Rabbit skeletal alpha-tropomyosin chains are in register.

2-Acetamido-2-deoxy-3-O-(D-2-propionyl-L-alanine)-D-glucopyranose ($\text{5}$) and 2-acetamido-2-deoxy-3-O-(D-2-propionyl-L-alanyl-D-isoglutamine)-D-glucopyranose ($\text{10}$) have been synthesized by condensation of benzyl 2-acetamido-4,6-O-benzylidene-3-O-(D-1-carboxyethyl)-2-deoxy-β-D-glucopyranoside ($\text{1}$) respectively with the L-alanine derivative $\text{2}$ and the dipeptide $\text{7}$, followed by debenzylidenation and hydrogenolysis. Compound $\text{10}$ is adjuvant active, whereas $\text{5}$ is inactive, so that $\text{10}$ is the smallest adjuvant active structure for the time being.     

Adjuvant activity of monomeric bacterial cell wall peptidoglycans

We have previously shown that lysozyme solubilized cell walls of Mycobacteria or Nocardiae can replace whole mycobacterial cells or Wax D in Freund's complete adjuvant and it was found quite recently that hydrosoluble peptidoglycans, free of neutral sugars, are also adjuvant active. We show now that the simplest fragment tested — the disaccharide tetrapeptide (I) — increases circulating antibodies to ovalbumin and induces a delayed hypersensitivity toward this antigen. Similar compounds obtained from the basal layer of the cell wall of $\text{E. coli}$ are also active. Thus the immunoadjuvant activity of soluble cell wall peptidoglycans is a property of the monomeric unit and is not restricted to acid fast bacteria.     

Lytic enzymes in sporulating Bacillus subtilis

Two specific lytic enzymes were found in sporulating $\text{B. subtilis}$ cells: a N-acetyl muramyl L-alanine amidase and a $\text{γ-D-glutamyl-(L)}\text{meso}$ diaminopimelyl endopeptidase. Both enzyme activities were measured using radioactive synthetic substrates. They are low in vegetative cells and increase during sporulation. The highest rates of increase are concomitant with cortex formation. In a mutant with delayed sporulation enzyme synthesis is also delayed. We suggest that both enzymes play a role in the synthesis of the specific cortex peptidoglycan.     

Activities of enzymes that metabolize platelet-activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in neutrophils and eosinophils from humans and the effect of a calcium ionophore

Enzymatic systems in human blood cells are described for the activation and inactivation of a biologically active phospholipid (1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine) with hypotensive, platelet-aggregating, and inflammatory properties. The results document the presence of alkyldihydroxyacetone-phosphate synthase (forms the $\text{O}$-alkyl linkage in lipids), 1-alkyl-2-lyso-$\text{sn}$-glycero-3-phosphocholine:acetyl-CoA acetyltransferase (produces the biologically active molecule), and 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine: acetylhydrolase (destroys the biological activity) in human neutrophils and eosinophils. Both the acetyltransferase and acetylhydrolase activities are increased severalfold after treatment of normal neutrophils with ionophore A23187; however, alkyldihydroxyacetone-phosphate synthase activity is not influenced by the ionophore. Eosinophils isolated from patients with eosinophilia have significantly greater activities of all the enzymes studied than the eosinophils isolated from normal individuals. Our results indicate the acetyltransferase responsible for 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine synthesis may serve an important role in human blood cells that release this biologically active phospholipid. Moreover, the acetyltransferase activity was found to be dramatically influenced by calcium flux.     

Carbohydrate structure and serological behaviour of "antifreeze" glycoproteins from an Antarctic fish.

The carbohydrate moiety of the “antifreeze” glycoprotein from $\text{Trematomus borchgrevinki}$ was found to be β-D-galactosyl 1–3 N-acetyl galactosamine by gasliquid chromatography. The glycoprotein inhibited anti-T antibody from human serum and $\text{Arachis hypogoea}$ lectin, but was inactive against $\text{Vicia graminea}$. Native “antifreeze” glycoprotein did not inhibit the agglutinins from $\text{Helix pomatia}$ or $\text{Cepaea hortensis}$, although after Smith degradation showed a strong inhibition towards them. Inhibition of the latter agglutinin demonstrates the carbohydrate-protein linkage to be α-linked. The presence of the Thomsen-Friedenreich antigen (T-antigen) on the “antifreeze” glycoprotein and its relation to tumour cell surfaces is briefly discussed.     

