Low Molecular Weight Polypeptides Research Articles

Porcine purple acid phosphatase: heterologous expression, characterization, and proteolytic analysis

Uteroferrin is an iron-binding glycoprotein, which is abundantly synthesized in porcine uterine glandular endometrium and believed to be involved in maternal/fetal iron transport. In the present study, uteroferrin has been cloned and functionally expressed using baculovirus-infected insect host cells Spodoptera frugiperda. The work also addresses the possible role of proteolytic cleavage to facilitate the release of uteroferrin-bound iron. The enzyme secreted in culture medium exhibits a molecular mass and catalytic properties similar to native porcine uteroferrin. The specific activity was estimated at 233 U/mg using p-nitrophenyl phosphate as substrate. Partial cleavage of the enzyme with trypsin resulted in a 1.7-fold enhancement in specific activity and a two-subunit polypeptide as observed in preparations of most mammalian purple acid phosphatases. Digestion with the aspartic protease pepsin resulted in a 2.5-fold enzyme inactivation correlated with the appearance of low molecular weight polypeptide fragments and the release of enzyme-bound iron.

Transgenic chickpea seeds expressing high levels of a bean -amylase inhibitor

We describe a robust and reproducible Agrobacterium-mediated chickpea transformation method based on kanamycin selection, and its use to introduce the bean αAI1 gene into a desi type of chickpea. Bean αAI1 was specifically expressed in the seeds, accumulated up to 4.2% of seed protein and was processed to low molecular weight polypeptides as occurs in bean seeds. The transgenic protein was active as an inhibitor of porcine α-amylase in vitro. Transgenic chickpeas containing α-AI1 strongly inhibited the development of Callosobruchus maculatus and C. chinensis (Col. : Bruchidae) in insect bioassays.

Interferon-gamma differentially regulates susceptibility of lung cancer cells to telomerase-specific cytotoxic T lymphocytes.

There is accumulating evidence that peptides derived from the catalytic subunit of human telomerase reverse transcriptase (hTERT) are specifically recognized by CD8+ cytotoxic T lymphocytes. We investigated the cytotoxicity of a human leukocyte antigen (HLA)-A*2402-restricted hTERT-derived peptide 461-469 (hTERT461)-specific CD8+ T-cell clone, designated as K3-1, established from a healthy donor by repetitive peptide stimulation. This clone exhibited cytotoxicity against 4 out of 6 HLA-A24-positive lung cancer cell lines with positive telomerase activity but not 4 HLA-A24-negative examples. When the target cells were pretreated with 100 U/ml of interferon (IFN)-gamma for 48 hr, the susceptibility to K3-1 increased with PC9 cells but unexpectedly decreased with LU99 cells. However, in both cell lines, the expression of molecules associated with epitope presentation such as HLA-A24, transporters associated with antigen processing, low molecular weight polypeptide 7 and proteasome activator 28 was similarly increased after IFN-gamma treatment. Results of CTL assays using acid-extracted peptides indicated that the epitope increased on PC9 cells but not on LU99 cells after IFN-gamma treatment. Semi-quantitative reverse transcriptase polymerase chain reaction disclosed that the expression of hTERT was attenuated in LU99 but not in PC9 cells, accounting for the decreased cytotoxicity mediated by K3-1. The attenuation of the hTERT expression and K3-1-mediated cell lysis after IFN-gamma treatment was also observed in primary adenocarcinoma cells obtained from pulmonary fluid of a lung cancer patient. Our data underline the utility of peptide hTERT461 in immunotherapy for lung cancer, as with other malignancies reported earlier, and suggest that modulation of hTERT expression by IFN-gamma needs to be taken into account in therapeutic approach.

Cowpea ( Vigna unguiculata) vicilins bind to the peritrophic membrane of larval sugarcane stalk borer ( Diatraea saccharalis)

