kDa Variant Research Articles

CFR-1 receptor as target for tumor-specific apoptosis induced by the natural human monoclonal antibody PAM-1

The human monoclonal antibody PAM-1 was isolated from a patient with stomach cancer. The germ-line coded IgM antibody identifies a recently described 130 kDa variant of CFR-1 (cysteine-rich fibroblast growth factor receptor 1). This CFR-1/PAM-1 receptor is post-transcriptionally modified and over-expressed on human epithelial tumors and carcinoma pre-cancer lesions such as H. pylori induced gastritis, intestinal metaplasia and dysplasia of the stomach, ulcerative colitis-related dysplasia and adenomas of the colon, Barrett metaplasia and dysplasia of the esophagus, squamous cell metaplasia and dysplasia of the lung and cervical intraepithelial neoplasia. Furthermore, the expression of CFR-1/PAM-1 correlates with the proliferation rate and increases with the grade of malignancy. This study demonstrates that the human monoclonal antibody PAM-1 inhibits cell growth and induces apoptosis, in vitro and in vivo. Both, the unique tumor-specific expression of the CFR-1/PAM-1 receptor and the growth inhibitory effect of the PAM-1 antibody makes this combination a good diagnostic and therapeutic tool for all kinds of epithelial cancers and precursor lesions.

Interaction of estrogen receptor α with protein kinase C α and c-Src in osteoblasts during differentiation

In cultured osteoblasts, protein kinase C (PKC) activity increases and estrogen receptor α (ERα) binding capacity decreases upon confluence. We investigated potential interactions between ERα and PKC isoforms and their confluence-induced modulations in clonal ROS.SMER#14 cells and primary osteoblasts. In sub-confluent ROS.SMER#14 cells, which express an exogenous plus small amounts of the endogenous ERα gene, the receptor appeared as two main bands of ≈66 and ≈46 kDa. In over-confluent, more differentiated cells, the cytosolic ≈66 kDa ERα appeared decreased and the ≈46 kDa variant increased. Enhanced expression and/or membrane translocation of PKCα and PKCε, but not PKCζ, was evidenced at over-confluence, along with transient increases in expression and kinase activity of c-Src, accompanied by membrane translocation of the kinase-activated enzyme. In contrast, negligible membrane translocation of PKCα and/or activated c-Src was observed in parental ROS 17/2.8 cells, which express low levels of full-length ERα. PKCα from over-confluent cells phosphorylated p60 c-Src in vitro, suggesting functional interaction between the two kinases. ERα co-immunoprecipitated c-Src and PKCα, mostly in its cleaved form (PKMα). An analogous interaction was observed in primary osteoblasts. However, in these cells, much more PKCα/PKMα was ERα-co-immunoprecipitated at over-confluence, a condition in which the shorter, ≈46 kDa ERα variant is increased. This interaction was enhanced by estradiol treatment or PKC down-regulation, but was unaffected by c-Src inhibition. These data highlight direct PKCα–c-Src–ERα interactions, which may be crucial in the modulation of estrogen responsiveness and the differentiation process in osteoblasts.

A frequent allele codes for a truncated variant of semenogelin I, the major protein component of human semen coagulum.

Human semen coagulum predominantly consists of high molecular mass complexes of the seminal vesicle secreted semenogelin I (SgI) and semenogelin II (SgII). Here we describe a previously unknown variant of the SgI gene that is present at an allele frequency of approximately 3% in the Swedish population. It gives rise to a protein with a molecular mass of 43 kDa, SgI(43), which compared with the 50 kDa variant, SgI(50), is lacking a tandem repeat of 60 amino acid residues that was probably deleted by homologous recombination. In spite of the size difference, SgI(43) has many properties in common with SgI(50), such as a very high iso-electric point and susceptibility to proteolytic degradation by prostate-specific antigen. Heterozygous carriers of the SgI(43) allele neither show impaired fertility nor do they significantly differ from individuals homozygous for SgI(50) with respect to sperm parameters such as semen volume, sperm count and fraction of motile spermatozoa.

