Analysis of the 4-hydroxytamoxifen (4-OHTAM) bound nuclear estrogen receptor from MCF-7 cells by limited proteolysis

The assumption that a different conformational form was induced in the nuclear estrogen receptor following binding by antiestrogens compared to estrogens was studied by analysing the proteolytic fragments of the receptor following limited digestion with chymotrypsin and trypsin. Nuclei were isolated from MCF-7 cells previously exposed to [3H] 4-OHTAM. The proteolytic digestion was performed either on the micrococcal nuclease hydrolysate or on intact nuclei. The molecular weights (Mr) were calculated from the sedimentation coefficients (S) determined on a sucrose gradient and from the Stokes radii (Rs) estimated by gel filtration. Digestion of the nuclei with micrococcal nuclease solubilized a receptor form of Mr = 155,000. This receptor form was degraded by chymotrypsin to a receptor of Mr = 63,000 which could not be further dissociated by 0.4 M KCl and 3 M urea. A similar receptor molecule was released by chymotrypsin from intact nuclei. Digestion of the micrococcal nuclease hydrolysate with trypsin degraded the receptor to a form of a Mr = 67,000 which could not be further dissociated by 0.4 M KCl and 3 M urea. Digestion of intact nuclei with trypsin followed by micrococcal nuclease, solubilized a receptor form of Mr = 80,000 which could be further dissociated with 0.4 M KCl and 3 M urea to a receptor form of Mr = 67,000. This trypsin degraded receptor form seems to be similar in Mr to the chymotrypsin degraded form. On the other hand different receptor fragments of Mr = 33,000 and Mr = 60,000 were excised by chymotrypsin and trypsin respectively from the estradiol ligated estrogen receptor. (Geier et al., J. steroid Biochem. 26 [1987] 35-40.) These results support the assumption of a different conformational form for the antiestrogen ligated receptor, compared to the estrogen ligated receptor since they were differentially susceptible to proteolytic degradation by chymotrypsin.     

Physical-chemical properties of the estrogen receptor released by deoxyribonuclease I

The physical-chemical properties of the nuclear estrogen receptor released by DNase I were characterized. Nuclei were isolated from MCF-7 cells previously exposed to 10-nM-[3H]estradiol. The parameters determined were: sedimentation coefficients (S) on a sucrose gradient, Stokes radii (Rs) by gel filtration on a Sephadex G-200 column and the binding ability to a DNA-cellulose column. The molecular weights (Mr) and frictional ratios (f/fo) were calculated from the S and Rs values. The properties of the receptor released by DNase I obtained from Worthington were compared to the properties of the receptor released by DNase I obtained from Sigma. Digestion with DNase I (Worthington) excised a receptor form which could be solubilized from nuclei by EDTA. This form sedimented at 5.2S with a Rs = 7.08 nm and a calculated Mr = 152.000. About 40% of this receptor form bound to a DNA-cellulose column. 0.4 M KCl dissociated this receptor form into a smaller form sedimenting at 4.2S with Rs = 4.64 nm and a calculated Mr = 80.000. The properties of the receptor solubilized by micrococcal nuclease followed by DNase I (Worthington) digestion were identical to the properties of the DNase I (Worthington) released receptor. Digestion with DNase I (Sigma) released a 3.2S receptor form, which diffused through the nuclear membrane and a 4-5S form which could be extracted from nuclei by EDTA. The 3.2S receptor had a Rs = 2.41 nm, a calculated Mr = 32.000 and less than 5% of it bound to a DNA-cellulose column. Digestion with micrococcal nuclease followed by DNase I (Sigma) solubilized a receptor form with identical properties to the 3.2S receptor. These results suggest that DNase I (Worthington) released a receptor form still associated with some molecules, probably chromatin proteins, which complexed it to DNA, while DNase I (Sigma) released the estradiol binding fragment of the receptor (meroreceptor) as a result of a proteolytic activity present in this preparation.     

