Intestinal lymph and plasma lipoproteins in the preruminant calf: partial resolution of particle heterogeneity in the 1.040-1.090 g/ml interval.

Our previous studies in the preruminant calf have provided evidence for the heterogeneity of lipoprotein particles in the 1.040-1.090 g/ml density interval in both plasma and postprandial intestinal lymph (Bauchart, D. et al., 1989. J. Lipid Res. 30: 1499-1514; and Laplaud, P. M. et al., 1990. J. Lipid Res. 31: 1781-1792). We therefore attempted to resolve this heterogeneity by use of heparin-Sepharose affinity chromatography. Experiments were performed on three calves; portal vein plasma and intestinal lymph were obtained simultaneously 10 h after a meal, i.e., at peak lipid absorption. In both fluids, the chromatographic profile presented three fractions, I, II, and III. Fraction I was characterized by the presence of cholesteryl ester-rich particles (approximately 35-37% of lipoprotein mass), which migrated electrophoretically as typical high density lipoproteins and exhibited Stokes diameters in the 130-160 A range; apoA-I was the predominant protein. In addition to this polypeptide, fraction II contained small amounts of a supplementary protein (Mr approximately 51,000), exhibiting heparin-binding properties. In the light of results reported in the literature, we suggest that this latter protein could correspond to beta 2 glycoprotein I. The chemical composition of each fraction II closely resembled that of the corresponding fraction I, while their electrophoretic migrations appeared slightly slower and their Stokes diameters slightly larger (155-165 A). Apart from the presence of small amounts of apoA-I, two high Mr proteins (Mr approx. 560,000 and 300,000) were typical of the apolipoprotein moiety of fractions III. The lower Mr form was present as a trace component only in fraction III originating from plasma; its proportion increased in lymph fraction III so as to approximately match that of the higher Mr (i.e., 560,000) protein. In both plasma and lymph, fraction III was electrophoretically heterogeneous, exhibiting a doublet of bands with migration and Stokes diameters (250 A) typical of low density lipoprotein particles. However, no evidence for the presence of a particle resembling lipoprotein[a] in fraction III could be obtained. In lymph only, fraction III contained a supplementary population of lipoproteins with migration intermediary between those of conventional low and high density lipoproteins and with Stokes diameters in the 190-200 A range. Other specific features of lymph fraction III included a sevenfold increase in its triglyceride content (8.5 +/- 3.4% vs. 1.2 +/- 1.1% in the corresponding fraction from plasma), to the detriment of cholesteryl esters, and a higher proportion of protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation and partial characterization of mitogenic factors from cementum.

Cementum is the mineralized structure through which soft connective tissues are attached to the teeth. It is a unique calcified tissue characterized by a low metabolic turnover, lack of blood supply, and presence of very few cells. However, it contains substances that influence the biological activities of fibroblasts of adjacent soft tissues. We have partially characterized cementum proteins that have mitogenic activity toward fibroblasts. Cementum was harvested from bovine teeth, and mitogenic factors were extracted in 0.5 M CH3COOH. Heparin-Sepharose chromatography separated the mitogenic activity into a major and a minor fraction eluted by 0.5 and 2.0 M NaCl, respectively. The distribution of cementum mitogens in heparin-Sepharose fractions was different from that of alveolar bone and other bones. The cementum mitogenic factor eluting with 2.0 M NaCl from a heparin-Sepharose column was shown to be basic fibroblast growth factor (bFGF) on the basis of inhibition by anti-bFGF antibody and Western blots. The 0.5 M NaCl fraction was purified by HPLC with use of a combination of a DEAE-3W column followed by TSK-250 and C18 columns. NaDodSO4-polyacrylamide gel electrophoresis revealed that the purified fraction contained two protein bands with Mr 22,000 and 19,000, and mitogenic activity was associated with the Mr 22,000 species. The activity of this mitogen, designated as CGF, was potentiated by small quantities of plasma-derived serum or epidermal growth factor. It was heat resistant, but was destroyed by reduction. Assays of CGF preparations revealed that they contained no detectable platelet-derived growth factor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heparin-binding apoproteins. Effects on lipoprotein lipase and hepatic uptake of remnants.

