Pyridoxal 5'phosphate binding site of Escherichia coli beta cystathionase and cystathionine gamma synthase comparison of their sequences

The phosphopyridoxyl peptides of beta cystathionase and cystathionine gamma synthase of Escherichia Coli were identified after reduction, carboxymethylation and proteolysis of the holoenzymes. Their comparison with those obtained from rat liver gamma cystathionase (Fearon, C.W., Rodkey, J.A. and Abeles R.H. 1982. Biochemistry 21 3790-3794.) showed a high degree of homology between the three PLP binding sites with the presence of the tripeptide sequence: Thr-Lys(Pxy)-Tyr in their structure. This homology suggests that these enzymes of methionine metabolism have probably the same origin.     

In vivo 31P NMR in crustacean muscles: fatigue and recovery in the tail musculature from the prawn Palaemon elegans

31P NMR has been used to observe the in vivo phosphometabolite concentrations in the tail musculature from the prawn Palaemon elegans, at rest and after escape swimming and subsequent recovery. Muscular fatigue corresponds to a 60% breakdown of phosphoarginine, and a 45% increase of sugar phosphates. The pHi fell from 7.10 to 6.86. During recovery, the sugar phosphates and arginine phosphate are replenished after 20 minutes. The ATP concentration did not change throughout the experiment. The pHi was restored within 20 minutes.     

Biochemical indicators of water stress in sunflower seedlings

The free proline and chlorophyll contents, and the chlorophyllase, peroxidase and nitrate-reductase activities were determined in sunflower seedlings grown under controlled conditions and submitted to water stress induced by 14 % polyethyleneglycolj (M_r = 4000) or isotonic NaCl solution. Combined free proline content and peroxidase activity may be used for detection of the factor inducing water stress.     

Evidence for a type-IV-related collagen in Drosophila melanogaster. Evolutionary constancy of the carboxyl-terminal noncollagenous domain

Type IV collagen, a major structural component of basement membrane, has been characterized only in vertebrates. It is unique among the collagenous proteins in that it forms specific lattice networks by end-to-end interactions. In particular, in mammals the C-terminal noncollagenous domain (NCl) of collagen IV was shown to be one of the major cross-linking sites in the network assembly. Here, we give the first direct evidence of type-IV-related collagen in invertebrates by sequence analysis of cDNA and genomic DNA clones for the 3'-end of a previously characterized Drosophila collagen gene. The data describe the C-terminal 190 amino acid residues of the triple helix and the entire noncollagenous domain (231 amino acids) of the chain encoded for by this gene. Comparison with data reported for human and mouse alpha 1(IV) chains reveals that triple-helix regions are quite different, while NC1 structures are very similar. This suggests different constraints on triple-helix and NC1 domains during evolution. Present data support the assumption that the NC1 structure originated from duplication of an ancestral sequence; the extent of both interspecies and intramolecular homologies suggests the maintenance in vertebrates and invertebrates of an ancestral specific function.     

Synthetic peptides used to locate the alpha-bungarotoxin binding site and immunogenic regions on alpha subunits of the nicotinic acetylcholine receptor

Synthetic peptides corresponding to 57% of the sequence of alpha subunits of acetylcholine receptors from Torpedo californica electric organ and extending from the NH2 to the COOCH terminus have been synthesized. The alpha-bungarotoxin binding site on denatured alpha subunits was mapped within the sequence alpha 185-199 by assaying binding of 125I-alpha-bungarotoxin to slot blots of synthetic peptides. Further studies showed that residues in the sequence alpha 190-194, especially cysteines-alpha 192, 193, were critical for binding alpha-bungarotoxin. Reduction and alkylation studies suggested that these cysteines must be disulfide linked for alpha-bungarotoxin to bind. Binding sites for serum antibodies to native receptors or alpha subunits were mapped by indirect immunoprecipitation of 125I-peptides. Several antigenic sequences were identified, but a synthetic peptide corresponding to the main immunogenic region (which is highly conformation dependent) was not identified.     

Patterns of spontaneous unit preoptic neurosecretory cell discharges in the rainbow trout, Salmo gairdneri

