Biosynthesis of the multiple forms of rabbit cardiac cathepsin D

Rabbit cardiac cathepsin D exists as multiple isomeric forms of Mr = 48,000 within cardiac tissue. Their mechanism of formation and their functional role in cardiac protein degradation are unknown. We have previously demonstrated that cathepsin D is initially synthesized as an Mr = 53,000 precursor that is processed by limited proteolysis within cardiac lysosomes to the Mr = 48,000 active forms of the enzyme. To determine if the multiple forms of active cathepsin D originate from a common precursor, isolated perfused Langendorff rabbit hearts were labeled in pulse (15 or 30 min) and pulse-chase (30 or 150 min) experiments with [35S]methionine. Newly synthesized cathepsin D was isolated by butanol/Triton X-100 extraction and immunoadsorption with anti-cathepsin D IgG-Sepharose, and the isomeric forms were separated by two-dimensional electrophoresis and fluorography. After 15- and 30-min pulse perfusions, 35S-labeled cathepsin D appeared as a single precursor form (Mr = 53,000, pI = 6.6). After 30-min pulse and 30-min chase, the precursor was modified to yield multiple precursor forms, all with molecular weight 53,000, but with differing pI values (6.6-6.0). After 30-min pulse and 150-min chase perfusion, multiple forms of both precursor and proteolytically processed active cathepsin D (Mr = 48,000, pI = 6.2-5.6) were detected. The 35S-labeled, proteolytically processed forms of active cathepsin D co-migrated with the major cathepsin D forms present in cardiac tissue. Subcellular fractionation and perfusions in the presence of chloroquine demonstrated that the multiple precursor forms of cathepsin D originated in a nonlysosomal intracellular compartment. Thus, the multiple forms of active cathepsin D originate from a common high molecular weight precursor, and their synthesis occurs prior to the limited proteolysis of the precursor in cardiac lysosomes.     

Biosynthesis of a lysosomal enzyme. Partial structure of two transient and functionally distinct NH2-terminal sequences in cathepsin D

Various biosynthetic forms of porcine spleen cathepsin D (Erickson, A. H. and Blobel, G. (1979) J. Biol. Chem. 254, 11771-11774), isolated by immunoprecipitation of in vivo- and in vitro-synthesized products, have been characterized by partial NH2-terminal sequence analysis. Two short lived and functionally distinct NH2-terminal sequence extensions, a "pre" sequence and a "pro" sequence, have been detected. Both sequence extensions are present in preprocathepsin D which is the primary translation product immunoprecipitated after translation of porcine spleen mRNA in a wheat germ cell-free system. Preprocathepsin D is not glycosylated and has an approximate Mr = 43,000. Its 20-residue pre sequence resembles the signal sequences of presecretory proteins in abundance of Leu residues (7 out of 20 residues). Addition of dog pancreatic microsomal vesicles to the translation system resulted in the cleavage of the pre sequence and yielded segregated and glycosylated procathepsin D (Mr = 46,000) that was indistinguishable from its in vivo-synthesized counterpart detected after pulse-labeling of cultured porcine kidney cells. Some of this in vivo-synthesized procathepsin D was secreted and persisted as such in the culture medium. The remainder was converted within a period of 15 min to 2 h to single chain cathepsin D (Mr = 44,000) by removal of a pro sequence which was estimated to be 44 residues. Its partial sequence showed considerable sequence homology to the 44-residue activation peptide of pepsinogen. It is possible, therefore, that the prosequence of procathepsin D serves as an activation peptide that keeps the enzyme inactive during intracellular transport to the lysosome. The enzymatically active single chain form of cathepsin D undergoes further cleavage into a light and a heavy chain (Mr = 15,000 and 30,000, respectively) over a period of 2-24 h after synthesis. The oligosaccharide moieties of procathepsin D and of the single chain and heavy chain forms of cathepsin D are cleaved by endoglycosidase H. Treatment of cells with tunicamycin arrests the biosynthetic pathway of cathepsin D at procathepsin D. The nonglycosylated procathepsin D is not proteolytically processed and its secretion is greatly inhibited.     