Apoprotein-free heparin-binding and non-binding chylomicrons were used as substrates to test the effects on lipoprotein lipase activity of (a) chylomicron protein I; (b) the mixture of proteins I, II and apoprotein E and (c) human beta 2-glycoprotein I. No activation of the enzyme was observed with any of those apoproteins. When rats were injected simultaneously with [3H]cholesterol-labelled heparin-binding chylomicrons (containing proteins I and II) and [14C]cholesterol-labelled non-binding chylomicrons, no differences were detected between the rates of removal from circulation of those two types of particles. Clearance of chylomicrons from circulation was accompanied by the incorporation of 3H and 14C labels into the livers at similar rates. It is concluded that proteins I, II and apoprotein E have no effect on the degradation of chylomicrons by lipoprotein lipase and that the hepatic recognition of remnants does not appear to be affected by proteins I and II.     

Isolation of a terminal cisterna protein which may link the dihydropyridine receptor to the junctional foot protein in skeletal muscle

The isolated dihydropyridine receptor and junctional foot protein were employed as protein ligands in overlay experiments to investigate the mode of interaction of these two proteins. As previously demonstrated by Brandt et al. [Brandt et al. (1990) J. Membr. Biol. 113, 237-251], the DHP receptor directly binds to an intrinsic terminal cisterna protein of Mr 95,000 (95-kDa protein). The junctional foot protein also binds to an Mr 95,000 protein showing similar organelle distribution to the 95-kDa protein which binds to the dihydropyridine receptor. The 95-kDa protein which binds to the dihydropyridine receptor was isolated to over 85% purity employing sequential column chromatography. Junctional foot protein and dihydropyridine receptor overlays of the column fractions at successive stages of isolation show an identical pattern of distribution, indicating that both probes bind to the same protein. When CHAPS-solubilized terminal cisterna/triads were passed through Sepharose with attached 95-kDa protein, the junctional foot protein was specifically retained, as evidenced by ryanodine binding. The junctional foot protein was incompletely released by 1 M NaCl. The alpha 1 subunit but not the beta subunit of the dihydropyridine receptor was also specifically retained, as evidenced by immunoblotting employing dihydropyridine receptor subunit-specific antibodies. A 170-kDa Stains-all blue staining protein, which appears to be bound to the luminal side of the terminal cisterna, was also retained on the 95-kDa protein column. From these findings, a model for the triad junction is proposed.     

Apolipoprotein A-IV: a protein occurring in human mesenteric lymph chylomicrons and free in plasma. Isolation and quantification.

1. Human mesenteric lymph chylomicrons were isolated from chylous ascites fluid by ultra-centrifugation and agarose/gel chromatography and their apoprotein composition was analysed by dodecylsulfate/polyacrylamide gel electrophoresis, analytical isoelectric focusing and immuno-chemically. Major components of mesenteric lymph chylomicrons were apoprotein A-I, proteins of Mr less than 15 000 including the C-group apoproteins and a protein of Mr 46 000. Minor components were apoprotein E and a protein of Mr approximately equal to 200 000 (B-like protein). This apoprotein composition was qualitatively identical with that of chylomicrons from intestinal lymph of the rat, but was distinctly different from plasma chylomicrons of humans with fasting chylomicronaemia. 2. The protein of Mr approximately equal to 46 000 has been isolated by preparative dodecylsulfate/polyacrylamide gel electrophoresis from human and rat lymph chylomicrons and was compared to a protein of identical Mr present in rat high-density lipoproteins (apoplipoprotein A-IV) and in the rho less than 1.006 g/ml serum lipoprotein fraction of individual humans with alimentary hypertriglyceridaemia. In both species the 46 000-Mr proteins isolated from lymph and serum were identical according to amino acid composition and isoelectric point in 6 M urea. The human proteins from both sources were also immunologically identical. The similarities in the molecular properties of the human apolipoprotein and rat apolipoprotein A-IV indicate that these proteins are homologous. 3. Plasma levels of human apolipoprotein A-IV determined by electroimmunodiffusion were 14.15 +/- 3.66 mg/100 ml (n = 59), but greater than 90% of the protein was unassociated with the major lipoprotein fractions. It is concluded, that apolipoprotein A-IV is a main protein component of human lymph chylomicrons, that is removed from the particles in the plasma compartment.     

