Analysis and prediction of the packing of α-helices against a β-sheet in the tertiary structure of globular proteins

The packing of α-helices and β-sheets in six $\text{α}\text{β}$ proteins (e.g. flavodoxin) has been analysed. The results provide the basis for a computer algorithm to predict the tertiary structure of an $\text{α}\text{β}$ protein from its amino acid sequence and actual assignment of secondary structure.The packing of an individual α-helix against a β-sheet generally involves two adjacent ± 4 rows of non-polar residues on the α-helix at the positions i, i + 4, i + 8, i + 1, i + 5, i + 9. The pattern of interacting β-sheet residues results from the twisted nature of the sheet surface and the attendant rotation of the side-chains. At a more detailed level, four of the α-helical residues (i + 1, i + 4, i + 5 and i + 8) form a diamond that surrounds one particular β-sheet residue, generally isoleucine, leucine or valine. In general, the α-helix sits 10 Å above the sheet and lies parallel to the strand direction.The prediction follows a combinational approach. First, a list of possible β-sheet structures (10⁶ to 10¹⁴) is constructed by the generation of all β-sheet topologies and β-strand alignments. This list is reduced by constraints on topology and the location of non-polar residues to mediate the sheet/helix packing, and then rank-ordered on the extent of hydrogen bonding. This algorithm was uniformly applied to 16 $\text{α}\text{β}$ domains in 13 proteins. For every structure, one member of the reduced list was close to the crystal structure; the root-mean-square deviation between equivalenced C^α atoms averaged 5.6 Å for 100 residues. For the $\text{α}\text{β}$ proteins with pure parallel β-sheets, the total number of structures comparable to or better than the native in terms of hydrogen bonds was between 1 and 148. For proteins with mixed β-sheets, the worst case is glyceraldehyde-3-phosphate dehydrogenase, where as many as 3800 structures would have to be sampled. The evolutionary significance of these results as well as the potential use of a combinatorial approach to the protein folding problem are discussed.     

Rotational dynamics of monoclonal anti-dansyl immunoglobulins

The rotational motions of monoclonal mouse anti-dansyl immunoglobulins were studied by nanosecond fluorescence emission anisotropic spectroscopy using a mode-locked argon-ion laser as the pulsed excitation source. Three homogeneous antibodies of the immunoglobulin Gl (IgGl) subclass containing different V regions were prepared. The fluorescence emission maxima of these antibodies (designated as DNS1, DNS2 and DNS3) are at 515, 480 and 500 nm, respectively. Their mean rotational correlation times, 〈φ〉, are 84, 109 and 96 ns, respectively. The binding of protein A or a monoclonal anti-allotype antibody to the F_c unit of DNS1 increased 〈φ〉 to 142 and 150 ns, respectively, whereas reduction of the disulfide bond between the heavy chains decreased 〈φ〉 to 48 ns. These nanosecond measurements show that the rotational motion of the F_ab arms in mouse IgGl is restricted.     

Glycerol Phosphate Dehydrogenase, Glucose-6-Phosphate Dehydrogenase, and Lactate Dehydrogenase: Activities in Oligodendrocytes, Neurons, Astrocytes, and Myelin Isolated from Developing Rat Brains

Abstract: Glycerol phosphate dehydrogenase (GPDH), glucose-6-phosphate dehydrogenase (G6PDH), and lactate dehydrogenase (LDH) activities were determined in Oligodendrocytes, neurons, and astrocytes isolated from the brains of developing rats. The activity of each enzyme was significantly lower in both neurons and astrocytes than in Oligodendrocytes. The GPDH activity in Oligodendrocytes increased more than 4-fold during development, and at 120 days cells of this type had 1.4-fold the specific activity of forebrain homogenates. The G6PDH activities in Oligodendrocytes from 10-day-old rats were 1.4-fold the activities in the forebrain homogenates. The activities of this enzyme in Oligodendrocytes were progressively lower at later ages, such that at 120 days the cells had 0.8 times the specific activities of homogenates. The Oligodendrocytes had 0.6 times the homogenate activities of LDH at 10 days, and this ratio had decreased to 0.2 by 120 days. These enzymes were also measured in myelin isolated from 20-, 60-, and 120-day-old rats. By 120 days the specific activities of G6PDH and LDH in myelin were <8% of the respective activities in homogenates. The GPDH activity in myelin was, however, at least 20% the specific activity in the homogenates, even in the oldest animals. It is proposed that LDH could be used as a marker for oligodendroglial cytoplasm in subfractions of myelin and in myelin-related membrane vesicles.     

