Mitochondrial Low-Molecular-Weight Heat-Shock Proteins and the Tolerance of Cereal Mitochondria to Hyperthermia

Relationships between the appearance of low-molecular-weight heat-shock proteins (LMW HSPs) in maize, winter wheat, and winter rye mitochondria and the tolerance of the mitochondria to hyperthermia (42°C, 3 h) were studied using one-dimensional SDS-PAGE, immunochemical methods, and polarography. Heat shock inhibited respiration to a greater extent in the wheat and rye than in the maize mitochondria. A single 20-kD LMW HSP was found both inside and on the surface of mitochondria isolated from heat-treated wheat and rye seedlings. After heating maize seedlings, two LMW HSPs (28 and 23 kD) appeared inside the mitochondria, and three proteins (22, 20, and 19 kD) appeared on their surface. We suppose that the latter three proteins play an essential role in the protection of mitochondria from hyperthermic damage. It seems likely that the diversity of the hyperthermia-induced LMW HSPs in plant mitochondria affects their thermal stability.     

Accumulation, stability, and localization of a major chloroplast heat-shock protein

Diverse higher plant species synthesize low molecular weight (LMW) heat shock proteins (HSPs) which localize to chloroplasts. These proteins are homologous to LMW HSPs found in the cytoplasm of all eukaryotes, a class of HSPs whose molecular mode of action is not understood. To obtain basic information concerning the role of chloroplast HSPs, we examined the accumulation, stability, tissue specificity, and intra-chloroplast localization of HSP21, the major LMW chloroplast HSP in pea. Intact pea plants were subjected to heat stress conditions which would be encountered in the natural environment and HSP21 mRNA and protein levels were measured in leaves and roots. HSP21 was not detected in leaves or roots before stress, but the mature, 21-kD protein accumulated in direct proportion to temperature and HSP21 mRNA levels in both tissues. All of the HSP21 in leaves was localized to chloroplasts; there was no evidence for its transport into other organelles. In chloroplast fractionation experiments, greater than 80% of HSP21 was recovered in the soluble chloroplast protein fraction. The half-life of HSP21 at control temperatures was 52 +/- 12 h, suggesting the protein's function is critical during recovery as well as during stress. We hypothesize that HSP21 functions in a catalytic fashion in both photosynthetic and nonphotosynthetic plastids.     

Expression of sunflower low-molecular-weight heat-shock proteins during embryogenesis and persistence after germination: localization and possible functional implications

We isolated and sequenced Ha hsp 17.9, a DNA complementary (cDNA) of dry-seed stored mRNA that encodes a low-molecular-weight heat-shock protein (LMW HSP). Sequence analysis identified Ha hsp17.9, and the previously reported Ha hsp17.6, as cDNAs encoding proteins (HSP17.6 and HSP17.9) which belong to different families of cytoplasmic LMW HSPs. Using specific antibodies we observed differential expression of both proteins during zygotic embryogenesis under controlled environment, and a remarkable persistence of these LMW HSPs during germination. Immuno-blot analysis of HSP17.9 proteins in two-dimensional gels revealed that the polypeptides expressed in embryos were indistinguishable from LMW HSPs expressed in vegetative tissues in response to water deficit; but they appeared different from homologeous proteins expressed in response to thermal-stress. Tissue-print immunolocalization experiments showed that HSP17.9 and HSP17.6 were homogeneously distributed in every tissue of desiccation-tolerant dry seeds and young seedlings under non-stress conditions. These results demonstrate developmental regulation of specific, cytoplasmic, plant LMW HSPs, suggesting also their involvement in water-stress tolerance.     

Characterization and Physiological Function of Class I Low-Molecular-Mass,Heat-Shock Protein Complex in Soybean

Examination of an ammonium sulfate-enriched fraction (70-100% saturation) of heat-shock proteins (HSPs) by nondenaturing polyacrylamide gel electrophoresis revealed the presence of a high molecular mass complex (280 kD) in soybean (Glycine max) seedlings. This complex cross-reacted with antibodies raised against soybean class I low-molecular-mass (LMW) HSPs. Dissociation of the complex by denaturing polyacrylamide gel electrophoresis showed the complex to contain at least 15 polypeptides of the 15-to 18-kD class I LMW HSPs that could be detected by staining, radiolabeling, and western blotting. A similar LMW-HSP complex was observed in mung bean (Vigna radiata L.; 295 kD), in pea (Pisum sativum L.; 270 kD), and in rice (Oryza sativa L.; 310 kD). The complex was stable under high salt conditions (250 mM KCI), and the integrity was not affected by 1% Nonidet P-40 and 3 [mu]g/ML RNase treatment. The size of the isolated HSP complex in vitro was conserved to 55[deg]C; however, starting at 37.5[deg]C, it changed to higher molecular forms in the presence of soluble proteins. The isolated HSP complex was able to protect up to 75% of the soluble proteins from heat denaturation in vitro.     

