Properties of three creatine kinases MM from porcine skeletal muscle

The properties of three fractions (FI, FII, and FIII) of porcine creatine kinase MM, which have been isolated by isoelectric focusing, were compared. Sugars were not detected in them. Their carboxyl-terminal sequences were identical and were determined to be -Thr-Lys by digestion with carboxypeptidases A and B. Immunodiffusion and competitive radioimmunoassay could not differentiate the three fractions from one another. Their amino-terminal sequences revealed that they had different primary structures. At residue 1, although all the three fractions had Pro, FI and FIII had an additional amino acid, Ser. At residue 23, only FI had Leu in addition to Ser, the amino acid common to the three fractions. These results indicate that differences among the three fractions of porcine creatine kinase MM are based on differences in the primary structures of the subunits in their dimer structures, and confirm the conclusion that FII is a homodimer and FI and FIII are heterodimers, which was reported in the preceding paper [Takasawa, T. & Shiokawa, H. (1983) J. Biochem. 93, 383-388].     

Susceptibility of muscle soluble proteins to degradation by mast cell chymase

We investigated the in vitro susceptibility of muscle soluble proteins to the major alkaline proteinase (chymase) from skeletal muscle tissue, an enzyme originating from intramuscular mast cells, but also present in certain muscle fibers. Cytoplasmic proteins from rat skeletal muscle tissue were fractionated into four groups according to their different isoelectric points: fraction A (pI 9.5-7.0), B (pI 7.0-5.6), C (pI 5.5-4.5) and D (pI 5.3-3.5). Chromatography of these fractions on octyl-Sepharose CL-4B revealed the presence of a higher percentage of hydrophobic proteins in fraction C and D as compared to fraction A and B. In vitro degradation of these protein fractions by chymase, isolated from rat skeletal muscle tissue, was monitored (a) by measuring the ability of these proteins to bind Coomassie G-250, and (b) by analyzing the digestion mixture in isoelectric focusing gels. Both methods revealed fraction B proteins to be degraded very rapidly. While there was also a significant breakdown of fraction A proteins, fraction C and D proteins were degraded only very slowly, if at all. These differences in degradability are not due to the presence of a proteinase inhibitor in fraction C and D. The results suggest that mast cell chymase preferentially degrades those groups of muscle soluble proteins, the constituents of which have neutral to basic isoelectric points and a relatively low surface hydrophobicity.     

Comparison of isoelectric focusing in Sephadex vs immobiline flatbeds for the recovery of follicle regulatory protein from porcine follicular fluid

We describe and compare the use of isoelectric focusing (IEF) in a granulated Sephadex matrix and in polyacrylamide immobilized pH gradients to separate an aromatase inhibitor (follicle regulatory protein: FRP) in preparative amounts from porcine follicular fluid (PFF). The starting material for IEF was derived from pFF after passage through agarose immobilized textile dye Orange A (0.5 KC1 eluent). Before IEF, some Orange A bound (OAB) material was further purified on a FPLC employing a Mono-Q anion exchange column. Previous use of chromatofocusing indicated that aromatase inhibitory activity is largely concentrated in OAB fractions with a pI in the ranges of pH approximately 4.5 and approximately 6.5. The current study revises these findings to provide a more precise measure of the isoelectric points in question to pH 4.73 +/- 0.05 and pH 6.41 +/- 0.06. The use of Sephadex was limited by gradient instability and the selection of pH ranges available. IEF using immobilized pH gradients had several advantages over Sephadex: 1) broader selection of gradients from 0.1 to 7.0 pH units; greater resolving power, and enhanced stability. The principal disadvantage of the immobiline system was the recovery of focused material from the gel matrix. The use of isoelectric focusing with immobilized pH gradients on a preparative scale to purify FRP from OAB resulted in a greater than 50% recovery with a substantial increase in specific activity (from ID50 approximately 300 micrograms/ml to 20 ng/ml).     

Oligoclonal IgG bands with and without measles antibody activity in sera of patients with subacute sclerosing panencephalitis (SSPE)

Oligoclonal IgG bands from SSPE sera were isolated by combination of Protein A-Sepharose 4B column and preparative isoelectric focusing gel procedures. Each eluted fraction, when examined in analytic IEF, showed two or three individual bands with isoelectric points close to one another, compared to approximately fifteen IgG bands seen in whole serum. When the bands were tested for measles antibody activity in immunofixation with measles virus followed by peroxidase staining, the bands eluted in pH region 8.5 to 9.3 were found to be measles specific, whereas those in pH 7.0 to 8.4 lacked significant measles activity. When eluted fractions containing groups of bands were absorbed with measles virus, the bands in pH region 8.5 to 9.3 were removed, whereas those in pH 7.0 to 8.4 region remained unchanged; this indicated that a number of oligoclonal IgG bands without measles virus activities are present in SSPE. The bands lacking measles-specific activity may be synthesized against other infectious agents or they may represent nonspecific activation of B cell clones.     

