Isolation and characterization of terminally differentiated chicken and rat skeletal muscle myoblasts

The ability of skeletal muscle myoblasts to differentiate in the absence of spontaneous fusion was studied in cultures derived from chicken embryo leg muscle, rat myoblast lines L6 and L8, and the mouse myoblast line G8. Following 48–96 hr of culture in a low-Ca²⁺ (25 μm), Mg²⁺-depleted medium, chicken myoblasts exhibited only 3–5% fusion whereas up to 64% of the cells fused in control cultures. Depletion of Mg²⁺ led to preferential elimination of fibroblasts, with the result that 97% of the mononucleated cells remaining at 120 hr exhibited a bipolar morphology and stained with antibodies directed against M-creatine kinase, skeletal muscle myosin, and desmin. Mononucleated myoblasts rarely showed visible cross-striations or M-line staining with anti-myomesin unless the medium was supplemented with 0.81 mM Mg²⁺, suggesting that Mg²⁺ plays a role in sarcomere assembly. Conditions of Ca²⁺ and Mg²⁺ depletion inhibited myoblast fusion in the rodent cell lines as well, but mononucleated myoblasts failed to differentiate under these conditions. Differentiated individual myoblasts from rat cell lines and from chicken cell cultures were obtained when fusion was inhibited by growth in cytochalasin B (CB). CB-treated rat myoblast cultures accumulated MM-CK to nearly twice the specific activity found in extensively fused control cultures of comparable age. Spherical cells which accumulated during CB treatment were isolated and shown to contain nearly eight times the CK specific activity present in nonspherical cells from the same cultures. Approximately 90% of these cells exhibited immunofluorescent staining with antibodies to skeletal muscle myosin, failed to incorporate [³H]thymidine or to form colonies in clonal subculture, and thus represent terminally differentiated rat myoblasts. Quantitative microfluorometric DNA measurements on individual nuclei demonstrated that the terminally differentiated myoblasts obtained in these experiments from both chicken and rat contain 2cDNA levels, suggesting arrest in the G₀ stage of the cell cycle.     

Stanniocalcin in terminally differentiated mammalian cells

Stanniocalcin (STC) is a glycoprotein hormone originally found in teleost fish, where it regulates the calcium/phosphate homeostasis, and protects the fish against toxic hypercalcemia. STC was considered an exclusive fish protein, until the cloning of cDNA for human (in 1995) and murine (in 1996) STC. We originally reported a high constitutive content of STC in mammalian brain neurons, and found that the expression of STC occurred concomitantly with terminal differentiation of neural cells. Since then, we have investigated the expression of STC in relation to terminal cell differentiation also in mammalian hematopoietic tissue, and fat tissues. In this review we summarize our findings on STC expression during postmitotic differentiation in three different cell systems; in neural cells, in megakaryocytes and in adipocytes. We also present findings, suggesting that STC plays a role for maintaining the integrity of terminally differentiated mammalian cells.     

The binding of ORC2 to chromatin from terminally differentiated cells

Nuclei from terminally differentiated Xenopus erythrocytes lack essential components of the prereplication complex, including the origin recognition complex (ORC) proteins XORC1 and XORC2. In Xenopus egg extract, these proteins are able to bind erythrocyte chromatin from permeable nuclei, but not from intact nuclei, even though they are able to cross an intact nuclear envelope. In this report we use both permeable and intact erythrocyte nuclei to investigate the role of cyclin-dependent kinase activity in modulating the binding of XORC2 to chromatin. We find that elevating the level of cyclin A-dependent kinase in egg extract prevents the binding of XORC2 to chromatin from permeable nuclei and that kinase inhibition reverses this effect. We also observe a nuclear transport-dependent accumulation of H1 kinase activity within intact nuclei incubated in the extract. However, inhibiting this kinase activity does not facilitate the binding of XORC2 to chromatin, suggesting that other molecules and/or mechanisms exist to prevent association of XORC proteins with replication origins within intact nuclei from terminally differentiated cells.     