Expression of a Conserved Family of Cytoplasmic Low Molecular Weight Heat Shock Proteins during Heat Stress and Recovery

Plants synthesize several families of low molecular weight (LMW) heat shock proteins (HSPs) in response to elevated temperatures. We have characterized two cDNAs, HSP18.1 and HSP17.9, that encode members of the class I family of LMW HSPs from pea (Pisum sativum). In addition, we investigated the expression of these HSPs at the mRNA and protein levels during heat stress and recovery. HSP18.1 and HSP17.9 are 82.1% identical at the amino acid level and are 80.8 to 92.9% identical to class I LMW HSPs of other angiosperms. Heat stress experiments were performed using intact seedlings subjected to a gradual temperature increase and held at a maximum temperature of 30 to 42 degrees Celsius for 4 hours. HSP18.1 and HSP17.9 mRNA levels peaked at the beginning of the maximum temperature period and declined rapidly after the stress period. Antiserum against a HSP18.1 fusion protein recognized both HSP18.1 and HSP17.9 but not members of other families of LMW HSPs. The accumulation of HSP18.1-immunodetected protein was proportional to the severity of the heat stress, and the protein had a half-life of 37.7 ± 8 hours. The long half-life of these proteins supports the hypothesis that they are involved in establishing thermotolerance.     

Purification and characterization of rat hepatic microsomal low molecular weight fatty acid ethyl ester synthase and its relationship to carboxylesterases.

We reported purification of a high molecular weight (HMW) (ca. 180 kD) and a low molecular weight (LMW) (ca. 60 kD) protein fractions from digitonized rat liver microsomes using ammonium sulfate precipitation followed by ion exchange and gel filtration column chromatography. Both fractions expressed fatty acid ethyl ester (FAEE) synthase as well as p-nitrophenyl acetate (PNPA)-hydrolyzing (esterase) activities. The HMW fraction was found to be a trimer with subunit molecular weight ca. 60 kD and structurally and functionally similar to rat hepatic microsomal carboxylesterase (CE, pI 6.1) and adipose tissue FAEE synthase. In this article, we report further purification and characterization of the LMW (minor) fraction expressing FAEE synthase activity and its structural and functional relationship to hepatic microsomal CEs. Using isoelectric focusing (IEF) followed by gel filtration-high-performance liquid chromatography (GF-HPLC), five proteins were purified, which expressed FAEE synthase as well as PNPA-hydrolyzing activity. The isoelectric point values of 6.5, 5.8, 5.6, 5.3, and 5.0 were found for the purified LMW proteins by IEF and each showed a peak corresponding to ca. 60 kD molecular weight by GF-HPLC, which expressed FAEE synthase as well as PNPA-hydrolyzing activity. Sodium dodecyl sulfate-polyacrylamide gel elecrophoresis (SDS-PAGE) analysis of the GF-HPLC purified LMW proteins revealed that these proteins are monomers (ca. 60 kD). All the purified LMW proteins cross-reacted with antibodies to rat adipose tissue FAEE synthase. Coelution of PNPA-hydrolyzing and FAEE synthase activity at each step of purification and cross-reactivity with rat adipose tissue FAEE synthase antibodies suggest that the purified proteins are related to various hepatic microsomal CEs. This conclusion is further supported by the homology of N-terminal amino acid sequence of the purified LMW proteins to various hepatic microsomal CEs and protease precursors. Therefore, LMW FAEE synthase activity most probably is expressed by various isozymes of hepatic microsomal CEs, which are also involved in the biotransformation of xenobiotic alcohols and amines.     