Protein kinases of rat liver endoplasmic reticulum. Solubilisation, partial characterisation and comparison with protein kinases of rat liver cytosol.

Protein kinase associated with rat liver microsomes was only partly extracted by treatment with 1.5 M KCl. The enzyme was solubilised by Triton X-100 or sodium deoxycholate at the same or slightly higher detergent concentrations than microsomal marker components. The enzyme activity increased 2-3 fold upon solubilisation. Three peaks with protein kinase activity (fractions MI, MII and MIII) were resolved on DEAE-cellulose chromatography. Fraction MIII but not fractions MI or MII was activated by adenosine 3':5'-monophosphate (cyclic AMP). All fractions catalysed the phosphorylation of protamine and histones but not that of casein or phosvitin. Fractions MI and MIII had a similar substrate specificity and phosphorylated histones at a relatively much higher rate than did fraction MII. The isoelectric points were 8.1 for fraction MI, 5.5 for fraction MII and 4.9 for fraction MIII. On incubation of fraction MIII with cyclic AMP it was split into two catalytically active components with pI 8.1 and 7.35. The component with pI 8.1 was predominant and corresponded to fraction MI. Five protein kinase peaks were resolved from rat liver cytosol by DEAE-cellulose chromatography. Three of them (fractions CIa, CIIb and CIII) had the same properties as each of the microsomal kinase fractions. A forth fraction (CIIa) was cyclic-AMP-dependent and had the same substrate specificity as fractions MI and MIII. Its pI was 5.1, and it was split into two components by cyclic AMP (pI 8.1 and 7.35). In binding studies fraction CIIb bound more efficiently to microsomes than fraction CIII, while fractions CIa, CIIa and the microsomal protein kinase fractions did not bind appreciably. When microsomes were treated with trypsin exposed protein kinase was inactivated and the latency of the remaining enzyme increased substantially. Most of fraction MII was inactivated by trypsin while fraction MIII was resistant. The possible orientation of protein kinase fractions MII and MIII in the microsomal membrane is discussed.     

Molecular heterogeneity of the subclasses of islet-activating protein (pertussis toxin)-sensitive GTP-binding proteins in porcine thyroid tissue

From porcine thyroid cell membranes, we purified five GTP-binding proteins (G-proteins); Nos. 1 to 3 have 41-kDa alpha-subunits, and Nos. 4 and 5 have 40-kDa alpha-subunits. They were chromatographically (Mono Q) separable and served as specific substrates for islet-activating protein (pertussis toxin). G-proteins 1 and 2 were indistinguishable from porcine brain Gi1 with respect to three criteria, i.e., mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), pI of the ADP-ribosylated alpha-subunit, and immunoreactivity. G-protein 3 was identified as Gi3 by immunoreactivity. The SDS-PAGE and isoelectric focusing (IEF) analyses identified G-proteins 4 and 5 as being chromatographically heterogeneous subtypes of Gi2 in comparison with a pure porcine brain preparation. The IEF analysis also disclosed that each of the Gi1, Gi2, and Gi3 subspecies isolated in the present study has a minor component characterized by a slightly lower pI of its alpha-subunit. We conclude that porcine thyroid tissue contains at least Gi1, Gi2, and Gi3, and that each is made up of heterogeneous populations.     

Analysis of photoaffinity-labeled aryl hydrocarbon receptor heterogeneity by two-dimensional gel electrophoresis

