Gly-238-Ser substitution changes the substrate specificity of the SHV class A beta-lactamases

The SHV-type beta-lactamase SHV-2A is related to SHV-1 by a Gly-238-Ser replacement. Strains carrying SHV-2A are resistant to the third generation cephems cefotaxime and ceftizoxime, whereas those that carry SHV-1 are sensitive to these drugs. We present a kinetic analysis of a SHV-1 and SHV-2A enzymes, with the goal of gaining insight into the role of residue 238 in hydrolyzing cefotaxime and ceftizoxime. SHV-2A shows altered kinetic properties for a number of other cephems that also have heterocyclic side chains at the amino position of the 7-aminocephalosporanic acid nucleus (R1 side chain), including a significantly higher kcat/Km than does SHV-1 for cephaloridine, cephalothin, and cefotiam. Two cephems with straight chain R1 substitutions, cephalosporin C and cephacetrile, are not hydrolyzed more efficiently by SHV-2A. These results indicate that the Ser-238-Gly substitution increases the affinity toward cephems with a heterocyclic ring in the R1 side chain. In addition, the data for ampicillin and benzylpenicillin show that addition of a nitrogen to the second carbon of the R1 side chain of a penem results in a lower kcat/Km for SHV-2A relative to SHV-1. These data strongly suggest that the previously proposed hydrogen bond formation between Ser-238 and the second carbon nitrogen of cefotaxime is not an important factor in hydrolysis by SHV-2A. We propose that the Gly-238 to Ser-238 replacement in SHV-2A has altered the hydrophobic pocket so that it can better accommodate cephems with bulky R1 side chains.     

Developmental regulation of NAD+ kinase in Neurospora crassa

The specific activity of NAD⁺ kinase (ATP:NAD⁺ 2-phosphotransferase, EC 2.7.1.23) from Neurospora crassa shows sharp peaks when the organism enters a new developmental stage of the asexual life cycle: the peaks are observed during hydration and germination of conidia, at the transition from exponential to stationary growth and at the photostimulated conidiation. As stimulation of NAD⁺ kinase activity by light in conidiating mycelium is not sensitive to translation inhibitors, the activiation of pre-existing molecules, rather than induction of protein synthesis de novo may be supposed. Enzyme electrophoresis revealed the presence of four forms of NAD⁺ kinase having different apparent molecular weights (I=333,000; II=306,000; III=229,000 and IV=203,000). Manifestation of the activity of individual forms of NAD⁺ kinase is developmentally controlled: form III is most abundant during vegetative growth, forms I and II prevail in conidia. At the conidial germination the increase of NAD⁺ kinase activity is associated with the activation of form III, whereas during photostimulation of conidiation form II is the most activated one. Therefore, certain molecular forms of the enzyme may be regarded as biochemical markers for different developmental stages of N. crassa.     

Specific protection of nucleotides in the lac operator from DMS methylation and DNase I nicking by crude bacterial cell extracts

Crude bacterial cell extracts prepared from an Escherichia coli lacIq strain were shown to protect specific nucleotides in the lac operator from methylation by dimethyl sulfate (DMS) or digestion by DNase I, whereas no protection was observed using extracts prepared from a nearly isogenic lacI- strain. These experiments show that it is not necessary to use purified regulatory proteins in experiments designed to localize sequences on DNA which interact with proteins. Therefore, crude cell extracts should be useful in DNA "footprinting" experiments to define regions of DNA which bind to unknown regulatory proteins.     

Influence of the surface potential on the Michaelis constant of membrane-bound enzymes: effect of membrane solubilization

We have purified from a membrane fraction of bovine brain a calmodulin-binding protein (calspectin) that shares a number of properties with erythrocyte spectrin: It has a heterodimeric structure with M_r 240 000 and 235 000 and binds to (dimeric form) or crosslinks (tetrameric form) F-actin. We show that calspectin (tetramer) is capable of inducing the polymerization of G-actin to actin filaments by increasing nucleation under conditions where actin alone polymerizes at a much slower rate. Thus, brain calspectin behaves in the same manner as erythrocyte spectrin, supporting the idea that, in conjunction with actin oligomers it comprises the cytoskeletal meshwork underlying the cytoplasmic surface of the nerve cell.     

