The three-dimensional structure of ω-amino acid:pyruvate aminotransferase from Pseudomonas sp. F-126, an isologous α₄ tetramer containing pyridoxal 5'-phosphate (PLP) as a cofactor, has been determined at 2.0 A resolution. The diffraction data were collected with a newly developed Weissenberg camera with a Fuji Imaging Plate, using synchrotron radiation. The mean figure-of-merit was 0.57. The subunit is rich in secondary structure and comprises two domains. PLP is located in the large domain. The high homology in the secondary structure between this enzyme and aspartate aminotransferase strongly indicates that these two types of enzymes have evolved from a common ancestor.

View full abstract

A part of the GTP γS-binding activity in murine thymocyte membranes was found to have affinity to a concanavalin A (Con A)-Sepharose column. The material was identified as G₁ (inhibitory GTP-binding protein) on the basis of the molecular weight and by islet activating protein-dependent ADP-ribosylation and anti- α1(α subunit of G₁) immunoblot-ting. However, when the membranes prepared from Con A-stimulated thymocytes were used, no GTP _γS-binding activity was detected in the Con A-bound fraction, suggesting that G₁ physically and specifically associated with Con A acceptors dissociates upon Con A stimulation. Furthermore, another GTP _γS-binding protein (25 kDa), which is quite similar to a novel phosphoinositide-specific phospholipase C (PI-PLC)-associated G protein in calf thymocytes (Wang, P., Toyoshima, S., & Osawa, T. (1988) J. Biochem. 103, 137-142), was detected among the Con A-Sepharose-bound proteins with the chemical cross-linking technique. When the 40 kDa and 25 kDa G proteins associated with Con A receptor(s) were isolated and their direct effects on the activity of partially purified PI-PLC as to phos-phatidylinositol 4, 5-bisphosphate hydrolysis were examined, the 25 kDa G protein was found to enhance the PI-PLC activity more effectively. On the other hand, pretreatment of cells with islet-activating protein completely abolished the inhibitory effect of Con A on the prostaglandin El and isoproterenol-induced increases of cellular cAMP. These results suggest that, in murine thymocytes, the 25 kDa GTP _γS-binding protein may be the G protein involved in Con A-induced phosphoinositide breakdown by PI-PLC, while the Con A receptor(s)-associated G₁ may be responsible for the inhibitory regulation of the cellular cAMP level.

View full abstract

Structural studies on a hereditary abnormal fibrinogen, fibrinogen Nagoya (Takamatsu, J., Ogata, K., Kamiya, T., Koie, K., Takagi, T., & Iwanaga, S. (1979) Thromb. Haemost. 42, 78), were performed to identify the abnormality responsible for the impaired polymerization of fibrin monomer. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, fibrinogen Nagoya showed the presence of an extra protein band with an apparent molecular weight of 49, 500 in addition to the normal three subunit chains. Amino acid sequence analysis of a peptide isolated from a lysyl endopeptidase digest of one of the CNBr fragments derived from fibrinogen Nagoya indicated that Gln-329 in the γchain had been replaced by Arg. This substitution can be explained by a single nucleotide change in the codon for Gln-329 (CAG→CGG). We conclude that Gln-329 in the γ-chain is indispensable for the normal polymerization of fibrin monomer.

View full abstract

The activation processes of two human pepsinogens A (pepsinogens 3 and 5) and progastricsin were compared with special attention to pepsinogens 3 and 5. Each zymogen was converted to pepsin in a stepwise manner through intermediate forms. In pepsinogens A, the major cleavage site was the Leu₂₃-Lys₂₄ bond and this cleavage was suggested to occur intramolecularly. When each of the pepsins A was added to the corresponding pepsinogen A exogenously, the latter was rapidly converted to pepsin, releasing the 47-residue intact activation segment. In this case, the Leu₄₇-Val₄₈ bond connecting the activation segment with the pepsin moiety was cleaved by an intermolecular reaction. On the other hand, when the pepsinogen A-pepstatin complex was attacked by each corresponding pepsin A added exogenously, significant cleavage by an intermolecular reaction occurred at the Asp₂₅-Phe₂₆ bond, generating the Phe₂₆-intermediate form. These shifts of the cleavage sites in pepsinogens A depending on the activation conditions are likely to correlate with the conformation of the activation segment. These results can be explained consistently in terms of a proposed molecular model of activation.

