5'-Nucleotidase in rat liver lysosomes

5'-Nucleotidase was found in purified rat liver tritosomes. When tritosomes were subfractionated into the membrane and soluble contents fractions, 73% of the total 5'-nucleotidase activity was found in the membrane fraction and 24% in the soluble contents fraction. Immunoblotting using specific polyclonal antibodies against the rat liver plasma membrane 5'-nucleotidase showed that the mobilities on SDS-polyacrylamide gel electrophoresis of both 5'-nucleotidases in the membrane and contents fractions were identical to that of the enzyme in the plasma membranes (Mr = 72,000). 5'-Nucleotidases in the membrane and contents fractions were sensitive to neuraminidase and converted into a form that was 4 kDa smaller after digestion, as observed in the case of plasma membrane enzyme. 5'-Nucleotidases, both from the membrane and contents fractions, were purified using immunoaffinity chromatography, and the isoelectric points, heat stability, and oligomeric structure of the purified enzymes were compared. Isoelectric focusing and the heat stability test indicated the resemblance of the soluble enzyme to the membrane-bound enzyme. However, the membrane-bound enzyme aggregated in the absence of Triton X-100, whereas the soluble enzyme behaved as a dimer. The topography of 5'-nucleotidase in the tritosomal membranes was studied using antibodies against 5'-nucleotidase and neuraminidase treatment. The inhibition of 5'-nucleotidase were not observed in the intact tritosomal fraction until the tritosomes had been disrupted by osmotic shock. These results show that the active sites and the oligosaccharide chains of 5'-nucleotidase are located on the inside surface of the tritosomal membranes.     

Calcium influx in a single rat basophilic leukemia cell as revealed with a digital imaging fluorescence microscope

Using a digital imaging fluorescence microscope, we have detected a rapid transient increase in the free cytosolic calcium concentration in a single rat basophilic leukemia cell (RBL-2H3) after antigen stimulation. Calcium ions were transported very rapidly (within 1 s) after a lag time (about 10 s at 37 degrees C) from the external environment into the cytoplasm. On the basis of the present experimental results we conclude that the gradual changes in the overall fluorescence intensity observed for a cell suspension are due to the distribution of different lag times shown by different cells as to the calcium influx through membrane calcium channels.     

Bovine high molecular weight kininogen. The amino acid sequence, positions of carbohydrate chains and disulfide bridges in the heavy chain portion

The existence of two types of circulating bovine plasma high molecular weight kininogen (HMWK) was predicted from analyses of complementary DNAs coding for this protein (Kitamura, N., Takagaki, Y., Furuto, S., Tanaka, T., Nawa, H., and Nakanishi, S. (1983) Nature 305, 545-549). The present protein-based study provided evidence in support of the proposed amino acid sequence derived from analysis of the cDNA clone, and the results confirm the existence of two types of circulating HMWK. Type I HMWK contains a heavy chain composed of 361 residues, while the heavy chain of type II HMWK contains 359 residues. The amino acid sequences of type I and type II HMWK determined in this study were identical to that inferred from the cDNA sequence with the exception of microheterogeneity observed in the cDNA at position 87 (Glu/Gln) and 168 (Lys/Arg). The heavy chain of type I HMWK contains 4 asparagine-linked carbohydrate chains at Asn-69, -150 (or -151), -179, and -186, while the heavy chain of type II HMWK contains these and an additional carbohydrate chain at Asn-264. In addition, a carbohydrate chain was found to be O-glycosidically linked to Thr-118 in both chains. Among nine disulfide linkages found in HMWK, eight intrachain disulfide pairs were established in the heavy chain. One interchain disulfide bridge occurs between the heavy chain and the light chain. This disulfide pairing, as well as repeating amino acid sequences observed in the heavy chain, provides strong evidence for the existence of three homologous domains in the heavy chain of bovine HMWK.     