The level of charge heterogeneity in the aryl hydrocarbon receptor (AhR) was examined by high-resolution denaturing two-dimensional (2D) gel electrophoresis. Hepa 1c1c7 cell cytosolic fraction was photoaffinity-labeled with 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin and applied to isoelectric focusing (IEF) tube gels. After optimization of focusing conditions a broad peak of radioactivity was detected in the apparent pI range of 5.2-5.7. IEF tube gels were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by visualization of the radiolabeled AhR by autoradiography; three distinct isoforms were detected. The same 2D electrophoretic isoform pattern was obtained when the AhR from Hepa 1c1c7 was photoaffinity-labeled in cell culture. BPrCl cells, a mutant line derived from Hepa 1c1c7 cells, contain an AhR that is unable to bind to DNA. Photoaffinity-labeled BPrCl cytosolic fractions were subjected to 2D gel electrophoretic analysis resulting in essentially the same molecular weight and isoform pattern as seen in Hepa 1c1c7 cytosol. This result would suggest that if a mutation is present in the BPrCl AhR it has not caused a significant change in its IEF pattern, although a small shift in the pI values was observed. Two-dimensional gel electrophoresis of photoaffinity-labeled cytosolic fractions from HeLa cells, the rat liver tumor cell line McA-RH7777, and buffalo rat thymus revealed three isoforms, essentially the same isoform pattern as in Hepa 1c1c7 cells. This would indicate that despite the considerable molecular weight polymorphism between species the level of charge heterogeneity is highly conserved.     

Heterogeneity of Escherichia coli-expressed human muscle creatine kinase

Creatine kinase (CK) plays an important role in maintaining a constant ATP:ADP ratio during periods of high energy usage. Elevated levels of CK give an early indication of myocardial infarction. The enzyme has four major isozymes with heterogeneity being observed for each of them. In many cases the source of the heterogeneity is unclear. However, some of the isoforms are known to result from exposure to serum proteases, and analysis of the plasma isoforms provides an estimate of the time of onset of the infarction. Somewhat surprisingly, isoelectric focusing (IEF) experiments provided evidence of heterogeneity in human muscle CK (HMCK) expressed in E. coli. To investigate this further, HMCK was purified to apparent homogeneity utilizing Blue Sepharose affinity chromatography and HiPrep Q anion exchange chromatography. Additional purification on a PBE 94 chromatofocusing column resulted in four fractions, three of which, HMCK I - III, were characterized. The three isoforms are all active and have similar kinetic parameters. They exhibited identical bands on SDS PAGE but different anodal mobility on non-denaturing gels. Modification of C-terminal and/or cysteine residues has been ruled out, and deamidation of asparagine or glutamine residue(s) is proposed to be the cause of isoform formation. In addition each of these isoforms showed a similar four-band pattern on a carrier ampholytes-based IEF gel. Two-dimensional IEF analysis showed that an equilibrium was established between the four bands, suggesting that the four components were unstable and generated only when the protein was subjected to IEF.     

Multiforms of mammalian adenylate kinase and its monoclonal antibody against AK1

An attempt has been made to determine the intracellular distribution of the multiforms of the adenylate kinase (AK) isoenzymes in mammalian tissues, to shed some light on their physiological roles, especially in energy metabolism. The adenylate kinase zymograms obtained from isoelectric focusing yielded two typical isoform patterns: (1) with a pI greater than or equal to 9 and 8.6, specific for bovine skeletal muscle, heart, aorta and brain, and (2) with a pI = 7.9 and 7.1, specific for liver and kidney. Pattern (1) was attributed to the cytosolic isoenzyme (AK1) as demonstrated by immunostaining with anti-AK1. Pattern (2) was attributed to the mitochondrial isoenzyme (AK2). These results were largely confirmed by chromatofocusing experiments. The AK1 isoenzyme was partially purified from the cytosol fraction of bovine aortic smooth muscle and had an apparent Mr of 23.5 kilodaltons. Its kinetic features are discussed from a comparative standpoint. Finally, the human serum AK1 isoform was also detected by Western blotting with a monoclonal antibody directed against crystalline porcine muscle AK1. These results are to form the basis of further studies on the 'aberrant' adenylate kinase isoenzyme from the serum of Duchenne muscular dystrophics.     

Multiple disc gel bands of mitochondrial creatine and adenylate kinases

Abstract— The activities and electrophoretic patterns of creatine and adenylate kinases in the mitochondrial and high speed supernatant fractions of adult mouse brain were determined. Approximately 22 per cent of the activities of both kinases is firmly bound to the mitochondria. On acrylamide gel electrophoresis of creatine kinase, in addition to the major band previously described, there were several other bands found. Although present in both the mitochondrial and supernatant fractions these additional protein bands with creatine kinase activity were significantly more intense in the mitochondrial fraction. There was only onesecondary band of adenylate kinase activity in the mitochondrial fraction but additional bands were found in the soluble fraction.     

