Production of β-mannosidase and β-mannanase by an alkalophilic Bacillus sp.

Summary An alkalophilic bacterium producing high amounts of the cell-associated -mannosidase and extracellular -mannanase was isolated from soil. The isolate (AM-001) that grew well in alkaline pH media was identified as a strain of Bacillus sp. The optimal cultivation temperature for enzyme production was 31° C for -mannosidase and 37° C for -mannanase with the optimum production medium composed of 1% konjac powder, 0.2% yeast extract, 2% Polypepton, 0.1% K₂HPO₄, 0.02% MgSO₄ · 7H₂O and 0.5% Na₂CO₃. Optimum pH and temperature for -mannosidase were 7.0 and 55° C, and for -mannanase were 9.0 and 65° C.     

Autoradiographic demonstration of gastrin binding sites in rat gastric mucosa

The location of 125I-iodotyrosyl gastrin I binding sites in rat gastric mucosa was studied. Peptide specificity was demonstrated by competitive binding studies through the addition of a large dose of cold human gastrin I or cholecystokinin-octapeptide. Autoradiography of the stomach tissue was carried out by freeze-drying, embedding in Epon, wet-sectioning with ethylene glycol, and dry-mounting the emulsion film by means of the wire-loop method to prevent loss of the labeled substance. Specific binding sites for gastrin were found on parietal and chief cells, whereas few binding sites were seen on the surface mucous or mucous neck cells. Binding sites on the parietal cells were dispersed in the cytoplasm, while those on the chief cells were found near the basal plasma membrane.     

Purification of Protein Body-I of Rice Seed and its Polypeptide Composition

Protein body type one (PB-I) was isolated and purified fromdeveloping rice grain by a combination of sucrose density gradientcentrifugation and treatment with pepsin. SDS-PAGE analysisshowed that isolated PB-I contains several polypeptide groups,the largest having an apparent molecular size of 13 kDa andtwo smaller ones of 10 kDa and 16 kDa. The 13-kDa group wasfound to be composed of two polypeptides of slightly differentmolecular sizes, 13a (larger component) and 13b (smaller component).Most of the 13a and 13b polypeptides were shown to be largelyprolamins, although there were also some salt- and alcohol-insolublepolypeptides with an apparent molecular size of 13 kDa. It wasconcluded that PB-I is the accumulation site of rice prolamin.It was further estimated that the protein amount in PB-I accountedfor about 20% of the total protein of rice endosperm. (Received March 20, 1987; Accepted September 8, 1987)     

Measurement of Serine Acetyltransferase Activity in Crude Plant Extracts by a Coupled Assay System Using Cysteine Synthase

Serine acetyltransferase (SATase) (EC 2.3.1.30 [EC] ) catalyzes theformation of Oacetyl-L-serine (OAS) from L-serine in the presenceof acetyl-CoA. A novel assay method was developed for measuringthis enzyme activity in extracts from plant tissues. The assayconsists of a coupled system in which the OAS formed is convertedto cysteine by the addition of cysteine synthase (CSase) (EC4.2.99.8 [EC] ). Cysteine thus formed is determined colorimetricallyand serves as a measure for SATase activity. This method israpid, simple and sensitive, and can be readily adapted formeasurement of SATase activity in crude tissue extracts or homogenates. (Received January 14, 1987; Accepted April 27, 1987)     

Effect of Osmotic Stress on Turgor Pressure in Mung Bean Root Cells

Turgor pressure in cells of the elongating region of intactmung bean roots was directly measured by using the pressure-probetechnique. After the external osmotic pressure had been increasedfrom 0 MPa to 0.5 MPa, turgor pressure rapidly decreased byabout 0.5 MPa from 0.65 MPa to 0.14 MPa and root elongationstopped. Subsequent turgor regulation was clearly confirmed,which followed the osmotic adjustment to maintain a constantdifference in the osmotic pressure between root-cell sap andthe external medium ( II). It took at least 6 h for turgor pressureto recover to an adjusted constant level of about 0.5 MPa dueto turgor regulation, but rootelongation resumed within onlyan hour after the osmotic treatment. Therefore, the resumptionof root elongation under osmotic stress could not have beendirectly connected with turgor regulation. Furthermore, sincethe amounts of decrease in turgor pressure just after applicationsof various degrees of osmotic stress could be interpreted inrelation to those in II, hydraulic conductivity between theinside and the outside of root cells must be large enough toattain water potential equilibrium rapidly in response to osmoticstress. We conclude that turgor pressure in the cells of theelongating region of mung bean roots is determined mainly by II because of water potential equilibrium. (Received January 27, 1987; Accepted May 21, 1987)     