View full abstract

In order to shed some light on the role of proteinases in the process of demyelination in the central nervous system, a calcium-dependent proteinase was purified to homogeneity from bovine spinal cord, and its action on myelin basic protein purified from the same tissue was investigated. Among the three major myelin basic protein fractions, Fraction I was resistant to the action of the enzyme whereas Fractions II and III were degraded in the same manner, giving two major bands on SDS-polyacrylamide gel electrophoresis. The hydrolysis products were fractionated by high-performance liquid chromatography and characterized. The results showed that the myelin basic protein (Fractions II and III) was rather selectively cleaved at the Val⁹³-Thr⁹⁴ bond and the Arg⁹⁶-Thr⁹⁷ bond in mutually exclusive ways, with minor cleavages at the Ala¹⁶-Ser¹⁷ and Gly⁶⁸-Ser⁶⁹ bonds, suggesting the implication in vivo of calcium-dependent proteinase in the limited proteolysis of myelin basic protein.

View full abstract

Structural studies were carried out on the O-polysaccharide fraction obtained from the lipopolysaccharide of Pseudomonas aeruginosa IID 1012, the standard strain of Homma serogroup K, by mild acid treatment. The O-polysaccharide was composed of L-rhamnose, N-acetyl-D-quinovosamine, and N-acetyl-D-galactosaminuronic acid. The results from analysis of fragments obtained by acid hydrolysis and Smith degradation of the O-polysaccharide, together with data on methylation analysis and nuclear magnetic reso-nance spectroscopic measurement of the polysaccharide, led to the most likely structure of the repeating units of the polymer chain, →4)D-GalNAcA(α1→3)D-QuiNAc(β1→2)L-Rha(αl→3)L-Rha(αl→, in which about 20% of the N-acetylgalactosaminuronic acid resi-dues were in an amide form and about 75% of the same residues were O-acetylated at C-3.

View full abstract

Structural studies were carried out on the O-polysaccharide fraction obtained by mild acid treatment of the lipopolysaccharide from Pseudomonas aeruginosa IID 1009 (ATCC 27585). TheO-polysaccharide was composed of L-rhamnose, N-acetyl-D-quinovosamine, and N-acetyl-L-galactosaminuronic acid in a molar ratio of 1 : 1 :1. The results from analysis of fragments obtained by hydrogen fluoride hydrolysis of O-polysaccharide, together with data on methylation analysis and nuclear magnetic resonance spectroscopic analysis, led to the most likely structure of the repeating units of the polymer chain →4)L-GalNAcA(αl →3)D-QuiNAc(α1 →3)L-Rha(α1 →, in which about 70% of the rhamnose residues were O- aeetylated at C-2. This structure coincides with that of the repeating unit of Lanyi 02 a, b polysaccharides.

View full abstract

When crude neurofilaments were dissolved in a solution containing 8 M urea and 1% β-mercaptoethanol (β-ME), the component proteins of the neurofilaments and other contaminating filaments were solubilized into monomeric forms. However, when reassem-bled filaments were solubilized again by the addition of urea to 8 M withoutβ-ME, several bands which seemed to be oligomeric forms of filament proteins were observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Among them, a band which appeared between microtubule-associated protein-1 (MAP-1) and fodrin was most remarkable. This band was also observed when a triplet mixture of the neurofilaments (NF-H, NF-M, NF-L) was reassembled. The molecular weight of this band wasestimated to be 280 kDa. In addition, much of this component was easily isolated on DE-52 column chromatography of the reassembled crude neurofilament proteins with buffers containing 6 M urea, while the low molecular weight component of the neurofilaments (NF-L, 70 kDa) was hardly detected. Furthermore, the isolated 280 kDa component was reduced to NF-L on the addition of β-ME to 1%. In contrast, the 280 kDa component was produced on dialysis of isolated NF-L against the assembly buffer. From these results, it is deduced that this component is the stable tetramer of NF-L which is produced through spontaneous inter-chain disulfide formation among protofilament tetramers.