Kinetics of Neutral Amino Acid Transport Through the Blood-Brain Barrier of the Newborn Rabbit

Abstract: Since protein synthesis in the developing brain may, under certain conditions, be limited by amino acid availability, the present studies were undertaken to characterize the kinetics of large neutral amino acid transport through the blood-brain barrier (BBB) of the newborn rabbit. The K_m, V_max, and K_D of the transport of eight amino acids were determined by a nonlinear regression analysis of data obtained with the carotid injection technique. Compared with kinetic parameters observed for the adult rat, the K_m, V_max, and K_D of amino acid transport were all two- to threefold higher in the newborn. Albumin was found to bind tryptophan actively in vitro , but had no inhibitory effect on tryptophan transport through the newborn BBB. Glutamine was transported through the BBB of the newborn at rates severalfold higher than are seen in the adult rat. However, glutamine transport was not inhibited by high concentrations of N -methylaminoisobutyric acid (NMAIB), a model amino acid that is specific for the alanine-preferring or A-system present in peripheral tissues. In conclusion, these studies show that the BBB neutral amino acid transport system of the newborn rabbit has a lower affinity and higher capacity than does the BBB of the adult rat. Under conditions of high plasma amino acids, the increased capacity of the newborn transport system allows for a higher rate of amino acid transport into brain than would occur via the lower capacity system present in the adult rat brain.     

Detection of Autographa californica and Heliothis zea baculovirus proteins by enzyme-linked immunosorbent assay (ELISA)

The structural proteins of Autographa californica (AcMNPV) and Heliothis zea (HzSNPV) nuclear polyhedrosis viruses were detected by indirect enzyme-linked immunosorbent assay (ELISA). The immunoassay detected less than 1 ng of AcMNPV protein. The extent of immunological relatedness between AcMNPV-occluded virus and AcMNPV polyhedral protein, AcMNPV-nonoccluded virus, Estigmene acrea granulosis virus, Amsacta moorei entomopoxvirus Heliothis zea NPV, and Lymantria dispar NPV was determined. No immunological relatedless was detected between HzSNPV, AcMNPV, and a persistent rod-shaped virus isolated from the Heliothis zea cell line (IMC-Hz-1). The polyhedral proteins of HzSNPV and AcMNPV were found to be immunologically identical.     

Reduction in Brain Glucose Utilization Rate after Tryptophol (3-Indole Ethanol) Treatment

Abstract: 3-Indole ethanol has been recently identified as the hypnotic agent in trypanosomal sleeping sickness, and because it is formed in vivo after ethanol or disulfiram treatment, it is also associated with the study of alcoholism. When administered intraperitoneally to rats (250 mg/kg) tryptophol induced a sleep-like state that lasted less than an hour (no righting reflex was apparent 2 min after injection, but it returned at 11 min in bovine serum albumin solution, and 47 min in 40% ethanol solution). In ethanol solutions, tryptophol reduced brain cortical glucose utilization by 55% to the basal brain metabolic rate, and this effect lasted less than 1 h. Synergistic effects of tryptophol and ethanol were suggested by the observation that in albumin solution, tryptophol reduced brain glucose utilization by 35%, but a normal rate was not observed until 2 h postinjection.     

Properties of Bovine Oligodendroglia Isolated by a New Procedure Using Physiologic Conditions

A new class of procedures, previously shown to permit the isolation of pure oligodendroglia from whole rat cerebrum, has been applied with equal or greater success for the bulk isolation of this cell type from bovine white matter. Thus, the generality of this approach has been demonstrated. The bovine preparations have a purity of greater than 90% intact, phase-bright oligodendroglia and are obtained in a yield of 8 x 10(6) cells per gram of white matter. Within 1 day it is possible to obtain a preparation containing 60 mg of protein from a single cell type. These cells show a higher degree of ultrastructural preservation of all cytoplasmic constituents than previously obtained. The values for protein (33 pg/cell), DNA (5.4 pg/cell), and lipid (5-6 pg/cell) are very similar to those obtained with an earlier procedure. The cell lipids are rich in galactolipid, which comprises 20% of the total. The activity of the "myelin-specific" enzyme, 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3.1.4.37), is 4.7 mumol/min/mg protein, similar to that obtained previously for isolated oligodendroglia and about 25-40% of that found in myelin. The activity of 5'-nucleotidase (EC 3.1.3.5) in the cells is about 10% of that in myelin or white matter.     

