Imbalance of purine metabolism in hepatomas of different growth rates as expressed in behavior of glutamine-phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase, EC 2.4.2.14).

The behavior of glutamine-phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase, EC 2.4.2.14) was determined in normal, differentiating, and regenerating liver and in a spectrum of hepatomas of widely different growth rates. The liver and tumor enzymes were measured in 100,000 x g supernatants prepared from 20% tissue homogenates containing 0.25 M sucrose and 1 mM MgC12. Kinetic studies were carried out on the amidotransferase in the curde supernatant from liver and rapidly growing hepatoma 3924A so that under optimum standard assay conditions only the enzyme amount would be the limiting factor. The kinetic results showed that certain properties of the amidotransferase from liver and hepatoma were similar. The liver and hepatoma enzyme exhibited apparent Km's for: glutamine, 1.7 and 2.3 mM; MgC12, 0.7 and 1.1 mM, and phosphoribosylpyrophosphate. S0.5 for 0.9 and 0.4 mM, respectively...

Amplification of growth inhibition by glucocorticoid on L5178Y and L1210 lymphoblasts in vivo.

One component of a factor in Proteus mirabilis (Factor 1) which specifically amplifies the induction of several liver enzymes by glucocorticoid in target cells also increases the growth inhibition of glucocorticoid on the ascitic form of L1210 cells and solid tumors of L5178Y lymphoblasts in vivo. The growth of L5U78Y and L1210 lymphoblasts was inhibited by a triamcinolone acetonide dose of over 0.5 to 1.0 mg/kg body weight. Factor 1 increased the inhibitory effect of a triamcinolone acetonide dose of less than 4.0 mg/kg bodyweight but had little effect on the effects of doses of over 4.0 mg. Factor 1 (10 biological units/kg body weight) itself also caused marked inhibition of the growth of these lymphoblasts without affecting the body weight or adrenal gland weight, its effect being equivalent to that of 3- to 4-mg/kg body weight doses of triamcinolone acetonide alone. There was no significant difference in the level of plasma total corticoids or that of plasma adrenocorticotropic hormone between rats treated with 0.9% NaCl solution or those treated with Factor 1, and Factor 1 had no cytotoxic effec on cultured L5178Y lymphoblasts. Thus, Factor 1 may amplify the effect of physiological level of glucocorticoid in mice sufficiently to inhibit the growth of these lymphoblasts without causing any significant side effects to the host animal.

Properties of fructose-1,6-bisphosphate aldolase inactivating enzymes in rat liver lysosomes.

The intralysosomal localization of the enzymes that catalyse inactivation of rat liver fructose-bisphosphate aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) to a form with antigenic activity was demonstrated. The inactivating enzymes like all other lysosomal markers tested except acid phosphatase, were readily solubilized by hypotonic shock. The inactivating enzyme activity was inhibited by PMSF, TPCK, TLCK and leupeptin, but not by pepstatin. On partial purification of the inactivating activity from the lysosomal fraction by DEAE-Sephadex (A-50) and Sephadex G-100 column chromatographies, it was copurified with lysosomal carboxypeptidase A and cathepsin B (EC 3.4.22.1). Studies on its substrate specificity and sensitivity to inhibitors indicated that cathepsin B and carboxypeptidase A are responsible for almost all the aldolase-inactivating activity in the lysosomal fraction.

Proteolytic modification of rat liver fructose-1,6-bisphosphate aldolase by administration of leupeptin in vivo.