View full abstract

Changes in the activities of Leu-and Arg-arylamidase in rat frontal and parietal cortices and the subcortical area (including thalamus, hypothalamus, and striatum) were examined in the 2nd, 4th, 8th, 12th, and 24th weeks of life. Average levels found in the subcortical region were greater than those in the cortical areas. The most marked changes in enzymatic activity in the course of brain development were found in the subcortical structure. Leu-arylamidase activity increased from the 2nd week up to the 8th week, returning to the 2nd week level at the 12th and 24th weeks. The maximum levels of Arg-arylamidase activity were found at the 4th and 8th weeks. These data suggest that proteolytic activity is involved in the postnatal development of rat brain.

View full abstract

The steady-state kinetic parameters of the enzymatic reduction of nitrite by spinach ferredoxin-nitrite reductase [EC 1.7.7.1] were measured under anaerobic conditions. The maximum velocity of ferredoxin-linked activity was essentially the same as for the methyl viologen-linked activity of the enzyme. The initial velocity patterns of the oxidation of reduced ferredoxin suggested a sequential reaction scheme by which nitrite and reduced ferredoxin bind to the free enzyme. The binding of nitrite and ferredoxin to the enzyme was also investigated by difference spectra produced by the complex formed by the enzyme with the substrates. Nitrite and ferredoxin each gave a 1: 1 complex with the enzyme. The dissociation constant (K_d) of the enzyme-nitrite complex agreed well with the K_m value for the ferredoxin-linked activity, whereas the K_d the enzyme-ferredoxin complex differed from the K_m value for the enzyme activity. It was concluded that our preparation of spinach ferredoxin-nitrite reductase differs from both the complex (M_r=85, 000) and the modified (M_r=61, 000) forms of the enzyme reported by Hirasawa et al.[J. Biol. Chem. 262, 12428-12433 (1987)].

View full abstract

Apolipoprotein E (apoE) is one of the protein moieties of the human serum lipoproteins. Three major isoforms of apoE (apoE2, apoE3, and apoE4) and minor variant isoforms (apoEl, apoE5, and apoE7) have been detected by isoelectric focusing. In this study we have cloned the apoE7 gene from a patient with the apoE3/E7 phenotype associated with hypertriglyceridemia and diabetes mellitus. DNA sequencing revealed that the apoE7 gene has two base substitutions (G →A) changing GIu_{244, 245}→Lys_{244, 245}, compared with the apoE3 gene. The replacement of the two amino acids is consistent with the result of isoelectric focusing of the apoE7 isoprotein, which shifts to four positively charged units compared with the apoE3 isoprotein.

View full abstract

Recently, we suggested that the effect of differentiation inducing factor (D-factor) which is found in the supernatant of macrophages, and induced the differentiation of a mouse myeloid leukemic cell line, M1, into macrophage-like cells, may be a result of the cooperative effects of tumor necrosis factor (TNF) and interleukin 1 (IL-1). In this study, we purified guinea pig (G.P.) TNF secreted from peritoneal macrophages and compared the antiproliferative and differentiative effects of the G.P. TNF with those of recombinant human TNF (rHuTNF). The purification scheme consisted of ultrafiltration, gel filtration-high performance liquid chromatography (HPLC), DEAE-HPLC, and reverse-phase HPLC. The cytotoxic activity of the purified substance was approximately 1.5 × 108U/mg. The isoelectric point was 5.2. The molecular weight was 40 to 45kDa as estimated by gel filtration and 18 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH₂-terminal amino acid sequence was determined to be Ser-Ala-Ser-Gln-Asn-Asp .... Approximately 76 or 71% homology between G.P. TNF and mouse or human TNF exists in the NH₂-terminal 21 residues. The purified G.P. TNF and rHuTNF demonstrated D-factor activity only in the presence of recombinant human IL-1α in M1 cells. We also determined the effect of TNF on two human myeloid leukemic cell lines (THP-1 and U937). The purified G.P. TNF and rHuTNF inhibited the growth of U937 cells, but did not induce their differentiation. In THP-1 cells, TNF slightly inhibited the growth and induced differentiation. In mouse cell lines G.P. TNF was more effective than rHuTNF for both the induction of cytotoxicity/antiproliferative effect and differentiation. The different efficacy of these two TNFs for the growth and differentiation of mouse and human cell lines may reflect the snecies difference.

