxymethyl derivative, the membrane pieces carrying the 15-kDa band were treated with trypsin and the released peptides were separated by reverse-phased HPLC. Amino acid sequence analyses revealed that the peptides corresponded to the partial sequences deduced from human genome coding for S100A3, a cysteine-rich calcium binding protein. The anti S100A3 serum, prepared by immunizing a synthetic peptide antigen, reacted with the 7-kDa and 15-kDa bands in immunoblotting analyses. Immunofluorescence microscopy showed intense labeling to the cuticular layer with the anti S100A3 serum. These results indicated that S100A3 was highly expressed in the human hair cuticle.
Asunto(s)
Proteínas de Unión al Calcio/análisis , Cabello/química , Proteínas S100 , Secuencia de Aminoácidos , Proteínas de Unión al Calcio/química , Extractos Celulares/química , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Humanos , Immunoblotting , Microscopía Fluorescente , Datos de Secuencia Molecular , Peso Molecular , Péptidos/química , Tripsina/metabolismoRESUMEN
1. Repeated oral administrations of tryptic hydrolysate of bovine milk casein (CEI) showed antihypertensive effect in spontaneously hypertensive rats. 2. Single oral administration of CEI antagonized the pressor response to angiotensin I. 3. Bovine milk casein hydrolysate inhibited the angiotensin I-converting enzyme (ACE) activity. Three peptides with ACE-inhibiting activity were isolated from CEI. 4. It is suggested that ACE-inhibiting peptides in the tryptic hydrolysate milk casein are absorbed from the intestinal tract and produce an antihypertensive effect.
Asunto(s)
Antihipertensivos , Caseínas/farmacología , Hipertensión/tratamiento farmacológico , Hidrolisados de Proteína/farmacología , Administración Oral , Secuencia de Aminoácidos , Angiotensina I/antagonistas & inhibidores , Inhibidores de la Enzima Convertidora de Angiotensina , Animales , Electrocardiografía/efectos de los fármacos , Corazón/anatomía & histología , Lípidos/sangre , Datos de Secuencia Molecular , Miocardio/patología , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas Endogámicas SHR , TripsinaRESUMEN
1. A purified preparation of myosin light-chain kinase (MLCK) was obtained from chicken gizzard, and it was shown to consist of two subunits; 130,000 (130 K)-dalton subunit and 17,000 (17 K)-dalton subunit. In amino acid composition the 130 K and 17 K subunits were identical with the 105 K and 17 K subunits of Dabrowska et al. (1977 and 1978), respectively. In disc gel electrophoresis, the 17 K subunit of our MLCK preparation responded to Ca2+ ions in the same way as bovine calmodulin, and differently from skeletal troponin C. There appeared to be one minor difference between 17 K subunit and calmodulin in the primary structure of the C-terminal region. 2. The Ca2+ and Sr2+ concentrations required for the three activities (ATPase and superprecipitation activities and MLCK activity) were measured. Two types of "reconstituted" myosin B were used; one contained 17 K subunit of gizzard MLCK and the other contained bovine brain calmodulin. The two types of "reconstituted" myosin B were practically identical with "natural" myosin B in the Ca2+ and Sr2+ requirements for the three activities measured above. 3. Both the extent and the activity of superprecipitation, and both the limited and steady activities of ATPase were measured. The MLCK activity was estimated in two ways; by urea gel electrophoresis and by measuring 32 P incorporation from [gamma-32P]ATP into myosin. The results thus obtained favor the kinase-phosphatase mechanism of calcium regulation of gizzard muscle contraction.
Asunto(s)
Actomiosina/metabolismo , Adenosina Trifosfatasas/metabolismo , Calcio/farmacología , Proteínas Quinasas/metabolismo , Estroncio/farmacología , Aminoácidos/análisis , Animales , Calmodulina/análisis , Precipitación Química , Pollos , Molleja de las Aves/enzimología , Cinética , Quinasa de Cadena Ligera de Miosina , Proteínas Quinasas/aislamiento & purificación , Conejos , RatasRESUMEN
Sepharose 4B conjugated with phosphorylated myosin light chains was used in affinity chromatography of a partially purified preparation of gizzard myosin light-chain phosphatase (MLCP) (Onishi et al. (1979) J. Biochem. 86, 1283-1290). The MLCP preparation thus purified contained, according to SDS gel electrophoresis, three components of 67,000 (67 K), 54,000 (54 K), 34,000 (34 K) daltons. In an accompanying report, Uchiwa et al. (J. Biochem. 91, 273-282 (1982)) described the purification of gizzard myosin light-chain kinase, which consisted of two subunits; 130 K and 17 K daltons. Using the purified preparations of MLCP and MLCK, it was demonstrated a) that reversible changes in the ATPase and superprecipitation activities occur as myosin light chains are enzymatically phosphorylated and dephosphorylated, and b) that addition of a very low concentration of Ca2+ and its removal cause reversible changes in the turbidity of actomyosin suspensions as well as in the state of phosphorylation of myosin light chains only when MLCK and MLCP are both present. These results provide strong support for the proposal (see Ikebe et al. (1977) J. Biochem. 80, 299-302) that MLCK and MLCP play a key role in the Ca2+ regulation in gizzard.