When leupeptin, a thiol protease inhibitor of microbial origin, was injected into rats, the activity of fructose-1,6-bisphosphate aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) in the liver decreased to about 60% of that in control rats. However, the concentration of aldolase protein in the liver extracts, measured with a specific antibody obtained with enzyme purified on a phosphocellulose column, remained unchanged. Injection of leupeptin also caused a marked increase in the activities of free lysosomal proteases, such as cathepsin B (EC 3.4.22.1), cathepsin L (EC 3.4.22.-), cathepsin D (EC 3.4.23.5) and lysosomal carboxypeptidase A in the cytosol fraction. A clear inverse relationship between aldolase and cathepsin B activities in the cytosol fraction was demonstrated. The possibility that the less active form of aldolase detected in the livers of leupeptin-treated rats was produced during homogenization was excluded by showing that the aldolase activity was not changed by addition of various protease inhibitors to the homogenization medium., When insulin was coinjected with leupeptin, increase in the activity of free cathepsin L and decrease of activity of aldolase produced by the injection of leupeptin was prevented. These findings indicate that modification of aldolase may be due to the action of a lysosomal protease(s). Enhanced sensitivity of lysosomes to osmotic shock was demonstrated in the livers of leupeptin-treated rats, suggesting that the lysosomal membrane is labilized by administration of leupeptin. Incubation of the purified aldolase with the lysosomal fraction produced the same changes in properties of aldolase as those observed in vivo on injection of leupeptin.

Amino acid sequence of rat liver cathepsin L.

The complete amino acid sequences of the heavy and light chains of rat liver cathepsin L (EC 3.4.22.15) were determined at the protein level. The heavy and light chains consisted of 175 and 44 amino acid residues, respectively, and their Mr values without glycosyl groups calculated from these sequences were 18941 and 5056, respectively. The amino acid sequence was also determined from the N-terminal sequences of the heavy and light chains, and the sequences of cleavage fragments of the heavy chain with lysylendopeptidase and cyanogen bromide. The fragments were aligned by comparison with the amino acid sequence deduced from the sequence of cDNA of rat preprocathepsin L. The sequence of rat liver cathepsin L determined at the protein level was identical with that deduced from the cDNA sequence except that in the heavy chain, residues 176-177 (Asp-Ser) were not present at the C-terminus and alanine was replaced by proline at residue 125. Asn-108 in the heavy chain is modified with carbohydrate.

Effect of proteinase inhibitors on intracellular processing of cathepsin B, H and L in rat macrophages.

The effects of various proteinase inhibitors on the processing of lysosomal cathepsins B, H and L were investigated in cultured rat peritoneal macrophages. The processing of newly synthesized pro-cathepsins B, H and L to the mature single-chain enzymes was sensitive to a metal chelator,1,10-phenanthroline, and a synthetic metalloendopeptidase substrate, Z-Gly-Leu-NH2, and insensitive to inhibitors of serine proteinases, aspartic proteinases and cysteine proteinases. Inhibitors of cysteine proteinases, E-64-d and leupeptin, inhibited the processing of the single-chain forms of cathepsins B, H and L to the two-chain forms. These results suggest that (a) metal endopeptidase(s) is (are) involved in the propeptide processing of cathepsin B, H and L, and that proteolytic cleavages of the mature single-chain cathepsins are accomplished by cysteine proteinases in lysosomes.

Phosphorylated cystatin alpha is a natural substrate of epidermal transglutaminase for formation of skin cornified envelope.

Both keratohyalin granules (KHG) and cornified envelopes were stained histochemically in an indirect immunofluorescent study by antiphosphorylated cystatin alpha antibody, indicating that phosphorylated cystatin alpha is a component of the cornified envelope proteins. When phosphorylated cystatin alpha (P-cystatin alpha) was incubated with epidermal transglutaminase (TGase) and Ca2+ ions, polymerized protein was produced by formation of epsilon-(gamma-glutamyl)lysine cross-linking peptide bonds between lysine residues of cystatin alpha and glutamine residues of suitable protein(s) in the enzyme preparation. However, phosphorylated and non-phosphorylated cystatins were polymerized to similar extents by the TGase. Immunofluorescent and immunoelectron microscopic observations revealed that P-cystatin alpha could be detected in vivo in the KHG and cornified envelopes. Treatment of sphingosine, a specific inhibitor of protein kinase C, markedly suppressed the incorporation of cystatin alpha into KHG. Thus phosphorylation of cystatin alpha by protein kinase C may play an important role in targeting cystatin alpha into KHG.

