Effect of a bovine hypothalamic factor on the release and biosynthesis of growth hormone.

Partial purification of growth hormone (GH)-releasing factor (GRF) by acid extraction followed by gel filtration on Sephadex G-25 has been attained from bovine hypothalami. When rat pituitaries were incubated in 2 ml Krebs Ringer-bicarbonate-glucose (KRBG) medium, a stimulatory effect of the GRF fraction on immunoreactive GH (IR-GH) release was observed, while that of the factor neither on GH synthesis nor release of the synthesized GH was demonstrated. Stimulation of the GH release was exerted maximally within 30 min of incubation. Cycloheximide and actinomycin D, at a concentration which inhibited protein and RNA synthesis to less than 5 and 20% of the control, respectively, were without effect on the stimulatory action of the factor on GH release. On the other hand, stimulation of GH synthesis was observed under incubation in 0.3 ml medium with the factor and enhancing effect of the factor on the IR-GH release was undetectable. These results suggest that stimulation of the release and synthesis of GH mediated by the hypothalamic GRF fraction is under influence of the pool size of incubation media.     

Two forms of α-glucosidase from sugar-beet seeds

Two forms of -glucosidase (EC 3.2.1.20), designated as I and II, have been isolated from sugarbeet (Beta vulgaris L.) seeds by a procedure including fractionation with ammonium sulfate and ethanol, carboxymethyl-cellulose column chromatography, and preparative disc gel electrophoresis. The two enzymes were homogeneous by polyacrylamide disc gel electrophoresis. Their molecular weights were 98,000 (I) and 60,000 (II). -Glucosidase I readily hydrolyzed maltose, isomaltose, kojibiose, maltotriose, panose, amylose, soluble starch, amylopectin and glycogen. -Glucosidase II also hydrolyzed maltose, kojibiose and maltotriose but hydrolyzed the other substrates only very weakly or not at all. -Glucosidase I hydrolyzed soluble starch at a faster rate than maltose. It produced isomaltose and panose as the main -glucosyltransfer products from maltose, whereas maltotriose was the main product of -glucosidase II. -Glucosidase I hydrolyzed amylose liberating -glucose. The neutral-sugar content was calculated to be 2.7% for -glucosidase I and 8.8% for -glucosidase II. The main neutral sugar was mannose in -glucosidase I, and glucose in -glucosidase II.     

Modification of available arginine residues in proteins by p-hydroxyphenylglyoxal

p-Hydroxyphenylglyoxal reacts with arginine residues in proteins to give a single product which can be quantitated spectrophotometrically. The reaction takes place under mild conditions, pH 7–9 and 25°C. Under these conditions up to complete modification of N^α-citraconyl-l-arginine was obtained within 60 min with less than 5% modification of other common amino acid side chains. The extent of modification in a protein can be determined at 340 nm using the molar absorption coefficient of 1.83 × 10⁴m^?1 cm^?1 for the product at pH 9.0 and 25°C following removal of excess reagent by gel filtration. Several proteins, previously shown to have essential arginines, were modified by p-hydroxyphenylglyoxal and the losses in arginines were determined spectrophotometrically. These results were in close agreement with those of previous investigators. Rhea ovomucoid, a glycoprotein without arginines but containing an essential lysine, was relatively unaffected.     

A cytoskeleton-related gene, uso1, is required for intracellular protein transport in Saccharomyces cerevisiae

The Saccharomyces cerevisiae mutant strains blocked in the protein secretion pathway are not able to induce sexual aggregation. We have utilized the defect of aggregation to concentrate the secretion-deficient cells and identified a new gene which functions in the process of intracellular protein transport. The new mutant, uso1, is temperature sensitive for growth and protein secretion. At the restrictive temperature (37 degrees C), uso1 mutant accumulated the core-glycosylated precursor form of the exported protein invertase in the cells. Ultrastructural study of the mutant fixed by the freeze-substitution method revealed expansion of the nuclear envelope lumen and accumulation of the ER at the restrictive temperature. Abnormally oriented bundles of microtubules were often found in the nucleus. The USO1 gene was cloned by complementation of the uso1 temperature-sensitive growth defect. DNA sequence analysis revealed a hydrophilic protein of 1790 amino acids with a COOH-terminal 1,100-amino acid-long alpha-helical structure characteristic of the coiled-coil rod region of the cytoskeleton-related proteins. These observations suggest that Uso1 protein plays a role as a cytoskeletal component in the protein transport from the ER to the later secretory compartments.     

The 55-kilodalton protein in an oriC complex fraction is glycogen synthase.

Three proteins with molecular masses of 35, 55, and 75 kDa were found in an oriC complex fraction after purification through CsCl density gradient centrifugation (W. G. Hendrickson, T. Kusano, H. Yamaki, R. Balakrishnan, M. King, J. Murchie, and M. Schaechter, Cell 30:915-923, 1982). Of these three proteins, the 55-kDa protein was determined to be glycogen synthase on the basis of the N-terminal amino acid sequence and the molecular weight. The oriC complex was formed in glgA mutant cells, which produce no detectable glycogen, as well as in wild-type cells. None of the 35-, 55-, and 75-kDa proteins were detected in the fraction from this mutant. The results indicate that these proteins were not constituents of the oriC complex.     