In this work, we show that vicilins from two Vigna unguiculata (cowpea) genotypes, Epace-10 and IT 81D-1045, which are susceptible and resistant to attack by the cowpea weevil Callosobruchus maculatus, respectively, associate with the peritrophic membrane (PM) from larvae of Diatraea saccharalis. Solutions with increasing concentrations of vicilins were incubated with PM of the larvae and subsequently analysed by electrophoresis with SDS. It was observed that the majority of the bands of approximately 50,000 Da (characteristic of vicilins) did not appear in the separating gel and only lower molecular weight polypeptides were seen. When vicilins were incubated with PM, and the solution was then heated after the incubation, the band pattern in the gel appeared completely different. It was observed that the vicilins were being hydrolysed by proteinases associated with the PM. When the incubated samples were heated after the reaction, the major bands reappeared, demonstrating that most of the vicilin molecules had bound to the PM of D. saccharalis. These results suggest that when the vicilins are in contact with the PM they are bound and also digested by the PM of this insect. The major and several minor proteinases from the PM were extracted with Triton X-100 and their activity and the inhibition of this activity were analysed by in gel assays. Based on the effects of proteinase inhibitors, the PM-associated activity is due to serine class proteinases. Larvae of D. saccharalis were fed on artificial diets containing purified vicilins from Epace-10 or IT 81D-1045 seeds. Vicilins from Epace-10 did not affect the larval development, while IT 81D-1045 vicilins reduced significantly the survival rate of the sugar cane borer.

Effect of transglutaminase-induced cross-linking on gelation of myofibrillar/soy protein mixtures

Microbial transglutaminase (MTGase)-catalyzed interaction and gelation of mixed myofibrillar (MPI)/soy (SPI) protein isolates were investigated at varying ionic strengths and MPI:SPI ratios, with or without SPI being preheated (80 °C). MTGase treatments in deionized water converted myosin heavy chain and actin into lower molecular-weight polypeptides, which gradually diminished as the ionic strength increased up to 0.6 M NaCl. A reduced intensity in the electrophoretic bands of soy proteins (7S and 11S except the basic subunits) was observed in all treatments, suggesting cross-linking with MPI. The enzyme treatment slightly increased the thermal transition (denaturation) temperatures of MPI/SPI but greatly enhanced ( P<0.05) the elasticity of the mixed protein gels when compared with untreated samples, independent of incubation time.

Expression and purification of human ribosomal proteins S3, S5, S10, S19, and S26

The cDNAs for the human ribosomal proteins S3, S5, S10, S19, and S26 were introduced into a pET-15b vector and recombinant proteins containing an N-(His) 6-fusion tag were expressed in high yields. To resolve the problem of frameshift during expression of S26 caused by the presence of tandem arginine codons in its mRNA that are rare in Escherichia coli, we substituted the rare AGA codon with the more frequent arginine codon (CGC) using a primer with this mutation for PCR amplification of S26 cDNA. All proteins were expressed mainly in the form of inclusion bodies and purified to homogeneity by metal affinity chromatography in one step (except for S3). Expression of the full-length S3 was accompanied by the formation of a low molecular weight polypeptide that was co-purified with S3 by metal affinity chromatography. Complete purification of S3 required an additional gel-filtration step. The proteins were refolded by stepwise dialysis. Both identity and purity of the proteins were confirmed by 2D PAGE. The proteins obtained could be used in a wide range of applications in biophysics, biochemistry, and molecular biology.

Impaired expression of proteasome subunits and human leukocyte antigens class I in human colon cancer cells.

The presentation of human leukocyte antigens (HLA) class I requires the coordinated expression of numerous components involved in antigen processing and antigen presentation. Tumor cells may alter the expression of these components to decrease HLA class I expression, allowing them to escape immune surveillance. The aim of this study is to investigate the expressions of these components, including proteasome subunits, and their involvement in the expression of HLA class I in human colon cancer cells. Four human colon cancer cell lines, HCT116, SW403, LoVo and DLD-1, were used to examine the expression of HLA class I by flow cytometry. Reverse transcription-polymerase chain reaction was performed to assess the expression of beta2-microglobulin, heavy chains, transporter subunits, immunoproteasomes subunits and proteasome activator 28 (PA28) subunits. Human leukocyte antigen class I was expressed highly in HCT116 and SW403 cells and weakly in LoVo cells, but was not expressed in DLD-1 cells. The DLD-1 cells were deficient in the expression of proteasome subunits including low molecular weight polypeptide proteasome subunit 2 (LMP2), multicatalytic endopeptidase complex-like-1 (MECL-1), PA28alpha and PA28beta, whereas other HLA class I-expressing cell lines expressed all components tested. gamma-Interferon (IFN-gamma) treatment of DLD-1 cells restored the expression of LMP2, MECL-1 and PA28beta, but not the expression of HLA class I. Enforced expression of PA28alpha induced the expression of HLA class I in IFN-gamma-treated DLD-1 cells, but not in untreated DLD-1 cells. These results suggest that the impaired expression of proteasome subunits is involved in the loss of HLA class I expression in human colon cancer cells.