A monoclonal antibody against the type II isotype of beta-tubulin. Preparation of isotypically altered tubulin

Mammalian brain tubulin consists of several isotypes of alpha and beta subunits that separate on polyacrylamide gels into three electrophoretic classes, designated alpha, beta 1, and beta 2. It has not been possible hitherto to resolve the different isotypes in a functional form. To this end, we have now isolated a monoclonal antibody, using as an immunogen a chemically synthesized peptide corresponding to the carboxyl-terminal sequence of the major tubulin isotype (type II) found in the beta 1-tubulin electrophoretic fraction. The antibody binds to beta 1 but not to alpha or beta 2. When pure tubulin from bovine brain is passed through an immunoaffinity column made from the anti-type II antibody, the tubulin that elutes in the unbound fraction is enriched greatly for the beta 2 electrophoretic variant. The tubulin that binds to the column appears to contain only alpha and beta 1, not beta 2. When these tubulin fractions are characterized by immunoblotting using the anti-type II antibody, the antibody binds only to the beta 1 band in the bound fraction, not to the beta 1 band in the unbound fraction. Using polyclonal antibodies generated against the carboxyl-termini of types I, III, and IV, we demonstrate that the beta 1 electrophoretic species is comprised of isotypes I, II, and IV, whereas the beta 2 variant is comprised exclusively of type III beta-tubulin. Further, we calculate that beta-tubulin in purified bovine brain tubulin is comprised of 3% type I, 58% type II, 25% type III, and 13% type IV tubulins.     

A Mr 80K hepatocyte surface protein(s) interacts with basement membrane components

We have identified a Mr 80K cell surface protein(s) from adult rat hepatocytes that binds basement membrane components, including collagen IV, heparan sulfate proteoglycan, and laminin. Freshly isolated hepatocytes were cell surface-labeled with 125I using the lactoperoxidase-catalyzed method, and detergent-solubilized membrane proteins were chromatographed on affinity columns prepared with purified basement membrane components. A Mr 80K protein was eluted with 0.15-1 M NaCl from a collagen IV column. Two proteins (Mr 80K and 38K) were eluted from a heparan sulfate proteoglycan column. The larger protein was also eluted from a column made with heparan sulfate side chains. Several proteins (Mr 80K, 67K, 45K, and 32K) bound to an affinity chromatography column made with the laminin A chain-derived synthetic peptide PA22-2, which is active for promoting cell attachment. When fractions eluted from these columns were analyzed by two-dimensional gel electrophoresis, the Mr 80K proteins showed similar patterns with a pI ranging from 8 to 9. The Mr 80K protein(s) may have an important role in the interaction of hepatocytes with basement membrane.     

Binding of beryllium to nuclear acidic proteins.

In vitro beryllium (Be) binding to rat liver nuclei has been reassessed (KAss = 2.0 X 10(6) M: n = 17 nmol Be/mg protein). Be also binds to rat liver nucleoli (KAss approx. 4 X 10(6) M: n = 10 nmol Be/mg protein). Examination of rat liver chromatin fractionated on a hydroxyapatite column shows that Be does not bind to histone or to the non-histone protein eluted by 0.05 M sodium phosphate. Be is strongly bound to the non-histone proteins eluted by 0.2 M sodium phosphate (KAss = 1.1 X 10(6) M: n = 55 nmol Be/mg protein) and also to the same extent to the fraction containing DNA which is subsequently eluted from the column. Evidence is provided that the latter binding is not due to DNA. The fractions containing the Be-binding proteins also contain the proteins which are phosphorylated to the greater extent.     