Binding of CaMKII to the giant muscle protein projectin: stimulation of CaMKII activity by projectin

Projectin is an integral high molecular mass protein of insect flight muscle binding to myosin and paramyosin. Yet, the role of projectin in insect flight muscle is not well understood. In this study we provide evidence for the interaction of projectin with the calcium sensor Ca 2+/calmodulin-dependent protein kinase II (CaMKII). Two CaMKII variants of 52 and 60 kDa, respectively, of locust flight muscle cells were shown by an anti-CaMKII antibody. Both variants were associated to myofibrils. The 52 kDa variant was also shown abundant in the cytosol. The cytosolic CaMKII variant was co-precipitated in vitro with externally added purified projectin in a dose-dependent manner. To specify the binding properties of CaMKII to projectin we used both purified projectin from the flight muscle of locust and CaMKII from rat forebrain, a naturally rich source of CaMKII. CaMKII is highly conserved even between insects and mammals. The binding of CaMKII to native projectin was demonstrated in vitro by the solid phase enzyme assay, immunoprecipitation, and ‘overlay’ binding. One mol projectin bound maximally 1.38±0.02 mol CaMKII in vitro with a K d of 3.08±0.09 nM. Application of in vitro autophosphorylated CaMKII revealed a decreased stoichiometry of binding to projectin (0.86±0.04 mol mol −1) accompanied by a lower affinity ( K d of 5.54±0.73) compared to non-autophosphorylated CaMKII. Furthermore, the CaMKII phosphotransferase activity was stimulated up to 2-fold by projectin. Even in the presence of calmodulin projectin enhanced the CaMKII activity moderately. Our data suggest that projectin represents a subcellular compartment for CaMKII to achieve its specificity, and activity in insect flight muscle cells.

PTP-PEST, a scaffold protein tyrosine phosphatase, negatively regulates lymphocyte activation by targeting a unique set of substrates.

There is increasing interest in elucidating the mechanisms involved in the negative regulation of lymphocyte activation. Herein, we show that the cytosolic protein tyrosine phosphatase PTP-PEST is expressed abundantly in a wide variety of haemopoietic cell types, including B cells and T cells. In a model B-cell line, PTP-PEST was found to be constitutively associated with several signalling molecules, including Shc, paxillin, Csk and Cas. The interaction between Shc and PTP-PEST was augmented further by antigen receptor stimulation. Overexpression studies, antisense experiments and structure-function analyses provided evidence that PTP-PEST is an efficient negative regulator of lymphocyte activation. This function correlated with the ability of PTP-PEST to induce dephosphorylation of Shc, Pyk2, Fak and Cas, and inactivate the Ras pathway. Taken together, these data suggest that PTP-PEST is a novel and unique component of the inhibitory signalling machinery in lymphocytes.

TARPP, a novel protein that accompanies TCR gene rearrangement and thymocyte education.

Studies on thymic T cell development have usually concentrated on cell surface molecules. However, intracellular proteins expressed only in thymocytes have never been described. Here we report the discovery of a novel thymocyte-specific protein, named TARPP, which represents a high molecular mass ( approximately 100 kDa) variant of the previously identified protein ARPP-21 ( approximately 21 kDa). TARPP is a cytosolic protein that is expressed at high levels in immature thymocytes. It appears concomitant with the commitment to T cell lineage, and its expression is switched off as a consequence of TCR engagement during positive selection. Such an expression pattern, correlating with the rearrangement of the TCR genes and thymocyte education, suggests a role for TARPP during this important phase of thymocyte development.