The nature of post-translational formation of MM creatine kinase isoforms

Isoforms (derived from the same isoenzyme but distinguished by differences in isoelectric point) of MM creatine kinase appear in plasma after myocardial infarction. They are formed by conversion of the tissue form of creatine kinase (MM-A, pI 7.80) to progressively more acidic species (MM-B, pI 7.50) and MM-C (pI 7.20) after release into the circulation. To define the changes responsible for myocardial MM creatine kinase isoform formation in humans and dogs, purified isoforms were treated with trypsin or cyanogen bromide. The digests were fractionated by reverse-phase high pressure liquid chromatography. Comparison of proteolytic maps showed that MM-A and MM-C were each characterized by a single, unique peptide peak. Maps of MM-B creatine kinase contained both of these peaks. Sequence analysis and comparison with the complete amino acid sequence of MM creatine kinase showed that the peptide unique to MM-A corresponded to the COOH-terminal tryptic or CNBr peptide. The peptide unique to MM-C was shown to have the same amino acid composition except for lysine (the COOH-terminal amino acid). Thus, isoform formation is characterized by the successive removal of the COOH-terminal lysine residue from one M subunit at a time resulting in the conversion of MM-A to isoforms MM-B and MM-C.     

Thermal stability of the multiple charge isoforms of inulase fromAspergillus niger

Summary The three fractions of inulase differing in isoelectric pH were isolated from a strain ofAspergillus niger by isoelectric focussing and characterized in their temperature stability. These fractions had distinguishable thermal stability. Among them, one fraction (pI 5.2) having main activity showed a high thermal stability comparable to that of the Novozyme 230 (Novo A/S, Denmark) at 60°C.     

Apolipoproteins as the basis for heterogeneity in high-density lipoprotein2 and high-density lipoprotein3. Studies by isoelectric focusing on agarose films

A method is described for the isoelectric focusing (IEF) of lipoproteins on thin films of agarose. Within a pH gradient of 4.60-5.30 both high-density lipoproteins 2 and 3 (HDL2 and HDL3) are resolved into more than 10 fractions which could be stained either for protein or for lipids. The isoelectric focusing patterns for HDL2 and HDL3 are similar although HDL2 appears richer in the more alkaline bands. Narrow film strips from the IEF separation of HDL2 and HDL3 were interfaced with various agarose plates containing antisera against apolipoproteins apoAI, apoAII and apoCIII either alone or in combination, to provide two-dimensional IEF immunoelectrophoresis patterns. This technique demonstrated that apoAI and apoAII were present throughout the IEF gel for both subclasses of HDL. It also provided evidence for the existence of lipoproteins containing both apoAI and apoAII and other lipoproteins present in the alkaline region of the gel which contained apoAI but no apoAII. ApoCIII was found mostly in acidic lipoproteins and was not distributed identically in HDL2 and HDL3. The lipoproteins separated by IEF on agarose were also analysed by two-dimensional IEF-SDS electrophoresis and the individual apolipoproteins were identified by reaction with antibodies to apolipoproteins AI, AII, CI, CII, CIII, D, and E. This technique confirmed that in IEF of HDL, apoAI extended throughout the spectrum of lipoproteins whereas apoE was only present in alkaline lipoproteins and apoD was only present in acidic lipoproteins. IEF on agarose of either HDL2 or HDL3 allowed us to collect eight different fractions, which have the same pI in either lipoprotein class. The apolipoprotein composition of each isolated band was analysed by electroimmuno-assays for apolipoproteins AI, AII, CI, CII, CIII, D, and E and the results expressed as the ratio of the measured apolipoprotein to measured apoAI. In both HDL2 and HDL3, acidic lipoprotein fractions were enriched in apoAII, apoCIII and apoD. ApoCII and apoCII were not similarly distributed in HDL2 and HDL3 subfractions whereas the apoCI distribution was similar in both classes. Noteworthy in all experiments was the difference in the distributions of apoCI, apoCII, and apoCIII in HDL2 and HDL3, which indicated that the existence of a lipoprotein containing simultaneously CI, CII and CIII can only account for a small fraction of these apolipoproteins. Therefore these experiments substantiate the theory of the protein basis of HDL heterogeneity and suggest that the majority of apolipoproteins are present in complexes which upon IEF result in lipoprotein fractions of identical pI for both HDL2 or HDL3.(ABSTRACT TRUNCATED AT 400 WORDS)     

Analysis of rat serum apolipoproteins by isoelectric focusing. II. Studies on the low molecular weight subunits.