Identification of novel calcium binding proteins of heart and brain 100,000 x g supernatant

The chelex competitive calcium binding assay has been used to assay the calcium binding activity of the 100,000 X g supernatant of bovine heart and brain. Chromatography of brain 100,000 X g supernatant on diethylamino-ethyl (DEAE) cellulose reveals the presence of two peaks of calcium binding activity, peak I eluting at about 0.05 M NaCl and peak II at about 0.18 M NaCl. Chromatography of peak I on Sephadex G-150 resolves a major and a minor peak of calcium binding activity, at Mr 40,000 and Mr 150,000. Chromatography of peak II (0.18 M NaCl) on Sepharose 6B produces two peaks of calcium binding activity, a broad peak of calcium binding activity composed of two molecular weight species of Mr 230,000 and Mr 420,000, and a sharp peak of calcium binding activity with Mr 75,000. Chromatography of the 100,000 X g supernatant of bovine heart on DEAE Cellulose reveals two peaks of calcium binding activity. Chromatography of the lower ionic strength peak on Sephadex G-150 resolved major and minor peaks of calcium binding activity at Mr 65,000 and 150,000, respectively. The results of this study suggest the presence of several calcium binding proteins, other than calmodulin, in these tissues.     

Rapid purification of native C protein from nuclear ribonucleoprotein particles

A rapid three step procedure is described for the purification of C protein from HeLa 40 S hnRNP particles. The procedure takes advantage of the salt resistant RNA binding of C protein, the size of the C protein-RNA complex, and the strong binding of C protein to an anion-exchange resin. Typically 120 micrograms of C protein is obtained from 4.0 X 10(9) cells with greater than 95% electrophoretic purity. Proteins C1 and C2 copurify in the ratio of 3.5 Cl to 1 C2. The purified C protein participates in hnRNP particle reconstitution and on this basis is judged to be native. The purified C protein binds to a gel filtration matrix at 0.5 M NaCl but at higher salt concentrations it elutes before the marker protein, apoferritin (Mr = 443,000). An abbreviated two step purification procedure utilizing anion-exchange chromatography is also described. This procedure results in relatively pure C protein, as well as a useful separation of the other hnRNP proteins.     

TGGCA protein is present in erythroid nuclei and binds within the nuclease-hypersensitive sites 5' of the chicken beta H- and beta A-globin genes

The developmentally regulated 5'-flanking DNase-I-hypersensitive site of the chicken beta H-globin gene in nuclei contains a subregion which is resistant to DNase I and which disappears when nuclei are extracted with 0.3 M NaCl, suggesting that there are salt-extractable proteins bound to sequences within this region. The 0.3 M NaCl extract contains two proteins which bind in vitro to these sequences. One of the binding sequences has an inverted repeat very similar to that bound by TGGCA protein. Partially purified TGGCA protein from chicken liver binds to this sequence in vitro giving exactly the same footprint as that obtained with erythroid nuclear proteins. Similarly TGGCA protein binds to an inverted repeat with the beta A-globin 5'-hypersensitive site giving a footprint identical to that obtained with erythroid nuclear protein extracts. From competition footprinting experiments and the electrophoretic mobility of the protein-DNA complex, it is concluded that the erythroid proteins previously described as binding to the beta H- and beta A-globin inverted repeats within the 5'-flanking hypersensitive sites both belong to the TGGCA protein family.     