Comparison of the physical properties of the nuclear progesterone receptor, bound to antiprogestin RU 486 or progestin ORG 2058, following limited proteolysis

The susceptibility of the progesterone receptor, liganded either by the antiprogestin RU 486 or by the progestin ORG 2058, to chymotrypsin and trypsin degradation was investigated. The nuclear fraction was isolated from T47D cells previously exposed either to 0.1 microM [3H]RU 486 or to 0.1 microM [3H]ORG 2058. The proteolytic digestion was performed on the micrococcal nuclease hydrolysate. The molecular weights of the receptor fragments were calculated, in high salt buffer, from the sedimentation coefficients determined on a sucrose gradient and from the Stokes radii estimated by gel filtration on an Agarose A-0.5 m column. Micrococcal nuclease solubilized receptor forms with molecular weights of 80,000 and 75,000 for the antiprogestin- or progestin-liganded receptor, respectively. Chymotrypsin degraded these receptor forms to fragments with molecular weights of 23,000 either for the antiprogestin- or progestin-liganded receptor. Similar molecular weights of 23,000 were calculated for the progesterone receptor liganded either by the antiprogestin RU 436 or the progestin ORG 2058 following trypsin cleavage. We conclude that the degradation pattern of the progesterone receptor liganded either by the antiprogestin RU 486 or the progestin ORG 2058 following chymotrypsin or trypsin digestion seems to be similar.     

Physical-chemical properties of the estrogen receptor solubilized by micrococcal nuclease

The physical-chemical properties of the nuclear estrogen receptor from MCF-7 cells were determined. The receptor was solubilized by micrococcal nuclease. Nuclei were isolated from cells previously exposed to 10 nM [3H]estradiol. The amount of receptor released was parallel to the extent of chromatin solubilized, which suggested that the receptor is homogeneously distributed on the chromatin. Following mild nuclease digestion the excised receptor sedimented as an abundant 6-7 S form and as a less abundant approximately 12 S species. The 6-7 S form represented the receptor excised in association with linker DNA, while the approximately 12 S may represent receptor bound to linker DNA which remained associated with the nucleosome. Increasing the extensiveness of digestion resulted in one receptor form sedimenting at 5.6 S. Additional digestion with DNase I did not affect the sedimentation coefficient of the receptor. Sedimentation of the micrococcal nuclease hydrolysate in a 0.4 M KCl sucrose gradient resulted in a 4.2 S receptor form. The same receptor form was extracted from undigested nuclei with 0.4 M KCl. Using Sephadex G-200 column chromatography we have determined the Stokes radii (Rs), molecular weight (Mr) and frictional ratio (f/fo) for the 5.6 S and 4.2 S receptor forms. For the 5.6 S form: Rs = 7.04 nm, Mr = 163,000 and (f/fo) = 1.80. For the 4.2 S receptor, Rs = 4.45 nm, Mr = 77,000 and (f/fo) = 1.46. The ability of the nuclease solubilized 5.6 S receptor to bind DNA was tested using DNA-cellulose column and highly polymerized DNA. About 40% of the applied receptor bound to the column and could be eluted by high salt concentrated buffer. The 5.6 S receptor form was sedimented on sucrose gradient by the highly polymerized DNA. These results suggested that the receptor is bound in chromatin as a dimer or as a monomer in association with other protein(s) which complexed it with DNA.     

Characterization of the progesterone receptor solubilized by micrococcal nuclease and DNase I digestion

In order to investigate the functional organization of the progesterone receptor in chromatin we characterized the physical-chemical properties of the receptor bound chromatin fragments released by micrococcal nuclease and DNase I digestion. The crude nuclear fraction was isolated from T 47 D cells, previously exposed to 0.1 microM [3H]ORG 2058. The parameters determined in low and high salt concentrated buffers were: sedimentation coefficients (S) on a sucrose gradient, Stokes radii (Rs) by gel filtration on a Sephadex G-200 column and the binding abilities to a DNA-cellulose column. The molecular weights (Mr) and frictional ratios (f/fo) were calculated from the S and Rs values. Micrococcal nuclease digestion solubilized a receptor form sedimenting as a single peak at 4.4 S with a Rs = 7.78 nm and an estimated Mr = 144,000. About 53% of the applied receptor bound to a DNA-cellulose column could be eluted by high salt concentrated buffer. 0.4 M KCl dissociated this receptor form into a smaller receptor sedimenting at 3.3 S with Rs = 5.53 nm and a calculated Mr = 76,000. A similar receptor form was extracted by 0.6 M KCl from the undigested crude nuclear fraction. DNase I digestion solubilized a receptor form sedimenting at 3.3 S with a Rs = 6.87 nm and a calculated Mr = 94,000. About 26% of the applied receptor bound to a DNA-cellulose column could be eluted by high salt concentrated buffer. Dissociation of this receptor form by 0.4 M KCl resulted in a receptor sedimenting at 2.8 S with a Rs = 6.53 nm and an estimated Mr = 76,000. These results suggest: The progesterone receptor in chromatin is associated with several molecules probably proteins which complexed it to DNA. Some of these molecules still associated with the progesterone receptor could be released by nucleases digestion. Micrococcal nuclease releases a larger portion of these molecules than those release by DNase I.     