LMP2 and LMP7 gene polymorphism in Mexican populations: Mestizos and Amerindians.

Low molecular weight polypeptide (LMP) genes are located within the major histocompatibility complex and have been associated with autoimmune diseases such as ankylosing spondylitis. In order to define the distribution of LMP genes in Mexican populations, the LMP2 and LMP7 polymorphism was analyzed in 312 Mexican individuals (95 Mexican Mestizos, 48 Nahuas, 56 Mazatecans, 50 Teenek, and 63 Mayos) belonging to different ethnic groups. In Mexican populations both Mestizos and Amerindians presented similar distribution of LMP2 and LMP7 polymorphisms, except Nahuas and Mayos who presented the higher frequencies of LMP2-H/H and the lowest frequencies of LMP2-H/R genotypes (P < 0.05 when compared with Mexican Mestizos). The LMP7-K/K genotype was absent in Nahuas, Teenek and Mayos and only one Mazatecan individual presented this genotype. Differences with other populations were found in Mexicans. An increased frequency of LMP2-H and a decreased frequency of LMP2-R alleles were observed in Mexican Amerindians (Nahuas and Mayos) when compared with Brazilian Amerindians (Kaingang and Guarani) and Caucasians (Spaniards) (P < 0.05). All Mexican populations (Mestizos and Amerindians) presented an increased frequency of LMP7-Q allele and a decreased frequency of LMP7-K allele when compared to Brazilian Amerindians (Kaingang), Caucasians (United States) and Asian (Japan) populations (P < 0.05). Genetic distances showed that Mexican Mestizos have an important relation with Spaniards and with all Mexican Amerindians. The present data corroborate the influence of Spaniard and Amerindian genes in the Mexican Mestizo population and could help to define the true significance of LMP polymorphism as genetic and evolutive marker in the Amerindian populations.

Low molecular weight polypeptides in virgin and refined olive oils

AbstractTwenty‐eight virgin olive oils—from different regions of Spain and prepared from olive drupes of different varieties—and six refined olive oils were analyzed to determine the presence of proteins in these oils. All oils studied showed the presence of proteins in the range of 7–51 μ/100 g of oil. There were no significant differences in protein content in oils from different varieties or between virgin or refined oils. In addition, all oils exhibited analogous amino acid patterns, suggesting a similarity among protein fractions obtained from different oils. A polypeptide with an apparent M.W. of 4600 Da was common to the isolated protein fractions. These results suggest that this polypeptide is a previously unknown minor component in olive oils. No clear influence of this component on oil stability was observed when oil stabilities were estimated as a function of phenol, tocopherol, phosphorus, and protein contents of the oils.

Characterization of materials ejected by excimer laser ablation of hydrated collagen gel

The materials ejected from the hydrated collagen gel by ArF laser (193 nm) ablation were investigated using time-resolved photography, atomic force microscopy (AFM), and FTIR attenuated total reflectance spectroscopy (FTIR-ATR). The time-resolved photography suggested that the characteristics of the ejected materials were affected by the laser fluence and the surface condition, and the driving force of the material ejection would be the cavitation bubble collapse. The AFM and FTIR-ATR measurements demonstrated that measurable-sized materials were ejected by the laser ablation and the structure of the collagen in the ejected materials was partially changed from the original. Some of the ejected materials were found to consist of low molecular weight polypeptides.

Lack of the Small Plastid-encoded PsbJ Polypeptide Results in a Defective Water-splitting Apparatus of Photosystem II, Reduced Photosystem I Levels, and Hypersensitivity to Light

Photosystem II is a large pigment-protein complex catalyzing water oxidation and initiating electron transfer processes across the thylakoid membrane. In addition to large protein subunits, many of which bind redox cofactors, photosystem II particles contain a number of low molecular weight polypeptides whose function is only poorly defined. Here we have investigated the function of one of the smallest polypeptides in photosystem II, PsbJ. Using a reverse genetics approach, we have inactivated the psbJ gene in the tobacco chloroplast genome. We show that, although the PsbJ polypeptide is not principally required for functional photosynthetic electron transport, plants lacking PsbJ are unable to grow photoautotrophically. We provide evidence that this is due to the accumulation of incompletely assembled water-splitting complexes, which in turn causes drastically reduced photosynthetic performance and extreme hypersensitivity to light. Our results suggest a role of PsbJ for the stable assembly of the water-splitting complex of photosystem II and, in addition, support a control of photosystem I accumulation through photosystem II activity.