In vivo binding partners of the Lens culinaris lectin

The immobilized lectin from the lentil (Lens culinaris) specifically binds two fractions out of the L. culinaris seed globulins. Both fractions are displaced from the lectin at low pH values. In addition, fraction I fails to interact at high ionic strengths, and fraction II in the presence of glucose or other lectin-specific sugars. The behaviour in zonal isoelectric precipitation and electrophoretical patterns indicate that both fractions represent subpopulations of the storage proteins. The interaction as demonstrated by affinity chromatography is corroborated by nephelometry: If the dissolved proteins (lectin plus fraction I or fraction II) are mixed under proper conditions the solutions become turbid. An even more pronounced interaction is observed if the lectin is reacted with both fractions at the same time. Seed albumins able to interact with the immobilized lectin include the dissolved lectin and two glycosidases (alpha-mannosidase, alpha-galactosidase) all of which are located in the protein bodies. A third glycosidase (beta-galactosidase) from outside of the protein bodies does not bind to the lectin. The results are discussed in view of the possibility that lectins may serve as packaging aids for other proteins in the protein bodies.     

DNA-binding proteins from Novikoff hepatoma cells.

In 0.05 M NaCl, 6-8% of the total soluble proteins from Novikoff hepatoma cells bind rapidly and reversibly to columns containing either heterologous or homologous DNA adsorbed to cellulose. These proteins can be eluted by buffer containing 2.0 M NaCl. 0.5-1% of the total protein exhibits a 7-17-fold preference for rat DNA over Escherichia coli DNA. 1-1.5% of the proteins bind DNA so strongly that elution cannot be effected by 4.0 M NaCl but can be accomplished by deoxyribonuclease I treatment of the columns. DNA-binding proteins eluted by 2.0 M NaCl were labeled with 125I or 131I and characterized by sodium dodecylsulfate-polyacrylamide gel electrophoresis and isoelectric focusing. These experiments indicate that DNA-binding proteins represent a discrete subset of the total soluble protein. Many similarities were noted between the major components of the homologous and heterologous DNA-binding fractions.     

Heparin-copper biaffinity chromatography of fibroblast growth factors

A novel method is described to separate and identify the various forms of fibroblast growth factor (FGF) based on their differential affinities for both heparin and copper. FGFs were extracted from bovine hypothalamus and purified by batchwise adsorption to heparin-Sepharose. The partially purified FGFs were then applied to an affinity column prepared by mixing equal portions of heparin-Sepharose and copper-Sepharose. The column was rinsed consecutively with the following four reagents: (i) 2 M NaCl, (ii) 0.6 M NaCl, (iii) 0.6 M NaCl plus 10 mM imidazole, and (iv) 0.6 m NaCl. FGFs were then eluted with a linear NaCl/imidazole gradient (from 0.6 m NaCl without imidazole to 2 M NaCl plus 10 mM imidazole). Fractions eluted from the column were analyzed by sodium dodecyl sulfate-gel electrophoresis with silver staining and electrophoretic immunoblot using site-specific antibodies against basic and acidic FGF. The results demonstrate that it is possible to resolve from hypothalamus at least two basic FGF species (with Mr values of 19,000 and 18,000) and three acidic FGF species (with Mr values of 18,000, 16,400, and 15,600). These findings indicate that heparin-copper biaffinity chromatography may have wide applicability in the study of the structure and activity of FGFs.     

Tryptic digestion of dynein 1 in low salt medium. Origin and properties of fragment A

