Rapid determination of substrate specificity of Clostridium histolyticum beta-collagenase using an immobilized peptide library.

The molecular basis of the substrate specificity of Clostridium histolyticum beta-collagenase was investigated using a combinatorial method. An immobilized positional peptide library, which contains 24,000 sequences, was constructed with a 7-hydroxycoumarin-4-propanoyl (Cop) fluorescent group attached at the N terminus of each sequence. This immobilized peptide library was incubated with C. histolyticum beta-collagenase, releasing fluorogenic fragments in the solution phase. The relative substrate specificity (k(cat)/K(m)) for each member of the library was determined by measuring fluorescence intensity in the solution phase. Edman sequencing was used to assign structure to subsites of active substrate mixtures. Collectively, the substrate preference for subsites (P(3)-P(4)') of C. histolyticum beta-collagenase was determined. The last position on the C-terminal side in which the identity of the amino acids affects the activity of the enzyme is P(4)', and an aromatic side chain is preferred in this position. The optimal P(1)'-P(3)' extended substrate sequence is P(1)'-Gly/Ala, P(2)'-Pro/Xaa, and P(3)'-Lys/Arg/Pro/Thr/Ser. The Cop group in either the P(2) or P(3) position is required for a high substrate activity with C. histolyticum beta-collagenase. S(2) and S(3) sites of the protease play a dominant role in fixing the substrate specificity. The immobilized peptide library proved to be a powerful approach for assessing the substrate specificity of C. histolyticum beta-collagenase, so it may be applied to the study of other proteases of interest.     

Ultrasonographic assessment of the endometrium in rhesus monkeys during the normal menstrual cycle

This study was undertaken to determine whether cyclical changes in the endometrium of the rhesus monkey could be observed by using ultrasound. Three indices of endometrial size were examined: the antero-posterior (or ventro-dorsal), longitudinal, and transverse diameters. Changes in the ultrasonic reflectivity of the endometrium were also assessed. We have attempted to correlate these endometrial parameters with the hormonal status of the animal. Ultrasonography was performed for an average of 12 consecutive days during 19 menstrual cycles. All ultrasonic recordings were normalized to the day of the estradiol (E2) peak (Day 0). We found that the reflectivity of the endometrium was dependent on the stage of the cycle: during the follicular phase, the endometrium appeared less echogenic (darker) compared to the myometrium; in the luteal phase, the endometrium was more echogenic (lighter). During the follicular phase (Days -9 to 0), there was a linear increase in the antero-posterior (p less than 0.001), longitudinal (p less than 0.05), and transverse (p less than 0.001) diameters. In the luteal phase (Days 1-15), no significant changes were observed in these diameters. An estimated endometrial volume (EEV) was obtained by the product of the antero-posterior, longitudinal, and transverse diameters. Each animal observed during the follicular phase (n = 14) exhibited a peak in the EEV, which correlated with the day of the E2 peak (p less than 0.01). From this study, we conclude that the sonographic appearance of the endometrium of the rhesus monkey reflects the cyclical changes that occur during the menstrual cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hemopexin-mediated heme uptake by liver. Characterization of the interaction of heme-hemopexin with isolated rabbit liver plasma membranes

Plasma membranes isolated from rabbit liver retain the ability to interact specifically with heme-hemopexin. In this system, apohemopexin does not compete effectively with heme-hemopexin for binding. The membranes bind heme-hemopexin complexes with high affinity (KD = 6.8 X 10(-7) M) and with an apparent capacity of 2.3 pmol/mg of membrane protein. These membranes also retain the ability to remove heme from heme-hemopexin. The release of heme reaches a plateau after 15-30 min at 30 degrees C and does not involve metabolic energy, proteolysis of hemopexin or pH gradients. The apohemopexin formed is rapidly released from the membranes. The accumulation of heme is saturable and is affected by pH and temperature with maximum uptake occurring between pH 5.5 and 6.5 and at 30 degrees C. Interestingly, much more heme (approximately 25 pmol/mg of membrane protein) is accumulated than hemopexin at saturation, implying that the receptor can turn over several times and that a heme-binding component exists in the rabbit liver plasma membrane.     