View full abstract

Structural analysis of 55 nearly full-length cDNA clones of rat liver alkaline phosphatase mRNAs revealed the presence of two totally different sequence stretches at the 5'-distal region starting from the position 88 nucleotides upstream of the initiation codon ATG. Since each of these two sequences, E1 and E2, was assigned on the rat genome about 36 kilobase pairs (kbp) and 10 kbp upstream of the common exon E3, respectively, they are presumably used as alternatively spliced exons. The distances between these sequences and E3 were unusually long, as compared with other intronic distances (0.4-4 kbp) observed between successive pairs of the eleven exons which are common to both types of mRNAs. The relative ratio of El-containing mRNA to E2-mRNA was about three in the liver after bile-duct ligation and colchicine treatment.

View full abstract

Interaction of human plasma α1-proteinase inhibitor (α1PI) with subtilisin BPN' was assessed by spectrophotometric determination of the inhibitory capacity and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). During the course of incubation of the enzyme and the inhibitor (E:I=1:7.5) at pH 8.0 about 17% of the enzyme activity which had been inhibited initially was regenerated, indicating a temporary type of inhibition. The results of the titration experiments indicate that 9.8 mol of the inhibitor is required to inhibit 1 mol of the enzyme completely. However, patterns of 5% disc SDS-PAGE under non-reducing conditions revealed only an equimolar complex (Mr 80K) of, α1PI with the enzyme and no other higher Mr component than the native inhibitor (Mr 56K). On the other hand, complete dissociation of the complex occurred under reducing conditions, producing an enzymatically modified inhibitor. When 5-21% gradient slab SDS-PAGE was employed, no complex formation was observed under either reducing or non-reducing conditions. With the gradient gel system, dissociation of the equimolar complex produced different forms of the inhibitor, that is, regeneration of an intact α1PI under non-reducing conditions and an enzymatically modified form under reducing conditions. All these results indicate that the complex formed between subtilisin BPN' and human α1PI is not so stable as that of the inhibitor with bovine chymotrypsin and that no covalent bond may be involved in the complex formation. The results also indicate that human α1PI is not an effective inhibitor of subtilisin BPN' and behaves like a substrate for the enzyme.

View full abstract

In order to establish the role of the extension peptide of the precursor of P-450 (SCC), a mitochondrial inner membrane protein, in the import into the organella, three deletion mutants of the precursor, in which the deletions were in the mature portion, were constructed. These mutant precursors were imported into mitochondria in vitro as efficiently as the original precursor, indicating that the extension peptide contains sufficient information for the import of the precursor into mitochondria. To investigate which portion of the extension peptide contains the mitochondrial targeting signal, various lengths of the amino-terminal portion of the extension peptide of P-450 (SCC) precursor were fused to the mature portion of adrenodoxin. The fusion proteins consisting of 44 and 19 amino-terminal amino acids and mature adrenodoxin were imported into mitochondria, whereas those containing 14, 7, and 2 amino-terminal amino acid residues were not. The importance of the amino-terminal portion of the extension peptide was confirmed by the deletion from the amino-terminal end of a fusion protein consisting of the amino-terminal 44 amino acid residues of P-450 (SCC) precursor and mature adrenodoxin, SCC44RAd. The amino-terminal deletions abolished the import of the fusion proteins into mitochondria. Substitution of all of the three basic amino acids, Arg (4), Arg (9), and Lys (14) in the extension peptide of SCC44RAd to Ser or Thr inhibited the binding of the fusion protein to mitochondria as well as its import. These results indicated that the amino-terminal 19 amino acid residues of the extension peptide of P-450 (SCC) precursor contain sufficient information for the import of the precursor into mitochondria, and that the basic amino acids in that particular portion of the extension peptide are essential for the binding of the precursor to mitochondria.