Dynein 1 was extracted from sperm flagella of the sea urchin Tripneustes gratilla with 0.6 M NaCl and dialyzed against 0.5 mM EDTA, 14 mM 2-mercaptoethanol, 5 mM imidazole/HCl buffer, pH 7.0, for 24-48 h. In some cases, fractions containing the alpha heavy chain and the beta/intermediate chain 1 complex (beta/IC1) were separated by density gradient centrifugation in the same solution. Treatment of the samples at a trypsin:protein ratio of 1:10 w/w for 32 min at room temperature yields a crude digest from which Fragment A is purified by density gradient centrifugation. The purified Fragment A consists of two principal peptides (Mr = 195,000 and 130,000) that cosediment with the peak of ATPase activity at 12.5 S, which is slightly faster than the 11 S of the original beta/IC1 complex. When digests of the separated alpha chain and of the beta/IC1 complex are followed as a function of time, the early cleavages of the two heavy chains (Mr = 428,000) resemble each other in that both lead to similarly sized peptides of Mr 316,000 and 296,000, but only in the beta/IC1 fraction does the digestion proceed to form Fragment A. The remainder of the beta chain, termed Fragment B, occurs as an Mr 110,000 peptide sedimenting at 5.7 S with no associated ATPase activity. Fragment A has a specific ATPase activity of 4.3 mumol Pi X min-1 X mg-1, with a Km of 29 microM in 0.1 M NaCl medium, and an apparent Ki for inhibition by vanadate of 1.2 microM in the absence of salt, and 22 microM in 0.6 M NaCl. Photoaffinity labeling with [alpha-32P]8-azidoadenosine 5'-triphosphate indicates that the ATP binding site on the beta chain of dynein 1 is located on the Mr 195,000 peptide of Fragment A. The possibility that Fragments A and B of the beta/IC1 complex may correspond to the head and tail regions of the tadpole-shaped particle seen by electron microscopy is discussed.     

Hepatitis B virus surface antigen binds to apolipoprotein H.

We have previously demonstrated that a plasma membrane-enriched fraction isolated from human liver is capable of binding recombinant hepatitis B surface antigen (rHBsAg) (P. Pontisso, M. A. Petit, M. Bankowski, and M. E. Peeples, J. Virol. 63:1981-1988, 1989). In this study we have separated the plasma membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and used a ligand-blotting technique to identify a 46-kDa rHBsAg-binding protein. This protein could be removed from the membranes with a weakly acidic buffer, implying that it is peripherally bound. Examination of human serum revealed that the 46-kDa binding protein is a serum protein. Isolation of plasma lipoproteins revealed that the binding protein is in part associated with chylomicrons and high-density lipoproteins, both of which are targeted to the hepatocyte during the normal course of lipid metabolism. The binding protein was identified as apolipoprotein H (apo H), also known as beta 2-glycoprotein I, on the basis of copurification of the rHBsAg-binding activity with the apo H protein and the ability of cDNA-expressed apo H to bind rHBsAg. Serum-derived HBsAg also binds to apo H, indicating that binding is not unique to rHBsAg. Binding is saturable, requires only the small S protein of rHBsAg, and is inhibited by excess rHBsAg, antibodies to HBsAg, and antibodies to apo H. The binding activity of apo H is destroyed upon reduction, indicating that 1 or more of its 22 disulfide bonds are required for interaction with rHBsAg. The possibility that an interaction between hepatitis B virus particles and lipoprotein particles may facilitate entry of the virus into hepatocytes is discussed.     

Purification of substrate proteins of casein kinases from the cytosol fraction of AH-66 hepatoma cells

We have attempted to purify endogenous substrate proteins for casein kinases I and II from the cytosol of AH-66 hepatoma cells. Utilizing the fact that only a few substrates are concentrated in the fraction eluted from DEAE-cellulose between 0.3 and 0.6 M NaCl, two substrates were purified from this fraction by DEAE-cellulose chromatography, hydroxyapatite chromatography, and HPLC on a DEAE-5PW column. The purified substrate proteins had molecular masses of 30.5 kDa and 31 kDa. The 31-kDa protein substrate was markedly phosphorylated by casein kinase II, but only slightly by casein kinase I. The radioactive phosphate incorporated into 31-kDa substrate by casein kinase II was 0.2 mol/mol of the protein and phosphorylation occurred on both threonine and serine residues. The 30.5 kDa protein was only slightly phosphorylated by casein kinase II, but not at all by casein kinase I.     