S-100 Protein in Clonal Astroglioma Cells Is Released by Adrenocorticotropic Hormone and Corticotropin-Like Intermediate-Lobe Peptide

S-100 protein in clonal GA-1 and C6 rat glioma cell lines was released in serum-free medium supplemented with adrenocorticotropic hormone (ACTH). The induction of S-100 protein release by ACTH was dose-dependent, showing a half-maximal release at about 5 microM, and the S-100 protein concentration in the medium increased sharply within 3 min, but slightly during further incubation. The S-100 protein release was apparently accompanied by a decrease in the membrane-bound form of S-100 protein in the cell. The S-100 protein release was induced not by the ACTH1-24 fragment, which exhibits the known effects of ACTH, but by the ACTH18-39 fragment, which is designated as corticotropin-like intermediate-lobe peptide (CLIP). These results indicate that the C-terminal half of ACTH is responsible for the S-100 protein release. The enhancement of S-100 protein release by ACTH was also observed in normal rat glioblasts. The release induced by ACTH was apparently specific to S-100 protein, because little release of the cytoplasmic enzymes, creatine kinase, and enolase was observed under the same conditions. High concentrations (5 mM) of dibutyryl cyclic AMP or dibutyryl cyclic GMP were also found to induce S-100 protein release; however, catecholamines (epinephrine, norepinephrine, isoproterenol, and dopamine), acetylcholine, and glutamic acid did not enhance the release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ovomucoid third domains from 100 avian species: isolation, sequences, and hypervariability of enzyme-inhibitor contact residues

Ovomucoids were isolated from egg whites of 100 avian species and subjected to limited proteolysis. From each an intact, connecting peptide extended third domain was isolated and purified. These were entirely sequenced by single, continuous runs in a sequencer. Of the 106 sequences we report (five polymorphisms and chicken from the preceding paper [Kato, I., Schrode, J., Kohr, W. J., & Laskowski, M., Jr. (1986) Biochemistry (preceding paper in this issue)]), 65 are unique. In all cases except ostrich (which has Ser45), the third domains are either partially or fully glycosylated at Asn45. The majority of the third domain preparations we isolated are carbohydrate-free. Alignment of the sequences shows that their structurally important residues are strongly conserved. On the other hand, those residues that are in contact with the enzyme in turkey ovomucoid third domain complex with Streptomyces griseus proteinase B [Read, R., Fujinaga, M., Sielecki, A. R., & James, M. N. G. (1983) Biochemistry 22, 4420-4433] are not conserved but instead are by far the most variable residues in the molecule. These findings suggest that ovomucoid third domains may be an exception to the widely accepted generalization that in protein evolution the functionally important residues are strongly conserved. Complete proof will require better understanding of the physiological function of ovomucoid third domains. This large set of variants differing from each other in the enzyme-inhibitor contact area and augmented by several high-resolution structure determinations is useful for the study of our sequence to reactivity (inhibitory activity) algorithm. It is also useful for the study of several other protein properties. In the connecting peptide fragment most phasianoid birds have the dipeptide Val4-Ser5, which is absent in most other orders. This dipeptide is often present in only 70-95% of the molecules and appears to arise from ambiguous excision at the 5' end of the F intron of ovomucoid. Connecting peptides from the ovomucoids of cracid birds contain the analogous Val4-Asn5 peptide. In laughing kookaburra ovomucoid third domain we found (in 91% of the molecules) Gln5A, which we interpret as arising from ambiguous intron excision at the 3' end of the F intron.     