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.     

In vivo metabolism of a new class of biologically active phospholipids: 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine,a platelet activating-hypotensive phospholipid

This report describes the in vivo metabolism of a new class of naturally occurring biologically active phospholipids (1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholines) that can cause hypotension and platelet aggregation. After intravenous injection in male rats, the acetylated ether phospholipid (1-[1′,2′-³H]alkyl) is rapidly cleared ($\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{?30}\text{s}$) from blood and its metabolites are found in a variety of tissues. The tissues containing the highest levels of radioactivity are lung, liver, spleen, and kidney. Chromatographic results showed that a considerable portion of the active lipid is not readily catabolized in some of the major tissues examined; however, inactive metabolites were also found, mainly 1-alkyl-2-lyso-$\text{sn}$-glycero-3-phosphocholine and 1-alkyl-2-acyl-$\text{sn}$-glycero-3-phosphocholine; the latter has a long chain fatty acid at the $\text{sn}$-2 position instead of the acetate. The findings are consistent with our earlier data that show these same tissues have the most active enzyme systems for metabolizing 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine.     

Lactose transport in Escherichia coli cells Dependence of kinetic parameters on the transmembrane electrical potential difference

We determine the kinetic parameters $\text{V}$ and $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ of lactose transport in Escherichia coli cells as a function of the electrical potential difference (Δψ) at pH 7.3 and $\text{Δ}\text{pH}\text{= 0}$. We report that transport occurs simultaneously via two components: a component which exhibits a high $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ (larger than 10 mM) and whose contribution is independent of Δψ, a component which exhibits a low $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ independent of Δψ (0.5 mM) but whose $\text{V}$ increases drastically with increasing Δψ. We associate these components of lactose transport with facilitated diffusion and active transport, respectively. We analyze the dependence upon Δψ of $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ and $\text{V}$ of the active transport component in terms of a mathematical kinetic model developed by Geck and Heinz (Geck, P. and Heinz, E. (1976) Biochim. Biophys. Acta 443, 49–63). We show that within the framework of this model, the analysis of our data indicates that active transport of lactose takes place with a H⁺/lactose stoichiometry greater than 1, and that the lac carrier in the absence of bound solutes (lactose and proton(s)) is electrically neutral. On the other hand, our data relative to facilitated diffusion tend to indicate that lactose transport via this mechanism is accompanied by a H⁺/lactose stoichiometry smaller than that of active transport. We discuss various implications which result from the existence of H⁺/lactose stoichiometry different for active transport and facilitated diffusion.     

Crystals of a functional 70,000 molecular weight subunit of hemocyanin from Limulus polyphemus

Crystals have been obtained of a subunit of Limulus polyphemus hemocyanin. The blue crystals have the symmetry of the space group R32 with hexagonal lattice parameters $\text{a = 115}\text{a}\text{?}$, $\text{c = 285}\text{A}\text{?}$. There is one 70,000 molecular weight subunit per asymmetric unit. Each subunit contains two non-heme copper atoms and can reversibly bind one oxygen molecule.     

Conformations and CD curves of cyclo(L- or D-Phe-L-Pro-Aca): cyclized models for specific types of β-bends

Cyclic tripeptides cyclo(L-Phe-L-Pro-Aca) (molecule $\text{3}$) (Aca, ?-aminocaproic acid) and cyclo(-D-Phe-L-Pro-Aca) (molecule $\text{4}$) are designed as models of specific types of β-bend. Energy calculation and ¹H and ¹³C NMR studies have indicated that peptides $\text{3}$ and $\text{4}$ form β-bend types VI and II', respectively. Circular dichroism spectra of $\text{4}$ have a double minimum negative band at the region of 200–230 nm like those of gramicidin S. The spectra of $\text{3}$, forming the cis peptide bond just before Pro, have a negative extremum at the 210–213 nm region. The spectra are used to estimate the contribution of various bend types in peptides.β-BendCD MeasurementConformational energy calculationCyclic peptideGramicidin SNMR measurement     