Tryptase TL2 in the membrane of human T4+ lymphocytes is a novel binding protein of the V3 domain of HIV-1 envelope glycoprotein gp 120.

A novel membrane-bound serine esterase in cultured human T4+ lymphocytes, recently purified and named tryptase TL2, binds specifically to the external envelope protein gp 120 of HIV-1, interacting with its V3 domain. This binding was selectively blocked by inhibitors of tryptase TL2 with a GPCR sequence in their reactive site, synthetic peptides corresponding with the sequences of the V3 domains of various HIV-1 strains with the GPGR sequence, and antibody against tryptase TL2, or neutralizing antibody against the V3 domain of HTLV-IIIB. These findings suggest that tryptase TL2 is a binding protein of the V3 domain of HIV-1 envelope glycoprotein.

Inhibition of growth and cysteine proteinase activity of Staphylococcus aureus V8 by phosphorylated cystatin alpha in skin cornified envelope.

The activity of a cysteine proteinase purified from Staphylococcus aureus V8 (SAV8) was inhibited by phosphorylated cystatin alpha (P-cystatin alpha) and by purified cornified envelope protein of newborn rat, a conjugated form of P-cystatin alpha. Immunohistochemical analysis demonstrated a marked decrease in P-cystatin alpha content in cornified envelope treated with sphingosine. The inhibition of papain activity by proteins from sphingosine-treated skin was much weaker than that exerted by proteins from the untreated skin. The suppression of SAV8 colony formation inoculated on the sphingosine-treated skin was examined. Colony formation on the sphingosine-treated skin was enhanced compared to that on normal skin. These findings suggest that P-cystatin alpha in the cornified envelope may have a bacteriostatic barrier function against bacterial infection, such as that with SAV8.

Epidermal growth factor as a new regulator of induction of tyrosine aminotransferase and tryptophan oxygenase by glucocorticoids.

Epidermal growth factor (EGF) dose-dependently enhanced the induction of tyrosine aminotransferase and tryptophan oxygenase by glucocorticoids in primary cultures of adult rat hepatocytes without itself having any effect on these enzymes in the absence of glucocorticoids. The amplifications were observed even with dexamethasone at high concentrations (10(-6) M-10(-5) M) that had a maximal effect. EGF had no effect on induction of tyrosine aminotransferase by glucagon or Bt2cAMP. The effect of EGF was also observed in adrenal-ectomized and submaxillary gland-ectomized rats. These results suggest that EGF is an endogenous amplifier of the action of glucocorticoids.

Biosyntheses and processing of lysosomal cysteine proteinases in rat macrophages.

The intracellular processing and release of three lysosomal cysteine proteinases, cathepsin B, H and L, by rat peritoneal macrophages were investigated by pulse-chase experiments. Newly synthesized procathepsins B (39 kDa), H(41 kDa) and L (39 kDa) after 15 min labeling were processed to the mature, single-chain enzymes within 1 h. The single-chain forms of cathepsin B, H and L were further processed to two-chain forms at different rates: conversion of cathepsin L to the two-chain form was rapid, whereas the conversions cathepsin B and H took at least 6 h. Macrophages released 30% of the procathepsins B and L, and 10% of the procathepsin H.

Identification of hematoxylin-stainable protein in epidermal keratohyalin granules as phosphorylated cystatin alpha by protein kinase C.

The hematoxylin-stainable protein (HSP) in keratohyalin granules of the newborn rat epidermis was found to have the same amino acid composition and the same inhibitory and immunological properties as cystatin alpha. However, only its pI value (4.7) differed from that of cystatin alpha (5.3). Alkaline phosphatase treatment of HSP changed its pI value from 4.7 to 5.3. This pI change was inhibited by EDTA, an inhibitor of alkaline phosphatase. Furthermore, 32P from [gamma-32P]ATP was incorporated into recombinant cystatin alpha by a protein kinase C (PKC) preparation in the presence of phosphatidyl serine and Ca2+ ions as co-factors. The incorporation increased dose-dependently with the added cystatin alpha and was inhibited significantly by H-7, a specific inhibitor of PKC. SDS-PAGE autoradiography of the 32P-labeled proteins showed that 32P was incorporated into the cystatin alpha. This incorporation was not observed by the action of cAMP-dependent protein kinase. Therefore, it is highly possible that the HSP is a phosphorylated cystatin alpha and that the phosphorylation is catalyzed specifically by PKC.