Effect of alpha-human atrial natriuretic polypeptide on anterior pituitary function in men

In order to examine the effects of alpha-human atrial natriuretic polypeptide (alpha-hANP) on the basal plasma concentrations of GH, TSH, LH, FSH and PRL in humans, synthetic alpha-hANP was infused into 10 normotensive, euvolemic, healthy volunteers. There were observed marked hypotensive, diuretic and natriuretic effects during the alpha-hANP infusion. The basal plasma concentrations of GH, TSH, LH and FSH, showed no significant change following the alpha-hANP infusion. However, significant suppression of the plasma PRL concentration was observed with the alpha-hANP administration. The mean plasma PRL concentration tended to be decreased during 20 min of alpha-hANP infusion, however, there the differences were not statistically significant. A significant reduction in the mean plasma PRL concentration (-20%, P less than 0.5) was observed 10 min after the end of infusion, following the reversion to the preinfusion level at 70 min after the end of infusion. Such a significant and delayed suppression was not seen in the case of placebo infusion. The data suggest that the circulating hANP may reduce the release of PRL.     

Transduction of human colony-stimulating factor-1 (CSF-1) receptor into interleukin-3-dependent mouse myeloid cells induces both CSF-1-dependent and factor-independent growth.

A retroviral vector encoding the receptor for human colony-stimulating factor-1 (CSF-1) was introduced into murine myeloid FDC-P1 cells which require interleukin-3 (IL-3) for their proliferation and survival in culture. Cells expressing the CSF-1 receptor (CSF-1R), selected by fluorescence-activated cell sorting in the continued presence of murine IL-3, formed colonies in semisolid medium and were able to proliferate continuously in liquid cultures containing human recombinant CSF-1. Thus, although they do not synthesize endogenous murine CSF-1R, FDC-P1 cells express the downstream components of the CSF-1 mitogenic pathway necessary for its signal-response coupling. After receptor transduction, slowly proliferating factor-independent variants that produced neither CSF-1 nor growth factors able to support the proliferation of parental FDC-P1 cells also arose. When the human CSF-1R was expressed in FDC-P1 cells under the control of an inducible metallothionein promoter, the frequencies of both CSF-1-responsive and factor-independent variants increased after heavy-metal treatment. In addition, a monoclonal antibody to human CSF-1R arrested colony formation by both the CSF-1-dependent and factor-independent cells but did not affect their growth in response to IL-3. Therefore, the induction of both the CSF-1-dependent and factor-independent phenotypes depended on expression of the transduced human CSF-1R.     

cDNA cloning of an mRNA encoding a sulfur-rich 10 kDa prolamin polypeptide in rice seeds

Using a rice maturing seed pUC9 expression library, we isolated a cDNA clone corresponding to 10 kDa sulfurrich prolamin by immunoscreening. A longer cDNA clone was obtained from a gtll library by plaque hybridization using this ³²P-labeled cDNA as a probe. A polypeptide sequence composed of 134 amino acids was deduced from the nucleotide sequence. A 24 amino acid signal peptide was assigned by computer calculation for the membrane spanning region and Edman sequencing of the purified mature polypeptide. Remarkably, 20% of methionine and 10% of cysteine were found in the mature polypeptide as well as high contents of glutamine, and hydrophobic amino acids. Part of the amino acid sequence was homologous with a conserved cysteine-rich region found in other plant prolamins. Two repeats of amino acid sequence were found in the polypeptide.     

Developmental changes in the effects of carbachol and morphine on cGMP contents of plasma, heart, and lung of mice

It is considered that carbachol increases plasma cGMP levels by acting on muscarinic receptors and morphine increases these levels by acting on opioid receptors, followed by stimulation of muscarinic receptors. We investigated the ability of carbachol and morphine to increase cGMP contents of plasma, heart, and lung and the guanylate cyclase activity of heart and lung homogenate in 1-, 2-, 3-, and 7-week-old mice. The increase in plasma cGMP levels induced by carbachol showed a peak at 2 and 3 weeks of age. The basal cGMP contents in heart and lung and their rise induced by carbachol, as well as the guanylate cyclase activity of these organs, were decreased in 7-week-old mice. The effects of morphine on the cGMP contents showed a similar developmental change, except for no effect in 1-week-old mice. These changes in the effects of carbachol and morphine may be the result of developmental changes of the muscarinic receptor--guanylate cyclase system and opioid receptors.