Proteolysis of factor Va by factor Xa and activated protein C

Bovine Factor Va, produced by selective proteolytic cleavage of Factor V by thrombin, consists of a heavy chain (D chain) of Mr = 94,000 and a light chain (E chain) of Mr = 74,000. These peptides are noncovalently associated in the presence of divalent metal ion(s). Each chain is susceptible to proteolysis by activated protein C and by Factor Xa. Sodium dodecyl sulfate electrophoretic analysis indicates that cleavage of the E chain by either activated protein C or Factor Xa yields two major fragments: Mr = 30,000 and Mr = 48,000. Amino acid sequence analysis indicates that the Mr = 30,000 fragments have identical NH2-terminal sequences and that this sequence corresponds to that of intact E chain. The Mr = 48,000 fragments also have identical NH2-terminal sequences, indicating that activated protein C and Factor Xa cleave the E chain at the same position. Sodium dodecyl sulfate electrophoretic analysis indicates that activated protein C cleavage of the D chain yields two products: Mr = 70,000 and Mr = 24,000. Amino acid sequence analysis indicates that the Mr = 70,000 fragment has the same NH2-terminal sequence as intact D chain, whereas the Mr = 24,000 fragment does not. Factor Xa cleavage of the D chain also yields two products: Mr = 56,000 and Mr = 45,000. The Mr = 56,000 fragment corresponds to the NH2-terminal end of the D chain and Factor V. Functional studies have shown that both chains of Factor Va may be entirely cleaved to products by Factor Xa without loss of activity, whereas activated protein C cleavage results in loss of activity. Since activated protein C and Factor Xa cleave the E chain at the same position, the cleavage of the D chain by activated protein C is responsible for the inactivation of Factor Va.     

Amino acid sequences of the human kidney cathepsins H and L

The complete amino acid sequences of human kidney cathepsin H (EC 3.4.22.16) and human kidney cathepsin L (EC 3.4.22.15) were determined. Cathepsin H contains 230 residues and has an Mr of 25116. The sequence was obtained by sequencing the light, heavy and mini chain and the peptides produced by cyanogen bromide cleavage of the single-chain form of the enzyme. The glycosylated mini chain is a proteolytic fragment of the propeptide of cathepsin H. Human cathepsin L has 217 amino acid residues and an Mr of 23720. Its amino acid sequence was deduced from N-terminal sequences of the heavy and light chains and from the sequences of cyanogen bromide fragments of the heavy chain. The fragments were aligned by comparison with known sequences of cathepsins H and L from other species. Cathepsins H and L exhibit a high degree of sequence homology to cathepsin B (EC 3.4.22.1) and other cysteine proteinases of the papain superfamily.     

Identification of renal cathepsin B as a human prorenin-processing enzyme

Prorenin, the inactive biosynthetic precursor of renin, is proteolytically cleaved in the renal juxtaglomerular cells to renin. The activity of renin is rate-limiting for generation of angiotensin II in the circulation. We identified a renal thiol protease which activates and accurately cleaves the 43-amino acid prosegment of human recombinant prorenin. In the current studies, 6.5 mg of this protease was purified from human renal cortex using a three-step procedure dependent upon Leu-Leu-arginyl affinity chromatography. This represented an overall 766-fold purification and resulted in three protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of molecular weights 30,000, 25,000, and 24,000. All three bands cross-reacted with an anti-human liver cathepsin B antibody upon immunoblot analysis; electrolution of each band and amino-terminal sequence analysis confirmed that the Mr 30,000 protein was mature cathepsin B and the Mr 25,000 and 24,000 bands were cathepsin B subunits. The pH optimum for the hydrolysis of pure human recombinant prorenin by pure renal cathepsin B was 6, and the Michaelis-Menten constant, Km, of the reaction was 1.4 x 10(-9) M. Immunostaining of human kidney using a sheep anti-human cathepsin B antibody demonstrated the presence of cathepsin B in the juxtaglomerular areas of the kidney, as well as in the renal proximal tubules. Electron microscopic immunohistochemistry using the same antibody demonstrated cathepsin B in dense secretory granules of the juxtaglomerular cells. Renin was also shown to be present in these granules. This study provides both biochemical and morphological evidence that renal cathepsin B is a human prorenin-processing enzyme.     