Purification and characterization of an arginine-specific carboxypeptidase from Mycoplasma salivarium.

The carboxypeptidase which had been shown to be present exclusively in nonfermentative mycoplasmas was found to be associated with cell membranes of Mycoplasma salivarium. The enzyme was released from the membranes with Triton X-100 and purified by ion-exchange chromatography on DEAE-Sephacel, affinity chromatography on arginine-Sepharose 4B, and chromatofocusing. The purified enzyme had a molecular mass of 218 kilodaltons, as estimated by gel filtration through Sepharose CL-6B, and yielded one band of activity in analytical disc-polyacrylamide gel electrophoresis performed in the presence of 0.5% (wt/vol) Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme treated in the presence or absence of 2-mercaptoethanol revealed one band with a molecular mass of 87 kilodaltons. The enzyme catalyzed selectively the cleavage of the C-terminal arginine residue of peptides such as N-benzoylglycyl-L-arginine, tuftsin, and bradykinin and was inhibited considerably by o-phenanthroline and EDTA but only slightly by NiCl2. The inhibition of the enzyme by EDTA was fully reversed by the addition of ZnCl2, whereas the addition of CoCl2 activated the enzyme.     

Histones of terminally differentiated cells undergo continuous turnover

In contrast to the widely accepted idea of the nearly absolute metabolic stability of histones, our experiments support the view that the histones of nonproliferating, terminally differentiated cells undergo continuous replacement. This conclusion is based on the incorporation of labeled amino acids into the histones of mouse kidney and liver cells after their intraperitoneal introduction. We have found that the intranuclear uptake of the histones made in the absence of replicative synthesis and their integration into chromatin proceed with striking delay. The metabolic rates of individual histones measured by calculating their half-lives suggest that each histone turns over at a specific rate. With regard to the basic chromatin structure, the nucleosome, such unequal turnover should mean that the histone core does not participate in this process as a single unit but rather as a protein mosaic in which each partner follows its own rate of removal. Additional experiments suggested that intact nucleosomes take part in the replacement, but the relative proportion of the nucleosomes involved should be limited. The nonnucleosomal H1A and H1 degree histones have been found to undergo faster replacement than the core histones. Moreover, in comparison to each other, these two histone subfractions are also replaced at a different rate. The results of autoradiography of isolated kidney and liver nuclei after continuous labeling with [3H]-thymidine suggest that the histone replacement is not associated with the repair of DNA.     

Purification and characterization of a developmentally regulated carboxypeptidase from Mucor racemosus.

A developmentally regulated carboxypeptidase was purified from hyphae of the dimorphic fungus Mucor racemosus. The enzyme, designated carboxypeptidase 3 (CP3), has been purified greater than 900-fold to homogeneity and characterized. The carboxypeptidase migrated as a single electrophoretic band in isoelectric focusing polyacrylamide gel electrophoresis (PAGE), with an isoelectric point of pH 4.4. The apparent molecular mass of the native enzyme was estimated by gel filtration to be 52 kDa. Sodium dodecyl sulfate (SDS)-PAGE under nonreducing conditions revealed the presence of a single polypeptide of 51 kDa. SDS-PAGE of CP3 reacted with 2-mercaptoethanol revealed the presence of two polypeptides of 31 and 18 kDa, indicating a dimer structure (alpha 1 beta 1) of the enzyme with disulfide-linked subunits. By using [1,3-3H]diisopropylfluorophosphate as an active-site labeling reagent, it was determined that the catalytic site resides on the small subunit of the carboxypeptidase. With N-carboben zoxy-L-phenylalanyl-L-leucine (N-CBZ-Phe-Leu) as the substrate, the Km, kcat, and Vmax values were 1.7 x 10(-4) M, 490 s-1, and 588 mumol of Leu released per min per mg of protein, respectively. CP3 was determined to be a serine protease, since its catalytic activity was blocked by the serine protease inhibitors diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, and 3,4-dichloroi Socoumarin (DCI). The enzyme was strongly inhibited by the mercurial compound p-chloromercuribenzoate. The carboxypeptidase readily hydrolyzed peptides with aliphatic or aromatic side chains, whereas most of the peptides which contained glycine in the penultimate position did not serve as substrates for the enzyme. Although CP3 activity was undetectable in Mucor yeast cells, antisera revealed the presence of the enzyme in the yeast form of the fungus. The partial amino acid sequence of the carboxypeptidase was determined.     