Autoantibodies to the mRNA destabilizing protein hnRNP-D/AUF1 in patients with systemic autoimmune diseases

The heterogeneus nuclear ribonucleoprotein (hnRNP) D, which is also known as AU-rich element binding factor 1 (AUF1), decreases stability of many short-lived mRNAs (including mRNAs of IL-1 and TNFa) by binding to adenosine and uridine rich sequences (ARE) contained in their 3'-untranslated regions. Previous studies had indicated this protein to be recognized by sera from patients RA, SLE and MCTD. To investigate this autoimmune response in greater detail 356 sera from patients (pts) with various rheumatic disorders were tested by immunoblotting employing the recombinant 45 kDa variant of D/AUF1 (the largest of the 4 known D/AUF1 proteins). Autoantibodies (aab) were detected in 20% of RA pts (n = 105), 34% of SLE pts (n = 70), 17% of MCTD pts (n = 31) and in 25% of pts with primary Sjoegren's syndrome (n = 21), but in less than 5% of 129 pts with other rheumatic disorders and not at all in healthy controls. Importantly, anti-D/AUF1 aab were already present in 25% of 60 patients with early RA of less than 6 months duration, whereas only one of 40 non-RA patients with other forms of early arthritis showed this antibody. Epitope mapping studies showed the aab to be directed to conformational epitopes in the N-terminal part of hnRNP-D/AUF1 known to be indispensable for the protein's function. However, aab did not interfere with RNA binding as assessed by gel shift assays employing the ARE of the TNFa mRNA. Instead, they were able to supershift protein-RNA complexes indicating binding sites for RNA and aab to be distinct. Thus, these findings suggest that anti-D/AUF1 aab target the mRNA decay complex which may form another large ribonucleoprotein target structure in systemic autoimmunity. We are tempted to speculate that increased formation of such complexes (e.g. due to overexpression of instable mRNAs such as those for IL-1 and TNFa as seen in RA) may lead to pathologic autoimmune reactions against D/AUF1 and other proteins of mRNA decay complexes.

Biogenesis of G-protein Mediated Calcium Signaling in Human Megakaryocytes

To understand how platelet signal transduction pathways develop during megakaryocytopoiesis, we isolated human stem cells from umbilical cord blood and cultured the cells in the presence of thrombopoietin (TPO). Based on the early expression of CD61 and late expression of CD42b, immature (CD61+/CD42b(low)) and mature (CD61+/ CD42b(high)) megakaryocytes were immunomagnetically purified and, together with stem cells (CD34+), characterized for Galpha-protein expression and agonist-induced [Ca2+]i increases. Megakaryocytopoiesis was accompanied by down-regulation of the 43 kDa and 46 kDa variants of G16alpha, constant expression of Gsalpha, and up-regulation of Gqalpha and Gialpha1/2. The increase in Gqalpha and Gialpha1/2 expression was accompanied by an increase in Ca2+ signaling triggered by thrombin and other agonists known to signal to Ca2+ via these G-proteins in platelets. The prostacyclin analog iloprost and TPO also induced [Ca2+]i increases, and the iloprost-induced Ca2+ response disappeared during maturation. These data reveal sharp changes in Ca2+ regulation during megakaryocytopoiesis.

Senescent human fibroblasts have elevated Ku86 proteolytic cleavage activity

A proteolytic activity capable of cleaving the Ku86 subunit of Ku protein to two polypeptides, with molecular masses of 69 and 17 kDa in vitro, is present in a human diploid fibroblast (HDF) cell line. The activity is elevated in late-passaged and senescent cells, and the cleaved 69-kDa product seems able to form complex with Ku70 to bind DNA ends. However, further studies indicate that cleavage of Ku86 could be inhibited by including leupeptin in the extraction buffer, and no 69 kDa variant was evident in the cell. In fact, the levels of Ku86, Ku70 and DNA-end binding activity of Ku remained unchanged during replicative senescence. Thus, this study reveals an intriguing protease in HDFs, and also indicates that inconsistent results of Ku86 expression will be obtained if the protease activity is not completely inhibited.

Stratum corneum thiol protease (SCTP): a novel cysteine protease of late epidermal differentiation.

