Characterization of human glucocerebrosidase from different mutant alleles.

Human cDNA was mutagenized to duplicate six naturally occurring mutations in the gene for glucocere-brosidase. The mutant genes were expressed in NIH 3T3 cells. The abnormal human enzymes were purified by immunoaffinity chromatography and characterized. The Asn370----Ser mutant protein differed from normal enzyme in its inhibition by both conduritol B epoxide and glucosphingosine demonstrating that the 370 mutant enzyme has an abnormal catalytic site. In addition, the 370 mutant enzyme is less activated by saposin C, but more stimulated by phosphatidylserine than the wild type enzyme. The Arg463----Cys mutant protein was normal with respect to conduritol B epoxide and glucosphingosine inhibition, but was less activated by both saposin C and phosphatidylserine. The Arg120----Gln mutant protein was catalytically inactive. The Leu444----Pro, the pseudopattern, and the Pro415----Arg mutants appear to have reduced amounts of enzyme protein in cells. The studies demonstrated that mutations in the gene for glucocerebrosidase have different effects on the catalytic activity and stability of the enzyme.     

Atrial and brain natriuretic peptide in adrenal steroidogenesis.

We elucidated the role of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in human and bovine adrenocortical steroidogenesis. The urinary volume, sodium excretion and cyclic GMP (cGMP) excretion and plasma cGMP were markedly increased by the synthetic alpha-human ANP (alpha-hANP) infusion in healthy volunteers. Plasma arginine vasopressin (AVP) and aldosterone levels were significantly suppressed. Both ANP and BNP inhibited aldosterone, 19-OH-androstenedione, cortisol and DHEA secretion dose-dependently and increased the accumulation of intracellular cGMP in cultured human and bovine adrenal cells. alpha-hANP significantly suppressed P450scc-mRNA in cultured bovine adrenal cells stimulated by ACTH. Autoradiography and affinity labeling of [125I]hANP, and Scatchard plot demonstrated a specific ANP receptor in bovine and human adrenal glands. Purified ANP receptor from bovine adrenal glands identified two distinct types of ANP receptors, one is biologically active, the other is silent. A specific BNP receptor was also identified on the human and bovine adrenocortical cell membranes. The binding sites were displaced by unlabelled ANP as well as BNP. BNP showed an effect possibly via a receptor which may be shared with ANP. The mean basal plasma alpha-hANP level was 25 +/- 5 pg/ml in young men. We confirmed the presence of ANP and BNP in bovine and porcine adrenal medulla. Plasma or medullary ANP or BNP may directly modulate the adrenocortical steroidogenesis. We demonstrated that the lack of inhibitory effect of alpha-hANP on cultured aldosterone-producing adenoma (APA) cells was due to the decrease of ANP-specific receptor, which caused the loss of suppression of aldosterone and an increase in intracellular cGMP.     

Dihydrofolate reductase as a new "affinity handle".

Dihydrofolate reductase (DHFR) has been demonstrated to be a versatile "affinity handle" for expression of recombinant proteins. The DHFR "handle" has advantages not only in terms of efficiency of expressing the fusion protein as a soluble form but also in stabilizing unstable polypeptides and facilitating purification of the expressed protein by means of methotrexate-bound affinity chromatography and by making use of the enzyme activity. Fifteen genes encoding different lengths of polypeptides of 5 to 44 amino acids were chemically synthesized and introduced into expression vectors, pTP70-1 or its derivatives. All the polypeptide genes were efficiently expressed in Escherichia coli cells as fusion proteins which show DHFR activity. The respective fusion proteins were highly purified from cell-free extracts by monitoring the DHFR activity at each purification step. The use of methotrexate-bound affinity chromatography was very effective. In order to cut out the polypeptides, the purified fusion proteins were treated with either BrCN or site-specific protease according to the spacer sequence. The objective polypeptide was purified by means of a reversed-phase high-pressure liquid chromatography (HPLC) system. Specific cleavage of the purified fusion protein actually yielded very few peptide fragments, so the assignment and isolation of the objective polypeptide were carried out without difficulty.     

Biological effects of exposure to high frequency electromagnetics on rabbits and guinea pigs]

To investigate the biological effects of exposure to feeble high frequency electromagnetism, skin surface temperature, blood vessel (arterioles and venules) diameter were examined, using infrared thermography, a laser doppler flowmeter, and a video microscope, respectively, in the ear of rabbits. After exposing the ear of rabbits to high frequency electromagnetism value of 9 MHz for 15 minutes, continued rising of local temperature was demonstrated. Though dilatation of arterioles was not seen. In addition, venules tended to dilate and blood flow also to increase, and microcirculation was accelerated at the site where electromagnetism was exposed. Hazardous effects of long term exposures of high frequency electromagnetism (9 MHz for 30 days, 8 hours/day) on guinea pigs were not observed in their behavior, food consumption, body and organ weights, hematological and biochemical values, macroscopic and microscopic findings on autopsy.     