Purification and characterization of a thermostable carboxypeptidase (carboxypeptidase Taq) from Thermus aquaticus YT-1.

A thermostable carboxypeptidase, which we named carboxypeptidase Taq, was purified from Thermus aquaticus YT-1 and characterized. The molecular weight of the enzyme was estimated to be about 56,000 and 58,000 on SDS-polyacrylamide gel electrophoresis and gel filtration, respectively, indicating that the enzyme has a monomeric structure. The optimum pH of the enzyme was 8.0, and the optimum temperature for the reaction was 80 degrees C. The enzyme activity was dependent on cobalt ion and was inhibited by metal-chelating reagents, indicating that the enzyme is a metalloenzyme. The enzyme had high thermostability independent of cobalt ion; about 90% of its activity remained even after treatment at 80 degrees C for 5 h. The enzyme showed broad substrate specificity, although proline at the C-terminus of peptides was not cleaved. The enzyme released amino acids sequentially from the C-terminus.     

Increased proteolysis in chromatin of terminally differentiated and quiescent cells

Endogenous proteolysis in chromatin of terminally differentiated, quiescent, and actively proliferating cells was studied by measuring the released acid-soluble radioactivity of [³H]tryptophan-prelabelled nuclear proteins, and by following the specific quantitative and qualitative changes in electrophoregrams of chromosomal proteins. The experiments suggest that the chromatin of differentiated mouse kidney and liver cells, as well as chromatin from Friend cells induced to commit terminal differentiation, exhibit increased proteolysis in comparison with that of chromatin isolated from actively proliferating cells. Enhanced proteolysis was found also for the slowly renewing and quiescent cells from adult mice. The control experiments designated to discriminate between the two possible alternatives explaining the difference—increased activity of the proteolytic enzymes associated with chromatin, or increased susceptibility of the chromosomal proteins to proteases—supported the latter alternative.     

Biosynthetic pathways of filaggrin and loricrin--two major proteins expressed by terminally differentiated epidermal keratinocytes

We have used immunoelectron microscopy to map the biosynthetic pathways of loricrin and filaggrin in epidermal keratinocytes at successive stages of differentiation in newborn mouse skin. The filaggrin epitope is first detected in large, irregularly shaped, keratohyalin granules (F-granules) in the stratum granulosum, and then distributed throughout the cytoplasms of the innermost layers of stratum corneum cells. We conclude that the poly-protein filaggrin precursor is first accumulated in F-granules, from which it is subsequently released and processed into filaggrin, and becomes associated with the densely packed bundles of keratin filaments inside stratum corneum cells. Its diminished visibility in the outer layers correlates with the known degradation of filaggrin to free amino acids. Loricrin is first detected in small round keratohyalin granules (L-granules), and subsequently at the periphery of cells throughout the stratum corneum. Labeling of purified keratinocyte envelopes establishes that this loricrin epitope is exposed only at their inner (cytoplasmic) surface. Thus loricrin is initially accumulated in L-granules, to be released at a specifically programmed stage of keratinocyte maturation, and incorporated into the covalently cross-linked lining of the cell envelope. Since loricrin is rich in cysteine, L-granules account for the sulfur-rich keratohyalin granules described earlier. Proposals are made to rationalize why, subsequent to synthesis, filaggrin precursor and loricrin should be segregated both from each other and from the rest of the cytoplasm.     