The origin of the carbon chain in the thiazole moiety of thiamine in Escherichia coli: incorporation of deuterated 1-deoxy-D-threo-2-pentulose

Non growing washed cells of Escherichia coli, derepressed for the biosynthesis of thiamine, have been incubated in the presence of glucose and either 1-deoxy-$\text{D}$-threo-2-pentulose $\text{1}$ or 1-déoxy-$\text{D}$-erythro-2-pentulose $\text{2}$ trideuterated on the methyl group. The incorporation of deuterium into the thiazole moiety of thiamine was measured by mass spectrometry. The label of the threo-compound was found in more than 40% of the thiazole biosynthesized in its presence; the label of the erythro-compound in less than 5%. Hence it is likely that the carbon chain of 1-deoxy-$\text{D}$-threo-2-pentulose is the precursor of the five carbons chain of the thiazole moiety of the thiamine molecule in E. coli.     

N-alkanes in normal and pathological human scale

When $\text{n}\text{-}\text{alkanes}$ have been found in mammalian tissues, they have been considered to be solely of exogenous origin, and have not been assigned any normal or pathological function. We have observed $\text{n}\text{-}\text{alkanes}$ regularly in normal stratum corneum (5.5 ± 0.2% total lipid), and found striking accumulations (> 25% total lipid) in some scaling diseases. Although the origin of these $\text{n}\text{-}\text{alkanes}$ is not known, evidence is presented that they do not arise from external contaminants, medications, sebaceous glands, and spontaneous or bacterial degradation. The presence of large quantities of $\text{n}\text{-}\text{alkanes}$ in human stratum corneum suggests that they may play a role in normal human epidermal function and in the pathophysiology of some epidermal diseases.     

1,25-Dihydroxyvitamin D3-glycoside: Identification of a calcinogenic principle of solanum malacoxylon

The water soluble calcinogenic factor present in the plant $\text{Solanum}$$\text{malacoxylon}$ is partially purified by selective extraction and chromatography on silicic acid and then hydrolyzed with a mixed preparation of glycosidases from the sea worm, $\text{Charonia}$$\text{lampus}$. Hydrolysis produces a chloroform soluble factor with biologic characteristics of 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃), the hormonal form of vitamin D. Purification of this factor is accomplished by chromatography on Sephadex LH-20, silicic acid, and Celite columns, yielding 3 μg of active material. During the isolation, bioactivity (as assessed by the ability of fractions to compete with labeled 1,25-(OH)₂D₃ for binding to a specific intestinal receptor protein) migrates exactly with authentic tritiated 1,25-(OH)₂D₃. The purified factor has an ultraviolet absorption spectrum identical to that of 1,25-(OH)₂D₃ and analysis via direct probe mass spectrometry yields a parent molecular ion of m/e 416 and a fragmentation pattern indistinguishable from synthetic 1,25-(OH)₂D₃ hormone. We therefore conclude that the vitamin D-like principle in $\text{Solanum}$$\text{malacoxylon}$ is a sterol-glycoside which contains the 1,25-(OH)₂D₃ molecule as its active sterol component.     

Evidence that Escherichia coli 30 S ribosomal subunit populations are conformationally heterogeneous.

We have estimated the number of sites on each protein of the 30 S ribosome which are accessible to chemical iodination. First, the total number of iodinatable sites was determined for the intact 30 S ribosome. The proteins were extracted, separated and the relative distribution of iodine in each protein determined. This distribution of iodine divided into the total sites per ribosome gave an estimate of the number of sites per individual protein.Second, the iodinated proteins were purified and their trypsin digestion products separated. The number of radioactive peptides was taken as a measure of the number of sites on that protein open to the iodination reaction. The number of iodinatable sites for each protein was found to be radically different by the two methods. In almost all cases, the number of unique, radioactively labeled peptides, derived from a given 30 S protein, far exceeded the total incorporation into that protein. We suggest that the best explanation for this unexpected discrepancy is that the 30 S ribosome population we used in these experiments is heterogeneous in its topography.In addition we have compared the topography by the chemical iodination procedure for ribosomes in two different conformations: active and inactive (see Zamir et al., 1971). We have found very little change in the chemical reactivity of the proteins when the ribosomes are in the two different conformations. The most notable changes involve proteins S10, $\text{S}\text{18}\text{S}\text{19}$ and especially $\text{S}\text{12}\text{S}\text{13}$.     