View full abstract

A 2, 200 base pair (bp) fragment containing the 5'-flanking region of the mouse DNA polymerase β gene was placed adjacent to and upstream of the chloramphenicol acetyl transferase (CAT)-coding region of the CAT vector. A transient expression assay of CAT activity in mouse NIH/3T3 cells transfected with this recombinant plasmid or a set of its 5'-deletion derivatives was carried out to identify a cis-acting regulatory element(s) for DNA polymerase β gene expression. Depending on the extent of the deletion, CAT activity was dramatically increased, indicating the existence of a negative regulatory region which could be divided into two distinct domains: removal of the first domain (NRE-I), nucleotides -1860 to -1580 (+1 denotes the position of 5'-most proximal transcription initiation site), caused two to three-fold stimulation of CAT activity, and removal of the second domain (NRE-II), nucleotides -828 to -456, stimulated CAT expression another two to three-fold. When an 1, 864-bp segment containing these negative regulatory elements (-2190 to -327) was inserted in the plasmid carrying the simian virus 40 early promoter and enhancer-directed CAT gene, it inhibited the CAT expression relatively independently of the orientation of insertion and the distance from the promoter-enhancer. We also mapped the promoter element of the DNA polymerase β gene to within a 133-bp DNA fragment from nucleotide position -100 to +33. Either the NRE-I region or the NRE-II region alone can inhibit DNA polymerase β gene promoter function.

View full abstract

The nucleotide ligation site of adenylylated glutamine synthetase, which contains a unique tyrosyl residue linked through a phosphodiester bond to 5'-AMP, was studied by digestion with three hydrolytic enzymes. The products on micrococcal nuclease digestion were adenosine and o-phosphotyrosyl glutamine synthetase. The K_m for this macromolecular substrate with the nuclease was 40 μM, at pH 8.9. The glutamine synthetase activity was not affected by deadenosylation with the nuclease, in contrast to SVPDE digestion, with which the glutamine synthetase activity was markedly increased. The K_m for the native adenylylated glutamine synthetase with the SVPDE was 36 μM, i. e., similar to that for the nuclease. When the isolated o-phosphotyrosyl enzyme was incubated with alkaline phosphatase at pH 7.2, the glutamine synthetase activity rapidly increased to the same level as that of the SVPDE treated enzyme. Furthermore, kinetic properties of the o-phosphotyrosyl glutamine synthetase were compared with those of the adenylylated enzyme. The optimum pH, apparent K_m for each of three substrates, glutamate, ATP, and NH₃, and V_max were in good agreement, as to either Mg²⁺- or Mn²⁺-dependent biosynthetic activity. From these results we can conclude that the regulation of glutamine synthetase activitysimply requires the piosphorylation of the tyrosyl residue in each subunit, without recourse to adenylylation.

View full abstract

Three serine proteinase inhibitors, MCTI-I, MCTI-II, and MCEI-I, were isolated from bitter gourd (Momordica charantia LINN.) seeds. MCTI-I and MCTI-II were inhibitors for trypsin and MCEI-I was an elastase inhibitor. Their amino acid sequences and the positions of disulfide bridges of MCTI-II were determined to be as follows.

View full abstract

The amino acid sequence of a short chain neurotoxin obtained from Bungarus fasciatus venom consists of 64 amino acid residues: Arg-Ile-Cys-Leu-Asn-Gln-Gln-Gln-Ser-Thr-Pro-Glu-Asp-Gln-Pro-Thr-Asn-Gly-Gln-Cys-Tyr-Ile-Lys-Thr-Asp-Cys-Gln-Asn-Lys-Thr-Trp-Asn-Thr-His-Arg-Gly-Ser-Arg-Thr-Asp-Arg-Gly-Cys-Gly-Cys-Pro-Lys-Val-Lys-Pro-Gly-Ile-Asn-Leu-Arg-Cys-Cys-Lys-Thr-Asp-Lys-Cys-Asn-Glu. The above result was obtained primarily from the amino acid analyses and sequencing of tryptic peptides accompanied with the necessary analyses and sequencing of the chymotryptic and lysyl endopeptidic peptides for alignment.