The cornified envelope of terminally differentiated human epidermal keratinocytes consists of cross-linked protein

A small proportion of the protein of stratum corneum of human epidermal callus is insoluble even when boiled in solutions containing sodium dodecylsulfate and a reducing agent. This protein is present in the cornified envelope, a structure located beneath the plasma membrane. When cornified envelopes were dissolved by exhaustive proteolytic digestion and the products analyzed by chromatography, approximately 18% of the total lysine residues were found as the cross-linking dipeptide ?-(γ-glutamyl) lysine.Labeled cornified envelope protein was synthesized by human epidermal keratinocytes allowed to differentiate terminally in culture. The extent of cross-linking, determined from the proportion of radioactive lysine in ?-(γ-glutamyl) lysine after exhaustive proteolysis, was similar to that in stratum corneum. The properties of the cornified envelopes (insolubility in detergent and reducing agents, and solubility following proteolytic digestion) are readily explained by a structure consisting of a cross-linked protein lattice.     

Purification and characterization of multiple isoforms of tropomyosin from rat cultured cells

We have previously shown that rat cultured cells contain five isoforms of tropomyosin (Matsumura, F., Yamashiro-Matsumura, S., and Lin, J. J.-C. (1983) J. Biol. Chem. 258, 6636-6644) and that these tropomyosins are differentially expressed upon cell transformation (Matsumura, F., Lin, J. J.-C., Yamashiro-Matsumura, S., Thomas, G. P., and Topp, W. C. (1983) J. Biol. Chem. 258, 13954-13964). To examine functions of tropomyosin in microfilament organization, we have purified and partially separated the multiple isoforms of tropomyosin by chromatography on hydroxylapatite. Analyses of cross-linked dimers produced by air oxidation have revealed that all isoforms except the tropomyosin isoform with apparent Mr of 35,000 form homodimers. Although these tropomyosins share many properties characteristic of tropomyosin, structural analyses at a peptide level and immunological analyses have shown that the five isoforms can be classified into two groups, i.e. tropomyosins with higher apparent Mr (Mr = 40,000, 36,500, and 35,000) and tropomyosins with lower apparent Mr (Mr = 32,400 and 32,000). The low Mr tropomyosins show less ability for head-to-tail polymerization and lower affinity to actin than the high Mr tropomyosins. We suggest that these differences in properties may be related to the changes in microfilament organization observed in transformed cells.     

Purification and characterization of a novel dipeptidyl carboxypeptidase from a Streptomyces species.

An extracellular protease derived from the culture broth of a microorganism, a Streptomyces species, produced Boc-Pro-Pro and diproline from Boc-Pro-Pro-Pro-Pro. The enzyme was purified 726-fold, with a yield of 2.6%, by ammonium sulfate fractionation, ion-exchange chromatography, and gel filtration chromatography. The molecular weight of the enzyme was determined to be 65,000 by gel filtration and 70,000 by SDS-PAGE. The enzyme released a C-terminal dipeptide from peptide substrates having a C-terminal proline and a penultimate proline or alanine residue, but did not hydrolyze angiotensin I or bradykinin. When the enzyme hydrolyzed Leu-Pro-Pro-Pro-Pro-Pro, it produced Leu-Pro-Pro-Pro and Pro-Pro before producing Leu-Pro. The enzyme thus seems to be a kind of dipeptidyl carboxypeptidase, its substrate specificity being very different from that of the well known dipeptidyl carboxypeptidases [EC 3.4.15.1] such as the angiotensin-converting enzyme.     