Phosphorylase phosphatase catalytic subunit. Evidence that the Mr = 33,000 enzyme fragment is derived from a native protein of Mr = 70,000

An active form of phosphorylase phosphatase of Mr = 33,000, referred to as the catalytic subunit for over a decade, was purified to near-homogeneity from rabbit skeletal muscle. Repeated immunization of a sheep produced immunoglobulins that blocked the activity of the phosphatase. These immunoglobulins were affinity-purified on columns of immobilized phosphorylase phosphatase and used as macromolecular probes in a "Western" immunoblotting procedure with peroxidase-conjugated rabbit anti-sheep immunoglobulins. Only one protein, of Mr = 33,000, was stained in samples of the immunogen, attesting to the specificity of the probes. However, the Mr = 33,000 phosphatase protein was not detected in muscle extracts or in partially purified preparations. Instead, a single protein of Mr = 70,000 was detected. Limited proteolysis, in particular by Staphylococcus aureus V8 protease and thermolysin, converted the immunoreactive protein from Mr = 70,000 to Mr = 33,000. Coagulation of the phosphatase preparation with 80% ethanol at room temperature rendered the Mr = 70,000 protein insoluble, but allowed extraction of the Mr = 33,000 protein from the precipitate. Thus, we conclude that the immunoreactive protein of Mr = 70,000 is the "catalytic subunit" of phosphorylase phosphatase with a catalytic domain of Mr = 33,000. Previous purification schemes have yielded only the fragment of Mr = 33,000 due to its relative resistance to proteolysis and coagulation. Gel filtration chromatography of the "native" form of phosphorylase phosphatase showed Mr approximately 230,000. Both the Mr = 70,000 catalytic subunit and a Mr = 60,000 protein related to inhibitor-2 were detected by immunoblotting in the same fractions that exhibited activity after treatment with Co2+ and trypsin. Only the Mr = 60,000 protein was degraded during this activation process. We propose that the native phosphorylase phosphatase is an elongated structure with two-fold symmetry, containing one catalytic subunit of Mr = 70,000 and one regulatory subunit of Mr = 60,000.     

Structural analysis of the hepatic glucagon receptor. Identification of a guanine nucleotide-sensitive hormone-binding region

[125I-Tyr10]Monoiodoglucagon [( 125I]MIG) was cross-linked to liver membrane glucagon receptors with hydroxysuccinimidyl-p-azidobenzoate, and the products were analyzed by sodium dodecyl sulfate-gel electrophoresis. Autoradiograms of the gel obtained after a 24-h exposure showed one major band at Mr = 63,000 that was sensitive to GTP and excess unlabeled glucagon. Exposure for 7 days showed labeling of an additional Mr = 33,000 species that was also sensitive to excess unlabeled glucagon. The Mr = 33,000 peptide can be obtained by subtilisin, trypsin, elastase, or Staphylococcus aureus V8 protease treatment of the [125I]MIG-occupied receptor in the membrane or in Lubrol-PX solution. In contrast, limited proteolysis of membranes containing vacant receptors results in labeling of a Mr = 24,000 peptide. The Mr = 24,000 peptide specifically binds [125I]MIG in a GTP-sensitive manner. The Mr = 33,000 peptide also retains GTP sensitivity since it releases bound [125I]MIG upon addition of GTP. Elastase treatment of the electroeluted Mr = 33,000 peptide yields the Mr = 24,000 and 15,000 fragments. The Mr = 15,000 peptide is the smallest fragment of the receptor as yet identified. Treatment of the Mr = 63,000 receptor with [125I]MIG cross-linked to it with endo-beta-N-acetylglucosaminidase F results in four distinct fragments with Mr values of 61,000, 56,000, 51,000, and 45,000; prolonged treatment resulted in the accumulation of the last two. Neither the Mr = 33,000 nor the Mr = 24,000 fragment appeared to be substrates for endo-beta-N-acetylglucosaminidase F. These data indicate that glucagon receptor is a glycoprotein of approximately 60,000 daltons which contains at least four N-linked glycans accounting for 18,000 daltons of its mass. Both its glucagon binding function and its capacity to interact with the stimulatory regulator of adenylyl cyclase are contained within a fragment of only approximately 21,000 daltons that does not contain any N-linked glycans. Hormone occupancy of the receptor results in a conformational change so as to expose a region that is susceptible to proteolysis by proteases of varying specificities to yield a peptide of approximately 30,000 daltons that also does not contain N-linked glycans.     