View full abstract

A cross-linked complex between plastocyanin and cytochrome f was prepared by incubation in the presence of a water soluble carbodiimide and its kinetic properties were studied. The optical spectra, oxidation-reduction potentials and isoelectric pH of plastocyanin and cytochrome f did not change upon the formation of the cross-linked complex. Studies on the ionic strength effect on the electron transfer rate from cross-linked plastocyanin to ferricyanide indicated that the negative charge on the reaction site of plastocyanin was masked upon the cross-linking. It was also suggested that the sign of the net charge near the cytochrome f heme edge changed from positive to negative upon the cross-linking. On the other hand, electrostatic interactions between cross-linked plastocyanin and P700 seemed to be essentially the same as those in the case of native plastocyanin, although the rate of electron transfer from cross-linked plastocyanin to P700 was severely reduced. We also measured the intra-complex electron transfer from cytochrome f to plastocyanin. This suggested that the covalently cross-linked complex is a valid model of the electron transfer encounter complex. Based on these results, the reaction sites of plastocyanin with P700 and cytochrome f were discussed.

View full abstract

The bindings of S-1 and the two heads of HMM with pyrene-labeled F-actin were studied using the change in light-scattering intensity or that in the fluorescence intensity of the pyrenyl group. At low ionic strength (50 mM KC1), both S-1 and HMM became bound tightly with F-actin (K_d, ⟨0.1μM) and both heads of HMM became bound to F-actin. The affinities of S-1 and HMM for F-actin decreased with increasing KC1 concentration. In 1 M KC1, the K_d, values of S-1 and HMM for F-actin were 11 and 0.58μM, respectively. Thus, HMM was bound to F-actin 19 times more tightly than S-1. We compared the extent of binding of HMM to F-actin measured by a centrifugation method with that measured by the fluorescence change of pyrenyl-group, and found that even in 1 M KC1, HMM became bound to F-actin with a two-headed attachment. We measured the kinetics of binding and dissociation of acto-S-1 and acto-HMM from the time course of the change in light-scattering intensity after mixing S-1 or HMM with F-actin at 1 M KC1 and that after mixing 1 M KC1 with acto-S-1 or acto-HMM formed at low ionic strength. The results could be explained by the following schemes:S-1+F-actin k₁_??_k_-1S-1-F-atin
HMM+F-actin k₁_??_k_-1HMM-F-actin (single-headed) k₂k_-2HMM=F-actin (double-headed)
where the values of k₁ and k_-1 for S-1 were 0.13 s^-1/μM and 1.8 s^-1, respectively. The values of k₁ and k_-1, for HMM were 0.45 s^-1/μM and 1.8 s^-1, respectively. The value of k_-2/k₂ was 0.15 and k_-2⟩3s^-1 (if k_-2=3.6 s-1, k₂=24 s^-1). Thus, the step from HMM-F-actin (single-headed binding) to HMM=F-actin (double-headed binding) does not depend on F-actin concentration, and the equilibrium of this step favors the double-headed binding.

View full abstract

The effect of acute exercise (treadmill running) on rat myocardium β-adrenergic receptors (β-AR) was studied. β-AR was identified in purified sarcolemmal membrane fractions and light vesicle fractions. In control hearts, the number of β-AR was 21.25 ± 2.25 and 20.89± 2.89 fmol/mg protein (mean ± SE) in sarcolemmal membranes and light vesicles, respec-tively. Immediately after a single bout of dynamic exercise, about 35% of β-AR was transferred from light vesicles to sarcolemmal membranes (p <0.05); concomitantly, isoproterenol-stimulated adenylate cyclase activity also significantly increased in sar-colemmal membranes (p <0.05). These results suggest that acute exercise provokes the translocation of β-AR from a presumably intracellular site (light vesicles) to functional membrane fractions (sarcolemmal membranes) in rat myocardium.