Induction and subsequent repair of DNA damage by fast neutrons in cultured mammalian cells

The induction and repair of DNA damage were studied by a DNA unwinding method in mouse L5178Y cells exposed to fast neutrons. DNA lesions induced by fast neutrons were classified into three types from their repair profiles: fast-reparable breaks (T1/2 = 3-5 min), slow-reparable breaks (T1/2 = 70 min), and nonreparable breaks. The repair rates of both fast-reparable and slow-reparable breaks were almost the same as those of corresponding damage induced by low-LET radiation. Neutrons induced a smaller amount of fast-reparable damage, an almost equal amount of slow-reparable damage, and a larger amount of nonreparable damage than those induced by equal doses of gamma rays or X rays. RBEs for fast- and slow-reparable damage were 0.3 and 0.9, respectively. The RBE for nonreparable damage was dose dependent and was 1.4 at the level of 100 breaks/10(12) Da DNA. Among the three types of lesions, only the nonreparable damage levels correlated with the linear-quadratic shape of the survival curves and with the enhanced killing effectiveness of neutrons (RBE = 1.7 at D0).     

Fine structure of myomyous junctions in the mouse skeletal muscles

The reaction product of acetylcholinesterase (AChE) activity is known to be specifically localized at a neuromuscular junction and a muscle-tendon junction of the striated skeletal muscles. In addition to the two junctions, we recently found some linear precipitates due to AChE activity running transversely across a fibre of the semitendinosus, rectus abdominis, gastrocnemius, tibialis anterior and diaphragm muscles in mice. Under an electron microscope, the linear precipitates were seen at the extracellular side of the muscle fibre endings. Most of the endings contacted each other to form a junction, which has been called the 'myomyous junction (M-Mj)'. The patterns of the M-Mj were grouped into three types: (1) a junction in which all contacts were firm, without any connective tissue, and invaginated deeply; (2) the ones in which numerous collagen fibres were visible in the space between the two separate opposing muscle fibres; (3) an intermediate type between (1) and (2), i.e. a junction with partial contacts. The muscle fibre ending forming M-Mj was constructed of finger-like processes like that of a muscle-tendon junction. However, the processes of a M-Mj adhered so closely to each other that no collagen fibrils could penetrate into their folds.     

Kyotorphin (tyrosine-arginine) synthetase in rat brain synaptosomes

Kyotorphin (Tyr-Arg) is a unique neuropeptide which produces analgesia by releasing Met-enkephalin from slices of the brain and spinal cord. Recent studies revealed that kyotorphin possesses the properties of neurotransmitter/neuroregulator. In the present study, we identified a kyotorphin synthetase in the soluble fraction of rat brain synaptosomes (synaptosol) and characterized it. The enzyme partially purified with Sephacryl S-300 showed an absolute requirement for ATP, MgCl2, tyrosine, and arginine. The optimal pH was 7.5-9.0 and the pI was determined to be 6.1-6.2 by isoelectric focusing. The Km was 25.6 microM for tyrosine, 926 microM for arginine, 294 microM for ATP, and 442 microM for MgCl2. The Vmax was 34.0 pmol/mg of protein/h. The apparent molecular size of this "kyotorphin synthetase" further purified by the DE52 column was 240,000-245,000 daltons, estimated using TSKgel G4000SW column chromatography. The enzyme reaction is represented by the following equation: Tyr + Arg + ATP + MgCl2 + kyotorphin synthetase----Tyr-Arg (kyotorphin) + AMP + PPi + MgCl2 + kyotorphin synthetase. The regional distribution and subcellular localization of the synthetase showed a close correlation to that of kyotorphin levels in the rat brain. The amounts of kyotorphin formed from amino acids by the synthetase in the dialyzed synaptosol was 3.0-4.0 times higher than that from precursor proteins by processing enzymes within the 30 min incubation.