Ca/Mg-dependent endonuclease from porcine liver. Purification, properties, and sequence specificity

A rapid and reproducible method for the purification of the Ca/Mg-endonuclease from porcine and rat liver and for the stabilization of the enzyme activity is presented. The optimum conditions for enzyme activity were determined. The enzyme degrades double-stranded DNA endonucleolytically. In the course of digestion of form I closed circular SV 40 DNA, the form II nicked circular DNA is the prominent intermediate product. Digestion of hen erythrocyte nuclei with added endonuclease produces a ladder of mono- and oligonucleosomal fragments similar to that generated by micrococcal nuclease digestion. Determination of the 5'-terminal nucleotides indicates the absence of a significant base specificity. Analyzing the cleavage pattern of end-labeled pBR322 restriction fragments on sequencing gels shows that the enzyme exhibits a weak preference for dinucleotides with A in the 5'-position; dinucleotides with 5%-C are less readily cleaved. Digestion of end-labeled pBR322 DNA, followed by electrophoresis in agarose gels produces a "smear"-like fragmentation pattern with weak superimposed bands, while micrococcal nuclease generates a different and much more distinct pattern. These data show that the sequence specificity of the enzyme is less pronounced than that of micrococcal nuclease and that the sites preferentially cleaved are not the same.     

Limited proteolytic digestion and dissociation of smooth muscle phosphatase-I modifies its substrate specificity. Preparation and properties of different forms of smooth muscle phosphatase-I

Smooth muscle phosphatase-I (SMP-I), a protein phosphatase purified from turkey gizzard smooth muscle, is composed of 2 regulatory subunits (Mr = 60,000 and 55,000) and a catalytic subunit (Mr = 38,000). Two other forms of this enzyme have been prepared and characterized. The free catalytic subunit, termed SMP-Ic, was prepared by ethanol treatment of SMP-I, and a form devoid of the 55,000-Da subunit, termed SMP-I2, was prepared by limited tryptic digestion. Exposure of SMP-I to proteases like trypsin and chymotrypsin results in a rapid degradation of the 55,000-Da polypeptide. Degradation of the catalytic subunit is observed only upon prolonged digestion. The 60,000-Da polypeptide appears to be resistant to the action of trypsin and chymotrypsin. SMP-I dephosphorylates myosin light chains but is not active toward intact myosin or heavy meromyosin. However, when the catalytic subunit is dissociated from both regulatory subunits or from the 55,000-Da polypeptide, the enzyme becomes active toward myosin suggesting that the 55,000-Da polypeptide inhibits the activity of the catalytic subunit toward myosin. In addition to alteration of the substrate specificity, the regulatory subunits also modulate the effect of divalent cations, like Mn2+, on the activity of the enzyme.     

A 10S particle released from deoxyribonuclease-sensitive regions of HeLa cell nuclei contains the 86-kilodalton-70-kilodalton protein complex

Digestion of HeLa cell nuclei with micrococcal nuclease or deoxyribonuclease I (DNase I) released the 86-kilodalton-70-kilodalton (kDa) protein complex in particles sedimenting at approximately 10 S in sucrose density gradients. Immunoaffinity-purified 32P-labeled complexes contained 86- and 70-kDa polypeptides with phosphorylated serine residues and DNA fragments, of which the largest was 110 base pairs long. Digestion of nick-translated nuclei with micrococcal nuclease released 32P-labeled 10S particles that were immunoaffinity-purified; they contained labeled 110-base-pair DNA fragments. The micrococcal nuclease digests were analyzed by two-dimensional electrophoresis, which separated nucleosomes in the first dimension and the associated proteins in the second. Western blots of the separated proteins showed that the 86-kDa-70-kDa complex was associated with the mono-, di-, and trinucleosomes. A more extensive electrophoretic separation revealed that the 10S particle from nick-translated nuclei migrated with a subfraction of the mononucleosomes that lacked H1 histones. These results suggest that the 10S particle which contains the 86-kDa-70-kDa complex is associated with an unfolded nucleosome that is present in DNase I sensitive regions.     