DNA repair kinetics in irradiated undifferentiated and terminally differentiated cells

The brains of male Fisher 344 rats bearing 80-150 mg intracerebral 9L/Ro tumors were irradiated with doses of 1,250-5,000 rads of x- or gamma-rays. At various times after irradiation, the cerebellum and tumor were excised, dissociated into single cells and the DNA from these cells sedimented through alkaline sucrose gradients in zonal rotors with slow gradient reorienting capability. Quantitation of the DNA repair kinetics demonstrated that the process in both tumor cells and neurons has a fast and slow phase. Although all other alternatives cannot be completely eliminated, we suggest that these two phases are most reasonably interpreted as representing repair of lesions in very accessible and less accessible regions of the genome rather than 1) repair of different types of lesions such as single- or double-strand breaks or 2) removal of immediate breaks and breaks induced during excision repair of latent base damage. The slow repair phase is saturable, but not inducible in both tumor cells and neurons. The data suggest that tumor cells restore their chromosomal DNA structure to the unirradiated state faster than neurons because 1) they contain more of the repair system per unit of DNA and 2) a larger proportion of their genetic material is comprised of very accessible regions. The data also suggest that the entire tumor cell genome may be accessible to the repair enzyme(s), while it is possible that a portion of the neuronal genome may be completely inaccessible.     

Purification and characterization of enkephalin convertase, an enkephalin-synthesizing carboxypeptidase

Enkephalin convertase, an enkephalin-synthesizing carboxypeptidase present in adrenal medulla chromaffin granules, has also been detected in brain and pituitary. To determine whether these three carboxypeptidase activities represent the same enzyme, we purified and characterized enkephalin convertase from adrenal medulla, whole brain, and whole pituitary. Enzyme from all three tissues co-purifies on DEAE-cellulose, gel filtration, concanavalin A, and L-arginine affinity columns, resulting in a 135,000-fold, 110,000-fold, and 2,800-fold purification for bovine adrenal medulla, brain, and pituitary, respectively. Purified enkephalin convertase appears homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showing a single band with an apparent molecular weight of 50,000 for enzyme isolated from all three tissues. Adrenal, brain, and pituitary enkephalin convertase are similarly inhibited by hexapeptide enkephalin precursors and active site-directed inhibitors. Both [Met]-and [Leu]enkephalin-Arg6 inhibit enkephalin convertase with Ki values between 50 and 80 microM, while [Met]-and [Leu]enkephalin-Lys6 are 3-fold less potent. Two active site-directed inhibitors, guanidinopropylsuccinic acid and guanidinoethylmercaptosuccinic acid, are potent inhibitors of all three enzymes with Ki values of 8-9 nM. A series of dansylated di-, tri-, and tetrapeptide substrates are hydrolyzed by enkephalin convertase with similar kinetic properties (Km, Vmax, and Kcat/Km) for the three enzymes. This evidence suggests that enkephalin convertase activity represents the same enzyme in adrenal medulla, brain, and pituitary. Enkephalin convertase may be involved in the production of other peptide neurotransmitters and hormones besides enkephalin.     

Mechanisms of radioresistance in terminally differentiated cells of mature retina

Retinopathy of animals is induced by many DNA-damaging agents. This fact shows that DNA lesions may initiate retinal degeneration. The aim of our work was to study the effects of gamma and proton irradiation and single administration of methylnitrosourea (MNU) on mice retina. We assessed morphological changes, DNA damage and repair, as well as expression of proteins (p53, ATM, PARP, FasR, and caspase 3) participating in apoptosis in retina. 14 Gy was the equitoxic dose for induction of DNA single-strand breaks by both gamma- and proton irradiation. However, protons were twice as effective as γ rays in induction of DNA double-strand breaks. All breaks have been repaired for ≤10 h. Irradiation resulted in increased expression of p53 and ATM. Seven days after irradiation, no signs of cell death and retinal degeneration were observed. Proton irradiation with 25 Gy resulted in destructive changes in retina localized mainly in the photoreceptor layer. These changes were accompanied by enhanced expression of proapoptotic proteins. A single systemic administration of MNU (70 mg/kg) increased intracellular levels of p53, PARP, FasR, and Caspase 3 followed by destructive changes in retina with sings of apoptosis in photoreceptors. Similarly to irradiation, a halved MNU dose did not exhibit a cytotoxic effect on retina. A high level of spontaneous DNA damage at apurine and apyrimidine sites were observed in mouse retina. The results show that there is a genotoxic threshold in initiation of retinal cell death in vivo. It is suggested that topoisomerase 2 translates primary DNA damage into a cytotoxic effect in retina.     