The selective role of cathepsins B and D in the lysosomal degradation of endogenous and exogenous proteins.

A selective inhibitor of cathepsin B, a derivative of E-64 (compound CA-074), and pepstatin-asialofetuin, a potent inhibitor of cathepsin D, were used for an in vivo study of the selective role of these proteinases in lysosomal proteolysis. Administration of compound CA-074 or pepstatinasialofetuin to rats caused only a slight shift of the lysosomal density and no increase in sequestered enzymes in the autolysosomal fraction, although cathepsin B or D activity in the liver was markedly inhibited. These treatments also had little effect on the inhibition of the degradation of endocytosed FITC-labeled asialofetuin. In contrast, leupeptin treatment caused marked inhibition of lysosomal degradation of endogenous and exogenous proteins. These results suggest a small contribution of cathepsins B and D to the initiation of lysosomal proteolysis.

Immunological significances of invariant chain from the aspect of its structural homology with the cystatin family.

The primary structure of p31 of invariant chain (Ii-chain) shows about 50% homology with those of the cystatin family which are endogenous cysteine protease inhibitors. The binding domains between Ii-chain and HLA-DR-7 were estimated from the structural homology between cystatin and Ii-chain and also between cathepsins and DR-7, respectively. The QL64-71 and GS76-88 of Ii-chain were estimated to be the binding domains with GG45-51 and VS57-63 of HLA-DR7, respectively. The purified human Ii-chain from spleen is capable of forming four molecular forms from monomer to tetramer by redox-potential dependent disulfide bond formation. The Ii-chain inhibits cathepsin L and H competitively as a dimer and the K(i) value for cathepsin L was 4.1 x 10(-8) M, but cathepsin B was not inhibited at all. The Ii-chain showed mainly a dimer (60 kDa) under the assay condition of cathepsins with cysteine and was not degraded by these cathepsins. The Ii-chain may play an important role in the regulation of antigenic peptide presentation to MHC class II.

Linkage between phosphorylated cystatin alpha and filaggrin by epidermal transglutaminase as a model of cornified envelope and inhibition of cathepsin L activity by cornified envelope and the conjugated cystatin alpha.

Lysine-rich phosphorylated cystatin alpha (P-cystatin alpha) from newborn rat epidermis is a good substrate for epidermal transglutaminase (TGase) and also one of the component proteins of cornified envelope in the stratum corneum. Since the filaggrin linker segment peptide was efficiently conjugated with P-cystatin alpha and was mediated by epidermal TGase in the presence of Ca2+ ions, filaggrin is a candidate for the glutamine-rich linkage protein to conjugate with lysine-rich P-cystatin alpha. A conjugated protein was found by epidermal TGase in the activated condition with Ca2+ ions and dithiothreitol. In contrast, the conjugated protein was not formed under chelated conditions with EDTA. The conjugated protein reacted positively with anti-P-cystatin alpha polyclonal antibody (PoAb). The conjugated protein and purified cornified envelope showed an inhibitory effect against papain and cathepsin L, but cathepsin B and H were not inhibited by these P-cystatin alpha conjugates. Although the component protein, P-cystatin alpha itself, inhibited cathepsin H strongly, these conjugated proteins inhibited specifically the cathepsin L family. The amino acid composition of cornified envelope protein and the conjugated protein of P-cystatin alpha and filaggrin linker segment peptide was not completely the same. The conjugated protein of P-cystatin alpha and filaggrin linker segment peptide showed the same inhibitory properties against cysteine proteinases as the cornified envelope. These findings suggest that the linkage protein between P-cystatin alpha and filaggrin linker segment peptide may be considered a model of cornified envelope, although skin cornified envelope may be conjugated with some additional proteins.