Analysis of conserved domains identifies a unique structural feature of a chloroplast heat shock protein

Summary A low molecular weight heat shock protein which localizes to chloroplasts has been identified in several plant species. This protein belongs to a eukaryotic superfamily of small HSPs, all of which contain a conserved carboxyl-terminal domain. To investigate further the structure of this HSP, we isolated and sequenced cDNA clones for the chloroplast LMW HSPs from Petunia hybrida and Arabidopsis thaliana. The cloning of chloroplast HSPs from these two species enabled us to compare the amino acid sequences of this protein from plant species (petunia, Arabidopsis, pea, soybean and maize) that represent evolutionarily divergent taxonomic subclasses. Three conserved regions were identified, which are designated as regions I, II and III. Regions I and II are also shared by cytoplasmic LMW HSPs and therefore are likely to have functional roles common to all eukaryotic LMW HSPs. In contrast, consensus region III is not found in other LMW HSPs. Secondary structure analysis predicts that this region forms an amphipathic -helix with high conservation of methionine residues on the hydrophobic face and 100% conservation of residues on the hydrophilic face. This structure is similar to three helices, termed methionine bristles, which are found in a methionine-rich domain of a 54 kDa protein component of signal recognition particle (SRP54). The conservation of regions I and II among LMW cytoplasmic and chloroplast HSPs suggests that these HSPs perform related functions in different cellular compartments. However, identification of the methionine bristle domain suggests that chloroplast HSPs also have unique functions or substrates within the special environment of the chloroplast or other plastids.Abbreviations HS heat shock - HSP heat shock protein - LMW low molecular weight     

Localization of small heat shock proteins to the higher plant endomembrane system.

Three related gene families of low-molecular-weight (LMW) heat shock proteins (HSPs) have been characterized in plants. We describe a fourth LMW HSP family, represented by PsHSP22.7 from Pisum sativum and GmHSP22.0 from Glycine max, and demonstrate that this family of proteins is endomembrane localized. PsHSP22.7 and GmHSP22.0 are 76.7% identical at the amino acid level. Both proteins have amino-terminal signal peptides and carboxyl-terminal sequences characteristic of endoplasmic reticulum (ER) retention signals. The two proteins closely resemble class I cytoplasmic LMW HSPs, suggesting that they evolved from the cytoplasmic proteins through the addition of the signal peptide and ER retention motif. The endomembrane localization of these proteins was confirmed by cell fractionation. The polypeptide product of PsHSP22.7 mRNA was processed to a smaller-M(r) form by canine pancreatic microsomes; in vivo, GmHSP22.0 polysomal mRNA was found to be predominantly membrane bound. In vitro-processed PsHSP22.7 corresponded in mass and pI to one of two proteins detected in ER fractions from heat-stressed plants by using anti-PsHSP22.7 antibodies. Like other LMW HSPs, PsHSP22.7 was observed in higher-molecular-weight structures with apparent masses of between 80 and 240 kDa. The results reported here indicate that members of this new class of LMW HSPs are most likely resident ER proteins and may be similar in function to related LMW HSPs in the cytoplasm. Along with the HSP90 and HSP70 classes of HSPs, this is the third category of HSPs localized to the ER.     

吐温80对体外HeLa细胞的杀伤抑制

研究吐温80对体外培养HeLa细胞的杀伤抑制。通过MTT法检测并观察了不同浓度吐温80、大肠杆菌内毒素(LPS)及前列腺素E1(PGEI)对体外培养HeLa细胞作用后细胞抑制率;将作用后细胞裂解液经SDS-PAGE及Western-blotting法测定其热休克蛋白70(Heat Shock Protein70,HSP70)表达。结果:0.02％吐温80具有明显杀伤HeLa细胞的作用,且作用后的HeLa细胞几乎无HSP70蛋白表达(正常HeLa细胞有一定量HSP70蛋白表达),但却有一种分子量为70kDa的蛋白高表达,表明吐温80显著抑制HSP70表达,以至HeLa细胞死亡,同时促使某一种蛋白大量堆积。大肠杆菌内毒素、前列腺素E1对HeLa细胞无明显抑制杀伤作用,且与吐温80无明显协同效应,同时亦对HeLa细胞HSP70表达无显著影响。推测:吐温80对HeLa细胞HSP70表达的显著抑制作用可能是其杀伤HeLa细胞的 作用机制之一,为理想的温热治疗肿瘤的协同合并作用药物,但其使细胞内大量某70kDa蛋白堆积现象有待进一步探讨。     

HSP16.6 Is Involved in the Development of Thermotolerance and Thylakoid Stability in the Unicellular Cyanobacterium, Synechocystis sp. PCC 6803

