Rapid turnover of mannitol-1-phosphate in Escherichia coli.

The phosphate moiety of D-mannitol-1-phosphate in Escherichia coli is subject to rapid turnover and is in close equilibrium with Pi and the phosphorus of fructose-1,6-bisphosphate. These three compounds account for the bulk of 32P label found in cells after several minutes of uptake of 32Pi and mannitol-1-phosphate represents some 30% of this label. Mannitol-1-phosphate occurs in E. coli grown on a variety of carbon sources, in the absence of D-mannitol, and is synthesized de novo even in mutants lacking mannitol-1-phosphate dehydrogenase. The mannitol moiety of mannitol-1-phosphate was not affected during the total chase of the P moiety, which exchanged with a half-life of about 30 s. These findings suggest that the rapid equilibration of the phosphorus is a function of an enzyme, possibly a component of the phosphotransferase system, capable of forming a complex that allows the exchange of the phosphate without the equilibration of the mannitol moiety with free mannitol.     

Role of actin binding protein phosphorylation in platelet cytoskeleton assembly

Actin binding protein from human blood platelets is shown to exist in the resting platelet as a phosphorylated protein and contains two residues of phosphate per 260,000 kd. Removal of one-half of these residues with E. coli alkaline phosphatase results in the loss of its ability to crosslink F-actin into a low speed sedimentable complex (its cytoskeleton) and to bind to an F-actin affinity column. Thus, phosphorylation-dephosphorylation of ABP may be an important regulatory mechanism by which the platelet regulates its shape via its cytoskeletal structure.     

Only site-directed antibodies reactive with the highly conserved src-homologous region of the v-abl protein neutralize kinase activity

Antisera specific for six regions of the v- abl protein were used to serologically characterize the Abelson murine leukemia virus tyrosine kinase. Chemically synthesized peptides corresponding to the predicted v- abl protein sequence and larger regions of the v- abl protein expressed as fusion proteins in bacteria were used as immunogens. The specificity of each antiserum was confirmed by immunoprecipitation analysis with defined deletion mutants of Abelson murine leukemia virus. Several of these v- abl -specific antisera display much higher titers and avidities than serum harvested from mice bearing Abelson murine leukemia virus-induced tumors, previously the only source of anti- abl -specific serum. Two antisera were found to block the in vitro autophosphorylation of the v- abl protein as well as its ability to phosphorylate a peptide substrate. Examination of the sites against which the kinase-blocking antisera were prepared revealed that both are in close proximity to the in vivo sites of tyrosine phosphorylation, which fall within the region of high homology with v-src and other tyrosine kinases. Antisera directed against other regions of v- abl did not inhibit kinase activity.     

How do food passage rate and assimilation differ between herbivorous lizards and nonruminant mammals?

Summary What digestive adaptations permit herbivorous nonruminant mammals to sustain much higher metabolic rates than herbivorous lizards, despite gross similarity in digestive anatomy and physiology? We approached this question by comparing four herbivorous species eating the same diet of alfalfa pellets: two lizards (chuckwalla and desert iugana) and two mammals (desert woodrat and laboratory mouse). The mammals had longer small and large intestines, greater intestinal surface area, much higher (by an order of magnitude) food intake normalized to metabolic live mass, and much faster food passage times (a few hours instead of a few days). Among both reptiles and mammals, passage times increase with body size and are longer for herbivores than for carnivores. The herbivorous lizards, despite these much slower passage times, had slightly lower apparent digestive efficiencies than the mammals. At least for chuckwallas, this difference from mammals was not due to differences in body temperature regime. Comparisons of chuckwallas and woodrats in their assimilation of various dietary components showed that the woodrat's main advantage lay in greater assimilation of the dietary fiber fraction. Woodrats achieved greater fiber digestion despite shorter residence time, but possibly because of a larger fermentation chamber, coprophagy, and/or different conditions for microbial fermentation. We conclude with a comparative overview of digestive function in herbivorous lizards and mammals, and with a list of four major unsolved questions.     

Abelson virus potentiates long-term growth of mature B lymphocytes.

Abelson murine leukemia virus (A-MuLV) infection of mouse bone marrow cells usually leads to transformation of pre-B cells. However, when the environment is modified by the continuous presence of lipopolysaccharide (LPS), two novel types of membrane immunoglobulin (mIg)-positive B cell lines are generated. Because the cells which give rise to these cell lines copurify with mIg-positive bone marrow cells, the cell lines arise as a result of A-MuLV interaction with a new type of in vitro target cell. The cell lines generated fall into two groups which differ in several phenotypic characteristics. Group 1 cells are more differentiated than the typical pre-B cell transformant in that they synthesize mIgM and appear to resemble virgin B cells. The group 1 cells do not secrete immunoglobulin and are independent of LPS for growth. In addition, these cell lines synthesize the Abelson P160 protein, contain integrated abl proviral DNA, and are highly tumorigenic in syngeneic animals. The group 2 cell lines differ markedly from both the group 1 cells and from typical, pre-B cell A-MuLV transformants. These cells are mIgG positive and secrete large amounts of immunoglobulin into the culture medium. The cell lines are comprised of both adherent and nonadherent cells and do not synthesize P160 or contain integrated v-abl sequences. The group 2 cells are nontumorigenic in syngeneic animals and require LPS for growth and viability. Both types of cells have remained in culture for over 2 years with no changes in their phenotypic characteristics. This A-MuLV infection system and the novel mIg-positive cell lines may serve as useful models for studying biochemical and molecular properties of mature B cells.     