Proteolytic enzymes play crucial roles in the formation of the stratum corneum barrier tissue and in its subsequent maturation. Despite this, the proteases involved in stratum corneum physiology are not well characterized. Hence, studies were performed to identify these proteolytic enzymes present in the peripheral layers of this tissue using a combination of tape stripping and zymography. Using this approach, a novel human cysteine protease was identified and characterized, and named stratum corneum thiol protease (SCTP). Gelatin zymography revealed that SCTP is composed of two variants with apparent molecular weights of 34 and 35 kDa which do not correspond to any previously described stratum corneum protease. Mechanistically SCTP belongs to the cysteine proteinase class as shown by: (1) acid protease activity, (2) a requirement for mild reducing conditions, and (3) the specific inhibition of activity by E64 and Z-phe-ala-diazomethylketone. Further analysis using concanavalin A affinity chromatography demonstrated that the two 34 and 35 kDa variants are both glycoproteins, which, after removal of the oligosaccharide sidechains with the specific enzyme N-glycopeptidase F, reveal a single active core protease of 32 kDa. SCTP did not crossreact with antibodies raised against the lysosomal cysteine proteases cathepsins B, H or L, thereby distinguishing it from the classical cysteine cathepsins. Localization studies revealed that SCTP is present at all depths in the stratum corneum, thereby precluding microbial contamination as the enzyme source. Moreover, it was also present at all body sites investigated, except for the hyperkeratotic palmoplantar stratum corneum. SCTP was found to be a product of late differentiation in cultured human keratinocytes; the enzyme was synthesized by differentiated calcium-switched cells and secreted into the medium, whereas nondifferentiated basal keratinocytes did not produce this protease. Moreover, human fibroblast cultures did not produce the enzyme, suggesting that SCTP is not produced by the dermis and hence is epidermal specific. The function of SCTP is unknown, but the observed gelatinolytic activity coupled with its secretion into the medium by cultured keratinocytes indicates that physiologically it is responsible for the degradation of extracellular structural proteins. Furthermore, the optimal activity at acid pH suggests that it can function in the acidic environment of the stratum corneum. It remains to be elucidated whether this enzyme has a role in desquamation.

Type 4 Cyclic Adenosine Monophosphate-Specific Phosphodiesterases Are Expressed in Discrete Subcellular Compartments during Rat Spermiogenesis*

The type 4 cAMP-specific phosphodiesterases (PDE4) are a family of closely related enzymes with similar catalytic domains and divergent amino- and carboxyl-terminus domains. Multiple PDE proteins with heterogeneous amino termini are derived from each gene. To understand the significance of this heterogeneity, the expression and localization of variants derived from PDE4A and PDE4D genes was investigated during spermatogenesis in the rat. RNase protection analysis with mRNA for testes at different ages of development showed that two transcripts (PDE4D1 and PDE4D2) are expressed at day 10 and 15 of age and become undetectable thereafter. An additional PDE4D transcript appears at day 30 and increased during testid maturation. This latter transcript codes for a long variant of the PDE4D gene and is expressed in germ cells as demonstrated by RNase protection with RNA from isolated pachytene spermatocytes and round spermatids. The presence of a corresponding PDE4D protein with a molecular mass of 98 kDa was established by immunoprecipitation and Western blot analysis with antibodies specific for PDE4D and by immunoaffinity chromatography purification of the 98 kDa variant from isolated germ cells. PDE4A transcripts were also expressed in pachytene spermatocytes and round spermatids. Two polypeptides encoded by these PDE4A transcripts were expressed in pachytene spermatocytes, reached a maximum in round spermatids, and declined thereafter. Immunofluorescence analysis demonstrated a localization of the PDE4D protein in the manchette and in a periacrosomal region of the developing spermatid, a localization confirmed by immunogold electron microscopy. Conversely, the PDE4A was mostly soluble in the cytoplasm of round spermatids. These data demonstrate that PDE4D and PDE4A variants are expressed at different stages and localized in distinct subcellular structures of developing spermatids. Different properties of the mRNAs derived from the two genes and localization signals are responsible for the temporal and spatial expression of the different PDE4 isoenzymes.

