Purification of GTP-binding proteins from bovine brain membranes. Identification of heterogeneity of the alpha-subunit of Go proteins

Using high-resolution Mono Q column chromatography, we purified 6 distinct peaks of GTP-binding proteins from bovine brain membranes. Five of them consisted of 3 polypeptides with alpha beta gamma-subunits and served as the substrate of islet-activating protein (IAP), pertussis toxin. The other one was purified as alpha-subunit alone and was also ADP-ribosylated by IAP in the presence of beta gamma-subunits. When each alpha-subunit was characterized by immunoblot analysis using various antibodies with defined specificity, the two of them were identified as Gi-1 and Gi-2, and other 4 appeared to be Go or Go-like G proteins. The alpha-subunits of immunologically Go-like proteins were apparently distinguishable from one another on elution profiles from the Mono Q column. Thus, there was a heterogeneity of the alpha-subunit of Go in the brain membranes.     

Photomovement in Dunaliella salina: Fluence rate-response curves and action spectra

We determined the action spectra of the photophobic responses as well as the phototactic response in Dunaliella salina (Volvocales) using both single cells and populations. The action spectra of the photophobic responses have maxima at 510 nm, the spectrum for phototaxis has a maximum at 450–460 nm. These action spectra are not compatible with the hypothesis that flavoproteins are the photoreceptor pigments, and we suggest that carotenoproteins or rhodopsins act as the photoreceptor pigments. We also conclude that the phototactic response in Dunaliella is an elementary response, quite independent of the step-up and step-down photophobic responses. We also determined the action spectra of the photoaccumulation response in populations of cells adapted to two different salt conditions. Both action spectra have a peak a 490 nm. The photoaccumulation response may be a complex response composed of the phototactic and photophobic responses. Blue or blue-green light does not elicit a photokinetic response in Dunaliella.Diagrams of the optical set-ups used for measuring the responses at the single-cell level and of the plans for building the phototaxometer described in this paper are available to the interested readerWe thank Mr. M. Kubota for a tremendous amount of technical assistance and Mr. R. Nagy for building the phototaxometer. We thank T. Kondo, Professor H. Imaseki and the members of the Laboratory of Biological Regulation, NIBB, for their help and support in various aspects of this research. This research was supported, in part, from grants from the Okazaki Large Spectrograph (Project Nos. 86-535, 87-518, 88-523), the Japanese Society for the Promotion of Science, and the College of Agriculture and Life Sciences at Cornell University to R. W.     

Cloning of a rabbit brain glucose transporter cDNA and alteration of glucose transporter mRNA during tissue development

A full-length cDNA clone that codes for glucose transporter protein was isolated from a rabbit brain cDNA library by using synthetic oligonucleotide probe derived from the sequence of human glucose transporter cDNA. The coding region shared 93.2% nucleotide and 97.0% amino-acid similarities with those of human glucose transporter and 89.4% nucleotide and 97.4% amino-acid similarities with those of rat transporter. Northern blot analysis revealed that glucose transporter mRNA is most abundant in the placenta and that it is also abundant in the brain. The fat tissue, heart, liver, and skeletal muscle of adult rats contained a very small amount of mRNA, while heart, liver, skeletal muscle and kidney of fetal rats contained a very high amount of glucose transporter mRNA. These results suggest that this type of glucose transporter might be closely related with cell proliferation and tissue development.     

In vitro release of growth hormone-releasing factor from rat hypothalamus: effect of insulin-like growth factor-1

The release of growth hormone-releasing factor (GHRF) from rat hypothalamus was investigated in vitro. After 60 min preincubation the released GHRF from sliced rat hypothalamic fragments during 60 min incubation was detected by a highly specific and sensitive radioimmunoassay for rat GHRF. The release of GHRF was Ca2+-dependent and enhanced by high concentration of K+. Insulin-like growth factor-1 (IGF-1) significantly decreased GHRF release to 65% and 84% of the control at concentrations of 10(-8) M and 10(-7) M, respectively. These results suggest that this in vitro system is useful for the investigation of the mechanism of GHRF release from the hypothalamus and that IGF-1 is probably involved in the feedback inhibition of growth hormone secretion by attenuating GHRF release from the hypothalamus besides countering the effect of GHRF on the pituitary.     