View full abstract

We examined the effects of cell aging and phenylhydrazine-induced oxidant damage on erythrocyte cathepsin E, which is present as a latent, membrane-associated enzyme in normal human erythrocytes. When young erythrocytes isolated from human mature erythrocytes by Percoll density gradient centrifugation were aged in vitro, the membrane-associated cathepsin E was progressively released from the membrane as an active enzyme. During the cell aging up to 100 h, about 40% of the membrane-associated enzyme was activated and solubilized. When phenylhydrazine was incubated with the erythrocytes, it also caused the activation and solubilization of cathepsin E in a dose-dependent and time-dependent manner. Exposure of erythrocytes to 2.5 mM phenylhydrazine for up to 2 h led to about 40% activation of the membrane-associated enzyme. Both aging and phenyl-hydrazine-treatment were accompanied with an increase in the association of the cytosolic proteins, primarily hemoglobin, with the membrane, which occurred prior to the release of cathepsin E from the membrane. A similar activation for the membrane-associated enzyme was observed with in vitro-aged hemoglobin-free membrane ghosts. Thus, the primary mechanism for activation of cathepsin E in the intact cells seems to be through lesion of the membrane framework that results from increased binding of hemoglobin to the membrane.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting employing polyclonal IgG antibodies for human spectrin and band 3 revealed that breakdown of the membrane proteins was enhanced in both aged and phenylhydrazine-treated cells. The relation between the cathepsin E activation and the membrane protein breakdown is discussed.

View full abstract

The specific cell surface receptors for lymphotoxin (LT) which are expressed on murine fibroblast L.P3 cells, a subline of L929 cells, were found to consist of a single class of specific high-affinity receptors with a dissociation constant (K_d) of 3.8×10^-10 M and a density of 5.8×10³ sites/cell. Similarly, murine fibroblast L929 cells, human melanoma A375 cells and human cervical carcinoma HeLa-S3 cells had about 7.2×10³, 3.5×10³, and 6.6×10³ sites/cell with K_d, values of 1.4×10^-10, 0.5×10^-10, and 1.1 ×10^-10 M, respectively. Among the LT receptor-positive cell lines, there was no direct correlation between the level of specific LT binding and the sensitivity to the cytotoxic or cytostatic effect of LT. Cross-linking of ¹²⁵I-LT to the cell surface receptors with disuccinimidyl suberate, followed by two-dimensional gel electrophoresis of the cell lysate, revealed two kinds of LT-LT receptor complexes with molecular weights of 70 and 97 kDa, and having the same pI value of 6.8. Cell-bound ¹²⁵I-LT was internalized within 1 h and degraded intracellularly, and finally secreted into the medium within a few hours. Appropriate concentrations of LT and interferon γ (IFNγ) showed synergistic cytotoxicity toward murine fibroblast L.P3 cells and human' monocytoma U937 cells, but these cytokines were only slightly cytotoxic individually. Preincubation of these cells with IFN γincreased the total number of LT receptors without any significant change in the dissociation constant or in the molecular weight. The binding of ¹²⁵I-LT to L.P3 cells was inhibited by both unlabeled LT and unlabeled tumor necrosis factor.

View full abstract

An exo-type cellulase (Ex-1) was extracted from Irpex lacteus (Polyporus tulipiferae) and purified essentially to homogeneity. This cellulase attacked cellulosic substrates in an exo-wise fashion to produce almost exclusively cellobiose. In contrast, Ex-1 was found to attack β-glucans having β(1→3)- and β-(1→4)-mixed linkages in a way similar to an endo-type cellulase. The products formed from barley glucan by Ex-1 were 3²-O-βD-cellobiosyl-cellobiose _??_3²-O-β-D-glucosyl-cellobiose >cellobiose _??_cellotriose _??_glucose in the early stage, but no laminaribiose was produced. An endo-type cellulase (En-1) obtained from the same fungus also hydrolyzed β-glucans but in a typical endo-wise fashion and the products from barley glucan were 3²-O-β-D-glucosyl-cellobiose_??_3²-O-β-D-cellobiosS71-cellobiose>cellobiose>laminaribiose; no glucose or cellotriose was produced. Thus, it seems likely that En-1 can attack any intramolecular linkage of β-glucan, while Ex-1 requires the presence of at least cellobiosyl residues adjacent to a β-(1→3)-D-linked glucosyl residue. This finding, together with the mode of hydrolysis of cellulosic substrates by Ex-1, suggests that the stereochemical structure of successive, β- (1→4)-cellobiosyl residues inserted by β-(1→3)-D-glucosidic linkage is permissible in the action of Ex-1, although this enzyme prefers the β-(1 →4)-linked cellobiosyl sequence.