Antigenic Variation in Malaria: a 3′ Genomic Alteration Associated with the Expression of a P. knowlesi Variant Antigen

Antigenic variation of malaria parasites was discovered in P. knowlesi, using a schizont-infected cell agglutination (SICA) assay to detect variant antigens expressed at the surface of infected erythrocytes. Later studies utilizing stable clones, Pk1(A+) and its direct derivative, Pk1(B+)1+, showed that SICA[+] clones express distinct parasite-encoded antigens of ∼200 kDa. Here we identify a P. knowlesi variant antigen gene and cDNA and demonstrate that it encodes the 205 kDa variant antigen expressed by B+ parasites. This gene belongs to a multigene family, which we term SICAvar. Its ten-exon structure with seven cysteine-rich coding modules is unique compared to P. falciparum var genes. Further, we highlight a 3′ genomic alteration that we predict is related to SICAvar gene switching.

Attenuated function of a variant form of the helix-loop-helix protein, Id-3, generated by an alternative splicing mechanism

The Id family of helix-loop-helix proteins function as negative regulators of DNA binding, basic helix-loop-helix proteins in the regulation of cell growth and differentiation. We report here on the identification of a 17 kDa variant of the 14 kDa Id-3 protein termed Id-3L (long version) which possesses a unique 60 amino acid carboxy-terminus generated by readthrough of a ‘coding intron’ and alternative splicing. Northern analysis revealed expression of a minor 1.1 kb Id-3L transcript together with the predominant 0.95 kd Id-3 transcript in the majority of adult human tissues analysed. The variant Id-3L protein is functionally distinguishable from conventional Id-3 since in in vitro DNA mobility shift assays, it was greatly impaired in its ability to abrogate binding of the basic helix-loop-helix protein, E47, to an E box recognition sequence.

Articular chondrocyte tenascin-C production and assembly into de novo extracellular matrix.

Tenascin-C is an oligomeric glycoprotein of the extracellular matrix that is expressed in a variety of processes including development, tissue remodeling, wound healing, cell adhesion/antiadhesion, and cell/matrix interactions. Tenascin has recently been acknowledged as a component of the extracellular matrix of articular cartilage, but its function remains unclear. In this study, bovine articular chondrocytes were grown in alginate beads for 35 days to examine the kinetics of tenascin synthesis and incorporation into de novo extracellular matrix. During the culture period, 6 harvest days were established in which culture medium was recovered, alginate beads were dissociated with an EDTA solution, and chondrocytes were collected and lysed by sonication. Total DNA determination performed on the cell lysates demonstrated chondrocyte survival and proliferation. Western blotting performed on the medium, EDTA/alginate, and lysate samples demonstrated the production of both the 220 and 320 kDa tenascin size variants and their differential compartmentalization within the culture system. Tenascin was incorporated into the alginate bead matrix at a constant rate of 3.8 micrograms/day. The 320 kDa variant was produced in higher quantity, but the 220 kDa fragment was twice as likely to be incorporated into the de novo matrix. Methylene blue/acid fuchsin staining and tenascin immunohistochemistry demonstrated the incorporation of tenascin into a progressively expanding matrix surrounding the chondrocytes. The results suggest a role for tenascin in the assembly of the chondrocyte matrix and as a soluble mediator of chondrocytes with possible diverse functions for the tenascin size variants.

Localization of a high molecular weight form of DNA topoisomerase I in amphibian oocytes.

Xenopus oocytes express a 165 kDa variant of DNA topoisomerase I (topo I) as opposed to the canonical 110 kDa form of somatic cells (Richard and Bogenhagen, Dev. Biol. 146: 4-11, 1991). By immunofluorescence microscopy using variant-specific antibodies we show that this high molecular weight form is associated with lampbrush chromosome loops and the inner regions of the amplified nucleoli. Inhibition of topo I-activity by either Camptothecin-treatment or microinjection of neutralizing antibodies resulted in loop retraction and the condensation of chromosomes and amplified nucleoli. These data indicate that the oocyte-specific 165 kDa form of topo I is involved in transcriptional processes mediated by RNA polymerase I and II and is therefore functionally equivalent to the somatic cell 110 kDa counterpart.