Extracellular antidromic potentials recorded from the neurosecretory cell body were characterized by the following criteria: constant latency, the ability to follow a high frequency rate of stimulation and the collision test. The latency of the antidromic potentials ranged from 12 to 24 ms (17.46 +/- 3.10 SD) which gave a mean conduction velocity of 0.19 m/s, typical of unmyelinated nerve fibers. Two components could be clearly distinguished in the antidromic potential. A small "A" spike which showed constant latency and a large "B" spike with a variable latency and amplitude. A delay of 6.5 ms between the two spikes could occur and sometimes the "B" spike was blocked leaving only the "A" spike. Four patterns of spontaneous activity seem to emerge: Type I (26% of units, M +/- SD = 0.77 +/- 0.32 sp/s) corresponds to a slow and irregular pattern of activity; Type II (28% of units, M = 1.58 +/- 0.47 sp/s) is hard to classify and may be related to an irregular bursting pattern of activity; Type III (28% of units, M = 2.59 +/- 1.19 sp/s) corresponds to a continuous pattern of activity; Type IV (18% of units) represents a rhythmic pattern of activity with an active phase of about 3 min (M = 2.42 +/- 0.90 min), a silent phase of about 4 min (M = 3.89 +/- 3.02 min) and a maximal frequency of unit discharge in the range 2-18 sp/s. No statistical differences exist for the mean dorsal aortic pressure (DAP) between the four types of neurosecretory cell activity.     

Intraventricular somatostatin-14, arginine vasopressin, and oxytocin: analgesic effect in a patient with intractable cancer pain

The analgesic effect of intraventricular somatostatin-14 (SOM-14), arginine vasopressin (AVP), and oxytocin (OT) were tested in one terminally ill cancer patient with a diffuse mesothelioma suffering intractable continuous and incapacitating thoracic pain. SOM-14 reduced pain by 90% for 48 min; AVP reduced pain by 95% for 75 min, and OT reduced pain by 88% for 77 min. The only notable side effects were seen after the administration of AVP, which induced anesthesia and flaccid paralysis of the lower limbs, from which the patient fully recovered after 20 h.     

Molecular cloning of cDNA for human prostatic acid phosphatase

A human liver cDNA library in λgt11 was screened with polyclonal antiserum to human acid phosphatase isoenzyme 2a/4. About eleven positive clones have been obtained. Two clones, λ Hap21 and λ Hap22 were further characterized: clone λHap21 contained a 0.8-kb cDNA insert and clone λHap22 a 1.8–2.0-kb insert. XbaI digestion of λHap22 generated two fragments of 1.0 and 0.9 kb. BglII digestion resulted in a 1.2-kb fragment and several smaller fragments of undetermined size. Clone 1 Hap22 contained all the genes carried by λ gt11(lac 5cI857nin 5Sam 100) and the 2-kb insert. An Escherichia coli(λHap22) lysogen was generated, and its acid phosphatase activity was approximately ten-fold higher than that in the control nonlysogenic lysate. Western-blot analysis of total proteins present in this E. coli(λHap22) lysate revealed that the non-induced λHap22 prophage directed the synthesis of an approx. 175-kDa protein. This protein was recognized by antibody to the human acid phosphatase isoenzyme 2a/4 and anti-β-galactosidase and was produced only upon induction with IPTG. These results indicated that AHap22 carried a major portion of the gene coding for the human acid phosphatase isoenzyme 2a and/or 4 and this protein fragment of acid phosphatase was sufficient to manifest enzymatic activity.     

Leishmaniasis in Bolivia. IV. The dog in the cycles of leishmaniasis in Bolivia

In Bolivia, the dog is involved in the cycle of visceral leishmaniasis (Leishmania (Le.) chagasi) in the Yungas (alt. 1,000-2,000 m), and also in the cycle of cutaneous leishmaniasis (Le. (V.) braziliensis) in the Alto Beni (alt. 400-600 m). But it plays a different role in the two cycles. In the Yungas focus, it is the main reservoir of Le. (Le.) chagasi and the source of contamination for man. In the Alto Beni focus, it is only a "victim-host", like man, of Le. (V.) braziliensis; the reservoir of which is unknown. Wild mammals are very likely to be involved.     

Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris AM2

An aminopeptidase was purified from cell extracts of Lactococcus lactis subsp. cremoris AM2 by ion-exchange chromatography. After electrophoresis of the purified enzyme in the presence or absence of sodium dodecyl sulfate, one protein band was detected. The enzyme was a 300-kilodalton hexamer composed of identical subunits not linked by disulfide bridges. Activity was optimal at 40 degrees C and pH 7 and was inhibited by classical thiol group inhibitors. The aminopeptidase hydrolyzed naphthylamide-substituted amino acids, as well as dipeptides and tripeptides. Longer protein chains such as the B chain of insulin were hydrolyzed, but at a much slower rate. The Michaelis constant (K(m)) and the maximal rate of hydrolysis (V(max)) were, respectively, 4.5 mM and 3,600 pkat/mg for the substrate l-histidyl-beta-naphthylamide. Amino acid analysis showed that the enzyme contained low levels of hydrophobic residues. The partial N-terminal sequence of the first 19 residues of the mature enzyme was determined. Polyclonal antibodies were obtained from the purified enzyme, and after immunoblotting, there was no cross-reaction between these antibodies and other proteins in the crude extract.