DNA polymerase activity in developmental stages of Drosophila melanogaster

An assay procedure was developed that allowed the first reproducible measurement of DNA polymerase activity in all developmental stages of Drosophila melanogaster. Evidence is presented that the same enzymatic species is present in extracts of embryos, pupae, and adults of both sexes and that this activity has many properties similar to vertebrate α-polymerases. Polymerase activity per individual is low in embryos and rises steadily through larval instars, reaches a peak in early pupae, declines through the late pupal period, and remains low in newly eclosed adults of both sexes. A dramatic increase is observed in adult females as mature oocytes are formed. This pattern of enzyme activity is completely coincident with changes in DNA levels during development, and suggests that the Drosophila enzyme, like vertebrate α-polymerases, functions in cellular DNA replication. Two mutagen-sensitive mutants, deficient in both replication on undamaged templates and postreplication repair, were found to have normal levels of this α-polymerase activity. Our results suggest that a single enzymatic species of α-polymerase holoenzyme exists in Drosophila and is common to all developmental stages of this organism.     

Effects of Detergents, Proteins, and Lipids on 2'':3''-Cyclic-Nucleotide 3''-Phosphodiesterase Activity

The myelin marker 2':3'-cyclic-nucleotide 34'-phosphodiesterase (CNPase) was isolated to a lipid- and phosphate-free stage. The effects of exogenously added lipids were tested on this preparation and compared to the known stimulation of the enzyme by detergents and proteins. CNPase could be stimulated 2-3 fold by these various agents which appeared to be additive in their effect. Enzyme-protein and enzyme-lipid interactions and possible medical use of the improved assay conditions for CNPase employed in the study are discussed.     

A reappraisal of the concept of metabolic cold adaptation in polar marine invertebrates

The concept of 'metabolic cold adaptation', namely that polar marine ectotherms are adapted in having an elevated basal metabolic rate, has been examined in the light of recent biochemical, physiological and ecological data for Antarctic marine organisms. It is now clear that marine invertebrates from Antarctic waters are characterized by slow growth rates, low basal metabolism and reduced annual reproductive effort, and there is thus no clear evidence of the traditional view of an elevated metabolic rate. By analogy with fish, protein synthesis rates are probably also low. This suggests that the major feature of cold adaptation is a reduction in the individual total annual energy intake in comparison with ecologically similar organisms from warm water. This allows a high standing crop of suspension feeders to develop, and low temperature is thus a significant factor in the successful widespread adoption of typical K-strategies in Antarctic marine invertebrates.     

Properties of acyl hydrolase enzymes from Phaseolus multiflorus leaves

The properties of acyl hydrolase enzymes purified from the leaves of Phaseolus multiflorus have been studied. Hydrolase I which deacylates phosphatidylcholine and oleoylglycerol had a pH optimum towards phosphatidylcholine of 5.3. Hydrolase II which deacylates glycosylglycerides and oleoylglycerol showed pH optima of 7.3 (monogalactosyldiglyceride, MGDG) and 4.3 (sulphoquinovosyldiglyceride, SQDG). Both enzymes showed activity peaks towards oleoylglycerol at pH 6.8 and 8.8. Unesterified fatty acids and Triton X-100 inhibited the rate of SQDG hydrolysis while bovine serum albumin increased activity. An apparent K_m for SQDG of 0.15 mM was found. Hydrolase II catalysed transmethylation of liberated fatty acids during the hydrolysis of oleoylglycerol when methanol was included in the assay system. A number of salts inhibited SQDG hydrolysis but their effect on oleoylglycerol was less consistent. The position of ester cleavage of oleoylglycerol was determined by the use of H₂¹⁸O. Cell-free extracts from P. multiflorus leaves degraded SQDG as far as sulphoquinovose.     

Regulation of nitrate reductase activity in higher plants

Nitrate reductase is one of the most important enzymes in the assimilation of exogenous nitrate—the predominant form of nitrogen available to green plants growing in soil. Activity of this enzyme in plants gives a good estimate of the nitrogen status of the plant and is very often correlated with growth and yield. Although it is difficult to explain the physiological significance and the mechanism of effects of several factors on the enzyme activity, in some cases suitable postulates have been advanced. In general, the enzyme activity in a plant tissue is a balance between its relative rates of synthesis/degradation and activation/inactivation. Factors may affect the overall activity by interfering with either of these processes.