Identification of latent procathepsin H in microsomal lumen: characterization of proteolytic processing and enzyme activation

Procathepsin H in kidney and liver microsomal lumen was identified to have a molecular mass of 41 kDa by immunoblot analysis. The proenzyme was then concentrated by applying the microsomal contents to a concanavalin A-Sepharose column. When the concanavalin A-adsorbed fraction was incubated at pH 4.0 at 20 degrees C, the activity measured with synthetic substrate increased 3.5 times over that of the control after 24 h incubation. Immunoblot analysis showed that acidic treatment caused the disappearance of procathepsin H. Thus the proenzyme might be processed to the mature enzyme under acidic conditions. The marked increase of enzymatic activity and the conversion of proenzyme were completely blocked with pepstatin which is a potent inhibitor of aspartic proteases. These results suggested that a protease for processing procathepsin H might be cathepsin D, a major lysosomal aspartic protease. Therefore, procathepsin H seems to be synthesized first in the enzymatically inactive form in endoplasmic reticulum and successively converted into the active form in lysosomes during biosynthesis.     

A homologous radioimmunoassay for guinea pig corticosteroid-binding globulin

A rapid, specific, and sensitive (requiring only 20 fmole of antigen equivalent to 0.007 microliter of serum) radioimmunoassay (RIA) was developed for the measurement of guinea pig corticosteroid-binding globulin (CBG). CBG was purified to homogeneity from guinea pig serum by affinity chromatography and used for immunization, as the standard and as the radiolabeled trace in the RIA. The antiserum to CBG was raised in rabbits. It was judged specific by immunoelectrophoresis and by comparison of RIA values with steroid-binding assay profiles obtained on serum separated on the basis of size and ion-exchange properties. The results of the radioimmunoassays agree with those of a steroid-binding assay run on identical samples. The sensitivity of the assay allows detection of CBG in serial serum samples, other biologic fluids such as milk, and cell culture supernatants.     

Gene organization in the region containing a new gene involved in chromosome partition in Escherichia coli.

A new mutation, parC, causing abnormal chromosome segregation was identified in two thermosensitive mutants of Escherichia coli. The thermosensitive growth of the mutants was corrected by pLC4-14 in the Clarke-Carbon collection. This plasmid carries a putative gene which can suppress the cell division defect due to ftsI (pbpB) and has hence been termed sufI (sui). The nearness of parC to metC was confirmed, and cotransduction frequency of parC was 59% with metC and 20% with glc. The parC-sufI region was analyzed by subcloning the chromosome region of pLC4-14. The parC and the sufI gene products were electrophoretically identified as proteins of 75 and 55 kilodaltons (kDa), respectively. The allelism of parC+ on pLC4-14 to parC1215 was confirmed by cloning parC1215. The sufI gene appeared to be dispensable for cell viability, and overproduction of its product caused suppression of ftsI. An essential gene coding for a 25-kDa protein was found between the parC and the sufI gene. These three genes were transcribed in the same direction and may be organized into an operon, with parC to the proximal side and with internal promoters at least for the distal genes. The localization of the gene products was examined in maxicells. The sufI protein was synthesized as a precursor which could be chased into a mature form. The major part of the mature form was found in the soluble fraction. The 25-kDa protein was found almost exclusively in the membrane fraction. The parC protein was associated with the membrane fraction in the presence of Mg2+ but found in the soluble fraction when Mg2+ was sequestered with EDTA.     

Purification and characterization of acid phosphatase in rat liver lysosomal contents

Acid phosphatase in rat liver lysosomal contents, C-APase I, was purified about 5,700-fold over the homogenate with 8.0% recovery, to apparent homogeneity as determined from the pattern on polyacrylamide gel electrophoresis in the presence and in the absence of SDS. The purification procedures included; preparation of crude lysosomal contents, DEAE-Sephacel ion exchange chromatography, hydroxylapatite chromatography, and gel filtration with Sephacryl S-300. The enzyme is composed of three identical subunits with an apparent molecular weight of 48K. The enzyme contains about 11% carbohydrate and the carbohydrate moiety was composed of mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine in a molar ratio of 20:3:11:1. Sialic acid was not detected in the enzyme. Antisera against the purified C-APase I were raised in goat and the C-APase I was rapidly purified with high yield (10%) by using the specific antibodies coupled to Sepharose 6B.     