A site of discontinuity in the interaction between DNA and histones in nucleosomes of sea urchin embryo chromatin.

Digestion of chromatin DNA in nuclei of sea urchin embryos with pancreatic nuclease and with micrococcal nuclease give additional details concerning the interaction between DNA and histones. A specific site of hydrolysis appears to be located on the nucleosome in such a position as to split the DNA unit length in two equivalent fragments of about 60–70 base pairs in length. The complete digestion of chromatin DNA appears to depend on the low stability of the nucleosome containing the split DNA fragments.     

The photoaddition of trimethylpsoralen to Drosophila melanogaster nuclei: a probe for chromatin substructure.

Derivatives of the furocoumarin, psoralen, can penetrate intact cells or nuclei and cross-link opposite strands of the chromosomal DNA under the influence of long wave-length ultraviolet light. The potential of trioxsalen (4,5',8-trimethylpsoralen) as a probe for chromatin structure has been investigated. The DNA in both embryo nuclei and tissue culture cells from Drosophila melanogaster was found to be about 90% protected from trioxsalen binding relative to purified DNA. Digestion of trioxsalen-treated nuclei by micrococcal nuclease and gel electrophoresis of the resulting DNA gave the same type of band pattern that is characteristic of native, untreated nuclei are digestion. Nuclease digestion was therefore used to examine the distribution of bound trioxsalen in the DNA. The resulting DNA fragments were analyzed both by radioactivity measurements and quantitative electron microscopy. The nuclease cleaved intact photoreacted nuclei in such a way that preferential excision of trioxsalen containing regions of the DNA occurred, but, when acting upon purified DNA that contained bount trioxsalen, it attacked the trioxsalen-free regions preferentially. It was thus concluded that trixosalen binds at the sites corresponding to the regular nuclease-sensitive regions of the chromatin in nuclei.     

Separation and characterisation of tryptic fragments from the adenosine triphosphatase of sarcoplasmic reticulum.

The two halves of the ATPase, M, 115,000, from sarcoplasmic reticulum produ-ed by limited trypsin treatment have been purified in sodium dodecylsulphate. The fragment of Mr60,000 has been purified by electrophoresis on cellulose acetate slabs and that of Mr 55,000 by gel filtration. The two halves of the 60,000 Mr fragment (Mr33,000 and 24,000) produced by more extensive trypsin treatment have also been purified by gel filtration in sodium dodecylsulphate. The sum of the amino acid analyses of the constituent tryptic fragments is in good agreement with that for the whole ATPase. The amino acid compositions of the two halves of the ATPase were strikingly similar. N-terminal analysis shows that the ATPase and its constituent tryptic polypeptides all possess a single N-terminal alanine implying no further cleavage of the polypeptide by trypsin. Attempts to solubilize selectively the tryptic fragments from the membrane by a variety of denaturing and solubilising agents under a variety of conditions have proved unsuccessful, suggesting that the interaction between the tryptic polypeptides is stronger than between the lipid and the protein. The possibility that the interaction between the tryptic polypeptides includes disulphide bonding has been eliminated.     

The variation with age of the structure of chromatin in three cell types from rat liver.

The organization of chromatin in three rat liver nuclear populations, namely diploid stromal, diploid parenchymal, and tetraploid parenchymal nuclei, which were separated by zonal centrifugation, was studied by digestion with micrococcal nuclease and pancreatic deoxyribonuclease in 3-week-old rats in which the parenchymal cells contain diploid nuclei and in 2-and 4-month-old rats with a high proportion of tetraploid nuclei. Digestion by micrococcal nuclease allowed the estimation of DNA-repeat length in chromatin. Parenchymal nuclei have shorter repeat length than stromal nuclei and DNA-repeat length increases with the age in all three nuclei populations. The kinetics of digestion by micrococcal nuclease showed that nuclei with shorter repeat length are more sensitive to micrococcal nuclease and that the sensitivity of chromatin decreases with age for all the types of nuclei in this study. The kinetics of digestion by pancreatic deoxyribonuclease showed that sensitivity of chromatin is related to the repeat length and that the sensitivity decreases with the ages.     