Purification of substrate proteins of casein kinases from the cytosol fraction of AH-66 hepatoma cells

We have attempted to purify endogenous substrate proteins for casein kinases I and II from the cytosol of AH-66 hepatoma cells. Utilizing the fact that only a few substrates are concentrated in the fraction eluted from DEAE-cellulose between 0.3 and 0.6 M NaCl, two substrates were purified from this fraction by DEAE-cellulose chromatography, hydroxyapatite chromatography, and HPLC on a DEAE-5PW column. The purified substrate proteins had molecular masses of 30.5 kDa and 31 kDa. The 31-kDa protein substrate was markedly phosphorylated by casein kinase II, but only slightly by casein kinase I. The radioactive phosphate incorporated into 31-kDa substrate by casein kinase II was 0.2 mol/mol of the protein and phosphorylation occurred on both threonine and serine residues. The 30.5 kDa protein was only slightly phosphorylated by casein kinase II, but not at all by casein kinase I.     

The distribution and dissociation of cyclic adenosine 3':5'-monophosphate-dependent protein kinases in adipose, cardiac, and other tissues.

In crude extracts of adipose tissue the protein kinase dissociates slowly at 30 degrees into regulatory and catalytic subunits in the presence of 700 mug per ml of histone or 0.5 M NaCl. If the kinase is first dissociated by adding 10 muM adenosine 3':5'-monophosphate (cAMP), reassociation occurs instantaneously after removal of the cAMP by Sephadex G-25 chromatography. In contrast, in crude xtracts of heart, the protein kinase dissociates rapidly in the presence of 700 mug per ml of histone or 0.5 M NaCl and reassociates slowly after removal of cAMP. These differences are accounted for by the existence of two types of protein kinases in these tissues, referred to as types I and II. DEAE-cellulose chromatography of extracts of adipose tissue produces only one peak of cAMP-dependent protein kinase activity (type II) which elutes between 0.15 and 0.25 M NaCl. Similar chromatography of heart extracts resolves enzyme activity into two peaks; a type I enzyme which elutes between 0.05 and 0.1 M and predominates (greater than 75% of total activity), and a type II enzyme which elutes between 0.15 and 0.25 M NaCl. The dissociation properties of the types I and II enzymes from heart and adipose tissue are retained after partial purification by DEAE-cellulose and Sepharose 6B chromatography. Rechromatography of the separated peaks of the cardiac enzymes does not change the elution pattern. Sucrose density gradient centrifugation and gel filtration studies indicate that the molecular weights of these enzymes are very similar. The type II enzyme isolated by DEAE-cellulose chromatography of heart extracts resembles the adipose tissue enzyme, i.e. it undergoes slow dissociation at 30 degrees in the presence of histone or 0.5 M NaCl. The adipose tissue kinase and the heart type II kinase are not identical, however, since they do not elute at exactly the same point on DEAE-cellulose columns. A survey of several tissues indicates the presence of type I and II protein kinases similar to the enzymes in adipose tissue and heart as determined by DEAE-cellulose chromatography of crude extracts and by dissociation of the enzymes with histone. The presence of MgATP prevents dissociation of type I enzyme from heart by 0.5 M NaCl or histone. The profile of the enzyme on DEAE-cellulose, however, is not changed...     

Proteins firmly bound to DNA in the E. coli nucleoid

The nucleoid isolated from E. coli cells was subjected to further deletion by treatment with 2 M NaCl. After disintegration of this nucleoid by ultrasonication, two fractions were obtained, i. e., a rapidly (RS) and slowly sedimenting (SS) ones. The protein, RNA and DNA patterns in the RS fraction are similar to that of the eukaryotic cell nuclear matrix. Electrophoretic analysis of total non-dissociating by 2 M NaCl proteins revealed that the RS and SS fractions predominantly contain proteins with Mr 31,27 and 23 kD. The protein with Mr = 31 kD is firmly bound to DNA, does not dissociate in the guanidine hydrochloride (4 M)-urea (5 M) mixture as well as in solution of 1% sodium-dodecyl sulphate and may be responsible for the chromosome binding to the E. coli membrane.