Diurnal rhythms of proopiomelanocortin-derived N-terminal peptide, beta-lipotropin, beta-endorphin and adrenocorticotropin in normal subjects and in patients with Addison's disease and Cushing's disease

In order to clarify the diurnal pattern of secretion of plasma immunoreactive (IR) proopiomelanocortin (POMC)-derived peptides, IR-N-terminal peptide (Nt), IR-beta-endorphin (Ep), IR-beta-lipotropin (LPH), and IR-ACTH (ACTH) in normal subjects and in patients with Addison's disease and Cushing's disease, we measured these 4 peptides in the same plasma obtained at 0900 h and then every three hours until 0600 h at the next day. All four peptides showed diurnal rhythms with the peaks at 0600 h, and the nadirs of ACTH, LPH, Ep and Nt were at 0000 h, 0000 h, 1800 h and 0300, respectively in normal subjects. In patients with Addison's disease, these four peptides also showed diurnal rhythms with the peaks at 0600 h for ACTH and Ep and at 0900 h for LPH and Nt, and the nadirs at 2100 h for ACTH and Ep and at 0000 h for LPH and Nt. The molar ratios of Ep/ACTH, LPH/ACTH and Nt/ACTH in plasma also presented diurnal variations in normal subjects and in patients with Addison's disease. On the other hand, in patients with Cushing's disease, ACTH, LPH and Nt showed no rhythmicity or change in molar ratios of Ep/ACTH, LPH/ACTH or Nt/ACTH. Only Ep showed diurnal variation. The molar ratios of Ep/ACTH, LPH/ACTH and Nt/ACTH in patients with Cushing's disease were significantly higher than those in normal subjects and in patients with Addison's disease at 0000 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Xyloglucan in the Cell Walls of Suspension-Cultured Rice Cells

The xyloglucan present in the 24% KOH extract of the cell wallsof suspension-cultured rice cells was characterized by fragmentationanalysis with Trichoderma viride cellulase and Aspergillus oryzaeß-D-glucosidase. The xyloglucan is composed mainlyof the following oligosaccharide units: Results showed that the xyloglucan of suspension-cultured ricecells is more extensively branched than is that of rice seedlings.Another structural characteristic of the former xyloglucan isthe presence of D-galactosyl-D-xylosyl side chains that arenot found in the latter. (Received June 15, 1984; Accepted January 11, 1985)     

Characterization of Fe2+-activated acid phosphatase in rat epidermis

A particulate acid phosphatase (EC 3.1.3.2, orthophosphoric monoester phosphohydrolase (acid optimum)) was extracted in 1 M KCl, from 2-day rat epidermis. The enzyme has a Mr of 32,000, but two forms, F1 and F2 with pI values of 8.6 and 8.3, respectively, were identified while the pI values of other acid phosphatases soluble in sucrose and Triton X-100 were all acidic. F1 and F2 also differed from other epidermal acid phosphatases because they were (a) activated by Fe2+ and reducing agents, (b) showed immunological cross-reactivity with purple acid phosphatase of rat spleen and (c) dephosphorylated phosvitin and alpha-casein even though they had rather high Km values.     

Triterpenes from Periandra dulcis roots

Three oleanane triterpenes were isolated from the roots of Periandra dulcis,and identified as 3β-hydroxy-25-al-olean-18-en-30-oic acid (periandric acid I), 3β-hydroxy-25-al-olean-12-en-30-oic acid (periandric acid II) and 3-oxo-25-hydroxy-olean-12-en-30-oic acid. The former two compounds (periandric acids I and II) were identical with the aglycones obtained by hydrolysis of periandrin I and II, respectively and the latter one was a new triterpene.