Characteristics of chromatin release during digestion of nuclei with micrococcal nuclease: Preferential solubilization of nascent rna at low enzyme concentration

• 1.1. Extensive digestion of nuclei with micrococcal nuclease (MNase), commonly used in the analysis of chromatin structure, results in the production of mono- and dinucleosomal chromatin fragments.
• 2.2. Digestion of nuclei from a range of cell types with low enzyme concentrations solubilized high molecular weight polynucleosomal fragments, some ⪢ 22 kb long.
• 3.3. Such digestion conditions also resulted in extensive solubilization of nascent RNA which contributed considerably to the nucleic acid content of the soluble fraction.
• 4.4. We conclude that the contribution of RNA to total nucleic acid content of the soluble fraction should be taken into consideration when nuclei are digested with low concentrations of MNase.

     

Mapping on the calf estrogen receptor of the binding domain for an antibody interfering with receptor activation

The localization on the calf estrogen receptor of the binding domain for B36 (an IgM antibody which prevents and reverses the effects of receptor activation) has been studied by means of controlled proteolysis of the receptor-estradiol complex using trypsin, chymotrypsin, and papain. We successively determined for intact and proteolyzed receptor-estradiol complex (i) the abilities of estradiol-binding species to aggregate in low salt medium, to bind to nonspecific DNA absorbed onto cellulose, and to interact with B36 antibody in sucrose gradients; (ii) the hydrodynamic properties of estradiol-binding species, by gel permeation chromatography and sucrose gradient centrifugation in high salt media and (iii) the molecular weights of B36-reactive species, by immunoblot analysis. Three tryptic receptor fragments of Mr 36,000, 34,000, and 33,000 and two chymotryptic fragments of Mr 36,000 and 33,000 included both the hormone- and B36-binding domains but did not interact with DNA, whereas at least two receptor fragments resulting from the action of chymotrypsin and papain bound estradiol with high affinity but interacted neither with DNA nor with B36. Taking into account these results and assuming that structure of the calf estrogen receptor is similar to those of sequenced estrogen receptors (which show a highly conserved organization with considerable homologies in the functional domains), we propose that the B36-binding domain is located either between the DNA- and hormone-binding domains (model I) or at the C-terminal end of the estrogen receptor (model II). The regions that include the main proteolytic cleavage sites of the receptor are also specified, and the abilities of the two models of the calf estrogen receptor to account for the effect of B36 on receptor activation are discussed.     

Subunit structure of alpha-satellite DNA containing chromatin from African green monkey cells.

alpha-Satellite DNA containing chromatin from African green monkey cells (CV-1 cells) has been used to study the question whether or not nucleosomes are arranged in phase with the 172 bp repeat unit of the satellite DNA. Digestion experiments with DNAase II led us to exclude a simple phase relationship between the nucleosomal and the satellite DNA repeats. Digestion of CV-1 nuclei with micrococcal nuclease and endogenous nuclease (s) produced a series of sharp bands in the satellite DNA register over a background of heterogeneous length fragments. This observation is explained by a preferential cleavage of certain nucleotide sequences by these nucleases and is not in contradiction to our conclusion that a simple phase relationship does not exist.     

Structure of eukaryotic chromatin. Evaluation of periodicity using endogenous and exogenous nucleases.

DNA isolated from (a) liver chromatin digested in situ with endogenous Ca2+, Mg2+-dependent endonuclease, (b) prostate chromatin digested in situ with micrococcal nuclease or pancreatic DNAase I, and (c) isolated liver chromatin digested with micrococcal nuclease or pancreatic DNAase I has been analyzed electrophoretically on polyacrylamide gels. The electrophoretic patterns of DNA prepared from chromatin digested in situ with either endogenous endonuclease (liver nuclei) or micrococcal nuclease (prostate nuclei) are virtually identical. Each pattern consists of a series of discrete bands representing multiples of the smallest fragment of DNA 200 +/- 20 base pairs in length. The smallest DNA fragment (monomer) accumulates during prolonged digestion of chromatin in situ until it accounts for nearly all of the DNA on the gel; approx. 20% of the DNA of chromatin is rendered acid soluble during this period. Digestion of liver chromatin in situ in the presence of micrococcal nuclease results initially in the reduction of the size of the monomer from 200 to 170 base pairs of DNA and subsequently results in its conversion to as many as eight smaller fragments. The electrophoretic pattern obtained with DNA prepared from micrococcal nuclease digests of isolated liver chromatin is similar, but not identical, to that obtained with liver chromatin in situ. These preparations are more heterogeneous and contain DNA fragments smaller than 200 base pairs in length. These results suggest that not all of the chromatin isolated from liver nuclei retains its native structure. In contrast to endogenous endonuclease and micrococcal nuclease digests of chromatin, pancreatic DNAase I digests of isolated chromatin and of chromatin in situ consist of an extremely heterogeneous population of DNA fragments which migrates as a continuum on gels. A similar electrophoretic pattern is obtained with purified DNA digested by micrococcal nuclease. The presence of spermine (0.15 mM) and spermidine (0.5 mM) in preparative and incubation buffers decreases the rate of digestion of chromatin by endogenous endonuclease in situ approx. 10-fold, without affecting the size of the resulting DNA fragments. The rates of production of the smallest DNA fragments, monomer, dimer, and trimer, are nearly identical when high molecular weight DNA is present in excess, indicating that all of the chromatin multimers are equally susceptible to endogenous endonuclease. These observations points out the effects of various experimental conditions on the digestion of chromatin by nucleases.     