Isolation and sequencing of a cDNA clone encoding rat liver lysosomal cathepsin D and the structure of three forms of mature enzymes

We isolated and sequenced a cDNA clone corresponding to the entire coding sequence of rat liver lysosomal cathepsin D. The deduced amino acid sequence revealed that cathepsin D consists of 407 amino acid residues (Mr 44,608) and the 20 NH2-terminal residues seem to constitute a cleavable signal peptide after which 44 amino acid residues follow as a propeptide. Two putative N-linked glycosylation sites and aspartic acid in the active site are as well conserved as those of human lysosomal cathepsin D. In the NH2-terminal sequence analysis of two isolated heavy chains of the mature enzyme, the termini were assigned as tryptophan (118th residue) and glycine (165th or 166th residue), respectively, hence demonstrates that the two heavy chains derive from a split of the single chain of cathepsin D at position between 117th and 118th or between 164th and 165th or 165th and 166th amino acids. We conclude that cathepsin D in rat liver lysosomes is a mixture of three forms composed of a single and two two-chain forms. However, the amounts of the two two-chain forms are low compared with that of the single chain form. Densidometric determination after SDS-PAGE revealed that the two two-chain forms account for less than 5% of the single chain form. There is a 82% similarity in amino acid level between rat and human liver lysosomal cathepsin D.     

Expression of human cathepsin D in Xenopus oocytes: phosphorylation and intracellular targeting

We have obtained expression of a cDNA clone for human cathepsin D in Xenopus laevis oocytes. Biosynthetic studies with [35S]methionine labeling demonstrated that most of the cathepsin D remained intracellular and underwent proteolytic cleavage, converting a precursor of Mr 47,000 D to a mature form of Mr 39,000 D with processing intermediates of Mr 43,000-41,000 D. greater than 90% of the cathepsin D synthesized by oocytes bound to a mannose 6-phosphate (Man-6-P) receptor affinity column, indicating the presence of phosphomannosyl residues. An analysis of [2-3H]mannose-labeled oligosaccharides directly demonstrated phosphomannosyl residues on cathepsin D. Sucrose-gradient fractionation, performed to define the membranous compartments that cathepsin D traversed during its biosynthesis, demonstrated that cathepsin D is targeted to a subpopulation of yolk platelets, the oocyte equivalent of a lysosome. Xenopus oocytes were able to endocytose lysosomal enzymes from the medium and this uptake was inhibited by Man-6-P, thus demonstrating the presence of Man-6-P receptors in these cells. Therefore, the entire Man-6-P dependent pathway for targeting of lysosomal enzymes is present in the oocytes. Xenopus oocytes should be a useful system for examining signals responsible for the specific targeting of lysosomal enzymes to lysosomes.     

Influx of bivalent cations can be independent of receptor stimulation in human endothelial cells.

The major active forms of cathepsins B and L were identified in Kirsten-virus-transformed mouse fibroblasts by the use of a specific radiolabelled inhibitor, benzyloxycarbonyl-Tyr(-125I)-Ala-CHN2. No other proteins were labelled, demonstrating the specificity of this inhibitor for cysteine proteinases. Cathepsins B and L were distinguished by the use of specific antibodies. One active form of cathepsin B, Mr 33,000-35,000, and two active forms of cathepsin L, Mr 30,000 and 23,000, were identified. The intracellular precursors of these proteins had higher Mr values of 39,000 and 36,000 for cathepsins B and L respectively, as shown by pulse-chase experiments with [35S]methionine-labelled proteins. These did not react with the inhibitor under our culture conditions. The precursor of cathepsin L was secreted whereas the precursor of cathepsin B was not, demonstrating that secretions of the two enzymes are regulated differently. In contrast with results found previously for the purified protein [Mason, Gal & Gottesman (1987) Biochem. J. 248, 449-454], the secreted precursor form of cathepsin L did not react with the inhibitor either, indicating that it is not active and therefore, as such, cannot be directly involved in tumour invasion. The secreted protein did react with the inhibitor when incubated at pH 3.0, showing that the protein can be activated, although this did not occur under our culture conditions.     

Functional expression of intrinsic factor-cobalamin receptor by renal proximal tubular epithelial cells.