View full abstract

1. CD spectra showed that the fowl protamine, galline, has an unordered structure rich in reverse turns in neutral solution. Eight reverse turns were predicted to be present in the galline molecule on the basis of its amino acid sequence. Spectrophotometric analyses revealed that galline efficiently bound to DNA in 0.25mM EDTA/10mM Tricine-HCl, pH 7.4, but hardly so in 30mM NaCl/3mM sodium citrate, pH 7.0. Citrate ions bound specifically to the galline molecule, causing a conformational change in it. As a result, galline could not interact with DNA. 2. The concentration of unbound galline in a mixture of DNA and galline in 100mM NaCl/50mM Tricine-HCl, pH 7.4, at 37° was determined by measurement of the intrinsic fluorescence of tyrosine residues of galline in the supernatant after ultracentrifugation of the mixture. The Scatchard plot showed positive co-operativity in the binding of galline to DNA and the binding parameters were determined: the co-operative binding constant (Kc)=3.3×10⁷M^-1, the co-operativity factor (q)=800, and the number of nucleotides of DNA occupied by one galline molecule (n)=28. The Kc and q values were intermediate between those for clupeine Z from herring sperm and S-methyl protamine from boar sperm. That is, the binding constants of protamine as to DNA decrease in the order of herring, fowl, and boar, while the co-operativities in binding increase in that order.

View full abstract

Mechanically isolated, structurally intact porcine zonae pellucidae composed of three
families of glycoproteins (PZP1-3) were digested with purified boar acrosin, and then the solubilized and unsolubilized fractions were separately analyzed by HPLC and/or SDS-PAGE. In isotonic solution, PZP1 was first degraded and then PZP2, whereas PZP3 was quite resistant to acrosin, reflecting the organization of these families in the zona structure. As the proteolytic hydrolysis proceeded, high-molecular-weight products appeared in the unsolubilized fraction. These products disintegrated on treatment with β-mercapto-ethanol. The zona materials solubilized by acrosin were separated into seven fractions by reverse phase HPLC. The total yield of the latter was only about 5% by weight. Thus, limited sites of the porcine zona were cleaved by the homologous sperm acrosin. Since five fractions contained peptides that were more hydrophilic than the original proteins, these peptides seemed likely to be present on the outer surface of the zona structure. SDS-PAGE of the unsolubilized fraction showed that acrosin cleaved the zona at many more sites in hypotonic solution than in isotonic solution. Thus, structural relaxation of the inner region of the zona was indicated to be induced under hypotonic conditions. However, no high-molecular-weight products were formed in hypotonic solution, suggesting that the native architecture of the zona is a prerequisite for their formation.

View full abstract

A synthetic gene containing the coding sequence for the human cysteine proteinase inhibitor, cystatin A, was obtained by enzymatic assembly of 20 oligodeoxyribonucleotides which had been chemically synthesized by the solid phase phosphoramidite method. It was cloned into an Escherichia coli plasmid. The expression plasmid for cystatin A was constructed by introducing the synthetic gene downstream of the tac promoter of an E. coli plasmid which is a derivative of pKK223-3 with high copy number. The gene was expressed in E. coli JM109 without IPTG-induction. The expression of cystatin A was detected by SDS-polyacrylamide gel electrophoresis of the E. coli JM109 lysate, followed by immuno-blotting using rabbit antiserum raised with human epidermal cystatin A and alkaline phosphatase-conjugated goat anti-rabbit IgG. The result showed that the molecular weight of the expression product is identical with that of the authentic protein and the antigenic properties are also the same. Furthermore, the expression product purified with a CM-papain Sepharose affinity column and FPLC system with a Mono-Q column showed the same inhibitory activity for various cysteine proteinases. Also, purified recombinant cystatin A was found to have identical amino acid composition, NH₂-terminal amino acid sequence, and peptide-map on reverse phase HPLC with those of the authentic inhibitor.

View full abstract