A re-examination of the effects of chymotrypsin and trypsin on the erythrocyte membrane surface topology

Silver/Coomassie blue staining of human erythrocyte membrane electrophoretograms permits simultaneous visualization and color differentiation of asialoproteins, sialoglycoproteins and lipids in the same gel. Using this technique evidence is provided that chymotrypsin cleaves glycophorin A as well as band 3. The chymotryptic fragmentation pattern of glycophorin A in situ intact cells was different from that generated by trypsin treatment. Chymotryptic cleavage of band 3 generated two Coomassie blue stained fragments at 62,000 and 38,000 Mr, whereas simultaneous cleavage of glycophorin A dimer and glycophorin A B heterodimer yielded yellow silver stained fragments at 68,000 and 47,000 Mr. Trypsin cleaved glycophorin A dimer (88,000 Mr) and monomer (38,000 Mr) to form membrane associated fragments of Mr = 40,000 and 18,000 respectively.     

Direct photolabeling of the cGMP-stimulated cyclic nucleotide phosphodiesterase

cGMP-stimulated phosphodiesterase (PDE) has been directly photolabeled with [32P]cGMP using UV light. Sequence analysis of peptide fragments obtained from partial proteolysis or cyanogen bromide cleavage indicate that two different domains are labeled. One site, on a Mr = 36,000 chymotryptic fragment located near the COOH terminus, has characteristics consistent with it being close to or part of the catalytic site of the enzyme. This peptide contains a region of sequence that is highly conserved in all mammalian cyclic nucleotide PDEs and has been postulated to contain the catalytic domain of the enzyme. The other site, on a Mr = 28,000 cyanogen bromide cleavage fragment located near the middle of the molecule, probably makes up part of the allosteric site of the molecule. Labeling of the enzyme is concentration dependent and Scatchard analysis of labeling yields a biphasic plot with apparent half labeling concentrations of about 1 and 30 microM consistent with two types of sites being labeled. Limited proteolysis of the PDE by chymotrypsin yields five prominent fragments that separate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at Mr = 60,000, 57,000, 36,000, 21,000, and 17,000. Both the Mr = 60,000 and 57,000 apparently have blocked NH2 termini suggesting that the Mr = 57,000 fragment is a subfragment of the Mr = 60,000 fragment. Primary sequence analysis indicates that both the Mr = 21,000 and 17,000 fragments are subfragments of the Mr = 36,000 fragment. Autoradiographs of photolabeled then partially proteolyzed enzyme show labeled bands at Mr = 60,000, 57,000, and 36,000. Addition of 5 microM cAMP prior to photolabeling eliminates photolabeling of the Mr = 36,000 fragment but not the Mr = 60,000 or 57,000 fragments. The labeled site not blocked by cAMP is also contained in a Mr = 28,000 cyanogen bromide fragment of the enzyme that does not overlap with the Mr = 36,000 proteolytic fragment. Limited chymotryptic proteolysis also increases basal activity and eliminates cGMP stimulation of cAMP hydrolysis. The chymotryptic fragments can be separated by either ion exchange high performance liquid chromatography (HPLC) or solid-phase monoclonal antibody treatment. A solid-phase monoclonal antibody against the cGMP-stimulated PDE removes the Mr = 60,000 and 57,000 labeled fragments and any intact, unproteolyzed protein but does not remove the Mr = 36,000 fragment or the majority of activity. Ion exchange HPLC separates the fragments into three peaks (I, II, and III). Peaks I and II contain activity of approximately 40 and 100 units/mg, respectively. Peak II is the undigested or slightly nicked native enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)