Previous studies from our laboratory (Seetharam, B., Levine, J. S., Ramasamy, M., and Alpers, D. H. (1988) J. Biol. Chem. 263, 4443-4449; Fyfe, J. C., Ramanujam, K. S., Ramaswamy, K., Patterson, D. F., and Seetharam, B. (1991) J. Biol. Chem. 266, 4489-4494) have identified and isolated a 230-kDa receptor from rat and canine kidney which binds with high affinity [57Co]cyanocobalamin (Cbl) complexed to gastric intrinsic factor (IF). Although these studies have identified a renal receptor which binds intrinsic factor-cobalamin (IFCR), it is not known whether the binding is specific for IF-Cbl and whether renal cells internalize [57Co]Cbl bound to IF and transport [57Co]Cbl across the cell. Using a variety of renal cells, our results show that IF-[57Co]Cbl binding activity is detected in proximal tubular-derived epithelial cells from opossum (OK) and porcine kidney (LLC-PK1) but not in distal tubular-derived cells from canine kidney cells (MDCK). Metabolic labeling studies with Tran 35S-label confirmed the presence of a 230-kDa IFCR in OK and LLC-PK1 cells. Cell surface labeling and binding studies demonstrated that IFCR is targeted to the apical membrane. This apical expression of IFCR in OK cells is inhibited by the microtubule-disruptive drugs, colchicine and nocodazole. Opossum kidney cells when grown on culture inserts are polarized and transport [57Co]Cbl only when bound to IF and not to other Cbl binders. Furthermore, the transport of [57Co]Cbl occurred unidirectionally from the apical to the basolateral surface. Treatment of cells with colchicine or nocodazole inhibited the surface binding of IF-[57Co]Cbl as well as the transcytosis of [57Co]Cbl by 70-75%. IFCR retained intracellualarly by incubation of cells with colchicine or nocodazole is degraded by leupeptin-sensitive proteases. Based on these results, we suggest that proximal tubular-derived epithelial cells transport [57Co]Cbl bound to IF in a saturable way via receptor-mediated endocytosis.     

Purification by affinity chromatography of the glycine receptor of rat spinal cord

The glycine receptor of rat spinal cord was solubilized with the nonionic detergent Triton X-100 and subsequently purified by affinity chromatography on aminostrychnine-agarose and wheat germ agglutinin-Sepharose. An overall purification of 1950-fold was achieved. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol revealed three glycine receptor-associated polypeptides of Mr = 48,000, 58,000, and 93,000. [3H]Strychnine was incorporated irreversibly into the Mr = 48,000 polypeptide upon UV-illumination. The dissociation constant (KD) of [3H]strychnine binding to the purified glycine receptor was 9.3 +/- 0.6 nM. The glycine receptor agonists glycine, beta-alanine, and taurine inhibited the binding of [3H]strychnine to the purified receptor. Gel filtration and sedimentation in sucrose/H2O and sucrose/D2O gradients gave a Stokes radius of 7.7 nm, a partial specific volume of 0.780 +/- 0.005 ml/g and a sedimentation coefficient s20,w of 8.2 +/- 0.2 S for the purified glycine receptor. From these data, a molecular weight of 246,000 +/- 6,000 was calculated for the glycine receptor protein.     

Monoclonal antibodies that coimmunoprecipitate the 1,4-dihydropyridine and phenylalkylamine receptors and reveal the Ca2+ channel structure

Monoclonal hybridoma cell lines secreting antibodies against the (+)-PN 200-110 and the (-)-demethoxyverapamil binding components of the voltage-dependent calcium channel from rabbit transverse-tubule membranes have been isolated. The specificity of these monoclonal antibodies was established by their ability to coimmunoprecipitate (+)-[3H]PN 200-110 and (-)-[3H]demethoxyverapamil receptors. Monoclonal antibodies described in this work cross-reacted with rat, mouse, chicken, and frog skeletal muscle Ca2+ channels but not with crayfish muscle Ca2+ channels. Cross-reactivity was also detected with membranes prepared from rabbit heart, brain, and intestinal smooth muscle. These antibodies were used in immunoprecipitation experiments with 125I-labeled detergent [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and digitonin] solubilized membranes. They revealed a single immunoprecipitating component of molecular weight (Mr) 170,000 in nonreducing conditions. After disulfide bridge reduction the CHAPS-solubilized (+)-PN 200-110-(-)-demethoxyverapamil binding component gave rise to a large peptide of Mr 140,000 and to smaller polypeptides of Mr 30,000 and 26,000 whereas the digitonin-solubilized receptor appeared with subunits at Mr 170,000, 140,000, 30,000, and 26,000. All these results taken together are interpreted as showing that both the 1,4-dihydropyridine and the phenylalkylamine receptors are part of a single polypeptide chain of Mr 170,000.     

