The essential light chains constitute part of the active site of smooth muscle myosin

Myosin, a major contractile protein, characteristically possesses a long coiled-coil alpha-helical tail and two heads. Each head contains both an actin binding site and an ATPase site and is formed from the NH2-terminal half of one of the two heavy chains (relative molecular mass, Mr, 200,000) and a pair of light chains; the so-called regulatory and essential light chains of approximately Mr 20,000 each. Recently we have identified Trp 130 of the myosin heavy chain from rabbit skeletal muscle as an active-site amino-acid residue after labelling with a new photoaffinity analogue of ADP, N-(4-azido-2-nitrophenyl)-2-aminoethyl diphosphate (NANDP). Nonspecific labelling was eliminated by first trapping NANDP at the active site with thiol crosslinking agents. Exclusive labelling of the heavy chains with no labelling of the light chains agreed with previous findings that the heavy chains alone contain the actin-activated Mg-ATPase activity of rabbit skeletal myosin. Here we report similar photolabelling experiments with smooth muscle myosin (chicken gizzard) in which 3H-NANDP is trapped at the active site with vanadate and which show that both the heavy chains and the essential light chains are labelled. The results indicate that both chains contribute to the ATP binding site and represent the first direct evidence for participation of the essential light chains in the active site of any type of myosin.     

Recognition of myelin-associated glycoprotein by the monoclonal antibody HNK-1

Myelin-associated glycoprotein (MAG) is a quantitatively minor component in both peripheral and central myelin sheaths that is thought to have a role in cell-cell interactions within the nervous system. We show here that a mouse monoclonal antibody, HNK-1, which is directed against human natural killer cells also recognizes an antigenic determinant of human central and peripheral nervous system white matter by immunoperoxidase staining of tissue sections. Immunoblot analysis of myelin proteins and purified extracted MAG indicates that the antigen recognized is MAG.     

Absence of cooperative haemoglobin-oxygen binding in Sphenodon, a reptilian relict from the Triassic

It is generally accepted that the sigmoidal nature of the haemoglobin-oxygen dissociation curve (ODC) is necessary for efficient oxygen transport in terrestrial vertebrates because it allows large volumes of oxygen to be bound or released for relatively small changes in the partial pressure of oxygen (PO2) in the blood. Furthermore, the amount of oxygen to tissues is increased by hydrogen ions produced from the dissociation of carbon dioxide in solution. The generality of these key features of cooperative oxygen binding and the Bohr effect holds for reptiles, birds and mammals, including representatives with special respiratory requirements for diving, burrowing and living at high altitude. Sphenodon punctatus is the sole surviving representative of the ancient order of 'beakhead' reptiles (order Rhynchocephalia) which were once widely distributed during the Triassic period before the spectacular radiation of dinosaur faunas. We have now investigated the oxygen transporting properties of blood from Sphenodon and find that the ODC is hyperbolic, with a high affinity for oxygen and very small Bohr effect. This combination of characteristics is unique among terrestrial vertebrates and accords with a low demand for oxygen and limited scope for aerobic activity.     

Phorbol ester and diacylglycerol mimic growth factors in raising cytoplasmic pH

There is now good evidence that cytoplasmic pH (pHi) may have an important role in the metabolic activation of quiescent cells. In particular, growth stimulation of mammalian fibroblasts leads to a rapid increase in pHi (refs 3-6), due to activation of a Na+/H+ exchanger in the plasma membrane, and this alkalinization is necessary for the initiation of DNA synthesis. However, the mechanism by which mitogens activate the Na+/H+ exchanger to raise pHi is not known, although an increase in cytoplasmic free Ca2+ ([Ca2+]i) has been postulated as the primary trigger. We now present data suggesting that the Na+/H+ exchanger is set in motion through protein kinase C, a phospholipid- and Ca2+-dependent enzyme normally activated by diacylglycerol produced from inositol phospholipids in response to external stimuli. Using newly developed pH microelectrodes and fluorimetric techniques, we show that a tumour promoting phorbol ester and synthetic diacylglycerol, both potent activators of kinase C (refs 12-15), mimic the action of mitogens in rapidly elevating pHi in different cell types. Furthermore, we demonstrate that, contrary to previous views, an early rise in [Ca2+]i is not essential for the activation of Na+/H+ exchange and the resultant increase in pHi. Finally, we suggest that an alkaline pHi shift, mediated by Na+/H+ exchange, may be a common signal in the action of those hormones which elicit the breakdown of inositol phospholipids.     

The Tetrahymena ribozyme acts like an RNA restriction endonuclease

A shortened form of the Tetrahymena self-splicing ribosomal RNA intervening sequence acts as an endoribonuclease, catalysing the cleavage of large RNA molecules by a mechanism involving guanosine transfer. The sequence specificity approaches that of the DNA restriction endonucleases. Site-specific mutagenesis of the enzyme active site alters the substrate sequence specificity in a predictable manner, so that endoribonucleases can be synthesized to cut at a variety of tetranucleotide sequences.     

Loss of a Harvey ras allele in sporadic Wilms' tumour

Genomic changes within chromosome band 11p13 appear to have a role in the initiation of Wilms' tumour. The human Harvey ras oncogene, c-Ha-ras 1, has been located by Jhanwar et al. immediately adjacent to this region at band 11p14 .1, although several groups have assigned the gene more distally at band 11p15 . We have examined tumour DNA from two cases of sporadic Wilms' tumour, and report here that in both cases one of the two constitutional c-Ha-ras 1 alleles was absent. One tumour had a reciprocal translocation between the short arm of chromosome 11 (at band 11p13), and the long arm of chromosome 12, with no visible loss of chromosomal material. The loss of a c-Ha-ras 1 allele in association with this translocation indicates that a submicroscopic deletion had occurred. The resulting hemizygosity may have had a role in tumour initiation. Our results indicate that the c-Ha-ras 1 gene and the 'Wilms' tumour locus' may be in close proximity. It would, therefore, be premature to exclude the possibility that these two sites are functionally related.     

Tests of the helix dipole model for stabilization of alpha-helices

Charged groups play a critical role in the stability of the helix formed by the isolated C-peptide (residues 1-13 of ribonuclease A) in aqueous solution. One charged-group effect may arise from interactions between charged residues at either end of the helix and the helix dipole. We report here that studies of C-peptide analogues support the helix dipole model, and provide further evidence for the importance of electrostatic interactions not included in the Zimm-Bragg model for alpha-helix formation.     

Effect of 5-hydroxytryptamine antibodies on pigment migration in the erythrophores of the fiddler crab,Uca pugilator: Further evidence for 5-hydroxytryptamine as a neurotransmitter that stimulates release of red pigment-dispersing hormone

Summary 5-Hydroxytryptamine (5-HT) antibodies inhibit red pigment dispersion in the fiddler crab,Uca pugilator. This observation supports the hypothesis that 5-HT stimulates release of red pigment-dispersing hormone.The 5-HT antibody preparation was a generous gift from Dr S. Spector, Roche Institute of Molecular Biology, Nutley, New Jersey.     

Stereoselectivity in mammalian chemical communication: Male mouse pheromones

Two male mouse pheromones, 3,4-dehydro-exo-brevicomin (DHB) and 2-sec-butyldihydrothiazole (SBT), are chiral molecules which were previously tested in their respective bioasays as racemic mixtures. The focus of this study has been to determine the absolute configuration of their natural forms and its relation to stereospecific biological action. DHB was established as the R,R-enantiomer possessing biological activity. Due to an extremely easy racemization of SBT under very mild conditions, enantioselectivity of its transmission and its action at the receptor site appear to be of secondary importance.