The low molecular weight proteins of rat apo HDL and apo VLDL have been isolated and analyzed by the technique of isoelectric focusing. Sephadex fractions from apo HDL (HS-3) and apo VLDL (VS-3) that contain these proteins reveal three major bands with apparent isoelectric points of pH 4.50, 4.67, and 4.74, as well as three minor bands at pH 4.43, 4.57, and 4.61. In addition, apo HDL has a major band at pI of 4.83. DEAE-Cellulose chromatography was used to prepare purified fractions of these components that were characterized by N-terminal analyses and molecular weight determinantions by SDS gel electrophoresis. The major low molecular weight components of apo HDL were focused on a slab gel and the bands were identified as A-II (pI 4.83), C-II (pI 4.74), C-III-0 (pI 4.67), and C-III-3 (pI 4.50). Neuraminidase treatment of apo HDL, followed by isoelectric focusing, suggested that the other bands, which have not previously been reported, may be additional forms of the C-III protein, differing only in their content of sialic acid.     

Heterogeneity of renin substrate released from hepatocytes and in brain extracts

Renin substrate was characterized in incubation medium of isolated hepatocytes, plasma, and brain extracts of the rat by isoelectric focusing and polyacrylamide gel electrophoresis. The isoelectric focusing (IEF) profile of renin substrate released into incubation medium of rat hepatocytes demonstrated two peaks with isoelectric points (pI) of 4.1 (minor peak) and 4.6 (major peak). Extracts of normal rat brain also showed two forms (pI 4.6 major form, and pI 5.1 minor form). In contrast, normal rat plasma contained a single broad peak of substrate with pI 4.5. On polyacrylamide gel electrophoresis (PAGE), the hepatocytes medium and brain extracts contained forms of substrate with reduced mobility as compared to the plasma form. Intraperitoneal injection of 17β estradiol (1 mg) or bilateral nephrectomy significantly elevated renin substrate levels in plasma and increased its release from hepatocytes, however, no change in the IEF or PAGE profiles was evident. There was no remarkable change of substrate concentration in the brain following these treatments. Molecular weights of renin substrate were 60,000–65,000 from all preparations. It remains to be established whether the different forms of renin substrate from hepatocytes represent precursor forms of circulating plasma substrate. The presence of distinct forms of brain renin substrate and the lack of an increase in brain renin substrate following nephrectomy or estrogen treatment suggest local synthesis and support the postulate of an independent renin-angiotensin system in the central nervous system.     

A protein kinase system from platelet rich plasma

Treatment of platelet rich plasma (PRP) at pH 5 results in the precipitation of a protein kinase system. The protein kinase is associated with the platelet fraction and is capable of phosphorylation of several plasma proteins. Analysis of the ³²P-labeled phosphoproteins by two dimensional gel electrophoresis showed the existence of three major phosphoproteins: 72K and 80K proteins with identical isoelectric points (pI) of 6.0 and another 72K protein with a pI of 6.8–7.0. This latter 72K phosphoprotein has recently been identified as the α-chain of fibrinogen. The identity of the other 2 proteins remains to be shown. The activity of the protein kinase is markedly enhanced by Mn²⁺, it phosphorylates calf thymus histone as an exogenous substrate and is independent of cAMP or cGMP. This protein kinase activity is inhibited competitively by ADP.     

Isolation of dipeptidyl peptidase IV (DP 4) isoforms from porcine kidney by preparative isoelectric focusing to improve crystallization

Abstract In the present studies we resolved the post-translational microheterogeneity of purified porcine dipeptidyl peptidase IV (DP 4) from kidney cortex. Applying SDS-homogeneous DP 4 onto an analytical agarose isoelectric focusing (IEF) gel, pH 4-6, activity staining resulted in at least 17 isoforms between pH 4.8-6.0. These could be separated into fractions with only two to six isoforms by means of preparative liquid-phase IEF, using a Rotofor cell. Starting off with three parallel Rotofor runs under the same conditions at pH 5-6, the fractions were pooled according to the specific activity of DP 4, pH and analytical IEF profile, and further refractionated without any additional ampholytes. Since excessive dilution of ampholytes and proteins was kept to the minimum, a second refractionation step could be introduced, resulting in pH gradients between 0.022 and 0.028 pH increments per fraction. By performing two consecutive refractionation steps, the high resolution necessary for the separation of DP 4 isoforms could be achieved. This represents an alternative method if isolation of isoforms with similar pI's results in precipitation and denaturation in presence of a narrow pH range. Furthermore, it demonstrates that preparative IEF is a powerful tool to resolve post-translational microheterogeneity of a purified protein required for crystallization processing.     