Effect of vitamin D deficiency on the composition and in vitro labeling of rat kidney proteins

Densitometric analysis of single-dimension gels consistently demonstrated that, in addition to rat renal calcium binding protein (CaBP) (Mr 28,000), two other kidney proteins of Mr 16,500 and Mr 18,000 were significantly enriched in their contents in the vitamin D-replete rat. Partial characterization of the Mr 18,000 and 16,500 proteins revealed that these proteins were heat-stable and distinct from calmodulin, as determined by their inability to undergo the calcium-dependent mobility shift in sodium dodecyl sulfate gels which is characteristic of calmodulin. The Mr 16,500 and Mr 18,000 kidney proteins did not cross-react with rat renal or rat intestinal CaBP antisera, as assessed by radioimmunoassay and Western blot analysis. A comparison of peptide maps of tryptic digests of these proteins and purified rat renal CaBP, as analyzed by high-pressure liquid chromatography, revealed no apparent homology. Protein synthesis studies using [35S]methionine and short-term tissue culture of kidney cortex fragments indicated that the most pronounced effect of vitamin D or 1,25 dihydroxyvitamin D3 was increased synthesis of the Mr 28,000 protein (3.2- to 4.6-fold increase compared to -D rats, P less than 0.001). Synthesis of a Mr 54,500 protein increased by 1.3- to 1.5-fold (P less than 0.05) and [35S]methionine incorporation into a Mr 66,000 protein decreased by 1.2- to 1.3-fold (P less than 0.05) in +D rats. This study represents the first detailed characterization of the effects of vitamin D on the composition and synthesis of rat kidney proteins. The data indicate that the most significant effect of vitamin D on kidney proteins is increased synthesis of the Mr 28,000 CaBP, suggesting that a major role of vitamin D in renal function is regulation of calcium transport at the distal tubule. However, dietary vitamin D or 1,25(OH)2D3 can influence the expression as well as the suppression of other specific kidney proteins.     

Cathepsins B and H from porcine spleen. Purification, polypeptide chain arrangements, and carbohydrate content

Procedures for the purification of cathepsins B and H from porcine spleens have been described. The purified porcine cathepsin B (Mr = 27,000) is predominantly a two-chain enzyme with a heavy chain (Mr = 22,000) and a light chain (Mr = 5,000). It also contains two minor forms of cathepsin B with different chain structures. Porcine cathepsin H is a single-chain enzyme with a molecular weight of 25,000. The carbohydrate analyses showed that these enzymes were glycoproteins. A glycopeptide containing 3 amino acids, 2 glucosamines, and 6 mannoses was isolated from cathepsin H. Proton NMR studies revealed that it contained a mixture of 4 high mannose-type of oligosaccharides characteristic of those found on lysosomal enzymes. The carbohydrate of cathepsin B consisted of a single residue of glucosamine and trace mannose. This sugar content is in agreement with the finding that about 80% of the porcine spleen cathepsin B contained a single N-acetylglucosamine while 20% of the enzyme contained a 5-sugar oligosaccharide (Takahashi, T., Schmidt, P. G. and Tang, J. (1984) J. Biol. Chem. 259, 6059-6062). Thus, the studies on carbohydrate contents also indicated the good purity of the enzymes.     

Effects of inhibition of N-linked glycosylation by tunicamycin on nucleoside transport polypeptides of L1210 leukemia cells

Membrane polypeptides (relative mass (Mr) 48,000--55,000) associated with the equilibrative transport of nucleosides were identified in cultured murine leukemia (L1210/C2) cells by site-specific photolabeling with [3H]nitrobenzylthioinosine ([3H]NBMPR). Growth of cells in the presence of tunicamycin resulted in the gradual conversion of 3H-labeled polypeptides to a form that migrated more rapidly (Mr 42,000--47,000) during sodium dodecyl sulfate (SDS)--polyacrylamide gel electrophoresis. When plasma membrane fractions were photolabeled and incubated with O-glycanase or endoglycosidase F, the [3H]NBMPR-labeled polypeptides migrated in SDS-polyacrylamide gels with the same mobility as native NBMPR-binding polypeptides, whereas incubation with either N-glycanase or trifluoromethane sulfonic acid converted [3H]NBMPR-labeled polypeptides to the more rapidly migrating form (Mr 41,000--48,000). These observations are consistent with the presence of N-linked oligosaccharides of the complex type on the NBMPR-binding polypeptides of L1210/C2 cells. Tunicamycin exposures that reduced incorporation of [3H]mannose into plasma membrane fractions by greater than 95% had little, if any, effect on either the affinity (Kd values, 0.1-0.2 nM) or abundance (Bmax values, 200,000--220,000 sites/cell) of NBMPR-binding sites, whereas uridine transport kinetics at 37 degrees C were altered in a complex way. Thus, although N-linked glycosylation is not required for insertion of the NBMPR-binding protein into the plasma membrane or for interaction of NBMPR with the high-affinity binding sites, it is important for function of at least one of the three nucleoside transporters expressed by L1210/C2 cells.     