Three-dimensional structure at 5 Å resolution of cytosolic aspartate transaminase from chicken heart

An X-ray study of orthorhombic crystals of cytosolic aspartate transaminase from chicken heart has been carried out at 5 Å resolution. The crystals belong to space group P2₁2₁2₁, with unit cell dimensions $\text{a = 62.7}\text{A}\text{?}\text{, b = 118.1}\text{A}\text{?}\text{, c = 124.5}\text{A}\text{?}$. The electron density map has been calculated on the basis of five heavy-atom derivatives. The model of the molecule derived from this map revealed clearly two subunits of similar structure related by a non-crystallographic dyad. The secondary structure of the protein comprises nine helical segments per subunit.The enzyme has been shown to be catalytically active in the crystal form. Removal of the coenzyme from the crystals made it possible to derive from the difference Fourier map the position of the active site in the enzyme molecule.Significant conformational changes have been observed which accompany the interconversion of intermediates of the enzymic reaction.     

Kinetics of subunit dissociation of partially oxygenated hemoglobin determined by haptoglobin binding

Human haptoglobin 1-1 binds very rapidly to hemoglobin dimers but not to tetramers. We have studied the binding kinetics of partially oxygenated Hb A to haptoglobin 1-1. Under the oxygenation conditions used for the measurement of the K₁ of oxygenation ($\text{Hb O}{}_2\text{Hb}\text{≤ 1\%}$, pO₂ ≤ 0.5 mm Hg), the dissociation kinetics were found to be 50 times faster than that of deoxy Hb A. This result suggested that the binding of one molecule of oxygen to hemoglobin tetramer changed the quaternary structure of the intersubunit α₁β₂ contact surface.     

Photosynthetic control and photophosphorylation in photosystem II of isolated spinach chloroplasts

Two cycles of photosynthetic control have been observed in isolated spinach chloroplasts in the presence of lipophilic class III electron acceptors, which may accept electrons at PS II. $\text{ADP}\text{O}$ ratios of 0.8 to 0.9 were recorded;rates of oxygen evolution were stimulated by phosphorylating reagents and uncouplers. Addition of the plastoquinone antagonist DBMIB decreased photosynthetic control, oxygen evolution and photophosphorylation. We believe that there is a coupling site associated with PSII which can be rate limiting. Comparison of the $\text{P}\text{2e}$ ratios observed with class I and class III electron acceptors leads us to propose that more than 0.6 and possibly approaching one molecule of ATP can be formed for every pair of electrons transported from water to PSII acceptors.     

A teleost (Tilapia mossambica) gonadotropin that resembles luteinizing hormone.

A purified gonadotropin preparation was obtained from pituitaries of a teleost fish ($\text{Tilapia}$$\text{mossambica}$). This gonadotropin was found to resemble LH in that it behaved identically to mammalian and non-mammalian LHs in several chromatographic systems, and stimulated testerone production in isolated rat Leydig cells. In this assay, specific for LH, the $\text{Tilapia}$ gonadotropin was less potent than mammalian LHs but significantly more active than avian, reptilian or amphibian LHs. The $\text{Tilapia}$ gonadotropin was found to be a glycoprotein; preliminary amino acid composition data show resemblances to both mammalian and non-mammalian LHs.     

Multiple forms of cytochrome P-450: resolution and purification of rabbit liver aryl hydrocarbon hydroxylase.

We describe the resolution and partial purification of two minor forms of cytochrome P-450 from liver microsomes of rabbits treated with 2,3,7,8-tetrachlorodibenzo-$\text{p}$-dioxin. Both forms have different electrophoretic mobilities when compared to the major form of cytochrome P-450 isolated from this source. The two cytochromes show different activities with several substrates. One form is very active in the hydroxylation of benzo($\text{a}$)pyrene when reconstituted with highly purified NADPH-cytochrome P-450 reductase.