Characterization of lineage restricted forms of a Xenopus CD45 homologue

The leukocyte common antigen, also known as CD45, is a structurally heterogenous molecule ranging in molecular weight from 180 to 220 kDa. CD45 belongs to a family of high molecular weight, cell surface glycoproteins expressed on all hematopoietic lineages with the exception of mature erythrocytes. In higher vertebrates, the highly conserved cytoplasmic domain of CD45 exhibits protein tyrosine phosphatase activity and has been implicated in lymphocyte activation through dephosphorylation of critical tyrosine residues on substrates associated with signal transduction pathways. The monoclonal antibody CL21 recognizes a high molecular weight determinant expressed on the surface of Xenopus leukocytes which was postulated to be a CD45 homologue. In order to determine if lymphocyte subpopulations expressed different molecular weight variants, splenic B cells were identified and isolated on the basis of surface IgM and the CL21 determinant expressed by these cells was compared to the determinant expressed by thymocytes. Immunoprecipitation revealed that IgM+B cells expressed a 220 kDa molecular weight variant whereas thymocytes and IgM-cells expressed a 180 kDa variant. Bone marrow myeloid cells, isolated on the basis of light scatter properties, expressed a determinant which ranged from 150 to 160 kDa. Dephosphorylation experiments utilizing p-nitrophenyl phosphate, 32P-labeIed Raytide [tyr(P)], or Kemptide [ser(P)] as substrates demonstrated that immunoprecipitated CL21 antigen exhibited tyrosine specific phosphatase activity which was inhibited by sodium orthovanadate. Thus, data based on the presence of enzymatic activity and lineage restricted molecular weight variants support the hypothesis that the CL21 determinant is the amphibian homologue of mammalian CD45, and suggest that both structural and functional elements of CD45 have been conserved during vertebrate evolution.

Proteins in tissue extracts which bind insulin-like growth factor binding protein-3 (IGFBP-3).

Membrane associated IGFBP-3 is now known to play a role in the modulation of IGF at the cellular level, but mechanisms involved in cell membrane binding are far from certain. In this study we report the identification and initial structural characterisation of proteins in a range of sheep and rat tissues which specifically bind recombinant human non-glycosylated IGFBP-3. Tissues were homogenised in Tris HCl (0.1 M, pH 7.4), containing proteolytic enzyme inhibitors and the residues re-extracted in buffer containing SDS and Triton X-100 (both 1% w/v) prior to analysis. These were subjected to SDS-PAGE, electro-blotted onto nitrocellulose and subjected to ligand blot analysis (LBA) using radioiodinated IGFBP-3 as ligand. LBA revealed a major band of binding activity migrating at 60 kDa in extracts of rat muscle while sheep muscle contained forms of 52 and 40 kDa as the principal species and ovine pancreatic extracts an abundance of the 40 kDa variant alone. Distribution of the binding activity appears tissue specific. Apart from skeletal muscle, pancreas and a small amount of the 52 kDa form in pituitary, analysis revealed no evidence of IGFBP-3 binding in a range of other sheep tissues including liver, kidney, spleen, intestine, adrenal, brain, mammary, uterus, ovary and plasma. The binding activity is TCA precipitable, trypsin digestible, dose responsive and relatively specific for IGFBP-3 since the related proteins recombinant human IGFBP-2 and purified caprine IGFBP-4 failed to bind.(ABSTRACT TRUNCATED AT 250 WORDS)

Annexin 3 is associated with cytoplasmic granules in neutrophils and monocytes and translocates to the plasma membrane in activated cells.