The low molecular weight (LMW) heat shock protein (HSP), HSP16.6, in the unicellular cyanobacterium, Synechocystis sp. PCC 6803, protects cells from elevated temperatures. A 95% reduction in the survival of mutant cells with an inactivated hsp16.6 was observed after exposure for 1 h at 47°C. Wild-type cell survival was reduced to only 41%. HSP16.6 is also involved in the development of thermotolerance. After a sublethal heat shock at 43°C for 1 h and subsequent challenge exposure at 49°C for 40 min, mutant cells did not survive, while 64% of wild-type cells survived. Ultrastructural changes in the integrity of thylakoid membranes of heat-shocked mutant cells also are discussed. These results demonstrate an important protective role for HSP16.6 in the protection of cells and, in particular, thylakoid membrane against thermal stress. Received: 14 October 1999 / Accepted: 16 November 1999     

Thermal stress evaluation of suspension cell cultures in winter wheat

Summary The objectives of this study were to compare thermotolerance in whole plants vs. suspension cell cultures of winter wheat, and to evaluate the synthesis of heat shock proteins in relation to genotypic differences in thermotolerance in suspension cells. Whole plant genetic differences in the development of heat tolerance were identified for three wheat genotypes (ND 7532, KS 75210 and TAM 101). Suspension cell cultures of these genotypes were used to evaluatein vitro response to heat stress. Viability tests by triphenyl tetrazolium chloride (TTC) and by fluorescein diacetate (FD) were utilized to determine the relationship of cellular response to heat stress (37°C/24 h, 50°C/1h). KS 75210 and ND 7532 are relatively heat susceptible. TAM 101 is heat tolerant. Both tests at the cellular level were similar to the whole plant response. Thus, cellular selection for enhancing heat tolerance seems feasible. Heat shock protein (HSP) synthesis of two genotypes, ND 7532 and TAM 101 were determined for suspension cultured cells. In suspension cultures, HSPs of molecular weight 16 and 17 kD were found to be synthesized at higher levels in the heat tolerant genotype (TAM 101) than the susceptible genotype (ND 7532), both at 34° and 37°C treatments for 2 hours and 5 hours. HSP 22 kD was synthesized more at 34°C for TAM 101 than ND 7532, but not at 37°C; whereas, HSP 33 kD was synthesized at 37°C at similar abundance for both genotypes, but not at 34°C.These results indicated that there is a differential expression of HSP genes in wheat suspension cells at different temperature stress durations and between heat tolerant and heat susceptible genotypes. It appears that the levels of synthesis of HSPs 16 and 17 kD are correlated with genotypic differences in thermal tolerance at the cellular level in two genotypes of wheat.     

HSP_(70)基因表达对高粱育性的影响(英文)

高粱细胞质雄性不育系３１９７Ａ（３Ａ）在常温条件下是不育的（Ｆｉｇｓ．１１＆２）,经热激（４５℃）诱导不同程度地恢复了育性（Ｆｉｇｓ．１３＆４）,为研究其不育机理提供了线索。热激２ｈ后,３Ａ中即可产生一类线粒体热激蛋白（ＨＳＰｓ）。其中,分子量为７０ｋＤ的ＨＳＰ７０含量最高,也最为稳定。不过,３Ａ中ＨＳＰｓ的稳定性弱于保持系３１９７Ｂ（３Ｂ）（Ｆｉｇ．２,Ｐａｎｅｌｓ１～４）。放线菌素Ｄ抑制ＨＳＰｓ的合成,而氯霉素无此作用（Ｆｉｇ．２,Ｐａｎｅｌｓ５＆６）,表明：ＨＳＰｓ是由核基因编码、在细胞质中合成、再跨膜转运到线粒体中的。３Ａ幼穗经热激后,线粒体的总蛋白量猛增了２．７倍（Ｆｉｇ．３）,达到３Ｂ的水平,育性亦变为可育的。Ｆｉｇ．４表明：ＨＳＰ７０反义链ｃＤＮＡ（Ｒ１）能进入到３Ｂ花药细胞中,并与靶ＲＮＡ（ＨＳＣ７０ｍＲＮＡ）结合,而对照、正义链ｃＤＮＡ（Ｄ）链无此反应。由此、再增加一个通用保守序列的反义链ｃＤＮＡ（Ｒ２）、共两个探针（Ｒ１、Ｒ２）,可以检测到：３Ａ在常温下没有能力合成ＨＳＣ７０ｍＲＮＡ（Ｆｉｇ．５）,而在热激条件下,转变为有能力（Ｆｉｇ．６）。启示：３Ａ在热激条件下由不育转变为可育     