Primary structure of human thromboxane synthase determined from the cDNA sequence.

Polymerase chain reaction techniques have been used to isolate a cDNA clone containing the entire protein coding region of thromboxane A2 synthase (EC 5.3.99.5) from a human lung cDNA library. The cDNA clone hybridizes with a single 2.1-kilobase mRNA species in phorbol ester-induced human erythroleukemia and monocytic leukemia cell lines. A second cDNA, differing only by an insert of 163 base pairs near the 3'-end of the translated region, was also found to be present in the same library. The proteins predicted from both nucleic acid sequences include the three polypeptide sequences determined from amino acid sequencing of the purified human platelet enzyme, five potential sites for N-glycosylation, and a hydrophobic region that may serve to anchor the synthase in the endoplasmic reticulum membrane. The longer predicted protein, designated thromboxane synthase-I, contains 534 amino acids, with a Mr of 60,684, whereas the shorter protein, designated thromboxane synthase-II, contains 460 amino acids and has a Mr of 52,408. Although thromboxane synthase-II lacks the conserved cysteine that serves as the proximal heme ligand in the other cytochromes, significant sequence similarities exist among thromboxane synthase-I and -II and several P450s, particularly those in family 3. The overall amino acid identity is considerably less than 40%, making it likely that thromboxane synthase represents a previously undefined family of cytochrome P450.     

A novel fluorescence method to monitor the lysosomal disintegration of low density lipoprotein.

A novel fluorescence method to monitor the lysosomal disintegration of low density lipoprotein (LDL) particles in living cells has been developed. The method is based on the fluorescence resonance energy transfer (RET) between two fluorescent molecules incorporated into LDL particles. NBD-cholesterol linoleate (NBD-CL) and octadecyl rhodamine B (R18) were incorporated simultaneously into LDL, as a RET donor and a RET acceptor, respectively. In this preparation of LDL (RET-LDL), efficient RET was observed, and after the disruption of the LDL particle by a Triton X-100 treatment, the relief of the RET was observed. RET-LDL was endocytosed by CHO cells via LDL receptors, and the RET-LDL particles were disintegrated after the uptake. The resultant relief of the RET upon the disintegration of the LDL was monitored by flow cytometry, and the amount of intact LDL in cells was estimated by calculation. The disintegration occurred with an about 25 min lag, and was inhibited by several lysosomal inhibitors. These results indicate that the disintegration was not a nonspecific event, but took place at the level of lysosomes. Since living cells can be analyzed by the present method, when coupled to flow sorting, it would permit the isolation of cells having different properties in the endocytic pathway of LDL.     

Solution structure of human growth hormone-releasing factor fragment (1-29) by CD: characteristic conformational change on phospholipid membrane

The conformations of synthetic human growth hormone-releasing factor fragment (1-29) in the presence and the absence of 1,2-dimyristoyl-sn-glycero-3-phosphorylglycerol liposome as well as in aqueous 2,2,2-trifluoroethanol solution were investigated by CD spectroscopy. The secondary structure of the peptide in each solution was analyzed by two methods. Both results show that the peptide has an unordered structure in the aqueous solution, whereas it folds into helical structure in the aqueous alcohol and in the phospholipid solution. In addition, although the peptide exists as almost complete helix in the 50 vol% aqueous alcohol (80-90% helicity), it does not reach full helicity even in the solution containing excess amount of phospholipid liposome (maximum 65-70% helicity). The conformational difference is explained by the characteristic amphipathy of the peptide, i.e., the necessity to twist the separated amphipathic helical parts in the interaction with the phospholipid membrane probably makes the helicity of the peptide decrease.     

Unique requirements for template primers of DNA polymerase beta from rat ascites hepatoma AH130 cells.

The optimal condition for the rat DNA polymerase beta activity with (rA)n . (dT)12-18 as a template-primer was determined. The activity was remarkably affected by the concentration of the primer, (dT)12-18' and the mixing ratio of (dT)12-18 to (rA)n. DNA polymerase beta requires higher primer concentration (Km = 11.1 microM with respect to 3'-OH of the primer) than DNA polymerase gamma (Km = 0.04 microM) or oncornaviral DNA polymerase (Km = 0.08 microM) and the enzyme represented the maximum activity in the base ratio of 2:1 with (dT)12-18 and (rA)n suggesting the difference in reaction mechanisms of these enzymes. Under the optimized conditions, the specific activity of the near homogeneous preparation of DNA polymerase beta was 1,000,000 units per mg protein.