Effect of triethylenepentaminehexaacetic acid on the renal damage in cadmium-treated Syrian hamsters

Cadmium (Cd)-induced nephropathy was treated by triethylene-pentaminehexaacetic acid (TTHA) in male Syrian hamsters. Hamsters injected three times a week with 3 mg/kg body wt CdCl₂ showed proteinuria, urinaryN-acetyl-β-d-inglucosaminidase (NAG), and fractional excretion of sodium (FENa) when compared to saline-injected control. Cd-treated hamsters injected ip with TTHA 10 mg/kg body wt five times a week showed reduction of renal damage, including reductions in urinary protein (from 6.7±2.2 to 4.3±0.5 mg/d) and NAG (0.17±0.06 to 0.04±0.02 U/d). Urinary excretion of Cd was significantly increased (from 87±51.3 to 3052±1485 mg/L) by TTHA administration. Cd concentration in renal cortical tissue was slightly reduced (26.4±3.0 to 21.8±2.7 mg/g. protein). Excretion of malondialdehyde (MDA) was increased only in Cd-injected hamsters (to 2.1±1.6 nM/L), and elevated MDA in renal cortical tissue was not reduced by the administration of TTHA (1041±105 vs 1104±358 nM/g protein). Glutathione (GSH) concentration in the renal cortex was significantly elevated after Cd administration and further increased after TTHA administration (5.5±2.1 to 9.8±2.0 μg/50 mg protein). There were no marked effects on creatinine clearance (Ccr) and hematocrit. Moreover, renal morphological changes were improved significantly by treatment with TTHA. We demonstrated the efficacy of TTHA in the treatment of Cd-induced nephropathy in hamsters. Although the precise mechanism of the TTHA effects on Cd-induced nephropathy has not been elucidated, it might involve GSH reducing the elevated MDA concentration in renal tissue.     

The distribution of alpha-melanocyte stimulating hormone (alpha-MSH) in the central nervous system of the rat: an immunohistochemical study. II. Lower brain stem

The distribution of immunoreactive alpha-melanocyte stimulating hormone (alpha-MSHI) in the rat lower brain stem was examined by indirect immunofluorescence or peroxidase- anti-peroxidase immunohistochemical method using an antiserum against synthetic alpha-MSH. The results confirmed the presence of alpha-MSHI fibers in the midbrain central gray matter and parabrachial area, and demonstrated a much more extensive distribution of these fibers in various parts of the lower brain stem areas previously thought not contain alpha-MSHI fibers. In addition, the commissural nucleus was identified as a new alpha-MSHI neurons-containing site. No alpha-MSHI neurons were seen in other regions of the rat lower brain stem.     

Substitution of leucine for tryptophan 412 does not abolish cytochalasin B labeling but markedly decreases the intrinsic activity of GLUT1 glucose transporter

GLUT1 glucose transporter cDNA was modified to introduce a single amino acid substitution of leucine for tryptophan 412, a putative cytochalasin B photo-affinity labeling site. Although the mutated transporter was expressed into plasma membranes of Chinese hamster ovary cells, glucose transport activity of the mutated transporter was observed to be only 15-30% of that of the wild-type GLUT1 when glucose transport activity was assessed by 2-deoxyglucose uptake at 0.1-10 mM concentrations. Analysis of glucose uptake kinetics depict that a mutation induced a 3-fold decrease in turnover number and a 2.5-fold increase in Km compared with the wild-type GLUT1. Importantly, cytochalasin B labeling was not abolished but decreased by 40%, and cytochalasin B binding was also decreased. In addition, the results obtained with side-specific glucose analogs suggested that the outer glucose binding site of the mutant appeared intact but the inner binding site was modulated. These results indicate 1) tryptophan 412 is not a cytochalasin B labeling site(s), although this residue is located in or close to the inner glucose binding site of the GLUT1 glucose transporter, 2) substitution of leucine for tryptophan 412 decreases the intrinsic activity of GLUT1 glucose transporter, which is definable as the turnover number/Km, to approximately 15% of that of the wild-type.     

The effect of apo E secretion on lipoprotein uptake in transfected cells.

To investigate the role of apolipoprotein E (apo E) secreted by peripheral tissues in local lipoprotein metabolism, we developed a cell strain that constitutively produced and secreted apo E. A fusion plasmid containing rat apo E genomic DNA under control of mouse metallothionein promotor was constructed and transfected into Chinese hamster ovary cells. A stable transformant designated CHO-MAEII constitutively secreted rat apo E mainly in the form of sialylated free protein. The secretion was further enhanced by metal induction up to 1 micrograms apo E/ml per 12 h. When incubated with 125I-labeled very low density lipoprotein (125I-VLDL) at 37 degrees C, CHO-MAEII took up and degraded 125I-VLDL with higher affinity than control cells. Furthermore, considerable amount of methylated 125I-VLDL was degraded by CHO-MAEII, while no methylated 125I-VLDL was degraded by control cells. No significant differences were found in the uptake of 125I-LDL. The data indicated that apo E molecules secreted by CHO-MAEII were transferred to 125-VLDL particles, which caused a higher affinity of these particles for LDL receptors on the cells. It is suggested that apo E secreted from peripheral tissues enhances the uptake of lipoproteins by themselves or by surrounding cells in the local environment which demand cholesterol and express LDL receptors. CHO-MAEII was a good model for these 'auto- or paracrine-like functions' of apo E.