Annexins are soluble proteins capable of binding to phospholipid membranes in a calcium-dependent manner. Annexin 3, a 33 kDa protein mainly expressed in neutrophils, aggregates granules in cell-free assays, and a 36 kDa variant of this protein, specifically expressed in monocytes, has recently been identified. To obtain further information on these proteins, we defined their subcellular localization in resting and activated cells by immunofluorescence microscopy. Both proteins were associated with cytoplasmic granules in resting cells. We obtained evidence to indicate that, in neutrophils which possess a heterogenous granule population, annexin 3 was more likely to be associated with the specific granules. In cells activated with phorbol 12-myristate 13-acetate or opsonized zymosan, the 33 kDa and 36 kDa proteins translocated to the plasma or the phagosome membrane. Upon stimulation with A23187, annexin 3 translocated to the plasma membrane only in neutrophils. We also report that while annexin 3 was associated with restricted membranes in intact cells, it binds indiscriminately to every membrane fraction in cell-free assay. In conclusion, association of both forms of annexin 3 with granules suggests that these proteins could be implicated in processes of granule fusion.

Detection of intracellular interleukin 2: Evidence for novel immunologically related forms of the lymphokine

At present, few data are available on intracellular interleukin 2 (IL-2) and its posttranscriptional regulation. Unlike other lymphokines, IL-2 does not accumulate within the cell, but is rapidly secreted following its production. The process of detection and biochemical characterization of intracellular IL-2 involved using a high producer subclone of the Jurkat T-lymphoma line as a source for IL-2, in combination with a two-step separation protocol and a sensitive detection method. Following phytohemagglutinin (PHA) 4β-phorbol 12-myristate 13 acetate (TPA) stimulation, a 14 kDa molecule could be visualized in Western blots by means of two monoclonal anti-IL-2 antibodies possessing different epitope specificities. This molecule exhibited biological activity of IL-2 as determined by a murine cytotoxic T-cell proliferation assay. In addition to this biologically active form of the lymphokine, a strongly immunoreactive protein with a molecular weight of 54 kDa (P54) was found in Jurkat cell lysates. Further biochemical characterization of this intracellular variant revealed an iso-electric point similar to that of secreted forms of IL-2. All attempts to split the 54 kDa molecule into smaller subunits failed, and no biological IL-2 activity could be measured in response to P54. However, the appearance of this high molecular weight variant followed clear-cut time kinetics. The highest concentration of P54 was found to occur after 2 h of stimulation. Thereafter its concentration decreased continuously, while the amount of the biologically active 14 kDa variant increased under ongoing stimulation. One possible explanation for these results is that P54 may represent an immature form of IL-2 that is tightly linked to a carrier molecule.

Human leucocyte glycosylasparaginase is an alpha/beta-heterodimer of 19 kDa alpha-subunit and 17 and 18 kDa beta-subunit.

Human lysosomal glycosylasparaginase (AGA; EC 3.5.1.26) consists of two glycosylated subunits, alpha and beta. Treatment with 3% SDS at 45 degrees C as part of a new purification scheme did not affect enzyme activity, but the alpha-subunit migrated an apparent 19 kDa peptide on SDS/PAGE instead of as a 24 kDa peptide, as observed without this SDS treatment. The N-terminal sequence was similar to that of the 24 kDa form, and, after reversed-phase h.p.l.c., the 19 kDa form was transformed to an apparent 24 kDa peptide on SDS/PAGE, indicating that their primary structures were identical. As the molecular mass of the alpha-subunit deduced from its cDNA was 19.5 kDa, the variation might be due to incomplete SDS coating of the 24 kDa form. This was confirmed by the tendency of the 24 kDa variant to polymerize even in the presence of SDS. The molecular mass of the beta-subunit was 17 and 18 kDa in accordance with previous reports. Chemical cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide resulted in the appearance of a 38 kDa peptide on SDS/PAGE which reacted with both the subunit-specific antisera on Western-blot analysis. On SDS/PAGE at pH 10.2 the active enzyme migrated as an apparent 43 kDa peptide. These results indicate that native human glycosylasparaginase is a heterodimer.