Isolation of the globular region of the subcomponent q of the C1 component of complement.

Digestion after heat treatment of the subcomponent q of the C1 component of complement by collagenase leads to the isolation of the globular region of the protein. This product ('heads') is composed of three chains giving an overall molecular weight of about 57000. About half of the collagen-like region present in C1 q is lost after digestion. The 'heads' are shown to be soluble and hemolytically active products.     

Central projections of fibers in the auditory and tensor nerves of cicadas (Homoptera: Cicadidae)

Summary The auditory and tensor nerves of cicadas are mixed nerves containing both afferent and efferent elements. In 17-year cicadas, and in Okanagana rimosa, the auditory nerve contains afferents from body hairs, from the detensor tympani-chordotonal organ, and some 1300–1500 afferents from the hearing organ. Within the fused metathoracic-abdominal ganglionic complex the receptors from both the auditory and tensor nerves form a neuropilar structure that reveals the metameric organization of this complex. A few fibers run anteriorly, projecting into the meso and prothoracic ganglia. Within the ganglionic complex a division of auditory nerve afferents into a dense intermediate and a more diffuse ventral neuropile is observed. In addition, a dorsal motor neuropile is outlined by arborizations of the timbal motor neuron. This neuron is one of several efferent cell types associated with the auditory nerve, and there is an indication that several efferent fibers innervate the timbal muscle. There is anatomical evidence for a possible neuronal coupling between the bilaterally symmetrical large timbal motor neurons. In general, central projections from the auditory and tensor nerves support evidence of a structural layering within the CNS of insects.     

A high-activity ATP translocator in mesophyll chloroplasts of Digitaria sanguinalis, a plant having the C-4 dicarboxylic acid pathway of photosynthesis

The effect of exogenous adenine nucleotides on CO₂ fixation and oxygen evolution was studied with mesophyll protoplast extracts of the C₄ plant Digitaria sanguinalis. Exogenous ATP was found to stimulate the rate of pyruvate and pyruvate + oxalacetate induced CO₂ fixation, as well as reverse the inhibition of CO₂ fixation by carbonyl cyanide m-chlorophenyl hydrazone and several electron transport inhibitors. The ATP-dependent stimulation of CO₂ fixation varied from 40 to 70 μmol CO₂ fixed/mg chlorophyll per h, suggesting that ATP was crossing the chloroplast membranes at rates of 80–140 μmol/mg chlorophyll per h, since 2 ATP are required for each CO₂ fixed. Fixation of CO₂ could also be induced in the dark by exogenous ATP, in which case ADP accumulated outside the chloroplasts. This suggests that external ATP is exchanging for internal ADP. In contrast, ADP and AMP were found not to traverse chloroplast membranes, on the basis that neither nucleotide inhibited CO₂ fixation or stimulated oxygen evolution that was limited by available ADP for phosphorylation. Further evidence that ATP can enter the chloroplasts was obtained by direct measurements of the increase in ATP in the chloroplasts due to addition of exogenous ATP in the dark. These studies yielded minimal rates of ATP uptake on the order of 30–40 μmol/mg chlorophyll per h. It is suggested that a membrane translocator exists that specifically transports ATP into the chloroplasts in exchange for ADP. The significance of these findings are considered with respect to the C₄ pathway of photosynthesis.     

The action of beta-galactosidase (Escherichia coli) on allolactose.

The parameters involved in the action of beta-galactosidase (EC 3.2.1.23) (Escherichia coli) on allolactose, the natural inducer of lac operon in E. coli, were studied. At low allolactose concentrations only galactose and glucose were formed, while at high allolactose concentrations transgalactolytic oligosaccharides were also produced. Detectable amounts of lactose were not formed. The V and Km values (49.6 U/mg and 0.00120 M, respectively) indicated that allolactose is as good if not a better substrate of beta-galactosidase as lactose. The pH optimum with allolactose (7.8-7.9) as well as its activation by K+ (as compared to activation by Na+) were similar to the case with lactose as substrate. The alpha-anomer of allolactose was hydrolyzed about two times as rapidly as was the beta-anomer.     

The purification and study of a honey bee abdominal sucrase exhibiting unusual solubility and kinetic properties.

A sucrase from honey bee abdomens was purified to a high state of homogeneity. It was unusual in that it was completely soluble in high concentrations of ammonium sulfate and because curved rather than rectilinear lines were obtained when initial velocity data for at least two substrates were plotted. The action of the enzyme towards a large number of glycosides showed that the enzyme was able to hydrolyze all α-glucosides tested except trehalose and starch. pH Optima of sucrose and p-nitrophenyl-α-d-glucopyranoside differed by 1.0 pH unit. The unusual kinetic patterns which were found seem to be unique to this disaccharidase and were shown to be the result of a combination of hydrolytic and transferolytic activity in which the initial substrate is also a very good acceptor molecule for the transferolytic process. The K_m value for hydrolysis was found to be about an order of magnitude lower than for other insect sucrases with the more usual type of kinetic action. Amino acid and amino sugar analyses showed that the sucrase was a glycoprotein which contained glucosamine and either mannosamine or galactosamine. The molecular weight of the enzyme was estimated to be 70,000 or higher and there was no evidence that the enzyme had subunit structure. An s⁰_20,w value of 5.3S was determined. The enzyme was quite stable to a series of denaturing conditions and sulfhydryl reacting agents had little effect on the activity.     