FACTORS LIMITING EDAPHIC ALGAL BIOMASS AND PRODUCTIVITY IN A GEORGIA SALT MARSH1,2

A “planted core” system was developed to test the effect of short term (1–2 weeks) experimental manipulation of environmental parameters on edaphic microalgae under field conditions. A large number of small cores (surface area = 7 cm²) were collected, randomized and replanted in the marsh in fiddler crab exclosures with appropriate experimental treatments. Daily enrichment of the cores with NH₄⁺ resulted in significant increases in edaphic primary productivity and levels of chlorophyll a in both summer and winter seasons in the short-Spartina marsh. Enrichment with a complete nutrient solution caused no further increases. Nutrient enrichment of creekbank sediments was much less stimulatory to the resident algal assemblage. In both sites, but especially in the creekbank, the removal of fiddler crab grazers resulted in significant increases in chlorophyll a and productivity. Experimental manipulation of light intensity showed that the average light intensity reaching the sediment surface was saturating for chlorophyll production in the short-Spartina marsh. A reciprocal transplant experiment involving unfertilized cores from the short-Spartina marsh and creekbank marsh demonstrated that NH₄⁺ inputs occurring in the creekbank site rapidly alleviated nitrogen limitation of edaphic algae from short-Spartina marsh. Algae in creekbank cores incubated in the short-Spartina marsh were unable to sustain high productivity once the original standing stock of NH₄⁺ declined.     

Structures of the Oligosaccharides of the Glycoprotein Coded by Early Region E3 of Adenovirus 2

Early region E3 of adenovirus 2 encodes a glycoprotein, E3-gp25K, that is a good model with which to study structure-function relationships in transmembrane glycoproteins. We have determined the structures of the oligosaccharides linked to E3-gp25K. The oligosaccharides were labeled with [2-³H]mannose in adenovirus 2-early infected KB cells for 5.5h (pulse) or for 5.5 h followed by a 3-h chase (pulse-chase). E3-gp25K was extracted and purified by chromatography on DEAE-Sephacel in 7 M urea, followed by gel filtration on a column of Bio-Gel A-1.5m in 6 M guanidine hydrochloride. An analysis of the purified protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that it was >95% pure. The oligosaccharides were isolated by pronase digestion followed by gel filtration on a column of Bio-Gel P-6, then by digestion with endo-β-N-acetylglucosaminidase H, followed by gel filtration on Bio-Gel P-6, and finally by paper chromatography. The pulse sample contained equal amounts of Man₉GlcNAc and Man₈GlcNAc and small amounts of Man₇GlcNAc and Man₆GlcNAc. The pulse-chase sample had predominantly Man₈GlcNAc and much less Man₉GlcNAc, indicating that processing of the Man₉GlcNAc to Man₈GlcNAc had occurred during the chase period. Thus, Man₈GlcNAc is the major oligosaccharide on mature E3-gp25K. The structures of these oligosaccharides were established by digestion with α-mannosidase, methylation analysis, and acetolysis. The oligosaccharides found had typical high-mannose structures that have been observed in other membrane and soluble glycoproteins, and the branching patterns and linkages of the mannose residues of Man₉GlcNAc were identical to those of the lipid-linked Glc₃Man₉GlcNAc₂ donor. Thus, adenovirus 2 infection (early stages) apparently does not affect the usual cellular high-mannose glycosylation pathways, and despite being virus coded, E3-gp25K is glycosylated in the same manner as a typical mammalian cell-coded glycoprotein.     

Proposal for Naming Host Cell-Derived Inserts in Retrovirus Genomes

We propose a system for naming inserted sequences in transforming retroviruses (i.e., onc genes), based on using trivial names derived from a prototype strain of virus.