Studies on the reactive site of the cystatin superfamily using recombinant cystatin A mutants. Evidence that the QVVAG region is not essential for cysteine proteinase inhibitory activities.

For study of the inhibition mechanism of the cystatin superfamily, cystatin A artificial mutants were obtained in which a well-conserved QVVAG region in the cystatin superfamily was changed to KVVAG or QVTAG and these mutants were then expressed in E. coli. For this, genes with these sequences were synthesized enzymatically from 11 oligodeoxynucleotides and expressed under the tac promoter gene of the E. coli plasmids. The products expressed were then purified on Sephadex G-50 and HPLC DEAE-5PW columns. The substitutions in cystatin A were confirmed by the amino acid compositions, N-terminal amino acid sequences and elution positions on ion-exchange chromatography of the products. The Ki values of these products for the cysteine proteinases, papain and cathepsins B, H and L, were determined in comparison with those of wild type recombinant cystatin A. Results showed that the cystatin A mutants had similar inhibitory activities to those of wild type recombinant cystatin A. Namely replacement of amino acids in the QVVAG sequence of cystatin A did not significantly affect the inhibitory activities on these proteinases. The results suggest that the QVVAG region is less important than the N-terminal region of cystatin for inhibitory activities on cysteine proteinases.

Gene structure of rat cathepsin H.

The gene structure of rat cathepsin H was determined. It comprises at least 12 exons of various lengths (32-433 bp) spanning in total more than 17.5 kbp. The gene structure does not correspond well to the functional unit of the proteinase. The region around the active site Cys residue, the most conserved region among cysteine proteinases, is split by an intron. This is a common characteristic among the gene structures of cysteine proteinases.

Properties and nature of a cysteine proteinase inhibitor located in keratohyalin granules of rat epidermis.

The pI 4.7, 14.5 kDa hematoxylin-stainable protein (HSP) from rat epidermis inhibited the activities of the cysteine proteinases papain, ficin, cathepsins B, H and L with similar inhibitory characteristics as recombinant cystatin-alpha. Proteinases of other classes were not inhibited. The inhibitory activity of HSP was heat stable in the wide pH range of 3.0-10.0. Polyclonal antibodies against HSP cross-reacted with cystatin-alpha and the molecular mass of HSP was similar to that of cystatin-alpha, though its isoelectric point was different. The in vivo location of both HSP and cystatin-alpha is on keratohyalin granules in epidermis as detected by indirect immunofluorescence technique using individual antibodies. Therefore it is highly probable that HSP is a cystatin-alpha derivative or a very similar proteinase inhibitor belonging to a family of cystatins.

Total synthesis of the cystatin alpha gene and its expression in E. coli.

A gene encoding cystatin alpha has been chemically synthesized, cloned and expressed in E. coli. The gene of 318 base pairs was assembled by enzymatic ligation of 19 oligonucleotides and cloned into a pBR322-derived expression plasmid down stream of the tac promoter. The expression product of the synthetic gene has been purified by Sephadex G-50 column chromatography and shown to have the same properties as those of the authentic protein isolated from rat epidermis.

Gene structure and 5'-upstream sequence of rat cathepsin L.

The structure of rat cathepsin L gene has been determined. The gene spans 8.5 kilobase pairs comprising 8 exons, and has an intron located near the active site cysteine residue. The gene structure does not correspond well to the functional units of the proteinase. These characteristics are found to be in common with the cysteine proteinase gene family. In the 5'-upstream region, one CAAT-box and four SP-1 binding sites, together with two AP-2 binding sites and CRE, but no typical TATA-box are found. Further, SP-1 and AP-2 binding sites and an octamer motif are also found in the 1st intron, suggesting a complex regulatory mechanism for the expression of the cathepsin L gene.