Mechanisms of radioresistance in terminally differentiated cells of mature retina

Retinopathy of animals is induced by many agents damaging DNA. This fact shows that DNA lesions may initiate retinal degeneration. The aim of our work was to study the effects of gamma and proton irradiation, and methylnitrosourea (MNU) on mice retina. We evaluated morphological changes, DNA damage and repair in retina, and expression of 5 proteins participating in apoptosis: p53, ATM, FasR, PARP and caspase 3 active. Dose of 14 Gy is equitoxic in terms of induction of DNA single strand breaks by both gamma and proton irradiation. But protons were 2 fold more effective than gamma-rays in induction of DNA double strand breaks. All breaks were repaired within < or =10 h. Irradiation resulted in increased expression of p53 and ATM. But no sings of cell death and retinal degeneration were observed during 7 days after irradiation. Proton irradiation in dose of 25 Gy resulted in increasing over time destructive changes localized mainly in photoreceptor layer of retina. These changes were followed by increased expression of proapoptotic proteins. A single systemic administration of MNU (70 mg/kg) increased intracellular levels of p53, PARP, FasR, caspase 3 active, which was followed by destructive changes in retina with sings of apoptosis of photoreceptors. As in the case of irradiation, the 2-fold dose reduction of MNU abrogated cytotoxic effect of MNU on retina. High level of spontaneous DNA damage such as apurine and apyrimidine sites were observed in mouse retina. The results of our study demonstrate the occurrence of genotoxic threshold in the initiation of retinal cell death in vivo. Topoisomerase 2 of retina is suggested to translate primary DNA damage to cytotoxic effect.     

Human carboxypeptidase M. Purification and characterization of a membrane-bound carboxypeptidase that cleaves peptide hormones

A membrane-bound neutral carboxypeptidase B-like enzyme was solubilized from human placental microvilli with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) and purified to homogeneity by ion-exchange chromatography and affinity chromatography on arginine-Sepharose. It gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent Mr of 62,000 with or without reduction. The enzyme is a glycoprotein as shown by its high affinity for concanavalin A-Sepharose and reduction in mass to 47,600 daltons after chemical deglycosylation. It has a neutral pH optimum, is activated by CoCl2, and inhibited by o-phenanthroline, 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, or cadmium acetate, indicating it is a metallopeptidase. The enzyme cleaves arginine or lysine from the COOH terminus of synthetic peptides (e.g. Bz-Gly-Arg, Bz-Gly-Lys, Bz-Ala-Lys, dansyl-Ala-Arg, where Bz is benzoyl and dansyl is 5-dimethylaminonaphthalene-1-sulfonyl) as well as from several biologically active substrates: dynorphin A(1-13), Met5-Arg6-enkephalin (Km = 46 microM, kcat = 934 min-1), bradykinin (Km = 16 microM, kcat = 147 min-1), Met5-Lys6-enkephalin (Km = 375 microM, kcat = 663 min-1), and Leu5-Arg6-enkephalin (Km = 63 microM, kcat = 106 min-1). Although the enzyme shares some properties with other carboxypeptidase B-like enzymes, it is structurally, catalytically, and immunologically distinct from pancreatic carboxypeptidase A or B, human plasma carboxypeptidase N, and carboxypeptidase H ("enkephalin convertase"). To denote that the enzyme is membrane-bound, and to distinguish it from other known carboxypeptidases, we propose the name "carboxypeptidase M." Because of its localization on the plasma membrane and optimal activity at neutral pH, carboxypeptidase M could inactivate or modulate the activity of peptide hormones either before or after their interaction with plasma membrane receptors.