The cell surface receptor for immunoglobulin E. Effect of tunicamycin on molecular properties of receptor from rat basophilic leukemia cells

Cell surface receptors for immunoglobulin E were isolated by repetitive affinity chromatography from rat basophilic leukemia cells biosynthetically labeled with L-[35S]methionine and D-[3H]mannose. Native immunoglobulin E receptor appeared as a very broad band in the 45,000 to 62,000 Mr region in sodium dodecyl sulfate polyacrylamide gels. However, from cells cultured in the presence of tunicamycin, a relatively narrow band with an apparent Mr of 38,000 was isolated. The 38,000 Mr band rebound to immunoglobulin E-Sepharose, was immunoprecipitated with antibodies to immunoglobulin E receptor, shared tryptic peptides with native receptor, and was labeled with L-[35S]methionine but not D-[3H]mannose, and thus appears to be immunoglobulin E receptor lacking N-linked oligosaccharides. It is demonstrated that N-linked oligosaccharides account for much of the apparent heterogeneity of native receptor in sodium dodecyl sulfate polyacrylamide gels and in two-dimensional gel electrophoresis. A receptor-associated protein with apparent Mr = 30,000, prominently labeled with L-[35S]methionine but not with D-[3H]mannose, did not have altered molecular properties when isolated from tunicamycin-cultured cells, and did not share tryptic peptides with receptor.     

Cell-associated proteoheparan sulfate from bovine arterial smooth muscle cells

Cell-associated proteoheparan sulfate has been isolated from bovine arterial smooth muscle cells preincubated with [35S]sulfate or a combination of [3H]glucosamine and [35S]methionine. The purified proteoheparan sulfate had an apparent Mr of 200,000 on calibrated Sepharose CL-2B columns. The glycosaminoglycan component (Mr approximately 30,000) was identified as heparan sulfate by its susceptibility to specific enzymatic and chemical degradation. After degradation of the proteoheparan sulfate by microbial heparitinase the resulting protein core had an apparent Mr of 92,000 on SDS-polyacrylamide gels. Its mobility was similar in the absence and presence of reducing agents indicating that the protein core consists of a single polypeptide chain. Pulse-chase experiments revealed that about 40% of the cell layer-associated proteoheparan sulfate was released into the medium, while the remainder was internalized and converted to smaller species through a series of degradation steps. Initially there was a proteolytical cleavage of the protein core generating glycosaminoglycan peptide intermediates with polysaccharides chains similar in size to the original. The half-life of the native proteoheparan sulfate was found to be about 4 h.     

A novel insulin-binding protein with a molecular weight of 30,000 daltons

Analysis of mouse Swiss/3T3 fibroblasts and rat hepatoma H35 cells using the affinity cross-linking method revealed multiple forms of 125I-insulin binding components (Mr greater than 300,000) in the absence of reducing agents. The same analysis, in the presence of reducing agents, revealed two major components (Mr = 125,000 and Mr = 30,000). The Mr = 125,000 component appeared to be the alpha-subunit of the high-affinity insulin receptor, whereas the small insulin-binding component of Mr = 30,000 was not a degradation product of the alpha-subunit but was apparently associated with the insulin receptor. We suggest that it is likely a novel component for regulating the function of insulin receptor.     

Evidence for a single steroid-binding protein in the rabbit progesterone receptor

The rabbit uterine progesterone receptor copurifies as two molecular weight (Mr) forms of about 105,000 and 78,000. To investigate whether these are different proteins, we have used protease digestion, reversible denaturation, and photoaffinity labeling in studies on the steroid-binding domain of the receptor. Digestion of the Mr 105,000 and 78,000 forms, photoaffinity labeled with [3H]R5020, with Staphylococcus aureus V8 protease revealed identical peptide fragments of Mr 43,000, 39,000, and 27,000-30,000. When receptor in cytosol was denatured, separated by electrophoresis, and then reconstituted, [3H]progesterone bound specifically to a single form at about Mr 105,000. After partial purification, the reversible denaturation procedure revealed both the larger and the smaller progesterone-binding species similar to the photoaffinity-labeled species in this preparation. Receptor in uterine cytosol prepared under mild conditions appeared as a predominant large molecular weight form on photoaffinity labeling with [17 alpha-methyl-3H]R5020, [6,7-3H]R5020, or [3H]RU27987. Further purification of this cytosol showed the generation of a smaller labeled species. These results from three different approaches reinforce the view that the rabbit progesterone receptor contains a single steroid-binding protein.     