Lamina-associated polypeptide 2 (LAP2) expression in fish and amphibians.

Somatic and germinal cells of 15 fish and 33 amphibian species were examined by SDS-PAGE followed by immunoblotting to determine the expression of LAP2 (lamina-associated polypeptide 2). LAP2 expression in frogs, salamanders and fish does not vary with the mode of reproduction. In fish and frog cells, a rim-like LAP2 positive region was detected around the nucleus by indirect immunofluorescence microscopy. The cell distribution and expression patterns of LAP2 in fish, frogs and salamanders are comparable with those found in Xenopus and zebrafish. The mammalian somatic cell pattern, which may also occur in gymnophione amphibians, includes LAP2alpha, beta and gamma as major isoforms, whereas LAP2alpha does not occur in cells of fish, frogs and salamanders. In fish, LAP2gamma is the major isoform of somatic cells, suggesting that LAP2gamma may be ancestral. However, in the rainbow trout, as in frogs and salamanders, LAP2beta was the major somatic isoform. Fish and frog sperm only express low molecular weight polypeptides. In contrast, fish and frog oocytes express an oocyte-specific LAP2 isoform of high molecular weight. In the toad Bufo marinus this isoform becomes upregulated in pre-vitellogenic oocytes of 150-200 microm in diameter. The absence of LAP2alpha and the differential expression of LAP2 isoforms in somatic and germ cells, as found in fish and frogs, may be ancestral vertebrate characters. In spite of differences in developmental time, the LAP2 isoforms of somatic cells are upregulated during gastrulation, suggesting that LAP2 may be implicated in the early development of fish and frog.

Cis elements for transporter associated with antigen-processing-2 transcription: two new promoters and an essential role of the IFN response factor binding element in IFN-gamma-mediated activation of the transcription initiator.

Expression of cell surface MHC class I:peptide complex requires coordinated expression of multiple genes such as MHC class I heavy chain, beta(2)-microglobulin (beta(2)m), transporters associated with antigen-processing (TAP)-1 and TAP-2, and proteosomal components low-molecular weight polypeptide (LMP)-2 and LMP-7. All of these genes are expressed at defined and distinct levels in normal tissues, and are inducible by IFN-gamma. While the cis elements involved in transcription of the MHC class I heavy chain, beta(2)m, TAP-1 and LMP-2 have been analyzed extensively, those for TAP-2 and LMP-7 have not been well studied. Here we systematically analyzed the cis elements for TAP-2 transcription. We found at least two independent elements that are sufficient to activate transcription of a reporter gene. One (hereby called TAP-2 P1) is located 5' to the TAP-2 exon 1, while the other (hereby called TAP-2 P2) is a transcription initiator residing in intron 1. Analysis of the 5' sequence of TAP-2 mRNA indicates that both promoters are active. Moreover, while the TAP-2 promoter region contains cis elements that can mediate TAP-2 induction by IFN-gamma, such as gamma-activation site and IFN response factor binding element (IRFE), only the IRFE is required for IFN-gamma induction of TAP-2 promoter in vitro. The IRFE appears to work as an enhancer for the initiator (P2). Together with another promoter recently identified by others, TAP-2 therefore has three independent promoters that can be differentially regulated.

Influence of zinc ions on protein secretion in a heavy metal tolerant strain of the ericoid mycorrhizal fungus Oidiodendron maius.

A heavy metal tolerant strain of the ericoid mycorrhizal species Oidiodendron maius, isolated from soil heavily contaminated with zinc, was previously shown to tolerate high concentrations of zinc and cadmium ions in the growth medium. We have investigated some of the specific molecular responses of this fungal strain to the presence of increasing concentrations of zinc ions in the growth medium. In particular, we show that zinc ions induce a general change in the array of secreted proteins, with a shift towards the production of more basic, low molecular weight polypeptides. Some of these proteins were microsequenced and identified through homology search in databases. Among them are hydrolytic enzymes (nuclease, proteinase, lysozyme) and two superoxide dismutase isoforms. The latter are antioxidant enzymes known to play a role in heavy metal response in plants, animals and microorganisms.