高温加速老化过程中甜菜种子线粒体小分

甜菜(BetavulgarisLcv.Loke)种子的人工老化(30℃,100％RH)导致种子的干重、生活力和活力逐渐丧失,线粒体的呼吸速率、细胞色素c氧化酶和苹果酸脱氢酶的活性下降。线粒体蛋白质随着种子老化而显著变化,小分子量热休克蛋白(LMWHSP)22从老化的0d到30d增加,然后从30d到90d下降。可以认为甜菜种子人工老化过程中线粒体活性和LMWHSP22的含量变化与种子活力密切相关。     

Antioxidant compounds EGB-761 and BN-520 21 attenuate heat shock protein (HSP 72 kD) response, edema and cell changes following hyperthermic brain injury

Influence of the extract of Gingko biloba (EGB-761) and one of its constituent Gingkolide B (BN-52021) on hyperthermia induced cellular damage and heat shock protein (HSP 72kD) response was examined in a rat model. Rats subjected to 4h heat stress at 38 degrees C in a biological oxygen demand (BOD) incubator (relative humidity 50-55%, wind velocity 20-25cm/sec) resulted in profound edema and cell injury in many parts of the cerebral cortex, hippocampus, cerebellum, thalamus, hypothalamus and brain stem. Immunostaining of HSP 72 kD showed marked upregulation in the damaged and distorted neurons located within the edematous area. Pretreatment with EGB-761 (50mg/kg/day, p.o.) and BN-520 21 (2mg/kg, p.o.) per day for 5 days significantly reduced HSP expression and attenuated cell damage. Our results show that EGB-761 and its component Gingkolide B (BN-52021) has the capacity to reduce edema and cell injury following hyperthermia and this effect of the compound is somehow associated with a reduction in cellular stress response as evidenced with a reduction in HSP expression.     

RAR1 and HSP90 form a complex with Rac/Rop GTPase and function in innate-immune responses in rice

A rice (Oryza sativa) Rac/Rop GTPase, Os Rac1, is involved in innate immunity, but its molecular function is largely unknown. RAR1 (for required for Mla12 resistance) and HSP90 (a heat shock protein 90 kD) are important components of R gene-mediated disease resistance, and their function is conserved in several plant species. HSP90 has also recently been shown to be important in mammalian innate immunity. However, their functions at the molecular level are not well understood. In this study, we examined the functional relationships between Os Rac1, RAR1, and HSP90. Os RAR1-RNA interference (RNAi) rice plants had impaired basal resistance to a compatible race of the blast fungus Magnaporthe grisea and the virulent bacterial blight pathogen Xanthomonas oryzae. Constitutively active Os Rac1 complemented the loss of resistance, suggesting that Os Rac1 and RAR1 are functionally linked. Coimmunoprecipitation experiments with rice cell culture extracts indicate that Rac1 forms a complex with RAR1, HSP90, and HSP70 in vivo. Studies with Os RAR1-RNAi and treatment with geldanamycin, an HSP90-specific inhibitor, showed that RAR1 and HSP90 are essential for the Rac1-mediated enhancement of pathogen-associated molecular pattern-triggered immune responses in rice cell cultures. Furthermore, the function of HSP90, but not RAR1, may be essential for their association with the Rac1 complex. Os Rac1 also regulates RAR1 expression at both the mRNA and protein levels. Together, our results indicate that Rac1, RAR1, HSP90, and HSP70 form one or more protein complexes in rice cells and suggest that these proteins play important roles in innate immunity in rice.     

The methionine-rich low-molecular-weight chloroplast heat-shock protein: evolutionary conservation and accumulation in relation to thermotolerance

The evolutionary conservation of the low-molecular-weight chloroplast-localized heat-shock protein (LMW chlpHsp) in vascular plants was examined using immunological methods. An antibody (Abmet) specific to the LMW chlpHsp was produced using a synthetic 28-residue peptide containing the most conserved elements of its unique "methionine-rich domain" as an antigen. This antibody detected a heat-inducible low-molecular-weight chloroplast protein in plants of six divergent Anthophyta species, including C3, C4, CAM, monocot, and dicot species. Abmet also detected a LMW chlpHsp in species from the Divisions Psilotophyta, Equisetophyta, Polypodiophyta, and Ginkgophyta. A preliminary examination of the relationship between accumulation of the LMW chlpHsp and habitat was also conducted. Seven Anthophyta species originating from both warm- and cool-temperature habitats were grown at 28C and then heat stressed at 40C. A positive qualitative relationship between the accumulation of the LMW chlpHsp and organismal thermotolerance in these species was observed; similar results were obtained separately with four nonAnthophyta species. The strong evolutionary conservation of this LMW Hsp and its localization to the chloroplast, and the correlation between production of this protein and plant thermotolerance, suggest that the LMW chlpHsp plays an important role in adaptation to heat stress.     