DNA polymerase alpha-primase from calf thymus. Determination of the polypeptide responsible for primase activity

Immunoaffinity-purified DNA polymerase alpha-primase complex from calf thymus consists of subunits with molecular weights of 148,000-180,000, 73,000, 59,000, and 48,000 (Nasheuer, H.-P., and Grosse, F. (1987) Biochemistry 26, 8458-8466). Primase activity was separated from the immobilized complex by washing extensively with 2 M KCl or, alternatively, by shifting to pH 11.5 in the presence of 1 M KCl. From both elution procedures, the primase activity was found to be associated with the polypeptides with molecular weights of 59,000 and 48,000. The specific activity, using either elution procedure, was 30,000 units/mg. Both polypeptides sedimented together at 5.7 S upon zonal centrifugation on a sucrose gradient. Primase activity was found in the flow-through fraction after DEAE-cellulose chromatography of the free primase. Analysis of this fraction by sodium dodecyl sulfate gel electrophoresis revealed only one band with a Mr of 48,000. Polyclonal antibodies were raised against the Mr 59,000 and 48,000 polypeptides. The anti-Mr 59,000 antibody affected the primase activity only marginally, whereas the anti-Mr 48,000 antibody inhibited the primase activity nearly completely. UV cross-linking of the DNA polymerase alpha-primase complex with alpha-32P-labeled GTP revealed a binding site at the Mr 48,000 polypeptide, but none at the other subunits of the complex. Taken together, these results suggest that the Mr 48,000 polypeptide bears the active site of the DNA primase activity. The Mr 59,000 polypeptide stabilizes the primase activity.     

Isolation and functional characterization of the active light chain of activated human blood coagulation factor XI

Human blood coagulation Factor XIa was reduced and alkylated under mild conditions. The mixture containing alkylated heavy and light chains was subjected to affinity chromatography on high Mr kininogen-Sepharose. Alkylation experiments using [14C]iodoacetamide showed that a single disulfide bridge between the light and heavy chains was broken to release the light chain. The alkylated light chain (Mr = 35,000) did not bind to high Mr kininogen-Sepharose while the heavy chain (Mr = 48,000), like Factors XI and XIa, bound with high affinity. The isolated light chain retained the specific amidolytic activity of native Factor XIa against the oligopeptide substrate, pyroGlu-Pro-Arg-p-nitroanilide. Km and kcat values for this substrate were 0.56 mM and 350 s-1 for both Factor XIa and its light chain, and the amidolytic assay was not affected by CaCl2. However, in clotting assays using Factor XI-deficient plasma in the presence of kaolin, the light chain was only 1% as active as native Factor XIa. Human coagulation Factor IX was purified and labeled with sodium [3H]borohydride on its carbohydrate moieties. When this radiolabeled Factor IX was mixed with Factor XIa, an excellent correlation was observed between the appearance of Factor IXa clotting activity and tritiated activation peptide that was soluble in cold trichloroacetic acid. Factor XIa in the presence of 5 mM CaCl2 activated 3H-Factor IX 600 times faster than Factor XIa in the presence of EDTA. In the absence of calcium, Factor XIa and its light chain were equally active in activating 3H-Factor IX. In contrast to Factor XIa, the light chain in this reaction was inhibited by calcium ions such that, in the presence of 5 mM CaCl2, Factor XIa was 2000 times more effective than its light chain. Neither phospholipid nor high Mr kininogen and kaolin affected the activity of Factor XIa or its light chain in the activation of 3H-Factor IX. These observations show that the light chain region of Factor XIa contains the entire enzymatic active site. The heavy chain region contains the high affinity binding site for high Mr kininogen. Furthermore the heavy chain region of Factor XIa plays a major role in the calcium-dependent mechanisms that contribute to the activation of Factor IX.