Distribution of HLA‐A, B alleles and polymorphisms of TAP and LMP genes in Korean patients with atopic dermatitis

Atopic dermatitis has been seen to result from multifactorial inheritance, with interaction between genetic and environmental factors. The genetic association may differ according to the ethnic backgrounds. The purpose of this study was to investigate the genetic factors in Korean atopic dermatitis patients by studying the human leucocyte antigen (HLA) class I association and polymorphisms of transporters associated with antigen presentation (TAP) and low-molecular-weight polypeptide (LMP) genes. HLA-A and B genotyping was performed in 53 atopic dermatitis patients and 184 healthy controls using the standard microlymphocytotoxicity technique. TAP1, TAP2, LMP2, and LMP7 gene polymorphisms were anaylzed using the polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP), PCR-amplification refractory mutation system (ARMS), and PCR-restriction fragment length polymorphism (RFLP). Allele frequency of HLA-A24 was significantly increased in patients with atopic dermatitis compared to controls (P < 0.05). HLA-B alleles showed no differences in distribution between patients and controls. Genotype, phenotype, and allele frequencies of TAP1 gene also revealed no differences in distribution between patients and controls. Analysis of TAP2 gene polymorphisms showed increased frequencies of the TAP2*C allele and TAP2*A/TAP2*C genotype in atopic dermatitis patients compared to controls (P < 0.05). Distribution of LMP2 and LMP7 gene polymorphisms was similar for patients and controls. This study demonstrates an association of atopic dermatitis with HLA-A24 and TAP2*C alleles in Korean patients. Discrepancy with the previous reports might be related to different patient characteristics and ethnic variations.

Yolk granules are differentially acidified during embryo development in the stick insect Carausius morosus.

Newly laid eggs of stick insects comprise a unique fluid ooplasm that is gradually partitioned into a number of yolk granules by invasion of secondary vitellophages. This study aimed at establishing how yolk granules become acidified in the course of embryonic development. Data show that acidified yolk granules are rather scarce and randomly distributed in vitellophages of early embryos, while they tend to increase gradually in number as development proceeds to completion. Yolk granule acidification is progressively more inhibited in the presence of increasing concentrations of chloroquine, monensin and bafilomycin. A pro-protease was identified cytochemically and by immunoblotting in yolk extracts of progressively more advanced embryos. A specific monoclonal antibody raised against this pro-protease helped to demonstrate that it is gradually processed to yield a lower molecular weight polypeptide as development proceeds to completion. This latter polypeptide was identified as a protease using electrophoresis in polyacrylamide gels containing yolk extracts. Simultaneous administration of a fluorescent substrate for cysteine protease and an acidotropic probe produced superimposable labelling patterns, suggesting that only acidified yolk granules possess a proteolytic activity. On the other hand, yolk granules probed simultaneously for acidification and latent pro-protease yielded labelling patterns partially superimposed. Pro-protease labelling is gradually lost as yolk granules are progressively more acidified during development. Distinct labelling patterns were also obtained in vitellophages processed for the simultaneous detection of pro-protease and protease, suggesting that the two activities are expressed by different yolk granule populations, and that one is gradually converted into the other as time goes by.

CBQCA assay of primary amine losses during hemodialysis

Nitrogen loss during hemodialysis is an important issue since all steps taken to improve clearance of urea will also increase loss of amino acids and other normally recyclable sources of nitrogen. Most dialyzers exclude albumin and other proteins, which react well with dyes commonly employed to measure protein, but do allow passage of mono- and oligopeptides that go undetected in protein assays. The CBQCA reagent provides a highly sensitive assay of primary amines that will detect amino acids, low molecular weight polypeptides, and high molecular weight proteins. We collected dialysate using a split stream technique from 28 chronic dialysis patients. With bovine albumin as the standard, the CBQCA assay reported 24.3±9.8 g albumin-equivalents (mean±S.D.) per dialysate whereas a Coomassie blue assay measured 1.19±0.78 g. The CBQCA assay values were substantially higher than previously reported amino acid losses. The CBQCA fluorescent assay for amines provides a simple assay for quantifying primary amine losses in dialysate fluid.