LacZ β‐galactosidase: Structure and function of an enzyme of historical and molecular biological importance

This review provides an overview of the structure, function, and catalytic mechanism of lacZ β‐galactosidase. The protein played a central role in Jacob and Monod's development of the operon model for the regulation of gene expression. Determination of the crystal structure made it possible to understand why deletion of certain residues toward the amino‐terminus not only caused the full enzyme tetramer to dissociate into dimers but also abolished activity. It was also possible to rationalize α‐complementation, in which addition to the inactive dimers of peptides containing the “missing” N‐terminal residues restored catalytic activity. The enzyme is well known to signal its presence by hydrolyzing X‐gal to produce a blue product. That this reaction takes place in crystals of the protein confirms that the X‐ray structure represents an active conformation. Individual tetramers of β‐galactosidase have been measured to catalyze 38,500 ± 900 reactions per minute. Extensive kinetic, biochemical, mutagenic, and crystallographic analyses have made it possible to develop a presumed mechanism of action. Substrate initially binds near the top of the active site but then moves deeper for reaction. The first catalytic step (called galactosylation) is a nucleophilic displacement by Glu537 to form a covalent bond with galactose. This is initiated by proton donation by Glu461. The second displacement (degalactosylation) by water or an acceptor is initiated by proton abstraction by Glu461. Both of these displacements occur via planar oxocarbenium ion‐like transition states. The acceptor reaction with glucose is important for the formation of allolactose, the natural inducer of the lac operon.     

The refined 2.15 A X-ray crystal structure of human liver cathepsin B: the structural basis for its specificity.

From the lysosomal cysteine proteinase cathepsin B, isolated from human liver in its two-chain form, monoclinic crystals were obtained which contain two molecules per asymmetric unit. The molecular structure was solved by a combination of Patterson search and heavy atom replacement methods (simultaneously with rat cathepsin B) and refined to a crystallographic R value of 0.164 using X-ray data to 2.15 A resolution. The overall folding pattern of cathepsin B and the arrangement of the active site residues are similar to the related cysteine proteinases papain, actinidin and calotropin DI. 166 alpha-carbon atoms out of 248 defined cathepsin B residues are topologically equivalent (with an r.m.s. deviation of 1.04 A) with alpha-carbon atoms of papain. However, several large insertion loops are accommodated on the molecular surface and modify its properties. The disulphide connectivities recently determined for bovine cathepsin B by chemical means were shown to be correct. Some of the primed subsites are occluded by a novel insertion loop, which seems to favour binding of peptide substrates with two residues carboxy-terminal to the scissile peptide bond; two histidine residues (His110 and His111) in this "occluding loop' provide positively charged anchors for the C-terminal carboxylate group of such polypeptide substrates. These structural features explain the well-known dipeptidyl carboxypeptidase activity of cathepsin B. The other subsites adjacent to the reactive site Cys29 are relatively similar to papain; Glu245 in the S2 subsite favours basic P2-side chains. The above mentioned histidine residues, but also the buried Glu171 might represent the group with a pKa of approximately 5.5 near the active site, which governs endo- and exopeptidase activity. The "occluding loop' does not allow cystatin-like protein inhibitors to bind to cathepsin B as they do to papain, consistent with the reduced affinity of these protein inhibitors for cathepsin B compared with the related plant enzymes.     

Phosphorylation of serine-15 of maize leaf sucrose synthase. Occurrence in vivo and possible regulatory significance.

Experiments were conducted to determine whether sucrose synthase (SuSy) was phosphorylated in the elongation zone of maize (Zea mays L.) leaves. The approximately 90-kD subunit of SuSy was 32P-labeled on seryl residue(s) when excised shoots were fed [32P]orthophosphate. Both isoforms of SuSy (the SS1 and SS2 proteins) were phosphorylated in vivo, and tryptic peptide-mapping analysis suggested a single, similar phosphorylation site in both proteins. A combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and automated Edman sequencing analysis unequivocally identified the phosphorylation site in the maize SS2 protein as serine-15. This site was phosphorylated in vitro by endogenous protein kinase(s) in a strictly Ca(2+)-dependent manner. A synthetic peptide, based on the phosphorylation site sequence, was used to identify and partially purify an endogenous Ca(2+)-dependent protein kinase(s) from the maize leaf elongation zone and expanding spinach leaves. Phosphorylation of SuSy in vitro selectively activates the cleavage reaction by increasing the apparent affinity of the enzyme for sucrose and UDP, suggesting that phosphorylation may be of regulatory significance. Conservation of the phosphorylation site, and the sequences surrounding it, among plant species suggests that phosphorylation of SuSy may be widespread, if not universal, in plants.