The 30-Base-Pair Deletion in Chinese Variants of the Epstein-Barr Virus LMP1 Gene Is Not the Major Effector of Functional Differences between Variant LMP1 Genes in Human Lymphocytes

One group of sequence variants of Epstein-Barr virus is characterized by a 10-amino-acid deletion within the CTAR-2 functional domain of the latent membrane protein, LMP1. A role for this deletion in enhancing the tumorigenicity of the viral oncogene in rodent fibroblasts was recently demonstrated. We examined the effect of this deletion upon LMP1 function in four human lymphoid cell lines by using three natural variants of LMP1: the prototype B95.8 gene and the CAO and AG876 genes, both of which have codons 343 to 352 of the B95.8-LMP1 deleted. These experiments revealed that LMP1-mediated upregulation of CD40 and CD54 was markedly impaired (by 60 to 90%) with CAO-LMP1 compared with B95.8-LMP1. In contrast, the function of AG876-LMP1 was indistinguishable from that of B95.8-LMP1 in two lines and was only slightly impaired in the other two lines. Activation of NF-κB by CAO-LMP1 was not impaired in any of the lines; rather, activation of an NF-κB reporter by CAO-LMP1 was consistently about twofold greater than the activation with B95.8- or AG876-LMP1. Therefore, while the CAO-LMP1 is functionally distinct from the prototype B95.8-LMP1 in human lymphocytes, the 10-amino-acid deletion appears not to be directly responsible. This conclusion was confirmed by using a B95.8-LMP1 mutant with codons 343 to 352 deleted and chimerae of CAO- and B95.8-LMP1 in which the CTAR-2 domains of these genes were exchanged. Sequences outside the CTAR-2 domain were implicated in the distinct functional characteristics of CAO-LMP1 in human lymphoid cells.     

The inhibition of phospholipase A2 by manoalide and manoalide analogues

Manoalide, a natural product from sponge, displays anti-inflammatory activity in vivo. Previous work has shown that manoalide is also a potent covalent inhibitor of the extracellular phospholipase A2 from cobra venom and that the inhibition correlated with a pH-dependent change in manoalide (Lombardo and Dennis (1985) J. Biol. Chem. 260, 7234-7240). Manoalide contains two rings and the opening of either would produce an alpha,beta-unsaturated aldehyde. The cobra venom phospholipase A2 may be able to catalyze the opening or isomerization of one of these rings, raising the possibility that manoalide is acting as a suicide substrate. To ascertain the role of the gamma-lactone ring in the inhibition, we have now investigated a synthetic manoalide analogue, 3(cis,cis-7,10)-hexadecadienyl-4-hydroxy-2-butenolide (HDHB) which contains only the alpha,beta-unsaturated gamma-lactone ring. We have found that the closed and open forms are in rapid equilibrium between pH 4 and 9 with the cyclic form being preferred at acidic pH values and the open cis form preferred at pH 9.5. When the pH is raised above 12, the alpha,beta double bond isomerizes to form trans-HDHB. Once the trans compound is formed, it is stable at all pH values and does not recyclize to the gamma-lactone ring. The observed pKa of 7.7 found for the inhibition of manoalide agrees well with the transition of the closed to the cis form of the gamma-lactone ring. Kinetic experiments with the HDHB compound show that under conditions in which the cis and closed form of the inhibitor are present in equal molar ratios, HDHB is not an irreversible inhibitor, but reversibly competes with substrate. However, the kinetics of this inhibition are complex and do not follow either pure competitive or non-competitive inhibition. The trans-HDHB exhibits similar complex kinetic but is several times more potent. The distinct differences between the behavior of manoalide and HDHB clearly indicate that while the gamma-lactone ring may play an important role in manoalide inhibition, it alone does not produce irreversible inhibition.