Regulation of the yeast metallothionein gene

To study regulation of the yeast CUP1 gene, we have employed plasmids containing the CUP1 regulatory sequences fused to the Escherichia coli galK gene. A comparison of galK expression from low- and high-copy-number CUP1/galK fusion plasmids demonstrated that both basal and induced levels of galactokinase (GalK) increase proportionately with plasmid copy number. Host strains with an amplified, single or deleted CUP1 locus were compared to look for effects of chromosomal CUP1 gene dosage on expression from the episomal CUP1 promoter. Basal GalK levels are similar in CUP1R and cupls hosts, but can be induced to higher levels in the cup1s than the CUP1R host. In contrast, in a strain deleted for the chromosomal copy of CUP1, synthesis of GalK is constitutive but can be induced to yet higher levels by copper. A hybrid vector, placing the CUP1 coding sequence under the control of a constitutive promoter, was constructed. Introduction of this hybrid CUP1 gene into the deletion host containing the CUP1/galK plasmid restores regulation. Thus, metallothionein, in trans, can effect repression of the CUP1 promoter. The possible roles of metallothionein and free copper in CUP1 regulation are discussed.     

Characterization of an ATP-Mg2+-dependent guanine nucleotide-stimulated adenylate cyclase from Neurospora crassa

A novel adenylate cyclase activity was found in crude homogenates of Neurospora crassa. The adenylate cyclase had substantial activity with ATP-Mg2+ as substrate differing significantly from the strictly ATP-Mn2+-dependent enzyme characterized previously. Additionally, the ATP-Mg2+-dependent activity was stimulated two- to fourfold by GTP or guanyl-5'-yl-imido-diphosphate (Gpp(NH)p). We propose that the ATP-Mg2+-dependent, guanine nucleotide-stimulated activity is due to a labile regulatory component (G component) of the adenylate cyclase which was present in carefully prepared extracts. The adenylate cyclase had a pH optimum of 5.8 and both the catalytic and G component were particulate. The Km for ATP-Mg2+ was 2.2 mM in the presence of 4.5 mM excess Mg2+. Low Mn2+ concentrations had no effect on adenylate cyclase activity whereas high concentrations of Mn2+ or Mg2+ stimulated the enzyme. Maximal Gpp(NH)p stimulation required preincubation of the enzyme in the presence of the guanine nucleotide and the K1/2 for Gpp(NH)p stimulation was 110 nM. Neither fluoride nor any of a variety of glycolytic intermediates or hormones, including glucagon, epinephrine, and dopamine, had an effect on ATP-Mg2+-dependent adenylate cyclase activity. However, the enzymatic activity was stimulated not only by GTP but also by 5'-AMP and was inhibited by NADH.     

Plasmids in Frankia sp.

A method to achieve cell lysis and isolate Frankia sp. plasmid DNA was developed. A screening of Frankia sp. strains belonging to different host compatibility groups (Alnus sp., Elaeagnus sp., Ceanothus sp.) showed that, of 39 strains tested, 4 (strains Cp11, ARgN22d, ArI3, and EUN1f) possessed plasmids ranging in size from 7.1 to 32.2 kilobase pairs as estimated from agarose gel electrophoresis and electron microscopy. A total of 11 plasmids were detected.     

Adenosine Triphosphatase Activity at the External Surface of Chicken Brain Synaptosomes

An ATP-hydrolysing activity on the external surface of intact synaptosomes from chicken forebrain has been investigated. The observed ATPase activity was not due to leakage of the intracellular ATPase activities, of artefacts resulting from breakage of the nerve endings during the incubation and isolation periods, or to possible contamination by other subcellular particles. Disruption of the synaptosomes resulted in an approximately 2.5-fold increase of the basal, Mg2+-dependent ATPase activity, suggesting that the plasma membrane was acting as permeability barrier to the substrate. ATP hydrolysis was maximal (0.8 mumol Pi/min/mg protein) at pH 8.2 in a medium containing either Mg2+ or Ca2+ ions. Ouabain (0.2 mM) and oligomycin (2 micrograms/mg protein) had no appreciable effect on this ATPase activity. Kinetic studies of the enzyme revealed an apparent Km value of ATP of approximately 4 x 10(-5) M. These data are consistent with the view that the observed ATP hydrolysis was being catalysed by an ectoenzyme, i.e., an enzyme in the plasma membrane of the nerve endings with its active site facing the external medium. The rapid hydrolysis of the released ATP is a suspected function for this ecto-ATPase.     

Effect of methylation of histidine-48 on some enzymatic and pharmacological activities of snake venom phospholipases A2

The effects on some pharmacological and enzymatic properties were determined following methylation of histidine at the enzymatic active site of the basic relatively toxic $\text{Naja}$$\text{nigricollis}$ and the acidic relatively non-toxic $\text{Naja}$$\text{naja}$$\text{atra}$ phospholipases A₂. Following methylation a very low residual enzymatic activity (0.4 -- 1% of control) was accompanied by a parallel loss in intraventricular lethality, anticoagulant potency, direct hemolytic action and ability to block directly and indirectly evoked contractions of the mouse phrenic nerve-diaphragm preparation. Since methylation does not impair the enzyme's ability to bind monomeric of micellar substrates or Ca²⁺, the results suggest that the pharmacologicallly active region of the molecule is different from the micellular substrate binding site but strongly influenced by the invariant histidine-48 located at the enzymatic active site.