Comparison of epitope specificity of anti-heat shock protein 60/65 IgG type antibodies in the sera of healthy subjects,patients with coronary heart disease and inflammatory bowel disease

Previously, we reported on the presence of antibodies to linear epitopes of human and mycobacterial 60 kD heat shock proteins (HSP) in the sera of healthy blood donors. Since many recent findings indicate that the levels of these antibodies may be altered in coronary heart disease (CHD) and also inflammatory bowel diseases (IBD), it seemed worthwhile to compare the epitope specificity of the anti-HSP60 and anti-HSP65 antibodies in the sera of patients with these diseases to those in healthy subjects. The multipin enzyme-linked immunosorbent assay method was applied with a large overlapping set of synthetic 10-mer peptides covering selected regions of human HSP60 and Mycobacterium bovis HSP65. Sera of 12 healthy persons (HP), 14 CHD, and 14 IBD patients with the same concentration of total anti-HSP60 and HSP65 IgG antibodies were tested. We have identified CHD-specific epitopes in the equatorial domain of the HSP60 protein but in neither region of the HSP65 molecule, indicating that the formation of anti-HSP60 antibodies is not or only partially due to the cross-reaction between human HSP60 and bacterial HSP65. IBD-specific epitopes were found in many regions of the HSP60 and in even more regions of the HSP65 molecule including an IBD-specific T cell epitope in region X as well. These findings indicate that the epitope specificity of the anti-human and anti-mycobacterial HSP60 antibodies associated with various diseases is different.     

Water relations and osmotic adjustment in Apium graveolens during long‐term NaCl stress and subsequent relief

Pea plants ( Pisum sativum L. cv. Feltham First) exposed to a heat stress of 37°C for 6 h accumulated two low molecular weight (LMW) heat shock proteins (HSPs) of molecular mass 22 kDa. The two LMW HSPs were associated with purified mitochondria. N‐terminal amino acid sequencing analysis indicates that the more basic of these proteins is a novel protein. The response of other cultivars of P. sativum to heat shock revealed that up to three 22‐kDa HSPs were expressed in a cultivar‐specific manner. Evidence presented suggests that the different 22‐kDa HSPs arise as a result of there being multiple 22‐kDa HSP genes. The expression of the most basic novel HSP was studied in the Feltham First cultivar using two dimensional SDS‐PAGE. Treatment of intact plants with chloramphenicol and cycloheximide prior to heat stress treatment indicated that the LMW HSPs were nuclear encoded and de novo synthesised. The response to heat shock was rapid with protein expression detected within 45 min and the protein remained in excess of 6 days following removal of the stress. The protein accumulated to very high levels with maximal expression being 2% of the total mitochondrial protein. The results are discussed in relation to the likely role of LMW HSPs in thermotolerance.     

Enhanced expression of a 27 kD protein during diethylnitrosamine-induced hepatocarcinogenesis in rats

Patterns of neosynthesized cellular proteins from normal rat liver and from diethylnitrosamine-induced neoplastic nodules and hepatocellular carcinomas were analyzed by radiolabeling and fluorography of two-dimensional gel electrophoregrams. Three proteins exhibited a significant and reproducible increase in labeling intensity in the nodules (n = 5) and in the tumors (n = 10) as compared to the normal liver (n = 10). Two of those proteins (MW 31 and 33 kD, pI of 5.25, 5.15 respectively) are secreted proteins and as yet, we have no clue as to their nature. The third one is an intracellular protein of 27 kD pI 5.5. Several similarities in physico-chemical properties (MW, pI, phosphorylated state, low methionine content) indicate that this 27 kD protein might be the 27 kD heat shock protein (27 HSP). This is further supported by our observation that the cadmium-induced 27 HSP comigrates with our 27 kD protein.