Characterization of purified His-tagged CP47-containing photosystem II complexes from a cyanobacterium, Synechocystis sp. PCC 6803

For the determination of the exact structure of PS II complexes, all the polypeptide constituents must be identified. For this purpose, highly active photosystem II (PS II) complexes were purified from a HT-3 mutant of Synechocystis sp. PCC 6803 whose CP47 has been His-tagged. Fluorescence measurements showed the intactness of the donor and acceptor sides of purified PS II complexes. It retained 80% of the initial activity of oxygen evolution even after incubation of the complexes for 2 weeks at 4°C in the dark. Using these highly purified PS II complexes, the polypeptide composition was analyzed by SDS-PAGE which can resolve polypeptides of low molecular weight. Separated polypeptides of the purified PS II complexes were subjected to N-terminal amino acid sequencing and matrix assisted laser desorption (MALDI) mass spectrometry. More than 20 Coomassie-stainable polypeptides were identified. All the polypeptides which was known as PS II components were found, except PsbN. Besides 19 polypeptides of well-known PS II member, some polypeptides, which had not been shown to bind to PS II complexes until now, were found. Among such polypeptides, the sll1638 gene product was found to be highly homologous to PsbQ. These PS II complexes contained the FtsH protein, which was assigned to be a protease of the D1 protein, and the Ycf9 (Orf62 in tobacco), which was suggested to be present in stromal thylakoids. The results obtained here showed the usefulness of this HT-3 strain for the investigation of the structure, function and biogenesis of PS II complexes.

Towards an Understanding of Biological Role of Colostrinin Peptides

The aim of this study was to elucidate the structure and possible function of colostrinin, also known as a proline rich polypeptide (PRP). The molecular weight of colostrinin was originally determined by gel filtration to be 17,200 daltons. In the presence of guanidinum chloride, however, the molecular weight was found to be about 6,000 daltons. Further studies utilizing high-performance liquid chromatography (HPLC) and mass spectroscopy revealed that colostrinin is a complex consisting of many low molecular-weight polypeptides. A total of 32 peptides were isolated from the original colostrinin preparation by HPLC and subjected to the N-terminal sequence analysis. The results of sequence analysis revealed significant homology of the peptides to three protein precursors: annexin, beta-casein, and a hypothetical beta-casein homolog. In addition, the sequence of several peptides showed no significant homology to any specific protein in the current GenBank database. The synthetic peptides of various lengths representing the N-terminal sequence of the colostrinin peptides were made to study some biological effects. Here we report that colostrinin and some of its component peptides are potent inducers of leukocyte proliferation and of certain cytokines. Also, a series of monospecific antibodies were produced in rabbits against the synthetic peptides. The antibodies were used to study the kinetic of antigen reduction in colostrum and mature milk following lambing. A threefold decrease was common for most antigens studied over the period of 72 h. Based on the results of these studies we postulate that colostrinin represents a diverse group of peptides produced in the mammary gland of mammals for the development of the optimal physiologic responses in offspring. Also, it is hoped that the beneficial use of colostrinin in Alzheimer's Disease (AD), recently reported elsewhere, will revive interest in its clinical application for treatment and/or prophylaxis of many age-related disorders.

Activation-dependent degradation of protein kinase C eta.

Prolonged activation of protein kinase Cs (PKCs) by long-term treatment of cells with phorbol ester tumor promoters down-regulates the expression of many PKCs. To investigate the molecular mechanisms involved in the down-regulation of PKC eta, we expressed the novel PKCs eta and &theta; and various mutant forms in baby hamster kidney cells. Upon overexpression, constitutively active PKC eta, but not wild type or kinase-dead PKC eta, underwent rapid degradation to generate several lower molecular weight polypeptides. When co-expressed with active kinases, kinase-dead PKC eta with a pseudosubstrate site mutation designed to give an active conformation was down-regulated while the wild type PKC eta was not. These results suggest requirements for kinase activity and an active conformation for down-regulation of PKC eta. Treatment with the proteasome inhibitors N-Ac-Leu-Leu-norleucinal and lactacystin led to accumulation of PKC eta proteolytic products and potentially ubiquitinated forms. While wild type PKC eta localizes mostly to the detergent-soluble fraction of the cell, a significant portion of full-length constitutively active PKC eta and of kinase-dead, active conformation PKC eta were found in the detergent-insoluble fraction. Several proteolytic fragments of constitutively active PKC eta also were found in the detergent insoluble fraction. These full-length and proteolytic fragments of PKC eta in the detergent-insoluble fraction accumulated further in the presence of proteasome inhibitors. These data suggest that active conformation PKC eta accumulates in the detergent-insoluble compartment, is degraded by proteolysis in the presence of kinase activity, and that the cleavage products undergo further degradation via ubiquitin-mediated degradation in the proteasome. Oncogene (2000) 19, 4263 - 4272