Purification of recombinant human growth hormone by isoelectric focusing in a multicompartment electrolyzer with Immobiline membranes.

Recombinant human growth hormone (r-hGH) expressed in Escherichia coli, was 70-80% purified by a combination of ion-exchange chromatography and metal ion affinity chromatography. For the last purification step, a multicompartment electrolyzer was used, containing three compartments delimited by isoelectric membranes and two additional anodic and cathodic chambers. The central compartment was situated between two membranes having isoelectric points (pI) of 5.08 (anodic) and of 5.16 (cathodic), i.e. equidistant from the pI value of hGH (pI 5.12). r-hGH was isoelectric between these two membranes and could not leave the central chamber, while more acidic and more cathodic impurities collected in the two lateral chambers under the influence of the electric field. The r-hGH, thus purified, exhibited a single band by isoelectric focusing (IEF) in immobilized pH gradients (IPG) and gave recoveries greater than 90%. The problem of isoelectric precipitation in a practically ion-free environment was alleviated by focusing in 30% glycerol added with 1% neutral detergent (Nonidet-P40). The latter was eliminated by passage through a Q-Sepharose column after collecting the pI 5.12 band from the electrolyzer. Also the pre-hormone (pre-hGH) can be purified in a similar manner (30% glycerol, 1% Nonidet P-40) between two membranes having pIs 4.77 (anodic) and 4.87 (cathodic) (pre-hGH pI 4.82). This paper demonstrates the possibility of purifying by a focusing process also poorly soluble proteins at the pI.     

Isoelectric focusing and crossed immunoelectrophoresis of heme proteins in the Escherichia coli cytoplasmic membrane.

Isoelectric focusing (IEF), agarose electrophoresis, and crossed immunoelectrophoresis (CIE) were used to resolve the heme-containing proteins of the Escherichia coli cytoplasmic membrane after solubilization by Triton X-100. Two bands in IEF stained for heme with pI values of 4.7 and 5.3. One of the bands, with an isoelectric point of pH 5.3, was present only when the cells were grown to late log or stationary phase and possessed N,N,N,'N'-tetramethyl-p-phenylene-diamine (TMPD) oxidase activity. The pI 4.7 band was present in cells harvested in both mid-log and stationary phases. Agarose electrophoresis, using larger samples, revealed the same two components apparent by IEF, and, in addition, a third component. The heme-containing fractions were extracted after agarose electrophoresis and subjected to further study. The component which was present in cells grown to stationary phase contained hemes b, a1, and d. The other two fractions contained only b heme. One of these corresponded to the component with pI 4.7 in IEF and had catalase activity. Antisera were raised against Triton X-100-solubilized cytoplasmic membranes and against the focused TMPD oxidase complex. With these anti-sera, CIE in the presence of Triton X-100 revealed four precipitin complexes containing heme. Three of these corresponded to the components identified by IEF and agarose electrophoresis. We demonstrate that the combined use of IEF and CIE is valuable for analysis of membrane proteins. In particular, this work represents a substantial initial step toward a structural elucidation of the E. coli aerobic respiratory chain.     

Changes in liver nuclear protein metabolism after a single dose of urethane to suckling mice

Treatment of 8-9-day-old C57BL/A mice with a single carcinogenic dose of urethane, at 1.2 mg/g body wt., resulted in an immediate decrease in liver DNA synthesis reaching a maximum at about 16-18 h after injection, the rate of synthesis returning to normal after 48 h. When the nuclear proteins were radiolabelled, the non-histone protein (NHP) fraction showed a significant decrease in specific activity 8-18 h after injection of urethane and slight increase in specific activity after 24 h. Histone and residual proteins did not show any significant change. The liver NHP were analysed by isoelectric focusing (IEF) and sodium dodecyl sulphate (SDS) electrophoresis in polyacrylamide gels. The latter technique failed to show any distinctive differences but IEF results indicated some quantitative and qualitative changes in protein content and synthesis were induced by the urethane treatment. The most noticeable change in the stained gels was an increase in a protein component having a pI of 7.35 and the appearance of new bands at pI's of 7.85 and 5.55 in the 18 h treated livers. However, the [3H]tryptophan labelling pattern indicated that this was not due to an increased synthesis of these components. 24 h after urethane there appeared to be an increased rate of synthesis of some of the major components of the mixture, particularly at the pI 5.65 region. Histone and residual protein fractions were also analysed by electrophoresis and showed no difference between treated and control livers.