Inhibition of phosphoenzyme formation from phosphate and sarcoplasmic reticulum Ca(2+)-ATPase by vanadate binding to high- or low-affinity site on the enzyme.

In the preceding paper, we suggested that 1 mol Ca(2+)-ATPase of sarcoplasmic reticulum (SR) contains 0.5 ml of high-affinity vanadate binding sites as well as 0.5 ml of low-affinity vanadate binding sites [Yamasaki, K. & Yamamoto, T. (1991) J. Biochem. 110, 915-921]. In the present study, we examined the effects of vanadate binding to the high- and low-affinity sites upon phosphorylation of the enzyme by inorganic phosphate (Pi). When vanadate was added to the reaction medium in which the Ca(2+)-ATPase had been phosphorylated by Pi in the absence of Ca2+, the steady-state level of phosphoenzyme (E2P) decreased due to inhibition of its formation. The decrease of E2P after addition of vanadate exhibited biphasic kinetics consisting of an initial fast decay process followed by a slower first-order decay process. The size of the fast E2P decay, which was estimated by extrapolating the slow phase decay to time 0, varied depending on the vanadate concentration with a dissociation constant of 17 microM, and reached maximum at 50 microM vanadate. The maximum value of the fast E2P decay was almost equal to the initial E2P level. The initial fast decay of E2P was competitively prevented by Pi with a dissociation constant of 7.4 mM, which was very close to Km for the E2P formation under similar conditions. These observations suggested that vanadate inhibits E2P formation by competition with Pi at a phosphorylation site on the Ca(2+)-ATPase. The slow first-order decay of E2P corresponded well to the vanadate binding to the high-affinity site of the Ca(2+)-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differences in pathological findings and growth hormone responses in patients with growth hormone-producing pituitary adenoma.

Plasma growth hormone (GH) responses to various stimuli were examined in 21 patients with GH-producing pituitary adenomas, classified into three types by the immunohistochemistry of cytokeratin and the glycoprotein hormone alpha-subunit distribution. Seven type 1 adenomas were exclusively composed of cells in which the cytokeratin formed a dot-like pattern; they were chromophobic to hematoxylin and eosin (H&E), occasionally positive for GH, and almost completely negative for the alpha-subunit. Thirteen type 2 adenomas were composed of cells with cytokeratin that had a perinuclear distribution; they were eosinophilic to H&E, and diffusely positive for both GH and the alpha-subunit. One patient had a type 3 adenoma which had a mixed pattern of intracellular cytokeratin distribution and was chromophobic and eosinophilic to H&E. Clinically, type 1 is characterized by earlier onset, larger tumor size, and more frequent aggressive extension. Paradoxical GH responses to TRH and OGTT were seen in 1 of 6 patients (16.7%) of type 1 and 8 of 9 patients (88.9%) of type 2, and 0% of type 1 and 62.5% of type 2, respectively. Type 2 cases showed higher plasma GH response to GH-releasing hormone, and a tendency to greater suppression of plasma GH by bromocriptine compared with type 1. Octreotide acetate administration revealed that the nadir/basal ratio of plasma GH levels was 42.9 +/- 6.6% in type 1 and 13.5 +/- 5.8% in type 2. These results suggest that there is a pathophysiological difference between these two distinct types of GH-producing pituitary adenomas.     

The hypotrophic axonopathy mutant in Japanese quail.

A new behavioral mutant showing either head or body quivering, or both, was found in Japanese quail. This trait was characterized by neurofilament deficiency in the axons of the cervical spinal cord and the optic and sciatic nerves and was named "hypotrophic axonopathy." This character was shown to be controlled by an autosomal recessive gene, for which the gene symbol hax was proposed.     

ATPase activity of SopA, a protein essential for active partitioning of F plasmid

Summary The SopA, B, C genes of the F plasmid play an essential role in plasmid partitioning during cell division in Escherichia coli. In this paper, the products of the sopA and sopB genes were isolated and their biochemical activities studied. [-³²P]ATP was cross-linked to the SopA protein by UV irradiation; this cross-linking was observed only in the presence of magnesium ion, and was competitively inhibited in the presence of non-radioactive ATP, ADP and dATP, but not other NTPs or dNTPs. In contrast, no ATP binding activity was detected for the SopB protein. The SopA protein showed a modest magnesium ion-dependent ATPase activity and this activity was stimulated in the presence of DNA. The ATPase activity in the presence of DNA was further stimulated by addition of the SopB protein. However, the SopB protein alone failed to stimulate the ATPase activity.     

Oleanane glycosides from Glycyrrhiza yunnanensis roots.

From the roots of Glycyrrhiza yunnanensis, collected in Yunnan, China, six new oleanane-type triterpene glycosides named yunganosides A1, B1, C1, D1, E2 and F2 were isolated together with hypaphorine. The structures of these glycosides were established by spectroscopic and chemical means.